IMMUNODIAGNOSTIC KIT AND METHOD FOR RAPID DETECTION OF HIV-1 AND HIV-2 ANTIBODIES

Co-Pending Applications

This application is a continuation-in-part application based on prior co-pending United States patent application Serial No. 08/179,512, filed on January 10, 1994, now abandoned, and PCT application Serial No. PCT/US 95/00305, filed January 9, 1995, still pending.

Field of the Invention

This invention relates to an immunoassay kit for rapid detection of HIV-1 and HIV-2 antibodies in biological fluid, and related method.

Background of the Invention

AIDS (Acquired Immunodeficiency Syndrome) is characterized by changes in the population of T cell lymphocytes. In the infected individual the virus causes a depletion of a subpopulation of T cells, called T helper cells, which leaves such individuals susceptible to opportunistic infections and some malignancies. The presence of the virus in the patient causes the immune system to elicit the production of anti-HIV-1

or HIV-2 antibodies. Tests to detect these anti-HIV-1 or HIV-2 antibodies are now widely used in order to identify infected patients and for the screening of blood derived products.

The synthetic peptides gP41, a transmembrane glycoprotein derived from HIV-1 and gP36 derived from HIV-2 represent major antigenic sites for anti-HIV antibodies. Fragments of these proteins and other HIV antigenic transmembrane glycoproteins such as gP38 and gP120 have been synthetically produced in the laboratory with the help of advanced genetic engineering technologies by others and are commercially available under these designations. The present invention is a rapid membrane- based immunodiagnostic assay for detection of HIV-1 and HIV-2 antibodies. The assay utilizes HIV peptide antigens on the membrane surface which are immunoreactive with antibodies in the fluid sample that are specific to HIV-l/HIV-2. After reaction such antibodies are visualized with a Protein A colloidal gold conjugate resulting in the display of visible red or red like spot on the membrane at the immunoreaction locus. Absence of antibodies in the fluid is indicated by a absence of color on the membrane.

Summary of the Invention

The present invention provides a test device and method for an accurate, rapid and simplified assay for detecting the

presence of antibodies to HIV-1 and HIV-2. It has been found that this assay provides high sensitivity and high specificity in detecting these antibodies in a single test in many cases less than three minutes after drawing a human blood sample. This makes it suitable for immediate field testing, without the need for laboratory facility involvement, and can be performed routinely by trained non-medical personnel.

The test device for the detection of antibodies to HIV-1 and HIV-2 comprises a filter device segment and a reaction cell. The filter device encases a filtration medium and is useful for removing red blood cells from a fluid such as a blood sample, if necessary, and is detachably attached to the reaction cell. Any filtered fluid such as serum can pass through the filter medium immediately into the reaction cell encountering the reaction membrane encased within the reaction cell. The reaction cell membrane is coated with antigen immunoreactive to HIV-1 and HIV-2 antibodies within the fluid. Upon the completion of filtration the filter device portion can be detached and discarded. After suitable treatment of the membrane which culminates in treatment of the membrane with an indicator such as Protein A colloidal gold conjugate, a red color may be viewed on the reaction cell membrane indicative of the presence of antibodies to HIV-1 or HIV-2.

The method comprises obtaining a fluid sample, filtering

the fluid sample, if necessary, through the filter device separating the red blood cells from the sample, coating the test or reaction cell membrane with antigenic glycoproteins of gP41, gP36, gP38 and gP120, contacting the sample with the coated membrane to allow for the immunoreaction of any antibodies with the antigen, conjugating the antibodies with a Protein A colloidal color indicator, viewing the membrane for presence of a red color, and confirming the existence or nonexistence of the subject antibodies.

Description of the Drawings

Fig. 1 exhibits the pouch, desiccant, pipette and fully assembled test device;

Fig. 2 exhibits the schematic placement of whole blood sample into the fully assembled test device; selected dimensions in Figure 2 are: a = 2.5 cm. width of reaction cell casing b = 3.5 cm. length of reaction cell casing c = 1.3 cm. height in the central section of the casing d = 1.0 cm. width of filter tab e = 2.0 cm. outer diameter of male funnel piece f = 3.0 cm. length of male funnel piece and tab g = 1.0 cm. diameter of funnel opening

Fig. 3 is a perspective view of the filter device separated from the reaction cell after filtration and immunoreaction and rotated to view the filter medium, the reaction cell membrane displaying a visually readable color indicating HIV antibody presence;

Fig. 4 is a cross-section view of the assembly of Fig. 2 taken along 4-4; selected dimensions in Figure 4 are:

1 = 1.6 cm. diameter of reaction cell casing opening n = 0.7 cm. depth of funnel to filter layer Fig. 5 is an exploded cross-section view of the reaction cell assembly of Fig. 3 taken along 5-5; selected dimensions in Figure 5 are: h = 0.7 cm. height of lower portion of reaction cell casing i = 0.6 cm. height of reservoir portion of reaction cell casing j = 0.6 cm. height of central upper portion of reaction cell k = 0.15 cm. thickness of mounted membrane at central location m = 0.8 cm. diameter of panel opening for interface of membrane and filter medium Fig. 6 is an exploded cross-section view of the filter device assembly of Fig. 3 taken along 6-6; selected dimensions in

Figure 6 are: o = 0.5 cm. inner diameter of male funnel piece at base p = 1.6 cm. diameter of female funnel piece at flange edge q = <2.0 cm. outer diameter of female funnel piece r = 1.2 cm. diameter of inner wall of female funnel piece s = 0.7 cm. inner diameter at base opening of female funnel piece

The design and approximate dimensions of the preferred embodiment of the test device are illustrated in Figures 1 through 6 and set-out above.

Detailed Description of Preferred Embodiment

The test device of the present invention can be used to screen a biological fluid sample for antibodies to HIV-1 and HIV-2. The biological samples which can be so screened include but are not limited to whole blood or components such as serum or plasma. Other biological fluids may be used. Preferably the test is used to screen whole human blood, serum, or plasma. The kit is comprised of a testing device having a filter portion and reaction cell portion, a pipette, and appropriate

solutions of Protein A gold colloidal conjugate, wash buffer, serum buffer, sealed in a pouch or container that is resistent to external moisture, with desiccant.

Some specific components of the kit are illustrated in Figure 1. The assembled filter test device 1 is illustrated in Figure 1. It is comprised of a plastic filter device 2, having a filtration funnel opening 2A in which the fluid to be filtered is placed, said filter device 2 is detachably attached to a plastic reaction cell 3 which has an upper casing 4 and a lower casing 5. The reaction cell 3 is assembled by friction fit of casings 4, 5. The pipette 6 desiccant 7 and pouch 8 are also illustrated.

Referring to Figure 3, the filter device 2 is held in place by friction fit within the aperture 4A of the upper reaction cell casing 4 by friction contact of the three flanges 15, symmetrically disposed around the outer wall of the female funnel piece 14, with the interface 28 between the flanges 15 and the upper reaction cell casing wall 4. Upon completion of the filtration of the fluid through the filter, the filter device can be removed from the reaction cell by the tab portion 12 (as illustrated in Figure 3) and discarded.

Figures 3 through 6 provide greater detail of the filter device and reaction cell, when the filter device is either seated within the aperture of the reaction cell or removed as

illustrated. The filter device is comprised of three general parts: a male funnel piece 13, a female funnel piece 14 and the filter medium 19. These parts, once assembled in appropriate fashion as by compression assembly, are not intended to be routinely disassembled during or after use or operation. The assembled filter device wherein the male funnel 13 and female funnel 14 pieces are mated (as illustrated in Figures 3 and 4) achieves several purposes. It fixes the filter medium 19 (with its two layers 22, 23) in a pinched position between the lower segments of male and female pieces 13, 14, and between their respective collar portions 21, 29 leaving the filter medium 19 in part disposed outside the edge of the female funnel rim 18 (Figure 3) , which is an alternative designation for the collar 29 (Figure 4) .

In the preferred embodiment the filter medium is comprised of two layers, pressed together, a top coarse layer 22 which is more porous and provides for more rapid filtration, and a bottom finer layer 23 for slower filtration. It is preferred that the finer layer 23 have porosity of about 122 cm ³ /s/cm ² . While the smaller pore size slows the overall filtration time, it improves the subsequent reactivity between the fluid antibodies and the antigen coated membrane.

In the preferred embodiment the coarse filter or upper filter 22 is comprised of 100% borosilicate glass fiber which

is white and unfinished in appearance and has a basis weight of 88 g/m ² , a thickness of 0.44 mm, a flow rate of 20 ml/min, air permeability of 1.4 cfm/ft ² , and retention of 0.7 μm. The lower filter or finer filter 23 is borosilicate glass fiber, also white, has a basis weight of 70 g/m ² , a thickness of .381 mm, a tensile machine direction of 6.7 kg/25mm, a tensile cross direction of 3.6 kg/25mm, a tear machine direction of 155 g., a porosity of 122 cm'/s/cm ² , and ash percentage of 95%. The filters can be obtained from Ahlstrom Filtration in Mount Holly Springs, Pennsylvania, and are designated as Grade 151 and .015" Manninglas 1200, respectively.

The male piece 13 and female piece 14 are compressed to fit along their interface 27A, which is between wall 17 of the male piece 13 and wall 27 of the female piece 14. Each of these walls terminates in the collar portions 21 and 29, respectively.

In the preferred embodiment the filter device is snugly fit within the upper reaction cell casing 4 such that the bottom filter layer 23 of the filter medium is in substantial contact with the reaction cell membrane 10, as illustrated in Figure 4. Figure 5 illustrates an exploded view of the reaction cell. Reaction cell 3 has an upper casing 4 and a lower casing 5. Inside the lower casing 5 is a reservoir 34 bounded by wall 33. Inside the reservoir 34 sits an absorbent

cotton pad 31, which is in substantial surface contact with porous adhesive gauze backing 30, which backing fixes the membrane 10 to a hard plastic panel 11, which panel has a reaction aperture 35 centrally positioned to directly contact and engage the lower filter layer 23 within aperture 35. This small opening or aperture 35 on the panel 11 exposes a localized portion of the membrane 10 to the assayed fluid. The vertical sequence, as illustrated in Figures 1, 3, and 4, of filter medium/membrane/backing/absorbent pad provides a path of continuous contact of absorbent materials for enhanced fluid diffusion and absorption.

In the preferred embodiment the membrane is comprised of nitrocellulose paper which is commercially available from Schleicher and Schuell GmbH. It can be first cut into suitably sized pieces for preparation and can be as small as necessary to fit within the dimensions of the test device, or can be reduced to appropriate size after membrane coating.

The membrane is preferably prepared as follows: gP41 is dissolved in 50mM sodium carbonate/bicarbonate (pH 9.3) to a final concentration of 20 μg/ml, and the nitrocellulose membrane is allowed to soak within the solution for about 2 hours at room temperature to coat the membrane. Then the membrane is air dried or vacuum dried. The membrane is then soaked in a similar solution of dissolved gP36 and

appropriately dried in the same manner as well. Finally, the membrane is soaked in a solution of gP38 and gP120 in a ratio of about three parts (micrograms/milliliter) gP38 to one part gP120 prepared in the same fashion, and then dried in the same fashion. These transmembrane glycoproteins gP 41, 36, 38 and 120 are commercially available and known by these designations. The complete nucleotide sequence of glycoproteins are listed in Nature 1985, 313:277-284 by Ratner, I. et al. It is preferred that each glycoprotein be at least 95% pure. In the event that any glycoprotein is not pure enough, then prior to coating it should be purified by any suitable means such as column chromatography.

In operation human whole blood is placed within the funnel opening 2A as illustrated in Figures 1 and 2. Anticoagulant such as EDTA is often added to the blood to eliminate the risk of clotting during the filtration and testing phases. Filtration of the blood sample is completed by passage of the fluid down the funnel successively engaging the filter medium top layer 22, and the bottom layer 23, filtering out the red blood cells. The filter device 2 may then be discarded by removing it from the reaction cell by using the tab 12.

After the blood has been filtered through the filter device and the filter device is discarded, the reaction cell membrane surface area of the nitrocellulose membrane is

accessible for further treatment and can be viewed, as illustrated in Figure 3. Two drops of conventional serum buffer, comprised of Tris buffer and protein stabilizer, are added directly to the membrane. After absorption of this buffer by the membrane, two drops of conventional wash buffer, comprised of Tris buffer, are added to the membrane to eliminate any discoloration of any kind such as that caused by ruptured or lysed red blood cells and hemoglobin discoloration. After absorption of the wash buffer, then two drops of Protein A gold colloidal conjugate are added. After absorption of the conjugate, two more drops of wash buffer are added. Shortly thereafter the red spot will appear, as illustrated in Figure 3, or will not appear on the membrane. The presence of the red spot indicates the positive identification of antibodies to HIV-1 and/or HIV-2.

This test is rapid and economic and reduces the quantity of reagents used in the manufacturing process, such as the glycoproteins, conjugate, and the buffers, while resulting in almost 100% specificity and reactivity. Stable and effective positive and negative controls can be included in the kit to verify periodically the stability of the reagents.

The following are additional features depicted by reference numerals in the drawings, and are set out to assist in the overall description of the preferred embodiment of the

test device:

Figure 4: 14A = outer rim of female funnel piece 16 = outer rim of male funnel piece 20 = interface between upper funnel void and funnel wall

24 = dotted line indicates female funnel outer wall behind flange

25 = void between female funnel piece and upper reaction cell casing

26 = interface between lower funnel void and funnel piece wall 32 = void between absorbent pad and reservoir wall

Figure 6: 36 = opening at base of male funnel piece

37 = opening at base of female funnel piece

38 = opening at top of female funnel piece

There are many variations and changes which will be within the spirit and scope of this invention, and the invention is limited only by the lawful scope of the following claims.