ANALYTICAL

BIOCHEMISTRY

189,80-83

(1990)

A General Procedure for Evaluation of Immunological Relevance of Synthetic Peptides: Peptides Synthesized on Paper in Enzyme-Linked lmmunosorbent Assay Viktor Krch%k, Josef VAgner, Jan No&k,* Alena Suchhnkov&* and Jaroslav Research Institute for Feed Supplements and Veterinary Drugs, 254 49 JE’loue’u Prahy; and *Institute of Sera and Vaccines, 101 03 Prague 10, Czechoslovakia

Roubal*

ReceivedFebruary1,199O

Multiple continuous-flow solid-phase peptide synthesis has been adapted for synthesis of peptides on a cellulose carrier (Whatman 3MM paper). Paper-bound synthetic peptides that represent antigenic determinants of particular proteins detected antibodies against the respective proteins in an enzyme-linked immunosorbent assay, The method is applied to the synthesis, and use in site-directed serology, of four peptides derived from the gp41 glycoprotein of HIV, the Epstein-Barr virusdetermined nuclear antigen-l and VCA proteins of the Epstein-Barr virus, and the early region of human papillomavirus type 11. o 1990 Academic prws, I~C.

In the study of principles of immunological regulation on a molecular basis, proteins are the most significant natural compounds and attract the particular attention of researchers in this field. The aptitude for humoral and cellular immunological responsiveness to proteins is located in certain domains referred to as B- and T-cell determinants for humoral and cellular responses, respectively (1,2). Because the synthesis of medium-size peptides (up to ca. 20 amino acid residues) by the Merrifield solid-phase method (3) is now a routine matter, synthetic peptides, whose amino acid sequences have been derived from proteins, have become the model compounds with which to study the immunological relevance of particular protein domains. Site-directed serology evaluates the immunological relevance of particular peptides with respect to the presence of B-cell determinants on the basis of the interaction of the corresponding synthetic peptides with antibodies that have been elicited against the whole proteins, e.g., antibodies against viral proteins in sera

of infected individuals. In practice, ELISA’ is the most commonly used technique. The methodology is based on the use of peptides, either free or coupled to a carrier protein, that are attached to the surface of wells of polystyrene ELISA plates. However, not every peptide will bind noncovalently to this surface, and coupling peptides to carrier proteins has limitations of its own (4). Moreover, there is a complicated moment in the synthesis and site-directed serology of synthetic peptides. The peptides are first synthesized on one carrier and then removed from it and attached to a different support to facilitate antibody screening. To simplify the procedure in testing peptides, Geysen et al. (5) have developed method for the synthesis of peptides on plastic rods suitable for direct ELISA screening. Recently, Modrow et al. (6) described the direct use of polystyrene-bound peptides in ELISA. In this paper we describe an alternative approach: the synthesis of peptides possessing antigenic determinants on cellulose disks and their use in site-directed serology for the detection of antibodies. MATERIALS

AND

METHODS

General Methods Solvents [DCM technical grade (Lachema, Brno), all others analytical grade (Fluka, Buchs)] were redistilled before use and stored over Type 4A molecular sieves (Fluka, Buchs). TFA (Riedel de Haen, Seelze) was redis1 Abbreviations used: ELISA, enzyme-linked immunosorbent assay; Boc, tert-butyloxycarbonyl; DCM, dichloromethane; DIC, N,N’-diisopropylcarbodiimide; DMA, N,N’-dimethylacetamide; DMF, NJ’-&methylformamide; EBNA, Epstein-Barr virus-determined nuclear antigen; Fmoc, fluorenylmethyloxycarbonyl; HIV, human immunodeficiency virus; HOBt, N-hydroxybenzotriazole; HPV, human papillomavirus; TFA, trifluoroacetic acid, EBV, Epstein-Barr virus; MeOH, methanol; t-Bu, t-butyl; aa, amino acids; OPD, o-phenylenediamine; VGA, viral capsid antigen.

80

Copyright All

rights

0

1990 of reproduction

0003-2697/90 $3.00 by Academic Press, Inc. in any form reserved.

PEPTIDES

ON

PAPER

IN

ENZYME-LINKED

tilled and used without drying. Other chemicals were used as supplied: DMA, N-methylpyrrolidone, DIC, HOBt, piperidine, dimethylaminopyridine (all Fluka, Buchs), swine anti-human IgG conjugated with horseradish peroxidase, OPD (USOL, Prague), and H202 (Lachema, Brno). Side-chain functional groups were protected as follows: Asp’, Glu, Ser, Thr, and Tyr by the t-butyl group; Cys by trityl; Arg by 4-methoxy-2,3,6trimethyl-benzenesulfonyl; and Lys by the Boc group (Bachem, Bubendorf). Peptide Sequences The amino acid sequence of the P-IX-4 peptide was derived from glycoprotein gp41 of HIV-l (aa 598-614) (7), peptide P-III-7 was derived from the VCA protein of EBV (aa 99-117) (8), peptide P-XVII-l is part of the EBNA-1 protein of EBV (9) (the sequence repeats three times: 150-168,176-195,271-289), and peptide P-XIX5 was derived from the early region (E7) of HPV type 11 (aa 43-52, V. Krchriik, in preparation). Attachment

of Fmoc-Gly

to Paper

Fifty disks cut from Whatman 3MM paper (diameter 13 mm) were immersed in a solution consisting of 1 mmol Fmoc-Gly, 1 mmol HOBt, 1 mmol DIC, and 0.2 mmol dimethylaminopyridine in 5 ml DMF and stirred very gently for 2 h. The disks were then inserted into a lo-ml polypropylene syringe equipped with a sintered polypropylene disk at the bottom, thoroughly washed with DMF, DCM, and methanol, and air-dried. To determine the degree of substitution, the Fmoc group was removed from one disk by a lo-min treatment with 1 ml of a 20% solution of piperidine in DMF (v/v). The loading was calculated from the absorbance of the solution at 301 nm (t = 8100). The loading of our paper disks was 0.75 pmol Gly/cm’. Peptide Synthesis Synthesis was performed on a manually operated synthesizer described in recent papers (10,ll). Each polypropylene flow reactor was charged with 10 paper disks, the flow reactors were connected to the synthesizer, and the synthesis was carried out essentially as described for multiple continuous-flow solid-phase peptide synthesis (11). p-Methylbenzhydrylamine resin was placed into the first flow reactor and served to trap impurities from solvents; no amino acid was coupled to this resin. One synthetic cycle, incorporating a Fmoc/t-Bu protection strategy, consisted of the following steps: 2 Amino acid abbreviations IUB Commission list (1984).

are used in accordance with Eur. J. Biochem. 183,9-37.

the IUPAC-

IMMUNOSORBENT

ASSAY

81

(i) deprotection, 50% piperidine in DMF (v/v) for 10 min (3 min regular flow rate, 7 min reduced flow rate of ca. 5 ml/min); (ii) washing, DCM; (iii) washing, DMF, to neutral effluent (checked by wetted pH paper); (iv) condensation: take 1 meq of protected amino acid in 2 ml DMA (Gin and Asn in N-methylpyrrolidone), add 1 meq HOBt in 0.5 ml DMA, add 1 meq DIC in 1 ml DCM, add 0.1 ml of 0.01 M solution of bromophenol blue (12) in DMA, disconnect concatenated flow reactors, and transfer the solution of HOBt ester into each reactor, wait until yellow color appears; (v) washing, DMF, 5 min. The flow rate during the washing steps was ca. 20-30 ml/min, and the overpressure was 50-80 kPa. After the last synthetic cycle the Fmoc protecting groups were removed (steps (i) and (ii)) and the side-chain protecting groups were split off by treatment with TFA containing 3% thioanisol, 3% ethanedithiol, and 3% o-cresol as scavengers for 2 h at ambient temperature. The disks were thoroughly washed with DCM and MeOH and airdried. Peptide-Based

ELISA

Each paper disk was cut into four parts. The quarters were incubated with 500 ~1 of bicarbonate buffer, pH 9.6, containing 1% BSA for 1 h to block nonspecific adsorption of immunoglobulins and washed thoroughly five times with PBS buffer (pH 7.2) containing 1 M NaCl and 1% Triton X-100 in a 2-ml plastic syringe equipped with polypropylene sinter. Each washing was performed with 10 ml of washing solution. The disks were then incubated for 1 h with 500 ~1of sera diluted 1:500 with washing buffer containing 1% BSA, washed five times, and incubated with swine anti-human IgG conjugated with horseradish peroxidase for 1 h. The disks were washed four times with washing solution, the color was developed by incubation with OPD and hydrogen peroxide in citrate buffer pH 5.0, the reaction was stopped by the addition of 500 ~1 of 2 M sulfuric acid, and the resulting absorbance was measured at 490 nm with a Dynatech Model MR 700 microplate reader at a two-wavelength mode. All operations were performed at room temperature. RESULTS

AND

DISCUSSION

To exemplify the synthesis of peptides on the cellulose support and the use of cellulose-bound peptides in sitedirected serology, we selected four peptides that had been shown to detect antibodies against the respective proteins in conventional ELISA, specifically a peptide derived from the HIV-l gp41 glycoprotein (7), an EBVVCA-derived peptide (8), an EBV-EBNA-l-derived

82

KRCHNAK TABLE

1

Amino Acid Sequences of Peptides Peptide code P-IX-4 P-III-7 P-XVII-1 P-XIX-5

Amino YLKDQQLLGI VPTI AHGDGRR AGAGGGAGGAGAGGGAGGAG QDAQPLTQHY

acid sequence WGCSGKL PSKQRTFI

peptide (9), and an E7-derived peptide from HPV type 11 (V. Krchnak, in preparation). The sequences are shown in Table 1. Cellulose disks were cut from Whatman 3MM paper and the carboxy-terminal amino acid Gly was attached to the hydroxyl groups of the cellulose via an ester bond using Fmoc-Gly-OBt ester in the presence of dimethylaminopyridine. The presence of a catalytic amount of dimethylaminopyridine is essential, since activation by the HOBt ester itself does not provide sufficient substitution of amino acids on the cellulose carrier. Synthesis of peptides on a cellulose carrier has already been described by Blankemeyer-Menge and Frank (13) and recently also by Eichler and co-workers (14). In the former work (13), the cellulose support was functionalized via an ally1 ester linkage that can be cleaved by Pd(O)-catalyzed isomerization without loss of protecting groups and yielding protected peptide. Eichler and co-workers (14) reacted the hydroxyl group of cellulose with Fmoc-protected amino acid chloride. Peptide cleavage was achieved by treatment with a 1 M NaOH or TFA/DCM solution with the peptide being attached via an acid-labile handle. In our approach we esterified cellulose hydroxyl groups by Fmoc-Gly-OBt in the presence of dimethylaminopyridine as catalyst. To determine the degree of loading, the Fmoc group was cleaved by a solution of piperidine in DMF and absorption of this solution was measured. The loading of ca. 0.75 pmol Gly/cm2 of paper is comparable with the loading of l-2 pmol/cm2 described by Eichler (14) with the functionalization performed via Fmoc amino acid chloride. The synthesis of peptides on Fmoc-Gly-functionalized cellulose was carried out by conventional chemistry, using the Fmoc protecting group for N-a-amino group protection and t-butyl-type side-chain protection. The Fmoc groups were removed by a solution of piperidine in DMF and the coupling reaction was performed by HOBt esters, DIC having been used for in situ preactivation. Diisopropylurea is soluble in a DCM-DMF mixture and eliminates the necessity of removing the insoluble urea that is formed when activating with dicyclohexylcarbodiimide. To monitor the course of the condensation, we employed our recently developed method that makes use of the acid-base indicator bromophenol blue (12). The

ET

AL.

disks were colored by the solution of bromophenol blue and HOBt in DMA. Deeply blue paper was then reacted with the HOBt ester of the Fmoc amino acid until the blue color disappeared. Synthesis was carried out in a manually operated synthesizer that had been constructed for multiple continuous-flow solid-phase peptide synthesis (11). After completion of the synthesis the side-chain-protecting groups were removed by a TFA solution in DCM and the paper was thoroughly washed and air-dried. The synthesis does not require any special chemistry and therefore paper-bound peptides can be synthesized in the same batch with standard synthesis on resin. The peptides described here have been synthesized by using the Fmoc/t-Bu protection strategy; however, the Boc/ Bzl protection strategy is compatible with the synthesis on paper as well. Several dozen peptides can be synthesized simultaneously, similar to the tea-bag synthesis developed by Houghten (15). To evaluate the synthetic efficiency and the purity of the peptides synthesized, they can be split off the support by l-h treatment with a 1 M NaOH solution in water (14). To compare our method with Geysen’s (5), we also synthesized the peptides on polyethylene rods. The condensation of protected amino acids was monitored by bromophenol blue; the coupling on the rods typically required an overnight reaction. Interaction of cellulose-bound peptides with antibodies was demonstrated by the conventional ELISA procedure that involved pretreatment with BSA solution to decrease nonspecific adsorption of immunoglobulins, incubation with diluted serum sample, thorough washing, incubation with enzyme-labeled swine anti-human antibodies, and development of color by reaction with an enzyme substrate. The ELISA results are shown in Table 2. The reactivity was expressed as the absorbance ratio of the paper-bound peptides to paper without peptide. The reaction of peptides bound to cellulose was invariably more sensitive than that of peptides synthesized on rods. The specific nature of this reaction was confirmed

TABLE

ELISA with Paper-Bound

2

Synthetic Antigens Reactivity”

Peptide code P-IX-4 P-III-7 P-XVII-1 P-XIX-5

virus

Protein

Location

Paper

Rod

HIV-l EBV EBV HPV-11

gP41 VCA EBNA-1 E7

598-614 99-117 b

5.4 4.8 3.5 4.2

3.5 2.0 2.3 3.1

n ELISA absorbance ratio: b The sequence is repeated

paper-bound three times:

43-52

peptide/paper. 150-168,176-195,271-289.

PEPTIDES

ON

PAPER

IN

ENZYME-LINKED

by preincubation of positive sera with free peptides (prepared independently on polystyrene resin): the absorbance ratio dropped significantly (results not shown). Synthesis of peptides on a paper support represents a method of general applicability to testing synthetic peptides for their antigenic properties and mapping the immunological relevance of particular domains in proteins. The synthesis does not require any special chemistry; the carrier-paper-is readily available; bromophenol blue monitoring documents smooth couplings; if necessary, the peptides can be split off the carrier; and the interaction with antibodies is evaluated by conventional ELISA with sensitivity higher than that for peptides on plastic rods. ACKNOWLEDGMENT We thank ene rods.

Professor

H. Mario

Geysen

for his kind

gift of polyethyl-

REFERENCES 1. Krchnik, V., Mach, O., and Mali, A. (1989) in Methods in Enzymology (Langone, J., Ed.), Vol. 178, pp. 586-611, Academic Press, Orlando, FL. 2. Cornette, J. L., Margalit, H., DeLisi, C., and Berzofsky, J. A. (1989) in Methods in- Enzymology (Langone, J., Ed.), Vol. 178, pp. 611-634, Academic Press, Orlando, FL.

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83

ASSAY

3. Barany, G., and Merrifield, R. B. (1980) in The E., and Meienhofer, J., Eds.), Vol. 2, pp. 2-254, New York.

Peptides Academic

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(Gross, Press,

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Schoofs,

G., and

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of the European

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J., and Mach,

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J., Saf&, P., and Lebl, 63,2542-2548. B., and Frank,

R. (1988)

M., and Bienert, 64,1746-1752.

R. A. (1985)

Proc. Natl.

Acad.

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Z’etrahe-

Znt. J. Peptide M.

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ProCollect.

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