Ž .Journal of Immunological Methods 257 2001 51–54www.elsevier.comrlocaterjim

Comparing tandem repeats and multiple antigenic peptides as theantigens to detect antibodies by enzyme immunoassayq

Peter Kim, Chou-Pong Pau)

DiÕision of AIDS, STD, and TB Laboratory Research, National Center for Infectious Diseases, Centers for Disease Control and PreÕention,1600 Clifton Road, Mail Stop D12, Atlanta, GA 30333, USA

Received 4 May 2001; accepted 5 June 2001

Abstract

Short synthetic peptides are useful alternatives to whole lysate or recombinant proteins as the antigens used forserodiagnosis of bacterial or viral infections. However, certain known antigenic peptides displayed low seroreactivities indirect enzyme immunoassay. This was believed to be due to the low coating efficiency, a constrained orientation, or loss offlexibility required for optimal antibody binding. Using a model peptide system derived from the V3-loop of HIV-1 gp120,

Ž .we demonstrated that low antigenicity could be overcome by using either tandem repeats TR or multiple antigenic peptidesŽ .MAPs which contained the same amino acid sequence as the monomeric peptide. In our model system, a four-branch MAPwas a better choice compared to the tandem repeats because of the MAP’s slightly higher sensitivity and lower cost ofproduction. q 2001 Elsevier Science B.V. All rights reserved.

Keywords: Enzyme immunoassay; Multiple antigenic peptide; Serodiagnosis

1. Introduction

Ž .Peptide-based enzyme immunoassays PEIAshave been widely used for serodiagnosis of bacterialor viral infections in recent years. Because of theenhanced specificity over whole-lysate-based assays,

q Use of trade names is for identification only and does notconstitute endorsement by the US Department of Health andHuman Services, the Public Health Services, or the Centers forDisease Control and Prevention.

) Corresponding author. Tel.: q1-404-639-3765; fax: q1-404-639-2660.

Ž .E-mail address: chp3@cdc.gov C.-P. Pau .

peptide-based serology is often used in differentialdiagnosis of closely related agents such as HIV-1

Ž .and HIV-2 Pau et al., 1993; Brattegaard et al., 1995Ž .and HTLV-I and HTLV-II Viscidi et al., 1991 .

However, the use of short peptides as the antigen toachieve better specificity may jeopardize assay sensi-

Ž .tivity Brattegaard et al., 1995 . This may be a resultŽof low coating efficiency Geerligs et al., 1988; Shin

.et al., 1996 , a constrained orientation, or loss offlexibility required for optimal antibody binding. Wehave investigated the use of tandem repeats and

Ž .multiple antigenic peptides MAPs to improve theassay sensitivity by eliminating these problems asso-ciated with monomeric peptides. A model systemusing V3-loop peptides derived from the HIV-1 sub-type B consensus sequence is presented.

0022-1759r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.Ž .PII: S0022-1759 01 00444-6

( )P. Kim, C.-P. PaurJournal of Immunological Methods 257 2001 51–5452

2. Materials and methods

2.1. Synthetic peptides

The model peptide system is derived fromthe consensus subtype B, V3-loop sequence of

ŽHIV-1 gp120. The monomeric peptide M1s.SIHIGPGRAFYTT has 13 residues with a free car-

boxyl- and a free amino-terminus. Peptides TR2 toTR5 are two to five tandem repeats of M1, respec-tively, and peptides MAP2, MAP4 and MAP8 are

Ž . Ž .multiple antigenic peptides MAPs Tam, 1988composed of two, four, and eight branches of M1,respectively.

All peptides were synthesized using FMOC chem-istry according to the manufacturer’s protocol on an

Žautomatic synthesizer Model 432A, Applied Biosys-.tems, Foster City, CA , partially purified by re-

verse-phase high-performance liquid chromatogra-

Ž .phy BioRad, Richmond, CA , lyophilized, and storeddesiccated at room temperature until use.

2.2. Serum samples

HIV-1 positive and negative control serum sam-ples were obtained from the American Red Cross.

2.3. Peptide enzyme immunoassay

Published procedures for PEIA, with slight modi-Ž .fication, were followed Pau et al., 1998 . Briefly,

peptides were dissolved in carbonate–bicarbonateŽ . Žbuffer 0.1 M, pH 9.4 Pierce Chemical, Rockford,.IL, USA to a final concentration of 0.05–10 mgrml,

and 100 ml of these solutions was used to coat themicrotiter wells by overnight incubation at 4 8C.Peptide-coated wells were washed twice with pH 7.4

Ž .phosphate-buffered saline PBS containing 0.05%

Ž . Ž . Ž . Ž .Fig. 1. Seroreactivity of tandem repeats TR top and multiple antigenic peptides MAPs bottom derived from consensus subtype BŽ .V3-loop sequence of HIV-1 gp120. Seroreactivity of the 13-residue monomeric peptide M1 was included for comparison. TR2 to TR5

comprised of two to five repeats, and MAP2 to MAP8 comprised of two, four and eight branches of M1, respectively. A coatingconcentration of 2.5 mgrml was used for all peptides.

( )P. Kim, C.-P. PaurJournal of Immunological Methods 257 2001 51–54 53

Ž . Ž . Ž .Fig. 2. Seroreactivities of M1 monomer , TR4 four repeats , and MAP4 four branches at varying coating concentrations with anŽ . Ž .HIV-positive top and HIV-negative bottom serum specimen. Sample dilution was 1:400.

Ž .Triton X-100 PBS-Tx , air dried, and stored desic-cated at y20 8C until use. Nonspecific binding sitesof the peptide-coated wells were blocked with 5%nonfat dry milk in PBS containing 0.1% Triton

Ž .X-100 milk buffer for 30 min at 37 8C just prior toperforming the assay. Sera were diluted in milkbuffer, and allowed to react with peptide-coatedwells for 1 h at 37 8C. Bound antibodies were

Ž .detected with goat anti-human IgG H q L -Ž .peroxidase conjugate BioRad, Hercules, CA and

tetramethyl-benzidinerhydrogen peroxide substratesŽ .BioFX, Owings Mills, MD , and optical densityŽ .OD at 450 nm was measured. Fold change inseroreactivity for a given peptide over M1 was esti-mated from the ratio of the reciprocal sample dilu-tions that resulted in similar ODs.

3. Results

Serologic reactivities of the tandem repeats of M1Ž . Ž .top and the MAPs bottom at varying sample

dilutions are shown in Fig. 1. At a coating concentra-tion of 2.5 mgrml, the reactivity of TR2 increasedby approximately 256-fold over M1, and the reactivi-ties of TR3 to TR5 further increased by another2-fold. The reactivity of MAP2 increased by approx-imately 512-fold over M1, and the reactivities ofMAP4 and MAP8 further increased by another 2-fold.

Serologic reactivities of M1, TR4 and MAP4 atvarying coating concentrations are shown in Fig. 2.Both TR4 and MAP4 sustained a stable and highreactivity at )0.5 mgrml with the positive controlŽ .top and did not react with the negative controlŽ .bottom at a sample dilution of 1:400. M1 was onlymarginally reactive at )1 mgrml, if the cutoff wasset at 2= the OD of the negative control at aparticular coating concentration.

4. Discussion

The low reactivity of the linear peptide M1 waspossibly due to its low binding efficiency to the solid

( )P. Kim, C.-P. PaurJournal of Immunological Methods 257 2001 51–5454

phase. However, increasing the coating concentrationof M1 up to 10 mgrml did not improve its sensitiv-ity. The increased molecular mass and size of thepolymeric peptides would likely increase the interac-

Ž .tion mainly hydrophobic between the peptide andthe solid phase. Other factors may also influence theassay sensitivity. It is known that the gp120 V3-loop

Ž .contains a type II turn at position 6 P of M1, whichŽ .is required for antibody binding Pau et al., 1994

and is also a critical part of the principal-neutralizingŽ .determinant LaRosa et al., 1990 . It is possible that

when M1 binds to the solid phase, its correct confor-mation is lost. For the polymeric peptides, since onlypart of the molecule is in contact with the solidphase, the other parts are free for antibody binding.Another possible explanation is the increase in anti-

Žgen density i.e. the number of binding sites per unit.surface area . This would also explain why the MAPs

are slightly more sensitive than the repeats. Theindividual peptide chains in the MAPs are moreclosely spaced than those in the repeats, and hence,occupy less surface area.

However, as indicated by our experimental re-sults, there is an upper limit for improvement. Whenthe multimeric peptide reached three repeats or fourbranches, further increases in chain lengths orbranches did not improve the assay sensitivity.

In conclusion, we have demonstrated that pooranalytical sensitivity of PEIAs that use shortmonomeric peptides as the antigen can be improvedsignificantly without sacrificing the assay specificityby using tandem repeats or MAPs. MAPs would be abetter choice because of their slightly higher sensitiv-ity and lower cost of production over tandem repeats.However, since every peptide has a different chemi-cal and physical property, we recommend that eachpeptide system be fully evaluated before being ap-plied to a particular assay.

References

Brattegaard, K., Soroh, D., Zadi, F., Digbeu, H., Vetter, K.M., DeCock, K.M., 1995. Insensitivity of a synthetic peptide-based

Ž .test Pepti-LAV 1-2 for the diagnosis of HIV infection inw xAfrican children letter . AIDS 9, 656–657.

Geerligs, H.J., Weijer, W.J., Bloemhoff, W., Welling, G.W.,Welling-Wester, S., 1988. The influence of pH and ionicstrength on the coating of peptides of Herpes simplex virustype 1 in an enzyme-linked immunosorbent assay. J. Immunol.Methods 106, 239–244.

LaRosa, G.J., Davide, J.P., Weinhold, K., Waterbury, J.A., Profy,A.T., Lewis, J.A., Langlois, A.J., Dreesman, G.R., Boswell,R.N., Shadduck, P. et al., 1990. Conserved sequence andstructural elements in the HIV-1 principal neutralizing deter-

wminant published erratum appears in Science 1991 FebŽ . x15;251 4995 :811 . Science 249, 932–935.

Pau, C.-P., Holloman, D.L., Lee-Thomas, S., Schochetman, G.,George, J.R., 1993. A combination immunoblot for the simul-taneous detection and differentiation of HIV-1 and HIV-2.Clin. Diagn. Virol. 1, 201–205.

Pau, C.P., Kai, M., Holloman-Candal, D.L., Luo, C.C., Kalish,M.L., Schochetman, G., Byers, B., George, J.R., 1994. Anti-genic variation and serotyping of HIV type 1 from four WorldHealth Organization-sponsored HIV vaccine sites. WHO Net-work for HIV Isolation and Characterization. AIDS Res. Hum.Retroviruses 10, 1369–1377.

Pau, C.P., Lam, L.L., Spira, T.J., Black, J.B., Stewart, J.A.,Pellett, P.E., Respess, R.A., 1998. Mapping and serodiagnosticapplication of a dominant epitope within the human herpesvirus 8 ORF 65-encoded protein. J. Clin. Microbiol. 36,1574–1577.

Shin, S.Y., Lee, M.K., Kim, S.Y., Hahm, K.-S., 1996. Use ofmultiple antigenic peptides as coating antigens in detection ofantibody. Mol. Cells 6, 169–175.

Tam, J.P., 1988. Synthetic peptide vaccine design: synthesis andproperties of a high-density multiple antigenic peptide system.Proc. Natl. Acad. Sci. U. S. A. 85, 5409–5413.

Viscidi, R.P., Hill, P.M., Li, S.J., Cerny, E.H., Vlahov, D.,Farzadegan, H., Halsey, N., Kelen, G.D., Quinn, T.C., 1991.Diagnosis and differentiation of HTLV-I and HTLV-II infec-tion by enzyme immunoassays using synthetic peptides. J.Acquired Immune Defic. Syndr. 4, 1190–1198.