MICROFICHE APPENDIX Not applicable.

TECHNICAL FIELD The invention relates to blood serology and in particular relates to recombinant blood group antigens for the detection of antibodies in a sample.

BACKGROUND OF THE INVENTION Although blood transfusion is a common medical procedure, it is a procedure that can be life-saving, if screening procedures to determine compatibility between donor and recipient are ensured so as to result in a minimal immune response. Generally, the recipient's serum is tested for compatibility with the donor's red blood cells prior to transfusion. The recipient's serum may be screened for the presence of anti-red blood cell antigen antibodies, wherein such antibodies may be directed against blood group A, B, Rhesus (Rh), Duffy (Fy), Kell, Kidd, Sex-linked, Lewis, MNS, P, Lutheran, or other antigens. Therefore, for example, serum containing anti- Duffy antibodies will react with red blood cells expressing the corresponding Duffy antigen, but will not generally react with other antigens expressed on the red

blood cell surface unless other antibodies are present in the serum. These antibodies may be of several classes, for example IgM or IgG, with the IgG class being the most clinically relevant.

Fy is a protein found on the surface of red blood cells and other cell types. The entire protein, including its membrane-spanning and intracellular domains, is 336 amino acids in length. Fy is polymorphic, with the two most common forms being Fy (a) or Fy (b). The former results when residue 42 of Fy's extracellular domain is Asp, i. e. Asp42; while Fy (b) results when residue 42 of Fy's extracellular domain is Gly, i. e. Gly42. Individuals possess two copies of the gene. Those who are Fy (a) Fy (b) heterozygotes are tolerant to this variation, but Fy (a) Fy (a) or Fy (b) Fy (b) homozygotes may mount an immune response against the other form of Fy if they come in contact with it. This contact can occur through blood transfusion or through maternal/fetal incompatibility.

Previously pregnant or previously transfused individuals immunized to Fy may mount a vigorous immune response in a blood transfusion that will at least reduce the effectiveness of the transfusion or at most result in serious illness or death due to severe systemic hemolysis.

Current technology for detection of anti-red blood cell antigens in patient samples has not changed much in the past forty or more years, and has not kept pace with advances in clinical biochemistry. A currently used technology relates to red cell agglutination assays,

wherein the use of commercial panels of human red blood cells, obtained from contracted individuals with certain patterns of red cell antigens is used, wherein the plasma or serum sample from the patient for whom a blood transfusion is needed is reacted with these cells. Cells and sera are mixed, centrifuged, and washed, in some cases with the addition of another anti-human antibody to help aggregate formation. The final red cell pellet is resuspended in saline, and its visual behaviour as it is resuspended is assessed by the operator (i. e. does it stream like grains of sand, or are there clumps visible).

In such current techniques, a qualitative assessment is made based on the operator's experience and, in some cases, based on reference to standardized photographs, with the experiment being made on several dilutions of plasma. The reverse test is also done, with recipient red cells and commercial human serum or plasma containing anti-red cell antibodies as the source of antibody.

Several disadvantages relating to this known method of detection are encountered. In particular, the accuracy of the test score is highly operator-dependent and requires considerable training. Therefore, an experienced technician is required, and the assessment of the test is subjective to that technician. In addition, there is no final record of the test apart from the technician's score, therefore, the test result is not archived for later reference. Moreover, such tests are not sensitive because they rely on the ability of the antibodies (in some cases aided by the addition of other reagents) to provoke a visual change in the blood. The

absence of such a change does not guarantee the absence of antibodies. The shortcomings of the present technology could have serious repercussions, as described in paragraph 0005. Other disadvantages to such a test relate to the need for repeated tests for different red cell antigens. This becomes a difficult and time consuming task in multiply transfused individuals, who may make antibodies against several red cell antigens, and therefore require blood transfusions tailored to their specific antibody profile.

Description of General Prior Art A general view of prior art is provided, wherein the documents noted relate to the general state of the art, and are not considered to be of particular relevance to the present claimed invention. Yazdanbakhsh et. al (Yazdanbakhsh K, Oyen R, Yu Q, Lee S, Antoniou M, Chaudhuri A, Reid ME. High-level, stable expression of blood group antigens in a heterologous system. Am J Hemato. 2000 Mar; 63 (3): 114-24) describe the limitations of current technology for detecting blood group antibodies in plasma or serum samples. The approach of this paper was to express Duffy, Kell, and Knops antigens as full-length membrane proteins in mouse erythroleukemic cells, generate stable cell lines, and use them to detect patient alloantibodies. A significant deficiency with this approach is that flow cytometry was used to perform these detections. This is an expensive and complicated technology and it is difficult to see how such an impractical test could be introduced into a clinical laboratory without extensive miniaturization and cost

reduction. Other groups, in studies cited in this report, described expression of Fy in other mammalian cell lines, and have found either similar limitations to those relevant to the Am J Hematol publication, or employed transient expression. In the latter approach, cells must be transfected with Fy-encoding DNA every time they are cultured, and this approach is even more limited in terms of testing applications. The present invention overcomes the limitations of the prior art.

Wasniowska et al (Wasniowska K, Czerwinski M, Jachymek W, Lisowska E. Expression and binding properties of a soluble chimeric protein containing the N-terminal domain of the Duffy antigen. Biochem Biophys Res Commun.

'2000 Jul 5; 273 (2): 705-11) report a less complicated and expensive route of expressing the Fy (a) and Fy (b) antigens in bacteria, wherein residues 3-60 of Fy (a) and Fy (b) was expressed using a bacterial signal peptide, residues 104-131 of human glycophorin A, and a hexahistidine tag, joined in order from N-to C-terminus.

This approach resulted in expression of the chimeric protein in the periplasm of, bacteria. Yields were not mentioned, but are expectedly less than the yields achieved in the presently provided approach. While the authors showed that monoclonal antibodies to Fy, or, in one case, to Fy (a) detected the proteins when immobilized on microtiter plates, they did not test patient alloantibodies. Their focus was on another aspect of Fy biology, its role as the Duffy Antigen Receptor for Chemokines (DARC), another name for the protein. Indeed, this role, and another pathological role as the receptor

allowing entry of the malarial Plasmodium pathogens, accounts for the vast majority of work referencing. Fy.

These references are cited for a general view of the relating general art, and are not considered to be of particular relevance to the claimed invention presently provide.

For example, USPA 20020193407 (Ronald M. Kim et al. , December 19,2002, entitled"Modulators of CCR5 chemokine receptor activity") or USPA 20020169305 (Kim Lee Sim et al. , November 14,2002, entitled"Binding domains from Plasmodium vivax and Plasmodium falciparum erythrocyte"mention the Duffy protein, but not in the context of its detection in transfusion medicine. USP 6,303, 390, and USP 5,213, 963 (Den Boer et al, October 16, 2001) entitled"Method for antigen and antibody determination in blood group serology"and USP 5,213, 963 relate to antibody determination in blood group serology, however, these applications specifically mentioned the use of intact red blood cells, and are additionally substantively different from the developments of the present invention. USP 6,017, 721, granted to Butz on January 25,2000, is entitled"Chromatographic method and device for preparing blood serum for compatibility testing"and USP 5,665, 558 describe a procedure added-on to the conventional testing described above. USP 5,985, 543 (Siegel, November 16,1999) does use a recombinant protein approach, but employs a recombinant antibody on the surface of a bacteriophage, and the approach provided is another red-cell-dependent approach that is clearly distinct from the embodiments of the

present invention. USP 5,776, 711 (Vyas et al. , July 7, 1998) entitled"Simultaneous human ABO and Rh (D) blood type or antibody screening by flow cytometry"is distinct and different from the present invention, and additionally relates to the Duffy antigens by flow. USP 5,683, 696, (Pogo et al. on November 4,1997) entitled "Cloning of the Duffy blood group antigen, gpD"and USP 5,578, 714 are different from the teachings of the present invention, and specifically relate to the gpD protein, which is the major subunit of the Duffy blood group antigen, and the use thereof in the detection and treatment of malaria. And moreover, USP 4,560, 647 and USP 4,328, 183 relate, for example, to the detection of antigen-antibody solutions in fluid, and do not relate to recombinant antigens, the solid-phase detection of blood groups and blood compatibility, but are specifically dependent on the use of red cells.

The present invention overcomes the deficiencies. in the prior art. In particular, the absence of antibodies in the recipient of a blood transfusion is essential to the success of that transfusion. This is the basis of the well-known need for ABO and Rh compatibility in blood transfusion. The less known red blood cell antigens can also complicate blood transfusion. While a severe acute hemolysis reaction is a possibility, a delayed hemolytic reaction is more common. The transfused blood will be destroyed, creating the need for another transfusion, which will be ineffective unless it lacks the antigen in question. These problems are particularly relevant to multiply-transfused individuals, such as

patients with sickle cell anemia, thalassemia, or, more commonly, forms of cancer. In general, the greater the numbers of transfusions, the more likely the formation of antibodies to different blood group antigens. This creates a problem for hospital blood banks, who have the primary responsibility for transfusing a matched product, and for transfusion services, who are called upon to provide increasingly rare products matching the recipient's red cells.

As. described above, the current invention overcomes deficiencies of the prior art, including the deficiencies of subjectivity and data storage. In the case of a patient with multiple antibodies, adsorption and various complicated tests, involving, for instance, varying the temperature of the assay may be required to tease out the patient's antibody profile using the currently known agglutination tests. In the case of Fy, the situation is further complicated by the fact that the Fy antigen in the commercial test red cells is labile, and is lost over time through the action of trypsin-like enzymes on the test cells.

The present invention overcomes the deficiencies of the prior art.

SUMMARY OF THE INVENTION The present invention provides novel recombinant red blood cell antigens. More specifically, the present invention provides novel recombinant Duffy antigens, namely recombinant Fy (a) and Fy (b) antigens. And more

particularly, the present invention provides Fy antigens wherein said Fy antigens of the present invention comprise the extracellular domain (ECD) of the Fy antigen, and more specifically, amino acid residues 1-65 of the Fy ECD or a functional fragment thereof.

Functional is intended to mean capable of being recognized by anti-Fy (a) or anti-Fy (b) antibodies.

The present invention comprises sequences relating to: SEQ ID NO : 1, nucleotide sequence of Fy (a) ECD (1-65), also noted in Fig. 2A, or any nucleotide sequence encoding a functional Fy (a) ECD antigen or antigenic fragment thereof.

SEQ ID NO : 2, nucleotide sequence of Fy (b) ECD (1-65), also noted in Fig. 2B, or any nucleotide sequence encoding a functional Fy (b) ECD antigen or antigenic fragment thereof.

SEQ ID NO : 3, nucleotide sequence of GST-Fy (a) ECD (1-65), or any nucleotide sequence encoding a functional GST- Fy (a) ECD fusion protein antigen or fusion antigenic fragment thereof.

SEQ ID NO : 4, nucleotide sequence of GST-Fy (b) ECD (1-65), or any nucleotide sequence encoding a functional GST- Fy (b) ECD fusion protein antigen or fusion antigenic fragment thereof.

SEQ ID NO : 5, nucleotide sequence of GST-Fy (a) ECD (36-52), or any nucleotide sequence encoding a functional GST- Fy (a) ECD fusion protein antigen or fusion antigenic fragment thereof.

SEQ ID NO : 6, nucleotide sequence of GST-Fy (b) ECD (36-52), or any nucleotide sequence encoding a functional GST- Fy (b) ECD fusion protein antigen or fusion antigenic fragment thereof.

SEQ ID NO : 7, protein sequence of Fy (a) ECD (1-65), also noted in Fig. or any functional antigenic protein fragment thereof.

SEQ ID NO : 8, protein sequence of Fy (b) ECD (1-65), also noted in Fig. 3B, or any functional antigenic protein fragment thereof.

SEQ ID NO : 9, protein sequence of Fy (b) ECD (36-52), also noted in Fig. 3B, or any functional antigenic protein fragment thereof.

SEQ ID NO : 10, protein sequence of Fy (b) ECD (36-52), also noted in Fig. 3B, or any functional antigenic protein fragment thereof.

SEQ ID NO : 11, protein sequence of GST-Fy (a) ECD (1-65), or any functional antigenic fusion protein fragment thereof.

SEQ ID NO : 12, protein sequence of GST-Fy (b) ECD (1-65), or any functional antigenic fusion protein fragment thereof.

SEQ ID NO : 13, protein sequence of GST-Fy (a) ECD (36-52), or any functional antigenic fusion protein fragment thereof.

SEQ ID NO : 14, protein sequence of GST-Fy (b) ECD (36-52), or any functional antigenic fusion protein fragment thereof.

The present invention embodies isolated nucleotide sequences in accordance with SEQ ID NOs : l-6, or said sequences in a vector suitable for expression thereof. The present invention also embodies all protein, protein fragments, fusion proteins, and fusion protein fragments that immunogenic fragments capable of binding their respective anti-Fy (a) or anti-Fy (b) antibodies specifically, for the detection of said antibodies in a sample.

The present invention embodies purified protein sequences in accordance with SEQ ID NOs : 7-14, or immunogenic protein fragments thereof. The present

invention also embodies the immobilization of said proteins, protein fragments, fusion proteins, and fusion protein fragments onto an immobilization medium, wherein said proteins are immunogenic antigens capable of binding their respective anti-Fy (a) or anti-Fy (b) antibodies specifically, for the detection of said antibodies in a sample.

The present method also provides a method of expressing proteins in accordance with the present invention. In a preferred embodiment, said proteins, and more preferably, said fusion proteins, i. e. GST-Fy (a) or GST-Fy (b) or any functional antigenic fragment thereof may be expressed in a bacterial system. The proteins and more preferably the fusion proteins of the present invention may also be expressed in a yeast system, wherein RSA may the expression protein fused thereto.

The present invention therefore also embodies any immobilization medium, or solid medium, comprising any of the proteins of the present invention, namely the proteins of SEQ ID NOs : 7-14, or any functional antigenic fragments thereof. In a preferred embodiment, said immobilization media may be an immunoblot, or a microtitre well, or any surface that allows for the irreversible adherence of said proteins thereto, for the purpose of detection of anti-Fy (a) or anti-Fy (b) antibodies specifically in a sample. In another preferred embodiment, said immobilization media may also comprise any other red blood cell antigen or fragment thereof, or fusion product thereof, wherein said fusion product may

be a GST- (red blood cell) antigen fusion or fragment thereof, such that an immobilization media in accordance with the present invention will allow for the detection of a plurality of anti-red blood cell antibodies, including anti-Duffy antibodies in a sample.

The present invention also provides a method of immobilization of the proteins of the present invention, namely the proteins of SEQ ID NOs : 7-14, or any functional antigenic fragments thereof onto an immobilization medium. Said method of immobilization may comprise immobilization on microtitre well plate, for use in ELISA, or on an immunoblot membrane for use in Western blotting.

The present invention additionally provides a method of detecting antibody in a sample, and more particularly anti-Duffy and/or anti-red blood cell antibodies in a sample, wherein the immobilized proteins of SEQ ID NOs : 7-14, or any functional antigenic fragments thereof, and/or any other red blood cell antigen or fragment thereof, or fusion product thereof, i. e. a GST- (red blood cell) antigen fusion or fragment thereof, specifically bind antibodies if present in said sample. For example, if said sample comprises human anti- Fy (a) antibody, and no human anti-Fy (b) antibody, the human anti-Fy (a) antibody will specifically bind to the respective antigenic protein,, i. e. GST-Fy (a) ECD (1-65), GST-Fy (a) ECD (36-52), or fragment thereof immobilized to the immobilization medium, wherein a second non-human anti-human IgG antibody conjugated to a visualization

agent is allowed to bind to said bound human anti-Fy (a) antibody, and the visualization allows for the detection of said binding and the confirmation of the presence, or absence, of said antibody in the test sample.

The present invention therefore provides a test kit for the detection of anti-Fy (a/b) antibodies, and/or the detection of any anti- (red blood cell) antibodies from a test sample. A test kit in accordance with the present invention preferably comprises an immobilized media in accordance with the present invention, i. e. an immunoblot or an microtitre well comprising any proteins of SEQ ID NOs : 7-14, or any functional antigenic fragments thereof, and/or any other red blood cell antigen or fragment thereof, or fusion product thereof.

For an ELISA-based, or immunoblot-based (Western) kit in accordance with the present invention, there is also provided a Blocking Buffer, Washing Buffer, a primary antibody, a secondary antibody,'an non-human anti-human antibody, and a visualization buffer.

And more preferably, the. present invention provides novel recombinant Fy (a) and Fy (b) fusion proteins, wherein the extracellular domain (ECD), or a functional fragment thereof, of the Duffy protein is joined to an expression protein to form a fusion protein antigen of the present invention. More specifically, residues 1-65 comprising the ECD of the Duffy protein, i. e. the ECD of either the Fy (a) or Fy (b) antigen, such that the ECD or a functional fragment thereof is joined in-frame to an expression protein that is well expressed

in bacteria, and preferably in E. coli, wherein said expression protein is preferably glutathione sulfotransferase (GST). In another preferred embodiment, a functional fragment may be residues 36-52 of the Duffy protein; wherein Fy (36-52) or Fy (36-62) may be an antigen of the present invention, and Fy (36-52) or Fy (36-62) fused to an expression protein may be a fusion protein in accordance with the present invention. Similar Western and ELISA results were obtained for Fy (36-52) (results not shown) as were obtained for the Fy (1-65) results provided in Fig. 4 and Fig. 5. Depending on the protein expression and purification system used, the expression protein that may be fused to the Fy ECD to form a fusion protein according to the present invention may be any suitable expression protein. For example, if a yeast expression system is used, then rabbit serum albumin (RSA) may be an expression protein that may be fused to the Fy ECD to form, for example, RSA-Fy (a) or RSA-Fy (b).

Similar results were obtained for these fusion proteins (results not shown) as were obtained for the fusion proteins provided in Fig. 4.

Accordingly, in a preferred embodiment, the present invention provides a recombinant preferred fusion protein, wherein a fusion protein of the present invention comprises the 228 residues of GST joined in- frame to residues 1-65 of Fy (a) (i. e. G42) or a functional fragment thereof, or to residues 1-65 of Fy (b) (i. e. D42) or a functional fragment thereof, which may be referred to herein as GST-Fy (a) or GST-ECDFy (a), and GST- Fy (b) or GST-ECDFy (b), respectively. Therefore, as noted

above, the present invention is not. limited to the fusion of residues 1-65 or functional fragments thereof of a Fy antigen to GST, wherein the present invention contemplates the fusion of residues 1-65 or functional fragments thereof of a Fy antigen to any expression protein that would facilitate the expression, purification and immobilization of the fused antigen, provided that the Fy antigen retains its binding affinity and specificity to its corresponding antibody.

For the purposes of the present specification, the following definitions are provided: Blood group antigen is intended to mean any protein or peptide antigen present on the surface of a red blood cell.

An anti-blood group antigen antibody is intended to mean an antibody which specifically recognizes and binds to an antigen present on the surface of a red blood cell. An anti-Fy antibody is intended to mean an antibody that specifically recognizes and binds to an Fy antigen.

Fy (a) antigen is intended to mean an Fy antigen comprising a Gly residue at residue 42 of Fy's extracellular domain. Fy (b) antigen is intended to mean an Fy antigen comprising a Asp residue at residue 42 of Fy's extracellular domain. It should be noted that the present invention identifies the variant residue in the Fy antigens as being at residue 42, rather than at, for example residue 44, since most immunohematologists refer to the more abundant mRNA transcript of Fy rather than

that originally reported by S. Iwamoto et al (Blood 1995; 85: 622-626). For the purposes of the present specification, an Fy (a) or Fy (b) antigen according to the present invention comprises the ECD of Fy (a) or Fy (b), respectively, wherein the ECD of Fy (a) or Fy (b) comprises amino acids 1-65 of the Duffy antigen. The present invention is not limited to an Fy antigen comprising all 65 amino acids of the ECD, and additionally contemplates functional fragments thereof, wherein said Fy antigens are fragment antigens that specifically bind corresponding anti-Fy antibodies with an equivalent or greater affinity than wild type Fy antigen.

GST-Fy (a) antigen is an Fy (a) antigen of the present invention fused in-frame to glutathione sulfotransferase (GST), and more specifically residues 1- 228 of GST. GST-Fy (b) antigen is an Fy (b) antigen of the present invention fused in-frame to GST, and more specifically residues 1-228 of GST. Although GST is a preferred fusion partner in the'present invention, the fusion antigens of the present invention are not limited to GST, and may be any fusion partner that allows for the expression, purification. and immobilization of Fy antigen in accordance with the preferred embodiments of the present invention.

Functional fragment is intended to mean serologically active peptide fragments capable of being recognized by antibody, and more preferably anti- (blood group antigen) antibodies, and more preferably, anti- Fy (a) or anti-Fy (b) antibodies. For example, the present

invention is not limited to an Fy antigen comprising all 65 amino acids of the ECD, and additionally contemplates functional fragments thereof, wherein said functional fragments are antigenic that specifically bind corresponding anti-Fy antibodies with an equivalent or greater affinity than wild type Fy antigen. For example, a Fy (a) functional fragment may comprise a fragment of Fy's ECD, for example residues 15 to 55, or residues 36- 52, so long as the fragment antigen retains or exceeds its antigenic specificity and affinity.

Blood group antigen is intended to mean the set of cell surface antigens found chiefly, but not solely, on blood cells. There exist many different blood group antigens on human blood cell surfaces, for example A, B, Rhesus (Rh), Duffy (Fy), Kell, Kidd, Sex-linked, Lewis, MNS, P, Lutheran, or any other known blood group antigens. Although not limited to human blood group antigens, according to preferred embodiments of the present invention, blood group antigens are preferably human blood group antigens. For the purposes of the present specification, any blood group antigen, and more particularly, any protein blood group antigen, displayed on the surface of red blood cells may be an antigen of the present invention. Wherein the extracellular domain, i. e. the region that is expressed on the surface of the red blood cell, ideally comprises a peptide antigen of the present invention, such that the ECD region of any blood group antigen may comprise a peptide antigen of the present invention. Moreover, the ECD region of any blood group antigen may be fused to an expression protein to

yield a fusion I antigen according to the present invention.

Immobilization medium is intended to mean any solid surface onto which Fy antigens of the present invention or Fy fusion antigens of the present invention may be irreversibly bound thereon, wherein irreversibly bound indicates that about 99% of the bound antigen is very tightly bound and will not be easily released from the immobilization medium. For example, it is known that protein irreversibly binds to nitrocellulose. For the purpose of the present specification, the immobilization medium may be any solid support matrix, such as a nitrocellulose membrane (which may be referred to herein as simply an immunoblot membrane), nylon membrane or combinations thereof, PVDF, microtitre plate well, or latex bead. The immobilization medium comprises bound antigen or fusion antigen, and more preferably binds GST- Fy (a) and GST-Fy (b) fusion antigens, and is used for the detection of anti-Fy antibodies in a test sample. The immobilization medium is a product of the present invention, and is a preferred component of a diagnostic or detection kit of the present invention.

Visualization agent is intended to mean any protein or enzyme capable of converting a colorless substrate into a colored product, or an unvisualized substrate in a visualized substrate, for the purpose of detecting antibodies bound to the recombinant blood group antigens described in the present invention. For example, a visualization agent may be a chromogenic or

chemiluminescent agent, or any agent that is capable of emitting light or colour.

Primary antibody is intended to mean an antibody which reacts specifically with an antigen or fusion antigen in the context of the present invention. A primary antibody can be a human antibody specific to a human red blood cell surface antigen, which may be the antibody present in a human plasma or serum sample being tested according to the present invention. Moreover, a primary antibody can also be an antibody specific to the expression protein fused to an antigen, for example an anti-GST antibody, as described below, which would then accordingly be used in control reactions.

Secondary antibody is intended to mean any antibody used to detect a primary antibody. Secondary antibodies are specific to the constant regions of primary antibodies and are obtained from a species different from that of the primary antibody. For example, in a test human sample wherein human anti-Fy antibody complexed to immobilized Fy antigen according to the present invention, a secondary antibody may be an non- human anti-human antibody which would bind the constant region of any complexed anti-Fy antibody. For example, in a control sample, a secondary antibody may be a antibody that binds the constant region of, for example, complexed anti-GST antibody. In accordance with preferred embodiments of the present invention, a secondary antibody is preferably conjugated to a visualization agent.

Chromogenic substrate is intended to mean a colorless substrate that is converted into a colored product by the action of a visualization agent.

Chemiluminescent substrate is intended to mean a substrate which does not emit light until acted upon by a visualization agent.

GST 1-228 is intended to mean the 228 amino acids of Schistosoma japonicum glutathione sulfotransferase encoded by the pGEX-5X-1 expression vector when used as described below, where these encoded amino acids terminate with the heptapeptide IEGRGIP joined in frame to a peptide antigen according to the present invention, for example, to Fy amino acids 1-65 as described below.

The present invention also provides a method of producing and purifying fusion antigens of the present invention, and more preferably GST-Fy (a) and GST-Fy (b) fusion antigens, wherein said method comprises a glutathione-Sepharose affinity column of immobilized glutathione that was used to purify GST, GST-Fy (a) and GST-Fy (b).

The present invention provides the use of the purified recombinant protein antigens or fusion antigens of the present invention on solid surfaces or immobilization media, such as immunoblot membranes of nitrocellulose or nylon, PVDF, microtitre plates or latex beads. In a preferred embodiment, immobilized recombinant proteins GST-Fy (a) and GST-Fy (b) are used to probe human

plasma samples for the specific and sensitive detection of anti-Fy (a) and anti-Fy (b) antibodies in a sample.

The present invention provides a method for the production and purification of recombinant antigens and fusion antigens of the present invention, wherein said method comprises: design of oligonucleotide primers appropriate for Polymerase Chain Reaction (PCR)-mediated amplification of Fy antigen-encoding DNA; PCR-mediated amplification ; ligation of the Fy antigen-encoding DNA into an expression vector; generation of transformed clonal bacteria harbouring the Fy DNA-inserted expression vector; induction of synthesis of the Fy-containing fusion protein by the bacteria; lysis of these bacteria; the purification of the Fy-containing fusion protein using immobilized glutathione affinity chromatography. An expression system of the present invention may comprise a bacterial expression system, preferably, using E. coli strain BL21, or a yeast expression system, preferably using Pichia pastoris strain X-33.

The present invention provides a method of producing an immobilization medium wherein said method comprises: the irreversible immobilization of recombinant antigens or fusion antigens onto an immobilization medium, wherein said immobilization comprises: (1) SDS- polyacrylamide electrophoresis of one or more proteins of the present invention; (2) equilibration of gel and membrane in Tris/Glycine/Methanol transfer buffer; (3) electroblotting in a wet cell transfer apparatus at sufficient power and time to transfer the

electrophoresed proteins onto an immobilization membrane; (4) blocking of the immobilization membrane using a blocking buffer; (5) drying of the membrane; (6) and storage of the membrane in a state suitable for later use. It should be noted that step running an SDS gel is not absolutely necessary when preparing an immobilization medium of the present invention. An immobilization medium of the present invention may be made simply by dotting antigen according to the present invention onto a solid surface, as would be the method of immobilization in a Dot-Blot or Slot-Blot or ELISA procedures. Moreover, it is also contemplated that the antigen can be denatured with SDS and then dotted on a solid surface or can simply be not denatured and accordingly dotted on a solid surface. Therefore, the immobilization according to the present invention is not limited to the running of a get, equilibration of said get and subsequent transfer onto a solid medium, such as an immunoblot, and immobilization can be simply effected by placing a peptide according to the present invention in or on a specific solid surface, and allowing the peptide to irreversibly bind to said surface.

As noted above, the immobilization medium of the present invention is preferably a nitrocellulose membrane, but is not limited to nitrocellulose, and may comprise a nylon membrane, a microtitre plate, PVDF, a latex bead or any other immobilization medium that allows for the binding of recombinant antigen or fusion antigen thereto, wherein said antigen is essentially irreversibly bound to said immobilization medium.

The present invention provides a method of detecting the presence of antibodies, and more preferably anti-Fy antibodies in a test sample, and more preferably a human serum or plasma sample, wherein said method comprises: (1) blocking of non-specific binding sites on the immobilization medium with a blocking buffer; (2) incubation of the blocked immobilization medium with a test sample; (3) washing of unbound materials with a wash buffer; (4) subsequent incubation of immobilization medium with a detection and visualization antibody ; (5) washing of unbound materials with a wash buffer; (6) and visualization of immobilization medium with a visualization medium.

In step (1) of the above detection method of the present invention, the blocking buffer may be referred to herein as"Block Buffer", and comprises 5% non-fat powdered skim milk (w/v) in TBS containing 0.05% Tween 20.

In step (2) of the above detection method of the present invention, the immobilized medium is allowed to incubate with a test sample, which may be diluted in a binding buffer, which may be identical to Blocking Buffer for a period of time suitable for the binding of anti-Fy antibodies in the test sample to the immobilized antigens or fusion antigens of the present invention. In preferred embodiments of the present invention, a test sample is preferably a serum or plasma sample, and more preferably a human serum or plasma sample.

In step (3) and step (5) of the above detection method of the present invention, the washing buffer may be referred to herein as"Wash Buffer", and comprises TBS (lOmM Tris-Cl pH 8.0, 150 mM NaCl) containing 0.05% Tween 20.

In step (4) of the above detection method of the present invention, subsequent to the washing off of unbound materials from the immobilization medium, the immobilization medium is allowed to incubate with a detection and visualization antibody for a period of time suitable for the binding of the detection and visualization antibody to anti-Fy antibodies from the test sample complexed to the immobilized antigens or fusion antigens on the immobilization medium surface of the present invention. In a preferred embodiment of the present invention, the detection and visualization antibody of the present invention is an anti-human antibody conjugated with alkaline phosphatase, horseradish peroxidase (HPR), or another chromogenic or chemiluminescent agent. To facilitate the method of the present invention a single detection and visualization antibody is used, that is, an antibody that is capable of binding to complexed anti-Fy antibodies (i. e. to the Fc region of bound antibodies from the test. sample) and is also conjugated with a detection moiety that facilitates the visualization of the bound antibody to the complexed antibody. However, the present invention additionally contemplates the use of more than one antibody for the separate and consecutive detection with one antibody and visualization with a second antibody. In preferred

embodiments of the present invention, the visualization and detection antibody of the present invention is Anti- Human IgG (H+L) antibody, Phosphatase labeled, (Kirkegaard & Perry Laboratories, Inc. ). However, the present invention is not limited to the noted preferred visualization and detection antibody and may be any anti- human antibody conjugated with a visualization agent.

In step (6) of the above detection method of the present invention, subsequent to the washing off of unbound materials from the immobilization medium, the immobilization medium is allowed to incubate with a visualization or detection medium for a period of time suitable for visualization. More specifically, the visualization or detection medium comprises the substrates or enzymes needed to catalyze the visualization moiety to produce colour or emit light.

More specifically, a preferred visualization or detection buffer of the present invention is 100 mM Tris-Cl pH 9.5, 100 mM NaCl, 5 mM magnesium chloride containing 0.165 mg/ml 5-bromo-4-chloro-3-indolyl phosphate and 0.33 mg/ml nitro blue tetrazolium.

Subsequent to the detection and visualization of bound anti-Fy antibodies using the immobilization medium of the present invention, the resulting immobilization medium showing the resulting banding pattern may be stored or archived for future reference. That is, the visualized immunoblot, film exposed by the blot, or an electronic image of the blot may be stored. Accordingly,

the products and methods of the present invention provide a tangible, storable result.

In a preferred embodiment of the present invention, there is provided a method of detecting a plurality of anti- (red blood cell antigen) antibodies on a single immunoblot membrane or a series of immunoblot membranes on which is. immobilized recombinant red blood cell antigen proteins or fusion proteins thereof. In a preferred embodiment, there is provided a method of detecting one, more than one, or all red blood cell antigens, wherein said antigens are selected from the group consisting of A, B, Rhesus (Rh), Duffy (Fy), Kell, Kidd, Sex-linked, Lewis, MNS, P, or Lutheran blood group antigens, or any other known blood group antigens, wherein one, more than one or all known red blood cell antigens may be immobilized onto a single immobilization medium, such as on a single immunoblot membrane at i different specific lanes, or spots, or in different wells on a microtitre plate. The method of detecting a plurality of anti-blood group antigen antibodies using a single immunoblot'of the present invention comprises: detecting the presence of antibodies, and more preferably anti-Fy antibodies in a test sample, and more preferably a human serum or plasma sample, wherein said method comprises: (1) blocking of non-specific binding sites on the immobilization medium with a blocking buffer; (2) incubation of the blocked immobilization medium with a test sample; (3) washing of unbound materials with a wash buffer; (4) subsequent incubation of immobilization medium with a detection and visualization antibody; (5)

washing of unbound materials with a wash buffer; (6) and visualization of immobilization medium with a visualization medium. Detection of a plurality of anti- (red blood cell-antigen) antibodies in a sample is essentially similar to the detection of a single anti- (red blood cell antigen) antibody, except that the results must be assessed in respect to which antigen is present in the various lanes of blot, or wells of a blot.

A control sample should always be run in parallel to the test sample so as to ensure the validity of the test result.

In accordance with the present invention, immobilization media of the present invention can be prepared and stored for later use or sale. More specifically, recombinant antigens or fusion antigens of the present invention or any other red blood cell antigens can be immobilized onto immobilization media of the present invention, more particularly nitrocellulose membranes, nylon membranes or combinations thereof, PVDF, latex beads or any other suitable media. More specifically, immobilized antigens or fusion antigens may be selected from the group consisting of GST-Fy (a) or GST-Fy (b) fusion antigens or functional fragments thereof, or with one or more, or all blood group antigens, namely A, B, Rhesus (Rh), Kell, Kidd, Sex- linked, Lewis, MNS, P, Lutheran, and functional fragments thereof, or other blood group antigens or fusion products thereof. Antigens may be fused to an expression protein, such as GST, wherein said immobilization media comprising at least one of said fusion antigens may be stored for

considerable lengths of time, and then used for the detection of specific antibodies in a sample.

In another preferred embodiment of the present invention, instead of detecting a plurality of anti- (red blood cell antigen) antibodies on a single immobilization medium, there is provided a method of detecting a plurality of anti- (red blood cell antigen) antibodies on a series of immobilization media, such as a series of immunoblot membranes immobilized with various red blood cell antigen fusion protein, wherein an initial immunoblot membrane may comprise the more common red blood cell antigens, such as antigens A, B, Rhesus (Rh), Duffy (Fy), Kell, Kidd, Sex-linked, Lewis, MNS, P, or Lutheran, and'a subsequent immunoblot membrane may comprise other known blood group antigens, such as antigens Diego, Cartwright, Ena, Gerbich, or any other known blood group antigens.

Therefore, another preferred embodiment of the present invention provides a multi-antigen immunoblot membrane comprising a plurality of blood group antigens bound to the immunoblot membrane. More specifically, there are at least 27 red cell membrane proteins that can be immunogenic, with several variable immunogenic sites per protein. Fortunately for patients, most of these antigens are exceptionally rare, however, it is imperative prior to blood transfusions that the presence of antibodies to such antigens be identified.

Accordingly, the present invention additionally provides a single immunoblot membrane comprising at least one,

more than one, or all known red blood cell antigens, or a series of immunoblot membranes comprising different antigens, wherein more common antigens are present on an initial immunoblot, and less common or related antigens are present on subsequent immunoblots, thereby allowing for the sequential detection of anti-red blood cell antibodies in a sample. An initial multi-antigen blot could reasonably include only 9 antigens, for example, antigens Rh, Fy, Kell, Kidd, Sex-linked, Lewis, MNS, P, and Lutheran antigens, may be immobilized on an initial immunoblot. It should be noted that the above noted preferred embodiment is not limited to immunoblot membranes, and the immobilization media of the present invention may be used to detect a plurality of anti- (red blood cell antigen) antibodies in a test sample using a single immobilization media or a series. For example, when the immobilization medium is an immunoblot membrane, the various red blood cell antigens or fusion antigens may be immobilized on different specific lanes or dots on the immunoblot or dot blot. For example, when the immobilization medium is a microtitre well, the various red blood cell antigens or fusion antigens may be immobilized on different specific wells on the microtitre plate.

The present invention simplifies the detection of anti-Fy antibodies and makes possible their detection with greater sensitivity. The present invention preferably provides an immunoblot (or Western blot) approach conceptually familiar to one skilled in the art of blood testing, but previously impossible to apply to

red cell antibody detection due in part to the scarcity of the natural antigens. The present invention also provides an ELISA method for the detection of anti-Fy antibodies. It is also noted that in the testing of numerous plasma samples using both Western blot and ELISA. For some samples, ELISA testing and looking at the ratio. of Fy (a) versus Fy (b) staining was superior to chromogenically visualized using Western Blots, where results were too faint to visualize or chemiluminescent visualization using Western Blots, where there was too much background staining to allow for clear visualization. It is nevertheless noted that testing using Western Blot or ELISA is contemplated by the present invention, and the preferred method of use may depend on the samples being tested. Both Westerns and ELISAs, and any other methods for such testing in the present art, are widely used in clinical bloods labs, for example, in hematology or viral screening.

Summary The present invention therefore provides an isolated nucleotide sequence comprising the nucleotide sequence of a blood group antigen; an isolated nucleotide sequence comprising the nucleotide sequence of a Duffy antigen; wherein said nucleotide sequence encodes the extracellular domain (ECD) of said antigen, or a functional fragment thereof; wherein said blood group antigen is a protein blood group antigen; wherein said nucleotide sequence is fused in-frame to a second nucleotide sequence encoding an expression protein, wherein the nucleotide sequence and second nucleotide

sequence-encode a fusion peptide antigen. Moreover, the present invention also provides an isolated nucleotide sequence comprising the nucleotide sequence of SEQ ID NO : 1, i. e nucleotide sequence of Fy (a); an isolated nucleotide sequence comprising the nucleotide sequence of SEQ ID NO : 2, i. e. the nucleotide sequence of Fy (b); an isolated nucleotide sequence encoding amino acids of the ECD of, Fy (a) or Fy (b) or functional fragments thereof; an isolated nucleotide sequence, wherein said ECD comprises amino acids 1-65 of the Fy (Duffy) antigen; an isolated nucleotide sequence encoding GST-Fy (a) or GST- Fy (b) fusion protein or functional fragments thereof; an isolated nucleotide sequence encoding an expression protein fused in-frame to a protein fragment comprising residues 1-65 of Fy (a) or Fy (b) or equivalent fragment proteins thereof; an isolated nucleotide sequence, wherein the expression protein is GST; wherein GST comprises a 228 amino acid expression protein or a functional fragment thereof.

The present invention also provides an isolated peptide comprising the amino acid sequence of a blood group antigen, or a functional fragment thereof; an isolated peptide comprising the amino acid sequence of a Duffy antigen; an isolated peptide wherein said peptide comprises the extracellular domain (ECD) of said antigen, or a functional fragment thereof; an isolated peptide, wherein said blood group antigen is a protein blood group antigen; an isolated peptide of any blood group antigen, preferably a Duffy antigen, and more preferably a peptide comprising the ECD domain of the Duffy antigen or

functional fragment thereof, wherein said peptide is fused in-frame to an expression protein; a peptide comprising the amino acid sequence of SEQ ID NO : 7, i. e encoding Fy (a); a peptide comprising the amino acid sequence of SEQ ID NO : 8 i. e encoding Fy (b); a peptide encoding amino acids of the ECD of Fy (a) or Fy (b), or an functional fragment thereof; wherein said ECD comprises amino acids 1-65 of the Fy (Duffy) antigen; a peptide encoding GST-Fy (a) or GST-Fy (b) fusion protein or an functional fragment thereof ; a peptide encoding an expression protein fused in-frame to a peptide or a functional fragment thereof; a peptide wherein the expression protein is GST, or RSA, or any expression protein that facilitates the expression, purification and immobilization of the Fy (Duffy) antigen; wherein GST comprises a 228 amino acid expression protein or a functional fragment thereof; peptide comprising the extracellular domain (ECD) of a Duffy antigen or functional fragment thereof; wherein said peptide (SEQ ID' NOs: 7-10) is fused in-frame to an expression protein (for example, in SEQ ID NOs: 11-14); wherein the ECD comprises amino acid residues 1-65 of a Duffy antigen; wherein the ECD region encodes antigen Fy (a) or. Fy (b) or a functional fragment thereof; a purified peptide comprising amino acids of the ECD of Fy (a) or Fy (b); wherein the ECD comprises amino acids 1-65 of a Fy (Duffy) antigen of a functional fragment thereof; a purified peptide comprising amino acids 1-228 of GST fused in-frame to amino acids 1-65 of Fy (a) or Fy (b).

The present invention also provides a plasmid comprising a nucleotide sequence of the present invention; wherein said plasmid is pGEX-5X-1.

The present invention also provides a cell comprising a plasmid according to the present invention; wherein said cell encodes a peptide according to the present invention; wherein said cell is BL21 or DH5alpha.

The present invention also provides an immobilization medium comprising a peptide according to the present invention, wherein said peptide is bound to said immobilization medium; wherein said peptide is irreversibly bound to said immobilization medium ; an immobilization medium comprising a peptide according to the present invention, and any other red blood cell antigen, or an immunogenic fragment thereof, bound to said immobilization medium; an immobilization medium according to the present invention wherein said immobilization medium comprises nitrocellulose, nylon or combinations thereof, PVDF, a microtitre well, a latex bead or any immobilization medium capable of irreversibly binding a peptide according to the present invention in a functionally active form; an immobilization medium comprising a peptide of the present-invention, and any other red blood cell antigen or fusion antigen, wherein said red blood cell antigens are selected from the group consisting of A, B, Rhesus (Rh), Duffy (Fy), Kell, Kidd, Sex-linked, Lewis, MNS, P, Lutheran, or any other known blood group antigens; wherein said red blood cell antigens are fused in-frame to an expression protein;

wherein said expression protein is GST or RSA, or any expression protein that facilitates the expression, purification and immobilization of the peptide of the invention; wherein said immobilization medium is a nitrocellulose immunoblot membrane.

The present invention additionally provides a kit comprising an immobilization medium of the present invention, Blocking Buffer, Washing Buffer, a primary antibody, a secondary antibody, an anti-human antibody, and a visualization buffer; wherein said Blocking Buffer comprises 5% [weight/volume] non-fat skim milk powder in Tris-buffered saline [TBS] supplemented with 0. 05% Tween 20 [TBST]; wherein said Washing Buffer comprises TBST; wherein said primary antibody is an antibody that binds to the immobilized expression protein; wherein said primary antibody is an anti-GST antibody; wherein said primary antibody is a non-human anti-GST antibody; wherein said primary antibody is a chicken anti-GST antibody; wherein said secondary antibody binds to the primary antibody; wherein said secondary binds to an anti-GST antibody; wherein said primary antibody and said secondary antibody are of different species; wherein said secondary antibody is a non-chicken anti-chicken antibody antibody; wherein said secondary antibody is a goat anti- chicken GST antibody; wherein said anti-human antibody is of a non-human; wherein said anti-human antibody is a goat anti-human antibody; wherein said anti-human antibody is an IgG antibody specific to the heavy and light chains of human antibodies (Anti-Human IgG (H+L) antibody, Phosphatase labeled, (Kirkegaard. & Perry

Laboratories, Inc. ) ; wherein said secondary antibody and said anti-human antibody are conjugated to a visualization moiety; wherein said visualization moiety is alkaline phosphatase, horseradish peroxidase or any other chromogenic or chemiluminescent agent; wherein said visualization buffer comprises alkaline phosphatase- activated chromogenic substrates, such as BCIP and NET salts, horseradish peroxidase or other enzymes capable of activating chromogenic or chemiluminescent substrates; wherein said immobilization medium may be visualized by exposure to X-ray film for visualization of the banding pattern; wherein said kit additionally comprises a control medium, wherein said control medium may comprise a control immunoblot membrane, a control well on a microtitre plate, or any control immobilization medium for running a control sample in parallel with a test sample on a test immobilization medium; wherein said control immobilization medium is a duplicate of the test immobilization medium, wherein the method of running a test sample and a control sample are duplicate methods run in parallel, but differ in that the primary and secondary antibodies used in a control sample comprise the primary and secondary antibodies, while a primary antibody in a test sample comprises antibody present in a human serum or plasma sample, and a secondary antibody comprises an anti-human antibody.

The present invention accordingly provides a method of making an immobilization medium according to the present invention, said method comprises: (1) when required, SDS-polyacrylamide gel electrophoresis of one

or more peptides of the present invention; (2) equilibration of gel and membrane in Tris/Glycine/Methanol transfer buffer; (3) electroblotting in a wet cell transfer apparatus at. sufficient power and time to transfer the electrophoresed proteins onto an immobilization membrane; (4) blocking of the immobilization membrane using a blocking buffer; (5) drying of the membrane; (6) and storage of the membrane in a state suitable for later use; or the peptides of the present invention may be placed directly in an immobilization medium or dotted on an immobilization medium or immobilization membrane, and allowed to bind to said immobilization medium; wherein said immobilization membrane or immobilization medium is a nitrocellulose membrane, a nylon membrane, or combinations thereof, or PVDF, or a microtitre well, or a latex bead; wherein electroblotting power and time is approximately 400 mA for 1-2 hours.

A method of making a peptide of the present invention is also provided, said method comprises: (1) design of oligonucleotide primers appropriate for Polymerase Chain Reaction (PCR) -mediated amplification of Fy antigen-encoding DNA; (2) PCR-mediated amplification; (3) ligation of the Fy antigen-encoding DNA into an expression vector; (4) generation of transformed clones harbouring the Fy DNA-inserted expression vector; (5) induction of synthesis of the Fy-containing fusion protein by a cellular expression system; lysis of cells; (6) the purification of the Fy-containing fusion protein using immobilized glutathione affinity chromatography ;

wherein said clones are transformed clonal bacteria ; wherein said expression system is a cellular expression system; wherein said expression system is in bacteria; wherein said expression system is in yeast; wherein said expression system is in E. coli strain BL21; wherein said expression system is in Pichia pastoris strain X-33.

The present invention also provides a method of detecting antibodies in a sample using an immobilization medium of any one of claims 39 to 46, said method comprising: (1) blocking of immobilization medium with a Blocking Buffer; (2) washing of immobilization medium with Wash Buffer; (3) incubation of a sample above the immobilization medium; (4) washing of immobilization medium with Wash Buffer; (5) incubation of immobilization medium with a secondary antibody; (6) washing of immobilization medium with Wash Buffer; (7) visual detection of the bound antibodies using Visualization Buffer; and (8) storage of visualized resulting immobilization medium (test and control samples); wherein said Blocking Buffer comprises 5% non-fat powdered skim milk (weight/volume) in Tris-buffered saline containing 0. 05% Tween 20; wherein said Washing Buffer comprises Tris-buffered saline (10 mM Tris-Cl pH 8. 0,150 mM NaCl) containing 0.05% Tween; wherein said sample may comprise a control or test sample; wherein said control sample comprises a primary antibody; wherein said test sample comprises a serum or plasma sample; wherein said test sample comprises a human serum or plasma sample; wherein said sample is a primary antibody in at test sample; wherein said primary antibody is an antibody that binds

to the fused expression protein; wherein said primary antibody is an anti-GST antibody; wherein said primary antibody is a non-human anti-GST antibody; wherein said primary antibody is a chicken anti-GST antibody; wherein said secondary antibody binds to the primary antibody; wherein said secondary binds to an anti-GST antibody; wherein said primary antibody and said secondary antibody are of different species; wherein said secondary antibody is an anti-primary antibody in a control sample; wherein said secondary antibody is an anti-human antibody in a test sample; wherein said secondary antibody is a non- chicken anti-chicken antibody antibody; wherein said secondary antibody is a rabbit anti-chicken GST antibody; wherein said anti-human antibody is of a non-human species; wherein said anti-human antibody is a goat anti- human antibody; wherein said anti-human antibody is an IgG antibody specific to the heavy and light chains of human antibodies; wherein said anti-human antibody is Anti-Human IgG (H+L) antibody, Phosphatase labeled, (Kirkegaard & Perry Laboratories, Inc; wherein said secondary antibody is conjugated with a visualization agent; wherein said visualization agent is a chromogenic or chemiluminescent agent; wherein said visualization agent is alkaline phosphatase or horseradish peroxidase; wherein said secondary antibody is an alkaline phosphatase non-human anti-human IgG antibody; wherein said secondary antibody is an alkaline phosphatase goat anti-human IgG antibody (H+L) specific (Anti-Human IgG (H+L) antibody, Phosphatase labeled, (Kirkegaard & Perry Laboratories, Inc; wherein said sample is diluted in binding buffer; wherein said Visualization Buffer is 100

mM Tris-Cl pH 9.5, 100 mM NaCl, 5 mM magnesium chloride containing 0.165 mg/1 5-bromo-4-chloro-3-indolyl phosphate and 0.33 mg/ml nitro blue tetrazolium; wherein said. antibodies being bound and detected in a test sample are anti-Fy antibodies; wherein said immobilization medium are. the wells of a microtitre plate; wherein said microtitre. plate may'comprise test wells and control wells; wherein said control well may comprise GST, GST fusion antigen or any control antigen for the verification of the test result; wherein said microtitre plate may comprise wells immobilized with any red blood cell antigen or fusion antigen selected from the group consisting of A, B, Rhesus (Rh), Duffy (Fy), Kell, Kidd, Sex-linked, Lewis, MNS, P, Lutheran, or any other known blood group antigen; wherein said visual detection of bound antibodies is completed using an automated plate reader; wherein said storage and archiving of resulting data printout from the plate reader quantifying the presence and amount of color in each well.

Also contemplated by the present invention is a substrate for determining the presence of anti-Fy antibodies in a sample; wherein said substrate is a peptide according to the present invention. A substrate comprising an immunoglobulin-binding component directed against anti-red blood cell antibodies; wherein said immunoglobulin-binding component is an antigen; wherein said immunoglobulin-binding component is a GST-Fy fusion antigen; wherein the GST-Fy fusion antigen comprises the ECD region of Fy fused in-frame to GST; wherein said substrate is irreversibly bound to a solid medium;

wherein said solid medium is an immunoblot membrane; wherein said solid medium is a nitrocellulose membrane; wherein said peptide is in a soluble or bound form; wherein said method comprises the adsorption of immunologically active peptides of to the present invention to a solid support; wherein said solid support is nitrocellulose, nylon, PVDF or combinations thereof, microtitre well, or a latex bead.

The present invention also contemplates a method of detecting antibodies in a sample, wherein said method comprises conjugation of a visualization agent to a peptide of the present invention, and incubating said conjugated peptides to an immobilization medium comprising antibody.

BRIEF DESCRIPTION OF THE DRAWINGS Further features and advantages of the present invention will become apparent from the following detailed description, taken in combination with the appended drawings, in which: Fig. 1 Schematic diagram of the reactants involved in detecting antibodies in a plasma or serum sample by binding to immobilized GST-Fy (a) or (b) recombinant antigens. This schematic indicates the reagents involved in the detection of antibodies, and more specifically, anti-Fy antibodies in a test sample; wherein the immobilization medium is represented as an immunoblot membrane comprising bound GST-Fy (a) and/or GST-Fy (b) fusion proteins wherein the GST portion of the

fusion protein is bound to the immobilization medium and the ECD 1-65 amino acid Fy portion of the protein or a functional fragment or equivalent thereof is fused to GST and is functionally displayed for binding of corresponding antibody; wherein the sample antibody is identified in Fig. 3 as human anti-Fy (a) or (b) antibody in test sample, wherein said sample is preferably a human serum or plasma sample; and wherein detection of the binding of human anti-Fy antibody to the bound fused antigen is detected using preferably a detection and visualization antibody represented herein as an alkaline- phosphatase conjugated goat-anti-human IgG (H L) antibody, wherein the goat-anti-human IgG antibody binds the constant region of the human antibody complexed to the immobilized antigen, and the alkaline phosphatase allows for the visualization of the bound antibody.

Although Fig. 3 identifies the visualization and detection antibody as alkaline-phosphatase conjugated goat-anti-human IgG, any visualization moiety can be conjugated to any anti-human antibody that binds human anti-Fy antibodies.

Fig. 2, Panel A: SEQ IN NO : 1, the nucleotide sequence of Fy (a) ECD, with added BamHI and XhoI, extracellular domain, used in'this invention. Panel B: SEQ ID NO: 2, the nucleotide sequence of Fy (b) ECD, with added BamHI and XhoI, extracellular domain, used in this invention. The nucleotide sequence of Fy (a) ECD (Panel A), and the nucleotide sequence of Fy (b) ECD (Panel B), with the added BamHI and XhoI, as described below and inserted into an expression vector. The bold and

underlined G in Panel A, and A in Panel B distinguishes the Fy (a) from the Fy (b).

Fig. 3, Panel A: SEQ ID NO: 7, the amino acid sequence of the Fy (a) extracellular domain used in this invention. The IP dipeptide is a portion of the GST protein. The underlined sequence corresponds to the first 9 amino acids identified in the original report of the Duffy gene sequence by S. Iwamoto et al. , (Blood 1995; 85: 622-626). These residues differ in another Duffy mRNA transcript that arises through alternative splicing; in that case, these nine residues are replaced by the hexapeptide MGNCLHR. The residue that defines Fy (a). is bolded and underlined, and is Asp44 using the MASSGYVLQ start, and Asp42 using the MGNCLHR start. Most immunohematologists call the variable residue 42 rather than 44. Panel B: SEQ ID NO: 8 The amino acid sequence of the Fy (b) extracellular domain used in this invention. The IP dipeptide is a portion of the GST protein. The . underlined sequence corresponds to the first 9 amino acids identified in the original report of the Duffy gene sequence by S. Iwamoto et al., Blood 1995; 85: 622-626.

These residues differ in another Duffy mRNA transcript that arises through alternative splicing; in that case, these nine residues are replaced by the hexapeptide MGNCLHR. The residue that defines Fy (b) is bolded and underlined, and is Gly44 using the MASSGYVLQ start, and Gly42 using the MGNCLHR start. Most immunohematologists call the variable residue 42 rather than 44. The amino acid sequence of the Fy (a) ECD (Fig. 3 Panel A) and Fy (b) ECD (Fig. 3 Panel B) corresponding to the nucleotide

sequences of Fig. 4. The amino acid sequences of Fig. 5, Panel A and B identify the Fy (a) and Fy (b) extracellular domain sequence used in the present invention respectively. The IP dipeptide is a portion of the GST protein. The underlined sequence corresponds to the first 9 amino acids identified in the original report of the Duffy gene sequence by S. Iwamoto et al., Blood 1995; 85: 622-626. These underlined residues differ in another Duffy mRNA transcript that arises through alternative splicing; in that case, these nine residues are replaced by the heptapeptide MGNCLHR. The residue that defines and differentiates the Fy (a) from Fy (b) is bolded and underlined, and is Asp44 or Gly44, respectively, using the MASSGYVLQ start, and Asp42 or Gly42, respectively, using the MGNCLHR start. As the latter heptapeptide start is the more abundant transcript, most immunohematologists refer to the variable residue as residue 42, as we have in the present invention, rather than residue 44; nevertheless the sequences identified in Fig. 5 Panel A and B are the Fy ECD amino acid sequences of the present invention, wherein in the preferred embodiment of the present invention the ECD region of Fy is fused to an expression protein, however, the present invention also contemplates the use of functional or equivalent ECD fragments thereof.

Fig. 4, Panel A: Coomassie Blue-stained 10% SDS- polyacrylamide gel (GEL) on which 300 ng of each purified protein preparation identified above each lane, has been resolved. Nitrocellulose blots (BLOT) of identical samples were probed with either (panel B) anti-GST

antibodies (Amersham Biosciences), or anti-Fy (a)- or anti-Fy (b) -containing human plasma samples (panel D and C respectively) obtained from Dominion Laboratories of the kind currently used in blood banks. In conventional titering assays, these were detectable up to a dilution of at most 1: 32 (1: 16 for the anti-Fy (b) ). In the immunoblots shown, the anti-Fy (a) or anti-Fy (b) plasma samples were diluted 1: 200 (50 1 in 10 ml of antibody binding solution containing skim milk powder, Tris- buffered saline and Tween 20). These immunoblots show that the antibodies specifically detected their cognate antigen. In other experiments we have tested dilutions up to 1: 10,000, at which band identification is still visible. Further refinements are likely if chemiluminescent, rather than chromogenic substrates are used for band visualization. As noted above, depending on the sample, ELISA testing may be preferred over chemiluminescent or chromogenic visualization using Western Blots. In addition, the amount of antigen electrophoresed per lane could be increased, since we can easily obtain milligrams of the GST-ECD-Fy proteins from 200 ml overnight cultures of E. coli in a matter of 8 hours work over two days.

Fig. 5, Panel A: Enzyme-Linked Immunosorbent Assay (ELISA) results using commercial anti-Fy (a) antiserum against microtiter plate wells coated with either 1. 0 pg of GST-Fy (a) (blue) or the same amount of GST-Fy (b) (maroon). The average optical density (OD) of signals coming from nine wells without specific coating (0.039) was subtracted from the values shown, which are

the mean of triplicate determinations the standard deviation (shown as an error bar). The ratios on the X- axis refer to the dilution of the antibody; Panel B: ELISA results using commercial anti-Fy (b) antiserum in the same manner described in the legend of Panel A; Panel C: ELISA results from assays identical to those described in Panel A, except a clinical anti-Fy (a) antiserum obtained from Canadian Blood Services was used to probe the immobilized recombinant antigens. Note the difference in scale that arises because this antiserum reacts more strongly than the commercial anti-Fy (a).

When looking at the results of all three graphics in Fig. 2, the results suggest that although the anti- Fy (a) or anti-Fy (b) specific antibodies react with both recombinant antigens, they bind more strongly to their cognate antigen. In other words, anti-Fy (a) binds to GST- Fy (a) more avidly than to GST-Fy (b), and vice versa. By comparing the relative binding of an unknown antibody to both recombinant antigens, a user could identify the antibody using ELISA, especially if using plate reader software capable of calculating ratios. The ease of visual interpretation of the immunoblot method also makes it a preferred embodiment of the present invention.

Accordingly, the use of ELISA or immunoblot analysis of test samples may depend on the test sample itself, wherein one analysis method may be preferred for some samples, while another may be preferred for other samples. It is possible, therefore, that conditions could be optimized which would make detection using ELISA more specific, therefore, based on the presently provided

data, an ELISA format could be used as another preferred method of detection according to the present invention.

Nevertheless, detection using an immunoblot membrane is a preferred embodiment.

It will be noted that throughout the appended drawings, like features are identified by like reference numerals.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT The Duffy protein is a glycoprotein expressed on the surface of erythrocytes (RBCs). The presence of either aspartic acid (D) or glycine (G) at residue 42 defines the Duffy blood group antigen system Fy (a/b), and complicates blood transfusion to Duffy-alloimmunized recipients. Current procedures to detect and identify anti-Fy and other blood group antibodies are subjective, qualitative, and require special training. The present invention, expresses truncated forms of the Duffy protein containing either the Fy (a) or Fy (b) antigen in transformed bacteria, thereby improving the sensitivity and reproducibility of detection of anti-Fy (a/b) antibodies.

The present invention provides a specific and sensitive method of detecting'antibodies in a patient sample, wherein strongly reactive antibodies at high dilution are easily detected, thereby reasonably predicting the detection of less abundant antibodies at lower dilution.

The present invention provides a novel way of producing proteins, and more preferably, fusion proteins that react with human antibodies directed against blood group antigens, and more preferably against the Duffy blood group antigen (Fy), referred to herein as, Fy antigen, Fy, or Duffy antigen. Fy isoforms, most commonly Fy (a) or Fy (b), differ in a single amino acid residue, specifically at residue 42, wherein Fy (a) comprises Asp42, while Fy (b) comprises Gly42 (i. e. D42G). To aid in the detection of anti-Fy antibodies in sensitized prospective blood transfusion recipients or donors, the present invention provides a recombinant fusion protein comprised of) 228 residues of glutathione sulfotransferase (GST) joined in frame to residues 1-65 of Fy D42 or Fy G42, i. e. Fy (a) or Fy (b), such that the fusion protein provided are GST-Fy (a) [also referred to herein as GST- ECD-Fy (a)] or GST-Fy (b) [also referred to herein as GST- ECD-Fy (b) or GST-Fy (b) ]. Residues 1-65 of Fy form the extracellular domain (ECD) of the protein. GST is a protein well expressed in E. coli, and is therefore well suited for economical and simple protein production.

Nevertheless, other expression systems could be used, and as such other fusion proteins could be used in accordance with the present invention, provided that such fusion proteins allow for the expression of Fy protein capable of binding corresponding antibody. For example, as further noted below, the present invention provides for the use of preferably GST, or thioredoxin. (Invitrogen), maltose binding protein (MBP) (New England Biolabs), DsbA, DsbC or NusA proteins (Novagen).'The fusion proteins were purified on an affinity column. In the case

of the GST fused antigens, an affinity column of immobilized glutathione was used to purify GST, GST- ECDFy (a), and GST-ECDFy (b). Use of the purified proteins on immobilization media that was used to probe human plasma or serum test samples for anti-Fy (a) and anti- Fy (b) revealed that the proteins could be used for specific and sensitive detection of antibodies.

In addition, the present invention provides a simplified objective test that can be automated and archived for future reference. In particular, for incorporation into automated Western blotting, the present invention advantageously provides internal controls in the form of the GST and other Fy antigens on each blot, along with an output that can be archived.

Moreover, currently available commercial red cell panels have a short half-life, in particular for the Duffy antigen, which can be degraded in storage. The present invention further overcomes this deficiency in the prior art, such that an immunoblot of the present invention could be'made and stored for months without loss of efficacy.

In another embodiment of the present invention, other red blood cell antigens could be immobilized on an immunoblot in accordance with the method of the present invention, such that, in addition to the detection of anti-Fy antibodies, other red blood cell antibodies could be detected on a single immunoblot, or a series of immunoblots. For example, we have expressed other red blood cell antigens, such as the JKA or JKB Kidd antigen,

in a system according to the present invention. A preferred embodiment of the present invention would be an immobilization medium, and more preferably an immunoblot, with multiple antigens, wherein a single immunoblot, or a series of immunoblots could be used to detect the presence of several anti- (red blood cell antigen) antibodies. In another preferred embodiment, an ELISA plate with multiple coated stable antigens may also be used to detect the presence of several anti- (red blood cell antigen) antibodies.

Expression and Purification of GST-Fy antigens: The extracellular domain (ECD) of the Duffy protein was PCR-amplified from a commercial pool of human leukocyte DNA (Clontech) using primers 29581 (5'CTAGGATCCC TATGGCCTCC TCTGGGTAT 3') and 29582 (5'CATCTCGAGT CAGGGCAGAG TCATC 5'). The resulting 215 bp product was restricted with BamHI and XhoI and inserted into the corresponding sites of the pGEX-5X-1 plasmid (Amersham).

Candidate subclones were screened for D42 or g42 codons.

Two such clones, directing the synthesis of a fusion protein of 228 residues of glutathione sulfotransferase (GST) joined in-frame to residues 1-65 of Duffy D42 or Duffy G42, were selected. The GST-ECDFy (a) or GST- ECDFy (b) proteins were purified on glutathione-sepharose and used in immunoblotting reactions with commercial plasma containing anti-Fy (a) or anti-Fy (b) antibodies.

The published sequence of the Duffy blood group antigen (Fy) was used to design two oligonucleotide primers, designated 29581 and 29582, for use in the

polymerase chain reaction (PCR). These were purchased from MOBIX, the Molecular Biology Institute (McMaster University). The purpose was to clone the 65 N-terminal amino acids of Fy, those that specify its extracellular domain (ECD). The sequence of the oligonucleotides was 5' CTAGGATCCC TATGGCCTCC TCTGGGTAT 3'and 5'CATCTCGAGT CAGGGCAGAG TCATC 3'. These were employed, using thermostable ThermalAce polymerase (Invitrogen, Carlsbad CA) and standard PCR protocols recommended by the manufacturer, to amplify a commercial, pooled human genomic DNA sample purchased from Roche Diagnostics (Laval, QC). The resulting PCR products were restricted with BamHI and XhoI (MBI Fermentas, Burlington, ON), and gel-purified using a QiaQuick gel extracting kit (Qiagen, Chatsworth, CA). The purified PCR product was ligated between the BamHI and XhoI sites of plasmid pGEX-5X-1 (Amersham Biosciences) using T4 DNA ligase and standard methods of plasmid DNA manipulation and transformation of E. coli strain DH5alpha. Individual colonies were picked, clonally expanded, and characterized by restriction analysis of mini-preparations of plasmid DNA, followed by automated DNA sequencing by MOBIX.

Because the source DNA was obtained from a pool of healthy individuals, the two variant forms of the Fy gene, Fy (a) and Fy (b) were expected to be found in the pool. Fy (a) is the Fy antigen with aspartic acid (D) at residue 42, while Fy (b) is the Fy antigen with glycine (G) at residue 42. Clones harbouring plasmids identical in all respects save at residue 42 were indeed identified and designated pGEX-GST-ECD-Fy (a) and pGEX-GST-ECD-Fy (b).

These plasmids had DNA sequences specifying the synthesis of glutathione sulfotransferase (GST) (228 amino acid residues) fused in-frame to amino acids 1-65 of the Fy (a) or Fy (b) ECD, with an expected molecular mass of approximately 33 kiloDaltons (kDa). The nucleotide sequence of Fy (a) and Fy (b) ECD is shown in Fig. 2 and the corresponding amino acid sequence is shown in Fig. 3.

Purified plasmid DNA from these clones was'then used to transform E. coli BL21, because this strain is superior for protein expression to DH5alpha, which is better for plasmid characterization.

BL21 cells harbouring pGEX-GST-ECD-Fy (a) and pGEX-GST-ECD-Fy (b) were inoculated into 0.5 L of Luria Broth containing 0.05 mg/ml sodium ampicillin and grown at 37 degrees with shaking until they reached an optical density at 600 nm (OD600) of 0.5. The expression of the GST-Fy-ECD fusion proteins was then induced by addition of isopropyl thiogalactoside (IPTG) to 0.1 mM, and the incubation was continued for 3 hours. The cells were harvested by centrifugation. The cell pellet was resuspended in phosphate buffered saline (PBS) supplemented with 1 mM phenylmethylsulphonylfluoride (PMSF) to inhibit proteases and lysed by sonication. The lysate was made 1% in Triton X-100, clarified by centrifugation, and passed over a 1.0 ml affinity column of glutathione-Sepharose (Amersham Biosciences). The column was washed with PBS until no OD280-absorbing material was found in the wash, then eluted with 10 mM reduced glutathione. Protein-containing fractions were pooled, concentrated, dialysed versus 10 mM Tris-Cl pH

7. 4/150, mM NaCl, and stored frozen prior to use in immunoblots, as described below.

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was initially used to characterize the novel recombinant Fy (a)- and Fy (b)- containing proteins. These were identified as the desired proteins based on the following observations: (a) induction with IPTG lead to the appearance of novel 33 kDa proteins in the profile of total bacterial proteins in cells harbouring the Fy plasmids, as compared to a smaller, 25 kDa protein by those harbouring the GST- encoding pGEX-5X-1 ; (b) these 33 kDa proteins, like GST, specifically bound to immobilized glutathione; (c) they reacted with specific affinity-purified chicken anti-GST antibodies raised and purified in our laboratory; and they, unlike the unfused GST, reacted with commercial anti-Fy (a) or anti-Fy (b) in a specific manner (see below). To one skilled in the art, and based on our previous work with recombinant proteins, this combination of DNA, protein and antibody results is considered more than sufficient to identify the recombinant proteins.

However, amino acid sequencing can nevertheless be determined to provide a complete and exhaustive characterization of the recombinant protein. In fact amino acid sequence analysis was completed for several samples, and all forms of fusion protein GST-Fy were confirmed to have the expected N-terminal amino acid sequence, thereby confirming the completeness of the present analysis.

In a preferred use of the present invention, an immunoblot (also known as a Western blot) immobilized with the novel GST-Fy-ECD fusion proteins of the present invention is used for the detection of anti-Fy antibodies in sample, wherein said sample is preferably a serum or plasma sample from a blood transfusion recipient or donor, and more preferably a human sample.

Anti-Fy antibodies present in plasma or serum can be detected using our novel recombinant Fy antigens.

Whole anticoagulated blood or clotted blood can be used as a test sample of the present invention; however the whole anticoagulated blood or clotted blood can only be used if a plasma or serum is prepared by centrifugation and separated from cells or clots prior to use. We used Western blots to demonstrate this finding. We first performed SDS-PAGE under reducing conditions, and electrophoresed 1.0 micrograms of total protein of purified GST-ECD-Fy (b), purified GST, and purified GST- ECD-Fy (a), in that order, in three adjacent lanes, to the left of a pre-stained protein molecular weight marker (Fig. 4). On gels stained with Coomassie Blue, the GST preparation appeared as one band. Both GST-ECD-Fy preparations contained a, major 33 kDa band, and two less abundant 31 and 29 kDa bands. Duplicate gels were electroblotted to nitrocellulose to form blots, using a Hoefer transblotting apparatus set to'0. 4 amps for 1-2 hours, which were then probed with antibodies using standard Western blotting protocols, using 5% non-fat skim milk powder in TBS supplemented with 0.05% Tween 20 (Sigma). All of these bands, in addition to three smaller

bands intermediate in size between 29 kDa and the size of GST, reacted with anti-GST antibodies when probed with 2 micrograms/ml affinity purified chicken anti-GST antibodies (done in parallel, and used as a control to ensure immobilization of fused antigen, and as an internal control to ensure validity of result), then rabbit-anti-chicken antibodies conjugated to alkaline phosphatase (Jackson Labs, Bar Harbour, Maine). The antibodies were then visualized using alkaline phosphatase-activated chromogenic substrates (using bromo-chloro-indolyl phosphate (BCIP) and Nitro-blue tetrazolium (NBT) salts), however other chromogenic or chemiluminescent substrates can be used.

In contrast, only the three bands corresponding to the most abundant GST-ECD-Fy (b) recombinant antigens were detected in immunoblots similar to those described above, but in which the antibody employed was a commercial anti-Fy (b) antibody purchased from Dominion Biologicals Limited (DBL) (Dartmouth, NS). This was diluted 1: 200 in 5% non-fat skim milk powder in TBS supplemented with 0.2% Tween 20 for blotting, and detected in the same manner as that described above for anti-GST, except that goat antibodies reactive against the constant region of all human antibodies, conjugated to alkaline phosphatase were employed.

A preferred method of the present invention for the detection of anti-Fy antibodies in a test sample using an immobilization medium of the present invention,

and more preferably using an immunoblot membrane of the present invention comprises the following method: (1) GST-Fy (a) or GST or GST-Fy (b) is immobilized on an immunoblot membrane (blot), preferably a nitrocellulose membrane after electrophoresis (SDS-PAGE) and electroblotting; (2) the blot is incubated in Blocking Buffer, (wherein said Blocking Buffer comprises 5% [weight/volume] non-fat skim milk powder in Tris-buffered saline [TBS] supplemented with 0. 2% Tween 20 [TBST] ) to block non- specific binding sites; (3) the blot is incubated with a serum or plasma sample (from a patient) that is diluted in Blocking Buffer, the sample may contain anti-Fy (a) or anti-Fy (b) antibodies which are allowed to bind to their corresponding immobilized antigen or fusion antigens on the immunoblot, wherein, anti-Fy (a) antibodies in the sample bind to the immobilized GST-Fy (a) and anti-Fy (b) antibodies in the sample bind to the immobilized GST-Fy (b); (4) the blot is washed with Washing Buffer (TBST); (5) to detect the presence of the bound anti-Fy antibodies, the blot is incubated with a commercially available anti-human antibody that is conjugated to alkaline phosphatase or any other chromogenic or chemiluminescent moiety and diluted in Blocking Buffer; (6) the blot is washed with Washing Buffer (TBST);

(7) bound antibodies-are subsequently visualized using alkaline phosphatase-activated chromogenic substrates such as BCIP and NBT salts. If analogous chemiluminescent substrates are used, the blot must be exposed to X-ray film in the dark in order to generate the final banding pattern. The enzyme and substrates used for detection may also be horseradish peroxidase or other enzymes capable of activating chromogenic or chemiluminescent substrates if they are conjugated to the anti-human antibodies ; and (8) the visualized immunoblot, film or picture thereof may be stored.

In the development of the products and methods of the present invention various antibodies were used. More specifically, we made a chicken anti-GST antibody which we used as a control antibody in the development of the present invention and used in parallel with test samples so as to ensure that immunoblot protocols (i. e. Western procedures) and reagents worked, thereby verifying the validity of the test result. Since the GST and the GST fusion proteins, namely GST-Fy (a) and GST-Fy (b) all comprise a GST portion, the use of anti-GST antibodies was a positive control in which all bands in all three lanes, i. e. GST-Fy (a), GST, and GST-Fy (b), should be detected. In contrast, when running a patient's plasma sample in parallel, the only bands that will light up are the lanes corresponding to GST-Fy (a) or GST-Fy (b) but not to GST. Running a GST blot in parallel to running a. test sample blot ensures that all Western blot procedures and reagents work, thereby providing a control when no signal

on the companion sample blot is visualized, so as to confirm that when a negative test sample is obtained, it is a true negative result and not a false negative.

Goat anti-chicken antibody was also used in the development of the present invention. The Goat anti- chicken antibody bound to the chicken anti-GST antibody complexed to the GST immobilized on the immunoblot. This goat anti-chicken GST antibody would correspond to the goat anti-human Fy antibody that is used in the preferred embodiment of the present invention for testing a patient sample. The goat anti-chicken GST antibody would accordingly be conjugated to a visualization agent.

Accordingly, a secondary antibody will react with all antibodies we made in chicken.

The preferred method of detecting antibodies, and more specifically anti-Fy antibodies in a test sample involved the use of various anitbodies in the of detecting antibody in a test sample. Depending on what anti- (red blood cell antigen) antibodies are present in the sample, the antibodies in the test sample will bind to their corresponding immobilized red blood cell antigens on an immobilization medium, such as an immunoblot, according to the present invention. Since a preferred embodiment of the present invention is a method of detecting anti-Fy antibodies in a test sample, and more preferably, in a human serum or human plasma sample, the anti-Fy antibodies in a test sample would be human antibodies. Therefore, the subsequent use of an anti- human antibody, which would bind to the Fc region of any

human antibody, would allow for the detection of human Fy antibodies complexed to the immobilized fusion antigens of the present invention. In a preferred embodiment, a goat anti-human antibody was used to detect complexed anti-Fy antibodies, but any anti-human antibody that binds human anti-Fy antibodies may be used. To facilitate the detection and visualization of complexed anti-Fy antibodies, it is preferred that the anti-human antibody used be conjugated to a visualization substrate. For example, the Phosphatase labelled Anti-human IgG (H+L) antibody (Kirkegaard & Perry Laboratories, Inc. ) is another preferred antibody used to detect human anti-Fy antibodies complexed to Fy antigen on an immobilization medium of the present invention, the above noted antibody is a goat anti-human antibody.

Exactly analogous results were obtained when a commercial anti-Fy (a) antibody was used; only the recombinant Fy (a) antigens were detected. These results are shown in the Figure 4.

In previous work with the GST expression system, we and others have noted that the proteins produced can be truncated by the action of bacterial proteases. This is the most likely explanation for obtaining multiple bands of a size intermediate between the expected full- length product and GST. Nevertheless, each of the three main bands has GST activity, reacts with both anti-GST and anti-Fy antibodies, and exhibits the expected residues of the N-terminus of GST by amino acid sequencing. Therefore is likely that they are missing

portions of the C-terminal end of the protein. Amino acid sequencing can be used to confirm this. Nevertheless, the three bands provide built-in redundancy and aid in visualizing the test results.

We have obtained similar results using 1: 1000 dilutions of the commercial antibodies, and the chromogenic substrates for developing Western blots described above. We have further been able to reduce the amount of antibody we can detect to 1: 10,000 using chemiluminescent substrates that expose X-ray film when placed next to the blot in a dark room for at most 15 minutes. Chemiluminescent substrates do not emit light until activated by alkaline phosphatase or horseradish peroxidase. Both our initial and latter approaches yield a blot that can be archived, with the X-ray film being more robust than the nitrocellulose. membrane.

The current approach is more sensitive than current tests, in which the ability to detect the same commercial antibodies in red cell agglutination tests, the current standard, was lost at an antibody dilution of 1/64.

We have also used this test to type three clinical samples obtained from our colleagues in Canadian Blood Services. All had initially been typed as anti- Fy (a), with signals lost past a dilution of 1/4 in one case, and 1/64 in two others. The latter two cases were strongly positive in our assay at 1: 1000 dilution, in a specific manner. Our test indicated that the ostensible weak anti-Fy antibodies were undetected. In fact, when

the standard test was repeated, no anti-Fy (a) antibodies were detected, thereby further confirming the sensitivity and specificity of our test, and in our tests the ability to correct an initial false positive that resulted using current technology.

Moreover, as little as 0.3 micrograms or as little as 3.0 micrograms of recombinant antigens per gel lane can be used on the blot with similar results.

Results: The purified GST-ECD-Fy (a) and GST-ECD- Fy (b) protein preparations contained a major 33 kDa band and two minor 31 and 29 kDa products on SDS gels. All three GST-ECDFy (a) or GST-ECDFy (b) bands reacted on immunoblots with anti-GST antibodies. In contrast, commercial anti-Fy (a) or anti-Fy (b) antibody preparations (Dominion Laboratories) bound only the corresponding GST- ECD-Fy (a) or GST-ECD-Fy (b) bands and not the unfused GST (not fused to the ECD-Fy (a) or ECD-Fy (b) antigen), when 300 ng of each protein was electrophoresed, and the anti- Fy (a) or anti-Fy (b) antibodies were diluted up to 1: 1000 and detected using alkaline phosphatase-conjugated goat anti-human antibodies visualised by chromogenic staining.

The same commercial antibodies agglutinated test RBCs if diluted <1: 32.

Therefore, the purified, recombinant Fy (a) or Fy (b) proteins provided a specific tool for the identification of plasma containing anti-Fy (a) and anti- Fy (b) antibodies, with a sensitivity >30X better than standard agglutination assays. Recombinant Fy (a) and Fy (b) proteins may also offer increased reagent stability

compared to test RBCs, which lose Fy (a) or Fy (b) through tryptic digestion in storage.

Therefore, in accordance with the present invention, we have expressed and purified novel recombinant proteins equivalent in function to the Fy (a) and Fy (b) antigens. Human antibodies in patient blood samples specifically bind to these recombinant antigens, allowing them to be detected and typed.

Comparative tests which compared the use recombinant antigens of the present invention in the methods of the present invention and the use of the recombinant antigens of the present invention in known red cell agglutination assays indicated that, because they bind anti-Fy antibodies effectively, it is a reasonable expectation that they could be used to remove anti-Fy antibodies from a plasma or serum sample, thus aiding in the identification of other antibodies present in the same sample by standard agglutination assays.

Accordingly, the recombinant antigens of the present invention may additionally be used to facilitate current known agglutination assays.

In another embodiment of the present invention, the recombinant antigens of the present invention may be used in other detection techniques, such as enzyme-linked immunosorbent assays (ELISAs). Accordingly, an immobilization medium of the present invention comprises microtitre wells. More specifically, we have also immobilized antigens of the present invention on a microtiter plate, the preferred tool for ELISA's. As

indicated in Fig. 4, ELISA may also be used to identify the presence of antibody in a sample, nevertheless, in a preferred embodiment; antibody detection on an immunoblot using Western analysis provides a straightforward visual interpretation of antibody detection. Although the present invention provides for the use of ELISA in the detection of antibody, ELISA conditions may need to be optimized so as to yield strong specific signals or processed to permit easy pattern recognition, for example by ratio calculations. Nevertheless, ELISA analysis is a method of antibody detection contemplated by the present invention.

The usefulness of the ELISA analysis method, with some difficult samples, is apparent on inspection of the data tabulated in Table 1. Samples were obtained from other immunohematology laboratories and their typing was compared to that obtained using the present invention in either blot or ELISA format. In most samples both tests gave the same result. However'calculation of the anti- Fy (a)/anti-Fy (b) ratio correctly identified the Fy antibodies present in several difficult samples containing other antibodies or high background.

Accordingly, although a preferred embodiment of the present assay is in an immunoblot format, an ELISA format or another antibody detection format may also be preferably used, so long as the immobilized recombinant antigens retain their natural ability to, specifically detect corresponding antibody. As in Fig. 5, the results from all three panels suggest that although

the anti-Fy (a) or anti-Fy (b) specific antibodies react with both recombinant antigens, they bind more strongly to their cognate antigen. In other words, anti-Fy (a) binds to GST-Fy (a) more avidly than to GST-Fy (b), and vice versa. By comparing the relative binding of an unknown antibody to both recombinant antigens, a user could identify the antibody. However, the ease of visual interpretation of the immunoblot method makes it the preferred embodiment at present. Nevertheless, optimized ELISA conditions or signal processing would allow for a more specific binding of antibodies to their specific cognate antigens.

As can be seen in Table 1 below, ELISA results are provided from assays identical to those in Figure 5, Panel A, except that eleven samples were obtained from other immunohematology laboratories. Anti-Fy (a) samples have ratios > 1 while anti-Fy (b) samples have ratios less than 1. In most samples, blots and ELISA ratios gave the same, correct result. However in difficult samples 4, C, and F, the ELISA ratio gave the same result as the conventional test from the outside laboratory. These results show that the use of the ELISA ratio is helpful in the minority of cases in which blotting cannot be interpreted.

Table 1 Sample Status Blot Absorbance ratio: [anti- (at storage) Fy (a): anti-Fy (b) ] (1/200) 1 anti-B anti-B 0.2 2 anti-A anti-A 2.8 4 anti-A neither 2.0 6 anti-B anti-B 0.2 7 anti-A anti-A 2.7 A anti-A anti-A 2.4 B anti-A anti-A 2.3 C anti-A- (GST) 1.3 D anti-A anti-A 1.6 E anti-A anti-A 1.9 F anti-A hi bkgd 1. 8 The present invention provides for the preferred expression of recombinant antigen in a bacterial system.

However, other compatible expression systems could be used to express the recombinant antigens of the present invention, so long as the antigens expressed are in a functional form. Examples of other bacterial expression systems are provided above. For example, the use of a yeast system, such as methylotropic yeast Pichia Pastoris (Invitrogen) may be used to express the proteins of the present invention. It is known to one skilled in the art that yeast are well characterized as being better capable of folding human proteins than bacteria. Accordingly, the present invention additionally provides for the expression of the proteins and fusion proteins of the present invention in a yeast expression system, wherein the expression protein fused to the ECD portion of the Fy antigen would be an expression protein suitable for protein expression in yeast. For example the use of

rabbit serum albumin (RSA) in yeast would be an ideal expression protein candidate for fusion to the Fy antigen wherein the use of RSA in yeast is analogous to the use of GST in bacteria. However, the use of a bacterial system provides for a straightforward and inexpensive production of sufficient amounts of antigen for immobilization on many blots. In a method of the present invention, milligrams of the GST-ECD-Fy proteins can easily be obtained from 200 ml overnight cultures of E. coli in 8 hours of work over two days. This amount is sufficient for the preparation of thousands of immunoblots, and scaling-up of this production could easily be achieved to 20 litre scales. Western blots can be stored dry for many months at room temperature, further adding to the potential utility of the present invention The preferred use of the invention is to detect anti-Fy antibodies in blood samples (specifically plasma or serum samples) from individuals to be transfused or from blood donations to be used for transfusion.

In the United States alone there is market for in vitro diagnostics estimated at $9 billion annually, with immunochemistry being the largest sub-market (27.7%) within this group, (Freedonia Group (http://freedonia. ecnext. com). Therefore, it is clear that a test in accordance with the present invention would be of significant importance for in vitro diagnostic tests. Although, the specific use of an anti- Duffy diagnostic test according to an embodiment of the

present invention would have a smaller market, and would be based in hospital or transfusion-service based clinical laboratories, the present invention is not limited to the detection of Duffy antigens, and accordingly may be used to detect other antigens, and more preferably other red blood cell antigens in a sample. In other embodiments of the present invention, the detection of Duffy antibodies, using recombinant Duffy antigens provided, along with other blood group antigens which, along with Duffy, could be incorporated into a multi-antigen immunoblot according to an embodiment of the present invention. Accordingly, the present invention provides an immunoblot-based method that can detect the presence of more than one antibody, wherein anti-Duffy antibodies are at least one of the antibodies being detected, and more preferably several anti-red blood cell antibodies are being detected in a single screening. This method of the present invention accordingly provides an objective process that may be partially or fully automated wherein results are obtained in a tangible storable form.

EXAMPLES EXAMPLE 1 Antibody determination of anti-Fy (a) or anti-Fy (b) antibodies using an immunoblot comprising immobilized Fy (a) or Fy (b) antigens.

The present invention provides an nitrocellulose membrane immobilized with GST-Fy (a) fusion protein, GST- Fy (b) fusion protein, a GST control, and any other control or size marker, wherein each of the immobilized

fusion proteins are irreversibly immobilized onto an immunoblot, which may be referred to herein as an GST- Fy (a) /GST-Fy (b) immunoblot, and for the purpose of this example, simply as the immunoblot or the immobilization medium. The immunoblot is primed according to standard immunoblot procedures (Western protocol). A sample, preferably a serum sample or plasma, and more preferably a human serum or plasma sample that may comprise Fy (a) or Fy (b) red blood cell antigens, is diluted in Blocking Buffer (as specified above or an equivalent blocking buffer) and is allowed to incubate above the immobilization medium at room temperature with agitation so as to allow sufficient time for the binding or complexing of anti-Fy (a) or anti-Fy (b) antibodies from the sample to the immobilized GST-Fy (a) or GST-Fy (b) fusion proteins respectively. Subsequent to binding, the immobilization medium is washed with a Wash Buffer of Tris-buffered saline containing Tween 20 [TBST].

Subsequent to the washing of unbound analytes, the immobilization medium is then incubated with alkaline phosphatase-conjugated anti-human antibodies (commercially available) diluted in Blocking Buffer, which will accordingly bind to the human anti-Fy antibodies that may have specifically bound to the Fy fusion antigens immobilized on the immunoblot. Subsequent to the binding of the commercial anti-human antibodies to the specifically complexed anti-Fy antibodies, the immobilization medium is washed with Washing Buffer, same buffer employed above. Subsequent to the washing of unbound antibodies, visual detection of the bound antibodies is completed with alkaline phosphatase-

activated chromogenic substrates. The final visualized immunoblot membrane may be archived for future reference.

In addition to the immunoblot described above, which is used to test for the presence of anti-Fy antibodies in human serum or plasma, the invention provides a duplicate control blot comprised of at least the same samples described above which will be probed in the same manner, except that chicken anti-GST antibodies will be the control sample that will be allowed to complex to the immunoblot, which would correspond to the test sample step of allowing antibodies, namely anti-Fy antibodies in a test human serum or plasma sample to complex to the immunoblot. The subsequent use of commercial anti-chicken antibodies conjugated to alkaline phosphatase would consequently be employed to detect the binding of bound chicken anti-GST antibodies in the control blot, which would correspond to the test sample step of allowing anti-human antibodies to bind to the human Fy antibodies complexed to the corresponding antigens on the immunoblot described above. The control and test sample should preferably be run in parallel.

Completion of the immunoblot protocol with this control blot, on which all proteins containing GST should be visualized, shall serve as a positive control. As noted above, although chicken anti-GST antibodies and anti- chicken antibodies conjugated to alkaline phosphatase are used in the above described Example, any non-human anti- GST antibody may be used in the control immunoblot, and any detection and visualization antibody corresponding to the anti-GST antibody species may be used in the control

of the present invention. Similarly, any anti-human antibody conjugated to a visualization agent may be used in the detection and visualization step in a test sample.

EXAMPLE 2 Antibody determination of anti-Fy (a) or anti-Fy (b) antibodies using an ELISA protocol, wherein microtitre plates coated with conjugated Fy (a) or Fy (b) antigens.

The present invention also provides a microtiter plate on which has been immobilized GST-Fy (a) fusion protein, GST-Fy (b) fusion protein, a GST control, and any other control marker, wherein each of the immobilized fusion proteins are irreversibly immobilized onto a microtiter plate, referred to for the purposes of this example, simply as the plate. The plate is primed according to standard ELISA protocols, typically by incubation with a Blocking Buffer, similar to the blocking buffer of Example 1. A sample, preferably a serum or plasma sample, and more preferably a human serum or plasma sample that may comprise Fy (a) or Fy (b) red blood cell antigens, is diluted in blocking buffer and is allowed to incubate in the microtiter plate wells so as to allow sufficient time for the binding or complexing of anti-Fy (a) or anti-Fy (b) antibodies from the sample to the immobilized GST-Fy (a) or GST-Fy (b) fusion proteins respectively. Subsequent to binding, the immobilization medium is washed with a Wash Buffer of Tris-buffered saline containing Tween 20 [TBST]. Subsequent to the washing of unbound analytes, the microtiter plate is then incubated with alkaline phosphatase-conjugated anti-human antibodies (commercially available) diluted in blocking buffer, which will accordingly bind to the human anti-Fy

antibodies that may have specifically bound to the Fy fusion antigens immobilized on the microtiter plate.

Subsequent to the binding of the commercial anti-human antibodies to the specifically complexed anti-Fy antibodies, the microtiter plate is washed with the same TBST Washing Buffer employed above. Subsequent to the washing of unbound antibodies, visual detection of the bound antibodies is completed with alkaline phosphatase- activated chromogenic substrates. The final visualized microtiter plate is then read in a plate reader capable of detecting and quantifying the colour produced by the chromogenic reaction, and archiving the results.

As noted in Example 1, a control well on the microtitre plate should be run in parallel with the testing of sample in test wells on the microtitre plate.

Similar control antibodies as those described above may be used in the present example. More specifically, in control wells separately comprising the GST-Fy (a), GST- Fy (b) and GST antigens, chicken anti-GST antibodies may be used to confirm immobilization of antigen to the well, and complexing of anti-GST antibody to the corresponding antigen, and anti-chicken antibodies conjugated to alkaline phosphatase may be used to detect and visualize bound antibody. As noted above, any non-human anti-GST antibody may be used in the control ELISA well, and any detection and visualization antibody corresponding to the anti-GST antibody species may be used in the control of the present invention. Similarly, any anti-human antibody conjugated to a visualization agent may be used in the detection and visualization step in a test sample.

EXAMPLE 3 Antibody determination of a plurality of anti-blood group antigens using an immunoblot comprising a plurality of conjugate blood group antigens.

This invention also provides for an immunoblot on which is immobilized a series of recombinant blood group antigens, including the Fy (a) and Fy (b) antigens described above. The immunoblot is tested in preferably the same manner as described in Example 1, however, the results obtained from a blot comprising a plurality of antigens must be assessed in respect of which antigen is present in which lane of the blot. A control blot is also performed to ensure the validity of the test, as described in Example 1.

EXAMPLE 4 Antibody determination of anti-red blood cell antigens using a microtitre plates coated with a plurality of recombinant blood group antigens.

This invention also provides a microtiter plate on which is immobilized a series of recombinant blood group antigens. The plate is tested in exactly the same manner as described in Example 3, except that its results must be assessed in respect of which antigen is present in which well of the microtitre plate, and control wells should also be performed to ensure the validity of. the test, as described in the above Examples.

EXAMPLE 5 Antibody determination of anti-Fy (a) or anti-Fy (b) antibodies using functionally equivalent Fy (a) or Fy (b) antigens.

The present invention preferably provides Fy (a) and Fy (b) antigens and fusion antigens comprising

residues 1-65 of the Fy ECD or fragments thereof.

Accordingly, in another embodiment of the present invention, there is provided smaller functional or equivalent Fy (a) or Fy (b) antigens or fusion antigens wherein said smaller functional or equivalent antigens are capable of binding anti-Fy antibodies in a sample.

For example, since most synthetic peptides commonly used in research are 30 amino acid residues or less in size, the present invention contemplates the use of smaller or equivalent Fy antigens, instead of using the complete ECD of Fy as the preferred antigen of the present invention.

In the present example of the invention, two peptides, corresponding to residues 36-52 of Fy, inclusive, for both Fy (a) and Fy (b) were synthesized, namely peptides DSFPGDYDANLEAAAP and DSFPGGYDANLEAAP, respectively, where the variable residue 42 is underlined. The use of these smaller Fy antigens, or any other antigens, provided that these smaller antigens are functionally equivalent or superior to the Fy (a) and Fy (b) antigens and fusion antigens of the present invention, is also contemplated in the present invention, wherein said smaller antigens may be made into fusion products, and may be immobilized onto an immobilization medium for the detection of anti- Fy antibodies in a sample. Although most smaller Fy antigens, for example those noted above, may not exhibit the correct protein conformation, and therefore are not embodied in the present invention, the present invention is not limited to Fy antigens comprising the entire ECD region, and includes all smaller functional equivalent or superior peptides. Nevertheless, the preferred Fy antigens of the present invention, namely the Fy (a) and

Fy (b) antigens comprising residues 1-65 of the ECD are the preferred antigens of the present invention.

EXAMPLE 6 Recombinant antigens made in yeast We used a Pichia pastoris expression system (Invitrogen), to express the recombinant antigens in yeast so as to obtain large quantities of appropriately folded fusion proteins, when a small protein or protein domain is fused to albumin has also been examined. We cloned and used rabbit serum albumin (RSA) as the fusion protein partner, analogous to the use of GST in bacteria.

P. pastoris can glycosylate proteins, and there are two sites of glycosylation in the Fy ECD, namely Asparagine 29 and Asparagine 35. Using a glycosylated fusion protein might improve detection, as the recombinant antigen more closely resemble its natural counterpart. If the RSA fusion performs equivalent to the GST-Fy-ECD proteins, then this will substantiate the use of RSA as a fusion partner, and the use of other equivalent fusion partners.

For example, as we have done with several other RSA fusion proteins, the RSA protein may be fused to an Fy -antigen of the present invention, for example as His6- RSA2-590-Fyl-65, and the RSA fusion protein may be purified on a nickel affinity chromatography.

In additional embodiments of the present invention, in addition to the use of GST, other fusion partners may be used to express Fy-ECD's in bacteria, as we have done using GST. For example, such fusion partners may be thioredoxin (Invitrogen) or the maltose binding

protein (MBP) (New England Biolabs) or the use of DsbA or DsbC or NusA proteins (Novagen).

EXAMPLE 7 Preparation of a nitrocellulose immunoblot membrane according to the present invention.

Anti-Fy antibodies present in plasma or serum can be detected using our novel recombinant Fy antigens.

Whole anticoagulated blood or clotted blood can be used as a test sample of the present invention; however the whole anticoagulated blood or clotted blood can only be used if a plasma or serum is prepared by centrifugation and separated from cells or clots prior to use. We used Western blots to demonstrate this finding. We first performed SDS-PAGE under reducing conditions, and electrophoresed 0.3 micrograms of total protein of purified GST-ECD-Fy (b), purified GST, and purified GST- ECD-Fy (a), in that order, in three adjacent lanes, to the left of a pre-stained protein molecular weight marker (Fig. 4). On gels stained with Coomassie Blue, the GST preparation appeared as one band. Both GST-ECD-Fy preparations contained a major 33 kDa band, and two less abundant 31 and 29 kDa bands. Duplicate gels were electroblotted to nitrocellulose to form blots, using a Hoefer transblotting apparatus set to 0.4 amps for 1-2 hours, which were then probed with antibodies using standard Western blotting protocols, using 5% non-fat skim milk powder in TBS supplemented with 0. 05% Tween 20 (Sigma). All of these bands, in addition to three smaller bands intermediate in size between 29 kDa and the size of GST, reacted with anti-GST antibodies when probed with 2

micrograms/ml affinity purified chicken anti-GST antibodies (done in parallel, and used as a control to ensure immobilization of fused antigen, and as an internal control to ensure proper method being carried out), then goat-anti-chicken antibodies conjugated to alkaline phosphatase (Kirkegaard & Perry Laboratories, Inc. ) The antibodies were then visualized using alkaline phosphatase-activated chromogenic substrates (using bromo-chloro-indolyl phosphate (BCIP) and Nitro-blue tetrazolium (NBT) salts), however other chromogenic or chemiluminescent substrates can be used.

It should be noted that any fusion peptides in accordance with the present invention may be immobilized as described above. For example, GST-Fy (a) ECD (1-65), GST- Fy (b) ECD (1-65), or any functional fragments thereof may be used, for example, GST-Fy (a) ECD (36-52), GST- Fy (b) ECD (36-52), or any other functional fragment capable of binding anti-Fy antibodies.

The embodiment (s) of the invention described above is (are) intended to be exemplary only. The scope of the invention is therefore intended to be limited solely by the scope of the appended claims.