Bioconjugate Chem. 1996, 7, 338−342
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Presentation of Peptide Antigens as Albumin Conjugates for Use in Detection of Serum Antibodies by Enzyme-Linked Immunosorbent Assay Zhiguang Yu,†,‡ John Mark Carter,§ Shaei-Yun Huang,†,‡ Henry Lackland,† Leonard H. Sigal,| and Stanley Stein*,†,‡ Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, New Jersey 08854, Chemistry Department, Rutgers University, Piscataway, New Jersey 08854, Cytogen Corporation, 307 College Road East, Princeton, New Jersey 08540, and Departments of Medicine, Pediatrics, and Molecular Genetics and Microbiology, UMDNJ-Robert Wood Johnson Medical School, One Robert Wood Johnson Place, New Brunswick, New Jersey 08903. Received January 16, 1996X
The use of linear peptides as antigens for detection of serum antibodies has been studied using a sequence of the Borrelia burgdorferi protein, flagellin, and Lyme disease sera as a model. It was found that a novel presentation of the peptide as a hapten on the carrier protein, bovine serum albumin, in the enzyme-linked immunosorbent assay format can be successfully applied to distinguish between Lyme disease and control sera.
INTRODUCTION
Diagnosis of many infectious diseases depends on detection of serum antibodies against determinants of the pathogenic microorganism. Our interest is in the serologic confirmation of the diagnosis of Lyme disease, which is caused by infection with the spirochete, Borrelia burgdorferi (Bb), transmitted by the deer tick (1). The most common Lyme disease diagnostic test is the enzymelinked immunosorbent assay (ELISA) (2), in which a bacterial lysate is adsorbed to the wells of a microtiter plate. Theoretically, in order to increase specificity (that is, decrease the number of false positives), it would be preferable to use defined peptide epitopes without adjacent sequences rather than entire bacterial proteins, which may be recognized by antibodies to proteins from other microorganisms. In some individuals without Lyme disease, such nonspecific binding may produce a false positive test result due to the presence of antibodies which cross-react with Bb proteins. Similarly, increased sensitivity (that is, fewer false negatives) might be secured by the use of more densely presented immunoreactive epitopes, rather than the entire proteins (sparsely populated epitopes). Although only a fraction of the antibodies elicited in an immune response to a protein may bind to a continuous epitope, as represented by a linear peptide, this may be sufficient for a diagnostic immunoassay (3). In furthering this technology, we present a generally applicable method for presentation of peptide antigens on ELISA plates as conjugates of bovine serum albumin. EXPERIMENTAL PROCEDURES
Antigen Preparation. Linear monomeric peptides and branched peptides were synthesized on PAL resin (Milligen/Biosearch, Woburn, MA) and Tentagel SRAM * Corresponding author: Stanley Stein, Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, NJ 08854. Telephone: 908-235-5319. Fax: 908-235-4850. † Center for Advanced Biotechnology and Medicine. ‡ Rutgers University. § Cytogen Corporation. | UMDNJ-Robert Wood Johnson Medical School. X Abstract published in Advance ACS Abstracts, April 1, 1996.
S1043-1802(96)00018-3 CCC: $12.00
Scheme 1. Conjugation of Cys-Containing Peptide to Bovine Serum Albumin
resin (Applied Biosystem, Foster City, CA), respectively, by Fmoc/t-butyl strategy, using a Model 9400 Excell instrument (Milligen/Biosearch, Milford, MA) or a Model 430A instrument (Applied Biosystems, Foster City, CA). Monomeric peptides were purified by high-performance liquid chromatography (HPLC) on a 2.2 × 25 cm C18 reverse phase column (Vydac 218TP1022, 10 µm, 300 Å pore size), using a linear gradient from 0.1% trifluoroacetic acid (TFA) (buffer A) to 100% buffer B (70% acetonitrile and 20% 2-propanol in buffer A) within 50 min, at a flow rate of 4.0 mL/min, whereas branched peptides were desalted by gel filtration chromatography (Sephadex G-15). Peptide structure and homogeneity were confirmed by laser desorption mass spectrometry, amino acid analysis, and/or Edman degradation on a Model 477 microsequencer (Applied Biosystems). Synthetic peptides were coupled to bovine serum albumin (BSA, Sigma, St. Louis, MO) as shown in Scheme 1. The BSA (2.5 mg/mL) was first activated by © 1996 American Chemical Society
Peptide−Albumin Conjugates for Use in ELISA
reaction with sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC, Pierce Chemical Co., Rockford, IL) (2.5 mg/mL) at 4 °C for 2 h in 20 mM sodium phosphate buffer (pH 7.0) containing 0.15 M sodium chloride and then freed of excess reagent by passage through a column of Sephadex G-75. The activated BSA was then reacted with the cysteinecontaining peptide at 4 °C overnight, in 0.1 M sodium phosphate buffer (pH 7.0) containing 0.15 M sodium chloride and 1 mM ethylenediaminetetraacetic acid (EDTA). The resulting peptide-BSA conjugate was freed of excess peptide by passage through a column of G-75. Conjugates were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) on 10% polyacrylamide gels (Novex, San Diego, CA) under reducing conditions and by amino acid analysis of aliquots hydrolyzed for 24 h in HCl vapor. ELISA. For coating the ELISA plate (Immulon 4, Dynatech Labs, Chantilly, VA), 100 µL of each antigen, at 25 µg/mL (unless otherwise indicated) in 0.1 M sodium phosphate buffer (pH 7.0) containing 0.15 M sodium chloride (PBS buffer), was added to each well in triplicate, and the wells were incubated overnight at room temperature. The wells were washed three times with PBS and then blocked for 1 h with 200 µL/well of 5% nonfat dry milk in PBS. For the assay, 100 µL of each serum sample, diluted 1:100 (unless otherwise indicated) in blocking buffer, was added per well and the wells were incubated for 2 h at room temperature. The plate was washed with PBS and incubated for 1 h with 100 µL/ well of peroxidase-conjugated goat anti-human IgG/IgM/ IgA antiserum (Cappel, Durham, NC) that had been diluted 1:4000 with a solution of 1% goat serum and 0.1% casein in PBS. After thorough washing with PBS, color was developed for 1 h (unless otherwise indicated) using 100 µL/well of 0.5 mg/mL ABTS substrate (Sigma), and absorbance was read on an MR 600 (Dynatech) plate reader at 410 nm. RESULTS
Epitope Selection. The first step in the development of a peptide-based ELISA is selection of the epitope sequences. This can be accomplished using a technology in which a series of peptides, comprising the sequence of an antigenic protein, are synthesized in a sequenceoverlapping manner on noncleavable pins (4). In a previous study using a small number of Lyme disease and control sera (5), antibody specificity to the immunodominant Bb protein, flagellin, was observed with peptides corresponding to a central region of the protein. This epitope was confirmed in an approach using fusion proteins, as well as the soluble peptide, EGVQQEGAQQPA (6). A region in the N-terminal portion of Bb flagellin, although reactive with Lyme disease sera, was cross-reactive with control sera (5). We synthesized a series of epitope-related peptides on pins varying in length from 6 to 13 residues and varying in the position of the N-terminal starting point. By testing nine individual Lyme sera, we found the optimal peptide sequence for detecting Lyme sera to be VQEGVQQEGAQQP. Peptide Presentation Formats. Different approaches for presentation of the flagellin epitope sequence on ELISA plates were investigated. A solution of each peptide (N-terminal-acetylated monomer, tandem repeat, 4-branched and 8-branched) was serially diluted, allowed to adsorb passively to the ELISA plate overnight, and then tested with a pool of nine flagellin positive Lyme disease sera and a pool of 10 control (non-Lyme disease) sera. No response was observed for the monomer, tandem repeat peptide, or 4-branched multiple antigenic
Bioconjugate Chem., Vol. 7, No. 3, 1996 339
peptide (MAP); only a minimal response was obtained for the 8-branched MAP, but it was not considered to be strong enough to distinguish between Lyme disease and normal sera (data not shown). The lack of response with the monomeric and branched peptides might be due to either poor retention on the plate or masking of the epitope. To improve peptide immobilization, the plate was first treated with glutaraldehyde, as previously reported (7). An N-terminal free MAP-4 peptide gave a dose-dependent response (Figure 1). At the same condition, N-terminalacetylated MAP-8 peptide still showed a minimal response (data not shown), demonstrating that the primary amine groups are involved in peptide immobilization. Furthermore, when 0.1 M glycine was added to the blocking reagent, the signal in the test with MAP-4 was totally quenched (data not shown), suggesting that the immobilization through glutaraldehyde is reversible. Since peptides are usually not immunogenic, they are covalently conjugated to carrier proteins for generation of antibodies (8). Accordingly, the presentation on an ELISA plate of peptide epitopes as haptens on a carrier protein was investigated. A facile approach is to use thiol-specific chemistry (Scheme 1). A cysteine residue, followed by two β-alanine residues (spacer), was incorporated at the C-terminus of the flagellin peptide during solid phase synthesis. In the first reaction of the conjugation protocol, the amino groups of bovine serum albumin (BSA), which was chosen as the carrier protein, were converted to thiol-reactive maleimido groups using the reagent sulfo-SMCC. The activated BSA was purified by gel filtration chromatography. In the second part of the protocol, the activated BSA was reacted with the cysteine-containing flagellin peptide, which had previously been purified by reverse phase HPLC. When the activation reaction was done with a 150:1 molar excess of SMCC to BSA (which is a 2.5-fold equivalent excess, based on the 60 primary amino groups in BSA), a coupling ratio of 13 molecules of peptide per molecule of protein was obtained, as determined by amino acid analysis (9) (Table 1). The calculation is based on quantitation of β-Ala in the peptide, and Tyr or Phe in the BSA. Thus, the peptide accounts for about one-fourth of the mass of the conjugate. When the first reaction was performed with a 60:1, 24:1, or 9.6:1 molar excess of sulfoSMCC to BSA, peptide/protein coupling ratios of 8, 4, and 2, respectively, were obtained (Table 1). In each second reaction, the molar ratio of peptide, which was measured by quantitative Ellman’s assay (10), to (activated) BSA was always 120:1 (2.0-fold equivalent excess). The peptide reagent was in excess, as confirmed in separate experiments in which increasing the amount of peptide in the second reaction did not change the final coupling ratio (data not shown). Analysis by polyacrylamide gel electrophoresis showed broad bands with an average molecular mass for each conjugate comparable to that calculated from the coupling ratio (Figure 2). The ELISA response for each peptide-BSA conjugate at different dilutions was tested using pooled Lyme disease and control sera, and a dose-dependent response was obtained (Figure 1). The maximal level of protein adsorption, which was reached when 2.5 µg per 100 µL was added to a well, was about 70 ng/well, as determined by bicinchoninic acid protein assay (11). This value is close to previous findings (12). Similar to the dependence of ELISA response on the quantity of conjugate adsorbed to the well, an increase in response with the relative content of antigenic peptide in the conjugate was observed (Figure 1). In separate experiments, the use of longer spacers, namely four or eight residues of γ-amino-
340 Bioconjugate Chem., Vol. 7, No. 3, 1996
Yu et al.
Figure 1. Comparison of different antigen presentations. The amount of BSA-peptide conjugate or MAP peptide applied per well is given. Each point represents the average of six determinations using a pool of nine Lyme disease sera or a pool of ten normal sera diluted 1:100 in blocking buffer and using a substrate development time of 25 min. Standard error bars are shown. GTA* indicates pretreatment of the plate with glutaraldehyde. The list is in the same order as the curves. Table 1. Determination of Coupling Ratios by Amino Acid Analysis BSA conjugate 9.6:1 sulfoSMCC:BSA 24:1 sulfoSMCC:BSA 60:1 sulfoSMCC:BSA 150:1 sulfoSMCC:BSA
a
amino acid
amount (pmol)
coupling ratioa
Phe Tyr β-Ala Phe Tyr β-Ala Phe Tyr β-Ala Phe Tyr β-Ala
571 369 93 701 457 197 757 473 398 918 613 830
2.2 2.4
average ratio
2.3 3.8 4.3 4.1 7.1 8.0 7.6 12.2 12.8 12.5
Coupling ratio ) (β-Ala/2)/(Tyr/19) or (β-Ala/2)/(Phe/27).
butyric acid or a polyethylene glycol heterobifunctional linker of 2 kDa (Shearwater Polymers, Huntsville, AL), resulted in progressively weaker ELISA responses with Lyme disease sera (data not shown). A lower ELISA response was also observed when the two β-alanine residues were not included (data not shown). The conjugate having the highest relative content of flagellin peptide (1:13 BSA:Fla) was added to each well at a level of 2.5 µg in 100 µL. The ELISA response, determined at a wide range of serum dilutions, was found to easily distinguish individual Lyme sera from normal controls (Figure 3). Since the readings for control sera also increased with concentration, the optimal discrimination between Lyme disease and non-Lyme disease sera may not necessarily be achieved at a high serum concentration. Another variable is the time of substrate incubation. Color development is essentially completed within 1 h (data not shown). On the basis of these results, a well loading of 2.5 µg of conjugate, a serum dilution of 1:100, and an incubation time of 60 min were
Figure 2. SDS-PAGE of BSA-peptide conjugates: lane 1, molecular mass standard; lane 2, BSA; lane 3, activated BSA; lanes 4-7, BSA-flagellin conjugates with activation ratios (sulfo-SMCC:BSA) of 9.6:1, 24:1, 60:1, and 150:1, respectively.
used to evaluate the flagellin epitope peptide with serum samples. Preliminary Studies on Serodiagnosis. Forty-one sera (Figure 4A), in which 16 were clinically diagnosed as Lyme disease, were tested with 1:13 BSA:Fla as the antigen. An additional 10 normal sera were used to establish a cutoff value as follows. The average absorbance reading of the ten control sera and the standard deviation from this average were calculated. The cutoff was set as the average plus three standard deviations. Any reading above this cutoff value was considered positive for Lyme disease. As seen in Figure 4A, 12 positive responses were obtained, 11 of which corresponded to Lyme disease sera, giving a sensitivity of
Bioconjugate Chem., Vol. 7, No. 3, 1996 341
Peptide−Albumin Conjugates for Use in ELISA
Figure 3. Serum dilution curves. Serum was diluted into blocking buffer. Analysis was with 2.5 µg of the BSA conjugate of flagellin peptide (1:13). Each point represents the average of triplicate assays using a suboptimal substrate development time of 10 min to keep all points on scale. The list is in the same order as the curves.
CDC negative serum (no. 26) was positive in our peptide ELISA (91% specificity). Clinically, this patient had the rash typical for Lyme disease, so the true nature of this serum is unclear. DISCUSSION
Figure 4. Preliminary serodiagnosis with BSA-flagellin (1: 13) as the antigen. Clinically diagnosed Lyme sera or CDC positive sera are represented by filled bars, whereas clinically normal sera or CDC negative sera are represented by empty bars. In our peptide ELISA, the index value of each serum was obtained by division of its absorbance by the cutoff value (0.51), which is the average (0.30) plus three standard deviations (3 × 0.07) of a group of ten normal controls (not shown).
69% (11/16). One false positive was observed among 25 normal samples, leading to a specificity of 96% (24/25). Another 46 sera (panel B), obtained from the Centers for Disease Control and Prevention (CDC), were also tested using the same BSA-flagellin peptide conjugate (Figure 4B). On the basis of the composite results of three CDC tests (Western blotting for IgG and for IgM and flagellin protein ELISA), 24 out of 35 Lyme sera were identified in our assay, giving a sensitivity of 69%. One
The use of small peptides as antigens for capturing antibodies in the serodiagnostic ELISA has the potential to limit false positive responses. In addition to the advantage of epitope specificity, synthetic peptide antigens can be carefully characterized from batch to batch, as opposed to cell lysates which may vary with passage of the bacteria. An obstacle to application of this approach, which is the subject of this article, is to devise a facile and general method for presentation of small peptides on the ELISA plate. Although monomeric peptides (13, 14) and multiple-branched peptides (15, 16) have been coated directly on the ELISA plate as antigens, their use in the present application was unsuccessful. The physical adsorption of antigen to the ELISA plate depends mainly on hydrophobic interaction. With small peptides, the force is frequently too weak to permit detection of the final antigen-antibody complex. Besides, passive coating of antigen often masks the epitope and underestimates the antibody level. This masking phenomenon has even been observed with a protein, unless presented as part of a fusion protein (17). Numerous methods have been developed to immobilize peptides to the polystyrene surface. The plate can be activated with UV or γ irradiation and then treated with glutaraldehyde. Peptides can then be linked to the plate through primary amino groups (18, 19). Peptides can also be immobilized as haptens on a carrier, such as BSA or polylysine (20, 21). Previously, these conjugations were prepared through carboxyl or amino groups under harsh reaction conditions, such as N-hydroxysuccinimide/ dicyclohexylcarbodiimide, glutaraldehyde, and bis-diazotized benzidine. Coupling with these reagents may completely abolish the antigenicity of the peptides (22),
342 Bioconjugate Chem., Vol. 7, No. 3, 1996
especially when Asp, Glu, or Lys residues are present. Therefore, these methods are not generally applicable. By synthesis of a cysteine-containing epitope peptide, conjugation to a carrier protein can be done in a two step reaction. Since the free thiol group preferentially reacts with maleimide, other residues in the peptide are not affected. Thus, this conjugation method is gentle and dependable. None of the epitope sequences identified in our pin-peptide scanning of several Bb protein sequences contained a residue of cysteine (data not shown), making the extra C-terminal cysteine residue a unique site for conjugation. Since many copies of the epitope peptide are present on each molecule of conjugate, several haptens will always be sterically available for antibody interaction after immobilization. In addition, several copies of peptides on the same carrier protein molecule may mediate multivalent interaction with antibody, potentially leading to enhanced avidity of interaction and greater reactivity in the immunoassay. BSA was chosen as the carrier, since it is a major component of serum and is essentially inert with respect to antibody cross-reactivity. Other advantages are its strong binding to the polystyrene microtiter plate surface, its availability in high purity, and its ease of handling. To prevent steric hindrance from the carrier protein, the coupling site was positioned away from the antigenic site with two spacer residues of β-alanine. Similarly, an optimal number of glycine residue spacers had been reported for branched peptide antigens (15). In our preliminary serological test for Lyme disease, using a single epitope from Bb flagellin as the antigen, a sensitivity of 69% and a specificity of 96 or 91% were obtained for panels A and B, respectively. It should be noted that a similar flagellin epitope sequence was presented as a glutathione transferase fusion protein, and a similar sensitivity for detection of Lyme disease was reported (23). A parallel comparison of this set of serum samples with a commercial ELISA kit (MarDx Diagnostics, Carlsbad, CA), using Bb whole cell lysate as the antigen, gave a higher sensitivity (81 and 88%) but a lower specificity (68 and 71%). This comparison demonstrates that a peptide ELISA may be useful for elimination of false positives in the serological confirmation of Lyme disease. We have identified some epitope sequences from several other immunodominant Bb proteins. These epitope peptides were synthesized, conjugated to BSA, and used to screen the two serum panels reported here. It was concluded that, if more epitopes from other Bb antigens are utilized, a higher sensitivity can be achieved (to be published). ACKNOWLEDGMENT
This research was supported in part by a Technology Transfer Merit Grant from the New Jersey Commission on Science and Technology. LITERATURE CITED (1) Barbour, A. G., and Fish, D. (1993) The biological and social phenomenon of Lyme disease. Science 260, 1610-1616. (2) Karlsson, M. (1990) Western Immunoblot and flagellin enzyme-linked immunosorbent assay for serodiagnosis of Lyme disease. J. Clin. Microbiol. 18, 2148-2150. (3) Van Regenmortel, H. V., and Pellequer, J. L. (1994) Predicting antigenic determinants in proteins: Looking for unidimensional solutions to a three-dimensional problems? Pept. Res. 7, 224-228. (4) Geysen, H. M., Meloen, R. H., and Barteling, S. J. (1984) Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid. Proc. Natl. Acad. Sci. U.S.A. 81, 3998-4002.
Yu et al. (5) Schneider, T., Lange, R., Ronspeck, W., Weigelt, W., and Kolmel, H. W. (1992) Prognostic B-cell epitopes on the flagellar protein of Borrelia burgdorferi. Infect. Immun. 60, 316-319. (6) Fikrig, E., Berland, R., Chen, M., Williams, S., Sigal, L. H., and Flavell, R. A. (1993) Serological response to the Borrelia burgdorferi flagellin demonstrates an epitope common to a neuroblastoma cell line. Proc. Natl. Acad. Sci. U.S.A. 90, 183187. (7) Ordronneau, P., Abdullah, L. H., and Petrusz, P. (1991) An efficient enzyme immunoassay for glutamate using glutaraldehyde coupling of the hapten to microtiter plates. J. Immunol. Methods 142, 169-176. (8) Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual. Immunizations, pp 53-135, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. (9) Tsao, J., Lin, X., Lackland, H., Tous, G., Wu, Y., and Stein, S. (1991) Internally standardized amino acid analysis for determining peptide/carrier protein coupling ratio. Anal. Biochem. 197, 137-142. (10) Riddles, P. W., Blakeley, R. L., and Zerner, B. (1979) Ellman’s reagent: 5,5′-Dithiobis(2-nitrobenzoic acid)-a reexamination. Anal. Biochem. 94, 75-81. (11) Wiechelman, K., Braun, R., and Fitzpatrick, J. (1988) Investigation of the bicinchoninic acid protein assay: identification of the groups responsible for color formation. Anal. Biochem. 175, 231-237. (12) Stevens, P. W., Hansberry, M. R., and Kelso, D. M. (1995) Assessment of adsorption and adhesion of proteins to polystyrene microwells by sequential enzyme-linked immunosorbent assay analysis. Anal. Biochem. 225, 197-205. (13) Peralta, J. M., Teixeira, M. G. M., Shreffler, W. G., Pereira, J. B., Burns, J. M., Jr., Sleath, P. R., and Reed, S. G. (1994) Serodiagnosis of Chagas’ disease by enzyme-linked immunosorbent assay using two synthetic peptides as antigens. J. Clin. Microbiol. 32 (4), 971-974. (14) Gnann, J. W., Jr., McCormick, J. B., Nelson, J. A., Mitchell, S., Nelson, J. A., and Oldstone, M. B. A. (1987) Synthetic peptide immunoassay distinguishes HIV type 1 and HIV type 2 infections. Science 237, 1346-1349. (15) Marsden, H. S., Owsianka, A. M., Graham, S., Mclean, G. W., Robertson, C. A., and Subak-Sharpe, J. H. (1992) Advantages of branched peptides in serodiagnosis. Detection of HIVspecific antibodies and the use of glycine spacers to increase sensitivity. J. Immunol. Methods 147, 65-72. (16) Tam, J., and Zavala, F. (1989) Multiple antigen peptide: A novel approach to increase detection sensitivity of synthetic peptides in solid-phase immunoassays. J. Immunol. Methods 124, 53-61. (17) Cartwright, G. A., Rothel, J. S., and Lightowlers, M. W. (1995) Conventional immunoassays underestimate anti-GST antibody titre. J. Immunol. Methods 179, 31-35. (18) Dagenais, P., Desprez, B., Albert, J., and Escher, E. (1994) Direct covalent attachment of small peptide antigens to enzyme-linked immunosorbent assay plates using radiation and carbodiimide activation. Anal. Biochem. 222, 149-155. (19) Boudet, F., Theze, J., and Zouali, M. (1991) UV-treated polystyrene microtitre plates for use in an ELISA to measure antibodies against synthetic peptides. J. Immunol. Methods 142, 73-82. (20) Price, M. R., Sekowski, M., Hoo, D. S. W., Durrant, L. G., Hudecz, F., and Tendler, S. J. B. (1993) Measurement of antibody binding to antigenic peptides conjugated in situ to albumin-coated microtitre plates. J. Immunol. Methods 159, 277-281. (21) Ball, J. M., Henry, N. L., Montelaro, R. C., and Newman, M. J. (1994) A versatile synthetic peptide-bound ELISA for identifying antibody epitopes. J. Immunol. Methods 171, 3744. (22) Briand, J. P., Muller, S., and Van Regenmortel, M. H. V. (1985) Synthetic peptides as antigens: pitfalls of conjugation methods. J. Immunol. Methods 78, 59-69. (23) Cretella, S., Gordon, S., Flavell, R. A., and Fikrig, E. (1995) Evaluation of a Lyme disease enzyme immunoassay using the 41-G fragment of flagellin. Eur. J. Clin. Microbiol. Infect. Dis. 14, 609-613.
BC960018S
