Field of the invention The present invention relates to a composition, a process for the preparation of said composition, the use of said composition in a diagnostic assay, and a process for immobilising an antigenic peptide onto solid supports

Background of the invention Since the advent of solid-phase peptide synthesis (SPPS) in the early sixties (Merrifield, 1963, J. Am. Che . Soc. 85, 2149), the SPPS method has been optimised to a method by which many different peptides can be synthesised in relatively short times. As a consequence, the use of synthetic peptides found widespread applications, such as in the field of epitope-mapping, specific diagnostic tests, the preparation and screening of anti-peptide antibodies, in studying antigen- antibody interactions, and in the field of synthetic vaccines. In many applications, peptide immobilisation onto a solid support, like i.e. polystyrene, as far as immuno- assay techniques are concerned, is a necessary procedure. In contrast to immobilised protein antigens, immuno-assays (like i.e. ELISA) using immobilised peptide antigens are often not satisfactory, since simple adsoφtion of a peptide onto a solid support frequently results in low coating efficiency. Alteration of the conformation upon immobilisation may result in a lowering of the accessibility of the peptide for antibody binding. Inefficient adsoφtion as a result of inappropriate hypophilic or hydrophilic properties of the peptides is another possibility. Most authors point out that relatively short peptides (e.g., less than 20 residues in length), lack sufficient secondary and tertiary structural features or do not have enough side-chains to bind well to plastic surfaces (Briand et al., 1985, J. Immunol. Methods 78, 59; Tam and Zavala, 1989, J. Immunol. Methods 124, 53; Dagenais et al., 1994, J. Immunol. Methods 222, 149). Accordingly, the ability of short peptides of diverse sequence and length to bind to a microtitre plate is subject to fluctuations. Larger peptides on the other hand can be composed of several epitopes and, consequently, be less discriminative and more cross-reactive in immunological assays.

Although optimising the coating conditions for each individual peptide (e.g. pH, ionic strength) yielded satisfactory results (Oshima and Atassi, 1989, Immunol. Investigations 18, 841; Beffy et al., 1994, Fund. Clin. Immunol. 2, 53), the conventional approach to overcome the poor coating efficiency is still the conjugation of the peptide to a protein carrier such as bovine serum albumin

(Briand et al., 1985; Price et al., 1993, J. Immunol. Methods 159, 277) or poly-L- lysine (Ball et al., 1994, J. Immunol. Methods 171, 37). Alternatively, methods which provide a covalent link between the support (like a polystyrene plate) and the peptide using direct coupling agents such as glutaraldehyde (Ordronneau et al., 1991, J. Immunol. Methods 142, 169) and carbodiimide (Søndergέrd- Andersen et al., 1990, J. Immunol. Mediods 131, 99) have become widespread as well. Some authors have pretreated their polystyrene plates with Alcian blue (Lagace et al., 1994, J. Immunol. Methods 175, 131), or UV-irradiation (Boudet et al., 1991, J. Immunol. Methods 142, 73) to facilitate immobilisation of the peptide antigen. A combined approach of chemical and physical activation was also investigated (Dagenais et al., 1994). Others have reported applications of the streptavidin-biotin system (Von Grϋningen et al., 1991, Biol. Chem. Hoppe-Seyler 372, 163; Ivanov et al., 1992, J. Immunol. Methods 153, 229) and the multiple antigen peptide (MAP) system (Tarn and Zavala, 1989; Caponi et al., 1995, J. Immunol. Methods 179, 193) for improved peptide immobilisation.

These referred approaches can all be described as either time-consuming, complex, or costly. Crosslinking procedures and peptide-protein conjugates have some additional disadvantages when non-directional coupling is involved with non- uniform coating as a consequence. Instability in time is another problem with peptide-protein conjugates, while the use of poly-L-lysine, and of UV-treatment tend to give high background readings.

Detailed description of the invention The present invention is concerned with a composition comprising an antigenic peptide immobilised on the surface of a solid support, wherein said peptide is linked to an acyl group of formula I:

R1-A-R2-CO-

wherein

Rl is lower alkyl, aryl or aralkyl;

R2 is a divalent radical derived from lower alkyl, aryl or aralkyl; and A is a sulfur containing functional group selected from -CO-S-, -S-, -SO-, -SO2- and -S-S-.

The introduction of said acyl group results in an overall enhancement of the coating efficiency of various peptides tested. Coating efficiency is defined as the peptide concentration in the coating solution, that is required to achieve 50% of the

maximum EIA signal (ECso). The improvement of the coating efficiency which is realised with the introduction of said acyl group is found to be between 2 and 5 orders of magnitude, as compared with the parent peptide.

This improvement substantially reduces the amount of peptide needed to develop a sensitive peptide-based immunoassay.

The term "lower alkyl", as used in the definition of formula I, refers to a branched or unbranched alkyl group having from 1-6 carbon atoms, like hexyl, isobutyl, propyl, isopropyl, ethyl, and preferably, methyl.

The term "aryl" refers to an aromatic group like phenyl and naphthyl. Also included in the definition of aryl are heteroaromatic groups like pyridinyl, pyrimidyl and thienyl. The aryl groups may be substituted by lower alkyl groups, O-lower alkyl, wherein lower alkyl is as previously defined, and by halogen. Preferred aryl groups are phenyl and 2-pyridinyl.

The term "halogen" refers to fluorine, chlorine, bromine or iodine.

The term "aralkyl" refers to a lower alkyl group, as previously defined, which is substituted with at least one aryl group, as previously defined.

Preferred divalent radicals derived from lower alkyl, aryl or aralkyl, terms which have each been previously defined, are methylene (-CH2-) and 1,2-ethanediyl (- CH2-CH2-).

A preferred embodiment of the present invention is directed to a composition wherein said acyl group is represented by Rl being a lower alkyl, R2 being derived from a lower alkyl, and A being -CO-S-.

Another preferred embodiment of the present invention is directed to a composition wherein said acyl group is represented by Rl being methyl, R2 being methylene, and A being -CO-S- (this group is also known as acetyl-thio-acetyl-group (Ata)).

A further preferred embodiment of the present invention is directed to a composition wherein said acyl group is represented by A is -CO-S- and: Rl being methyl and R2 being 1,2-ethanediyl (acetyl-thio-propionyl (Atp)), by Rl being phenyl and R2 being methylene (benzoyl-thio-acetyl (Bta)), or by Rl being phenyl and R2 being 1,2-ethanediyl (benzoyl-thio-propionyl (Btp)).

Another preferred embodiment of the present invention is directed to a composition wherein said acyl group is linked at the N-terminus of the peptide.

A further preferred embodiment of the present invention is directed to a composition wherein said solid support is a polystyrene support.

The results, presented in the examples and figures, clearly demonstrate that the introduction of the acyl groups according to the present invention improve the coating efficiency of peptides of various length and sequence, and, hence, the amount of peptide required for physical adsoφtion can be drastically reduced to achieve a sensitive peptide-immunoassay such as an ELISA.

The term "peptide" as used herein refers to a molecular chain of amino acids with a biological (immunological) activity, and does not refer to a specific length of the product. Thus inter alia, proteins, fusion-proteins or -peptides, oligopeptides, nucleopeptides and polypeptides are included. Preferred are peptides with a length of 2-30 amino acids. Also modifications by replacement of amino acids by non- natural amino acids and other building blocks to obtain a biologically active molecule are included.

The term "solid support" as used herein refers to polymeric substances such as polystyrene, polyvinylchloride (PVC), nylon, vinypolymers and nitrocellulose, or to glass. The surface of the solid phase may have the form of the inner wall of a microtest well or a cuvette, a tube or capillary, a membrane, filter, test strip or die surface of a particle such as, for example, a latex particle, a dye sol, a metal sol or metal compound as sol particle.

The preparation of the peptides according to the invention is effected by means of one of the known organic chemical methods for peptide synthesis or with the aid of recombinant DNA techniques.

DIRECT CHEMICAL SYNTHESIS

Direct synthesis of a peptide including an acyl group as presented: The organic chemical methods for peptide synthesis are considered to include the coupling of the required amino acids by means of a condensation reaction, either in homogeneous phase or with the aid of a so-called solid phase. The condensation reaction can be carried out as follows: a) condensation of a compound (amino acid, peptide) with a free carboxyl group and protected other reactive groups with a compound (amino acid, peptide) with a

free amino group and protected other reactive groups, in the presence of a condensation agent; b) condensation of a compound (amino acid, peptide) with an activated carboxyl group and free or protected other reaction groups with a compound (amino acid, peptide) with a free amino group and free or protected other reactive groups. Activation of the carboxyl group can take place, inter alia, by converting the carboxyl group to an acid halide, azide, anhydride, imidazolide or an activated ester, such as the N-hydroxy-succinimide, N-hydroxy-benzotriazole or p- nitrophenyl ester.

The most common methods for the above condensation reactions are: the carbodiimide method, the azide method, the mixed anhydride method and the method using activated esters, such as described in The Peptides, Analysis, Synthesis, Biology Vol. 1-3 (Ed. Gross, E. and Meienhofer, J.) 1979, 1980, 1981 (Academic Press, Inc.).

Preparation of suitable fragments of above-mentioned peptides according to the invention using the "solid phase method" is for instance described in J. Amer. Chem. Soc. 85, 2149 (1963) and Int. J. Peptide Protein Res. 35, 161-214 (1990). The coupling of the amino acids of the peptide to be prepared usually starts from the carboxyl end side. For this method a solid phase is needed on which there are reactive groups or on which such groups can be introduced. This can be, for example, a copolymer of benzene and divinylbenzene with reactive chloromethyl groups, or a polymeric solid phase rendered reactive with hydroxymethyl or amine- function.

A particular suitable solid phase is, for example, the p-alkoxybenzyl alcohol resin (4-hydroxy-methyl-phenoxy-methyl-copolystrene-l % divinyl-benzene resin), described by Wang (1974; J. Am. Chem. Soc. 95, 1328). After synthesis the peptides can be split from this solid phase under mild conditions. After synthesis of the desired amino acid sequence, detaching of the peptide from the resin follows, for example, with trifluoroacetic acid (Fmoc), or with trifluoromethanesulphonic acid (t-boc). The peptide can also be removed from the carrier by transesterification with a lower alcohol, preferably methanol or ethanol, in which case a lower alkyl ester of the peptide is formed directly. Likewise, splitting with the aid of ammonia gives the amide of a peptide according to the invention.

The reactive groups which may not participate in the condensation reaction are, as stated, effectively protected by groups which can be removed again very easily by

hydrolysis with the aid of acid, base or reduction. Thus, a carboxyl group can be effectively protected by, for example, esterification with methanol, ethanol, tertiary butanol, benzyl alcohol or p-nitrobenzyl alcohol and amines linked to solid support.

Groups which can effectively protect an amino group are the ethoxycarbonyl, benzyloxycarbonyl, t-butoxy-carbonyl (t-boc) or p-methoxy-benzyloxycarbonyl group, or an acid group derived from a sulphonic acid, such as the benzene- sulphonyl or p-toluene-sulphonyl group, but other groups can also be used, such as substituted or unsubstituted aryl or aralkyl groups, for example benzyl and triphenylmethyl, or groups such as ortho-nitrophenyl-sulphenyl and 2-benzoyl-l- methyl-vinyl. A particularly suitable ε-amino-protective group is, for example, the base-sensitive 9-fluorenyl-methoxycarbonyl (Fmoc) group [Caφino & Han, 1970, J. Amer. Chem. Soc. 92, 5748].

A more extensive account of possible protecting groups can be found in The Peptides, Analysis, Synthesis, Biology, Vol. 1 - 9 (Eds. Gross, Udenfriend and Meienhofer) 1979 - 1987 (Academic Press, Inc.).

It is necessary also to protect the ε-amino group of lysine and advisable for the guanidine group of arginine. Customary protective groups in this connection are a Boc-group for lysine and a Pmc- or Pms- or Mbs-group or Mtr-group for arginine.

The protective groups can be split off by various conventional methods, depending on the nature of the particular group, for example with the aid of trifluoroacetic acid or by mild reduction, for example with hydrogen and a catalyst, such as palladium, or with HBr in glacial acetic acid.

Covalent linkage of two or more peptides in a hybrid- or combi-peptide can for instance be carried out through solid phase peptide synthesis, using the methods described above, of a peptide sequence wherein the amino acid sequences of the individual peptides are aligned. It is understood that a linker sequence may be inserted between the individual peptides sequences. Such a linker sequence may for instance be a stretch of 2-5 residues of glycine. A hybrid- or combi-peptide can also be prepared through solid phase synthesis using the fragment condensation approach. The latter method, in which the fragments (the sequences of which may correspond with the sequences of the individual peptides of the invention) are separately prepared and purified, is preferred in the synthesis of the longer hydrid- or combi-peptide sequences. The

methodology for the preparation of longer peptides is known in the art, and for instance described in The Peptides, Analysis, Biology, Vol. 1-9 (vide supra). Alternatively, hybrid- or combi-peptides can be prepared through conjugation of appropriately modified peptides of the present invention. In a preferred method for the conjugation of two different peptide sequences which are devoid of the amino acid cysteine, the peptides are derivatized to contain an additional residue of cysteine at either the carboxyl- or the amino-terminal end. One of the peptides is subsequently activated at the single cysteine thiol function with 2,2'-dithiodipyridine. The resulting pyridyl-dithio-peptide derivative is then reacted with the second peptide containing the cysteine thiol group to yield a hybrid peptide in which the individual peptides are linked through a disulfide bond.

INDIRECT COUPLING

Indirect coupling of peptide to an acyl group as presented: As already indicated above, the peptides according to the invention can likewise be prepared with the aid of recombinant DNA techniques. This possibility is of importance particularly when the peptide is incorporated in a repeating sequence ("in tandem") or when the peptide can be prepared as a constituent of a (much larger) protein or polypeptide or as a fusion protein with, for example, (part of) β- galactosidase. This type of peptides therefore likewise falls within the scope of the invention. For this puφose, as a constituent of a recombinant DNA, a nucleic acid sequence is used which codes for a peptide according to the invention and which, furthermore, is substantially free from nucleic acid segments.

This latter method involves the preparation of the desired peptide by means of bringing to expression a recombinant polynucleotide with a nucleic acid sequence which is coding for one or more of the peptides in question in a suitable micro¬ organism as host. The peptides expressed by the host can be purified, if necessary, and coupled to acyl-groups according to the present invention moieties by known methods (use can be made of the chemical methodology that has been developed in the field of protein-protein conjugation. An overview is given by Means and Feeney (Bioconj. Chem. 1, 2-12, 1990)). Also oligonucleotide molecules can be linked to an acyl group according to the present invention.

Another embodiment of the present invention is directed to a process for the preparation of a composition comprising an antigenic peptide immobilised on the

surface of a solid support, wherein an aqueous solution of said peptide which is linked to an acyl group of the formula:

R1-A-R2-CO-

wherein

Rl is lower alkyl, aryl or aralkyl;

R2 is a divalent radical derived from lower alkyl, aryl or aralkyl; and A is a sulfur containing functional group selected from -CO-S-, -S-, -SO-, -SO2- and -S-S-, is brought into contact with a solid support at a pH range of 8.0-10.0.

Another embodiment of the present invention is directed to the use of a composition according to the present invention, in a diagnostic assay.

Another embodiment of the present invention is directed to a process for immobilising an antigenic peptide onto solid supports, wherein said peptide is linked to an acyl group of the formula:

R1-A-R2-CO-

wherein

Rl is lower alkyl, aryl or aralkyl;

R2 is a divalent radical derived from lower alkyl, aryl or aralkyl; and A is a sulfur containing functional group selected from -CO-S-, -S-, -SO-, -SO2- and -S-S-, after which the modified peptide is brought into contact to said solid support.

Another embodiment of the present invention is directed to a test kit wherein a composition according to the present invention is used.

Materials and methods

Abbreviations:

Abbreviations and symbols for amino acids and peptides are in accordance with the recommendations of the IUPAC-IUB joint Commision on Biochemical Nomenclature as given in Eur. J. Biochem. (1984), 138: 9 - 37. Further abbreviations are: NMP, N-methyl-pyrrolidone; Fmoc, N-9- fluorenylmethyloxycarbonyl; PS, polystyrene.

In order to clearly indicate the acyl-groups used in the experiments, the generic formula R1-A-R2-CO- with the different side groups is not used. The general names of the groups are indicated as: Ata (acetyl-thio-acetyl: represented by A is - CO-S-, Rl is methyl and R2 is methylene), Atp (acetyl-thio-propionyl: A is -CO-S- , Rl is methyl and R2 is 1,2-ethanediyl), Bta (benzoyl-thio-acetyl: A is -CO-S-, Rl is phenyl and R2 is methylene), Btp (benzoyl-thio-propionyl: A is -CO-S-, Rl is phenyl and R2 is 1,2-ethanediyl).

Monoclonal antibodies Five mouse monoclonal antibodies of which the epitope was previously determined were selected, and were all of the IgGl subclass. Anti-human chorionic gonadotropin (hCG) monoclonal antibodies OT-3A and OT-1A, and Mab OT- C100/8A-1 (specific for the C100 part of the hepatitis C-virus, HCV) were produced using a hollow-fibre dialysis system (Schόnherr, 1987, Dev. Biol. Stand. 66, 211). The anti-hCG Mabs were further purified by protein A affinity chromatography as described by Van Sommeren et al. (1992, Prep. Biochem. 22, 135)[binding buffer: 1.5 mol/1 glycine, 3 mol/1 NaCl, pH 8.9]. The mouse Mab GCIR 1202, specific for the PP52 part of the cytomegalovirus (CMV), was purchased from the Goodwinn Institute (Plantation, Florida). Murine monoclonal antibody HB.OT95A was produced by injecting Balb/c mice with E. coli derived recombinant HBeAg in Freund's complete adjuvans. The best responding mouse received an intravenous dose of the recombinant antigen dissolved in PBS. Fusion and selection were performed according to standard methods. Reactive clones were recloned to 100% clonality. Murine monoclonal antibody HB.OT95A was deposited with the European Collection of Animal Cell Cultures (ECACC), Porton Down (UK), under deposit No.95090611.

Synthesis and purification of acylated peptides

The synthesis of the peptides was carried out by an automated procedure on a Perkin Elmer/Applied Biosystems Inc. 433A peptide synthesizer, using standard FastMoc 0.25 mmol procedures with UV-monitoring and feedback option. Some of the peptides were synthesized in a semi-automated manner on an in-house built multiple peptide synthesizer using standard Fmoc/tBu-chemistry with in situ activation by carbodiimide. An overview of techniques, protecting groups, linkers, and solid supports in the SPPS 'Fmoc chemistry' is given by Fields & Noble (Int. J. Peptide Protein Res., 35, 161, 1990). Fmoc amino acid derivatives were obtained from Bachem (Bubendorf, Switzerland). Desaminophenylalanine was purchased from Merck (Mϋnchen, Germany). The peptides were synthesized on a TentaGel S RAM Fmoc resin (RAPP Polymere, Tubingen, Germany) via the

Fmoc/tBu chemistry. The linker is of a Rink-amide type, which automatically yields a C-terminally amidated peptide. During solid phase peptide synthesis the amino acid side-chains were protected with acid-labile protecting groups: the ε- aminogroup of lysine with Boc, the δ-guanidino group of arginine with 2,2,5,7,8- pentamethylchroman-6-sulphonyl (Pmc), the γ-carboxyl group of glutamic acid and the β-carboxyl group of aspartic acid with OtBu, the γ-amide group of glutamine and the β-amide group of asparagine with trityl (Trt), histidine and cysteine with Trt, the β-hydroxyl group of serine and threonine with tBu, and tyrosine with tBu. All reactants were dissolved in NMP. The cleavage of the Fmoc groups was carried out with 25% (vol/vol) piperidine in NMP during at least 2 consecutive cycles of 1.5 min (semi-automatic: 3 consecutive cycles of 3 min). Coupling of the first Fmoc amino acid derivative (Fmoc-Aaa-OH, 4 eq. 1 mmol) was performed by in situ activation with 2-(lH-benzotriazol-l-yl)-l,l,3,3-tetramethyluroniumhexafluor o phosphate (HBTU), 1-hydroxybenzotriazole (HOBt, 4 eq, 1 mmol) and diisopropylethylamine (DIPEA). In the semi-automatic procedure coupling of the first Fmoc amino acid derivative (Fmoc-Aaa-OH, 2.5 eq.) was performed by in situ activation with diisopropylcarbodiimide (DIPCDI) and 1-hydroxybenzotriazole (HOBt, 2.5 eq). After coupling of each amino acid derivative (at least 20 min) no check for completion of the acylation reaction was carried out. In the semi- automatic procedure bromophenol blue was used as indicator for completion of the acylation reaction. The acylation reaction was followed by a capping-step with acetic anhydride in NMP. The N-terminal Ata-group was introduced via an active ester coupling with Ata-N-hydroxysuccinimide. The N-acetyl-peptide was obtained by acetylation of the N-terminus with acetic anhydride. The fully protected peptides were cleaved from the resin during a 2-hr reaction at room temperature under nitrogen with 5% thioanisole (vol/vol), 3% ethanedithiol (vol/vol), 2.5% water (vol/vol), and 2% anisole (vol/vol) in trifluoroacetic acid (87.5% vol/vol) followed by precipitation in diethylether. The crude peptides were washed twice with diethylether, dried at the air, dissolved in water/acetonitrile (3:1) and lyophilized. The composition of the peptides were confirmed by mass and amino acid analysis. The HPLC analysis and purification (when necessary) were carried out on a Beckman Gold HPLC system. HPLC analyses were performed on a RP-C2/C18 column (Supeφack prepS, 4x250 mm, Pharmacia) at a flow rate of 1 ml/min, using a 3 min isocratic elution with 0.1 % trifluoroacetic acid in acetonitrile followed by a 30 min linear gradient from in water (100%) to 75 % 0.1 % trifluoroacetic acid in acetonitrile. Peptides were detected by UV measurement at 206 nm. The synthesised peptides (summarised in table II) were of more than 75% purity (obtained by purification when necessary). The molecular weight, HPLC -purity and retention times of these peptides are listed in table II.

ELISA-procedure

Polystyrene microtitre plates (Greiner, Frickenhausen, Germany) were sealed during every incubation (to prevent evaporation) and washed (Washer Microelisa system 400, Organon Teknika, Boxtel, The Netherlands) four times after each incubation with PBS-Tween (6.7 mmol/1 phosphate buffer pH 7.2; 0.13 mol/1 NaCl; 0.05% (v/v) Tween-20). During every experiment blanks were included to check for non-specific binding of the monoclonal antibody and the sheep anti-mouse IgG conjugate. The plates were freshly coated for each experiment. The synthetic peptides (parent or N-acylated), serial diluted (135 μl) in 0.05 mol/1 bicarbonate coating buffer (pH 9.6) in concentrations of 1 mg/ml to 1 ng/ml, were allowed to passively adsorb to a well of the microtiter plate during overnight incubation at room temperature with constant shaking (600 rpm, TPM-2 shaker; Sarstedt, Nϋmbrecht, Gemany). Non-specific adsoφtion of the monoclonal antibody and/or conjugate was prevented by performing a subsequent incubation with aliquots of 135 μl of bovine serum albumin (2.0 g/1; Organon Teknika, Boxtel, The Netherlands) in Tris-buffer (pH 7.4) for 2 h at room temperature, after the first washing procedure. 100 μl of the appropriate monoclonal antibody solution, diluted in sample diluent (20% v/v normal goat serum in 1% (v/v) triton in 7.6 mmol/1 sodium phosphate buffer (pH 7.4) with 120 mmol/1 NaCl), was added to each well for 1 h at room temperature with constant shaking. Subsequently, after washing, the wells were incubated for 30 min at room temperature under constant shaking with 100 μl of sheep anti-mouse IgG, conjugated to horse radish peroxidase according to the procedure of Wilson and Nakane (1978), and diluted 1/5000 in sample diluent. The final washing procedure was followed by color development which was initiated by adding 100 μl of the substrate solution (0.45 mmol/1 3,3', 5,5'- tetramethylbenzidine, 1.5 mmol/1 H2O2 and 0.1 mol/1 sodium acetate pH 5.5). The reaction was terminated after 30 min incubation in the dark, by adding 100 μl of 1 mol/1 sulphuric acid solution. Absorbance values at 450 nm were measured on a Microelisa reader 510 (Organon Teknika, Boxtel, the Netherlands).

All results given are the mean of at least duplicate determinations. The intra and interassay variation were within normal range. The day to day variation was also acceptable although the effects of batch to batch differences, frequent lyophilization (refers to water content) could sometimes be observed as a maximal variation of half an order of magnitude in EC50 values.

Ala-scanning method

The Ala-scanning method is frequently used to determine the contribution of individual amino acids to the binding interaction between peptide and antibody.

Therefore every amino acid of the peptide sequentially is replaced by the amino acid alanine. When the natural peptide sequence contains an alanine itself, it is replaced by aspartic acid.

In this way residues are found which can be replaced by alanine without impairing ELISA reactivity. Presumably, they do not correspond to critical residues that contribute to the energy of interaction. For example, this pattern of replaceability is important for modelling peptide-antibody interaction.

EXAMPLES:

Example 1 :

Comparison of N-terminally acylated peptide 3A [amino acid sequence: RLPGPSD (B-hCG)] with parent (free N ^α amino-group) peptide:

Preliminary experiments were carried out to assess the optimal ELISA conditions. By using a 0.05 M carbonate coating buffer of pH 9.6 (tested in a pH-range of 3.6 to 10.0) immobilisation of the parent peptide by passive adsoφtion was optimal. Storage of the peptide solution up to 24 hours at 4 °C had only a negligible effect on the sensitivity of the ELISA.

The effects of the antigenic properties of various N-acyl groups according to the present invention were tested by comparing the coating efficiency (the term surface reactivity or binding capacity is also used in the same context) of N-acylated peptide 3A to the parent peptide 3A in the following ELISA-format. The (N-acylated) peptide was coated in a serial dilution in the range of 1 mM to 1 nM and a fixed concentration of antibody was presented to the immobilised peptide. Comparison of coating efficiency between parent and N-acylated equivalents is made in terms of the amount of peptide coat concentration required to achieve 50% of the maximum ELISA-signal (ECso). The coating efficiencies of the parent peptide 3A, the N ^α acetyl-peptide 3A, and the N" Ata-peptide 3A were determined. The results are presented in Figure 1 :

Fig. 1A shows that the coating efficiency of peptide 3A can be substantially improved when N-acylated with an Ata-group. The concentration of peptide required to produce the dose-response curve with an equal amount of antibody could be reduced from 390 μM to 18 nM which is a reduction of more than four orders of magnitude.

Fig. IB shows that the surface reactivity of the peptide 3 A coating cannot be improved by N-acylation with an acetyl-group.

Example 2:

Time series:

In a next series of experiments, the time of coating was varied (88 h - 10 min). The superior ELISA-performance of the Ata-linked peptide in comparison to the parent peptide 3A is evident from Figure 2. Furthermore, when the time of coating is above 4 hours, the coating efficiency of the Ata-peptide increased at a faster rate compared to the parent peptide 3 A.

Example 3:

Four other peptide sequences were N-acylated with the Ata-group to further explore die general applicability of the Ata-group and analogues tiiereof. All peptides clearly differed in amino acid sequence, length, and hydrophobicity (see also table I and II). The peptides are: - peptide 1A [amino acid sequence: DTPILPQ (β-hCG)], - peptide 5-1-1 [amino acid sequence:

DREFLYREFDEM (HCV-NS4-C100)],

- peptide CMV-1 [amino acid sequence: GGSLSSLANAGGLHDDG (CMV-PP52)]

- peptide HBeAg [amino acid sequence: LEDPASRDLVVNYVNTN (HBeAg)].

The adsoφtion of the parent peptide 1A to the solid phase did not result in any ELISA-reactivity up to a coating concentration of 1 mM (Fig. 3A). However, the binding of die specific antibody could be detected when an N-acylated group according to die invention was introduced to the peptide, indicating poor coating efficiency for the parent peptide 1A. Also the parent peptide 5-1-1 did not exceed the background signal unambiguously in contrast to the N-acylated equivalents (Fig. 3B). Fig. 3 (A t/m C) shows that the Ata-group and analogues tiiereof systematically increase the surface reactivity of all three peptides in comparison to their parent counteφarts but each to a different extent (see table III).

Example 4:

Ala-scanning of the N-acyl linked HBeAg-peptide (sequence of HBeAg peptide in Table I). Coating efficiency was tested with parent peptides and Ata-linked peptides. The ELISA was assessed by coating of the peptides (5 μg/ml) and binding of HB.OT95A (1:4000). Detection was performed with a Rabbit-anti-Mouse HRP- labeled antibody (1: 1000).

The result from this Ala-scanning indicated that the following amino acids are essential for binding of the antibody HB.OT95A:

Leucine at position 1, Glutamic acid at position 2, Aspartic acid at position 3, Proline at position 4, Alanine at position 5, Arginine at position 7, Aspartic acid at position 9, Valine at position 11, Asparagine at position 13.

Conclusion:

The presented results support the finding that the use of high molecular weight carriers (e.g., MAP or BSA) or covalent activation of polystyreen (PS) can be circumvented in the immobilisation procedure of small synthetic peptides. Application of small N-acylgroups, such as the Ata-group or analogues thereof, to the peptide is sufficient to improve the coating efficiency at least two orders of magnitude. This substantially reduces the amount of peptide needed to develop a sensitive peptide-ELISA. Besides saving time and costs the use of an Ata-group or analogues thereof at the N-terminal end of the peptide has the additional advantage that the solubility of the peptides improves due to the addition of extra polarity.

TABLE I

PEPTIDES USED IN THIS STUDY

^a represents peptides from the C-terminal part of the β-chain of human chorionic gonadotropin.

HCV-NS4-C100 refers to the C100 part of the NS4 region of the Hepatitis-C virus genome. ^c CMV-PP52 refers to the PP52 part of the Cytomegalovirus. UL44 refers to the correct reading frame. ^d represents a part of the non-particulate e-antigen (HBeAg) secreted by the hepatitis B-virus.

TABLE II

PEPTIDE CHARACTERISTICS

Peptide N-linked Molecular Retention time ⁹ HPLC- origin moiety weight (in min) purity (in (in Dalton) %)

3A H- 740 12.6 84

3A Ac- 781 13.5 100

3A Ata- 856 14.9 100

1A H- 781 15.0 91

1A Ata- 898 16.6 100

5-1-1 H- 1601 16.6 78

5-1-1 Ata- 1717 18.7 76

CMV-1 H- 1527 15.1 77

CMV-1 Ata- 1643 17.2 81

Retention time is determined by reverse phase column analysis

TABLE III

EC50 VALUES DETERMINED IN ELISA WITH PEPTIDES N-TERMINALLY LINKED WITH DIFFERENT ATA-ANALOGUES. FACTOR OF IMPROVED BINDING CAPACITY IN COMPARISON TO THE PARENT PEPTIDE.

^a Ata, acetyl-thio-acetyl; Atp, acetyl- thio propionyl; Bta, benzoyl-thio-acetyl; Btp, benzoyl- thio-propionyl. ^b These peptides showed no response in the ELISA. To set off against the results of with the

N-terminally linked a peptide coatconcentration of 1 mM was chosen as the estimated EC50 value.

Fig. 1. Coating efficiency of (N Ata) peptide 3A.

A: ELISA was performed using dilution series of parent peptide 3A (-§- ) and N ^α Ata-peptide 3 A ( - - ). Binding was determined using an anti-hCG Mab OT-3A concentration of 1 μg/ml. B: ELISA was performed using dilution series of parent peptide 3A (-f-) and N" Acetyl-peptide 3A Hfc-). Binding was determined using an anti-hCG Mab OT-3A concentration of 10 μg/ml.

Fig. 2. Effect of coating time on coating efficiency of parent peptide 3 A and its N" Ata-peptide 3 A. The coating efficiency decreased when the coating time was decreased from 88 h to 10 min via intermediate coating times for both parent peptide 3A from 19.5 h (-ft-), 4 h (-#-), 1 h (H-) to 10 min (-f-), and for N ^α Ata-peptide 3A from 19.5 h (-fr), 5 h (-A-), 1 h (-$►-) to 10 min (-#-). Binding was assessed with an anti-hCG Mab OT-3A concentration of 1 μg/ml.

Fig. 3. Coating efficiency of (N" acyl group) peptides.

A: ELISA-reactivity of anti-hCG Mab OT-1A (1 μg/ml) with parent peptide 1A (- ), and peptide 1A N ^α -acylated with the Ata-group (—1—).

B: ELISA-reactivity of anti-HCV Mab C100/8A-1 (1: 1000) with parent peptide 5- 1-1 (-φ-) and peptide 5-1-1 N ^α acylated with the Ata-group (-+-)•

C: ELISA-reactivity of anti-CMV Mab GCIR 1202 (1:2000) with parent peptide CMV-1 (-§-) and peptide CMV-1 N ^α acylated with the Ata-group (-|-).

Fig. 4. Ala-scanning of the N-acylated HBeAg-peptide (sequence of HBeAg peptide in Table I). Coating efficiency was tested with parent peptides and N ^α Ata- peptides. The amino acid (one-letter code) directly at the bottem of the bar represents the amino acid in the HBeAg sequence which is substituted by alanine.