Clin. exp. Immunol. (1992) 89, 115-119
Monoclonal antibody epitopes of mycobacterial 65-kD heat-shock protein defined by epitope scanning A. H. HAJEER, J. WORTHINGTON*, K. MORGAN & R. M. BERNSTEIN Department of Rheumatology and *ARC Epidemiology Unit, University of Manchester Medical School, Manchester, UK
(Acceptedfor publication 4 March 1992)
SUMMARY The binding sites for MoAbs to the 65-kD heat-shock protein (hsp65) of mycobacteria have been investigated by epitope scanning. Five hundred and twenty-six 8-mer peptides representing the complete sequence of Mycobacterium tuberculosis hsp65 were synthesised in duplicate using the Epitope Scanning Kit (CRB Ltd.). The epitopes of six MoAbs raised to the hsp65 of M. tuberculosis or M. leprae were investigated. We have identified the epitope of a new MoAb (DC1 6); this epitope is continuous, hydrophilic in nature and 11 amino acids long. We have also confirmed the location of the epitopes of three MoAbs (IIH9, ML30 and IIC8). Thus the epitope scanning technique has proved suitable for the detection of continuous epitopes of hsp65.
Keywords B cell epitopes monoclonal antibodies mycobacterial hsp65 epitope scanning INTRODUCTION
MATERIALS AND METHODS
The 65-kD heat-shock protein (hsp65) is a major antigen of mycobacteria that induces specific antibody  and T cell immune responses  during mycobacterial infections. Humoral and cellular immune responses to hsp65 have also been demonstrated in rheumatoid arthritis [3-6] and ankylosing spondylitis . T cell clones recognizing hsp65 can mediate or protect from disease in the rat model of adjuvant arthritis [8,9]. Hsp65 is ubiquitous and highly conserved between species, with at least 40-50% identity between the human and mycobacterial proteins . The hsp65 proteins from Mycobacterium bovis and M. tuberculosis are identical, whereas the M. leprae protein differs at 36 amino acid residues. Several MoAbs have been produced to mycobacterial hsp65. These have been used to detect species-specific antigenic determinants  and to locate hsp65 in tissues . Epitopes recognized by several of these MoAbs have been determined using synthetic peptides  and genetically engineered fragments of the protein . In general, the epitopes have been located on stretches of 9-22 amino acids, but some (presumably conformational) have eluded detection . We describe for the first time the identification of the binding site of a new monoclonal anti-M. tuberculosis hsp65 antibody (DC16) using the epitope scanning technique . Using the same technique we have confirmed the sequence of the epitopes of three monoclonals for which binding sites have been described previously.
Monoclonal antibodies Six MoAbs to hsp65 were available for this study: monoclonals 11H9, ML30, 1IC8 and IIIE9 were raised to M. leprae, whereas DC16 and TB78 were raised to M. tuberculosis. The MoAbs IIH9, ML30, 1IC8, IIIE9 and TB78 were provided by the UNDP/World Bank/WHO Special Programme for Research Training in Tropical Diseases. DC 16 was obtained from Dr J. Ivanyi (Tuberculosis Unit, Hammersmith Hospital, London, UK). A HLA DR-specific MoAb (6B6H 1) was used as a negative control (Dr Jane Worthington, ARC Epidemiology Research Unit, University of Manchester, UK).
ELISA to detect antibodies to recombinant M. bovis hsp65 All MoAbs were tested by ELISA for their ability to recognize mycobacterial hsp65. Microtitre ELISA plates were coated with 100 pl/well of 5 yg/ml recombinant M. bovis hsp65 (Dr van der Zee, RIVM, The Netherlands) in carbonate buffer, pH 9-6 at 4°C overnight. Unsaturated binding sites were blocked with 200 p1/well 2% bovine serum albumin (BSA) in PBS pH 7 2. The wells were then washed three times with PBS containing 0 05% Tween 20 (PBS/T). Monoclonal antibodies were diluted 1: 1000 in the blocking buffer and incubated in the wells for 2 h at room temperature (100 p1/well). The plates were then washed three times with PBS/T, incubated with 100 p1/well peroxidaseconjugated goat anti-mouse immunoglobulins (Dako Ltd.) diluted 1: 1000 in the blocking solution for 2 h at room temperature. After washing with PBS/T they were incubated with freshly prepared substrate, 0 5 mg/ml azino-di-3-ethylbenzthiazodinesulphonate (ABTS) in 0-08 M citric acid, 0 1 M disodium hydrogen phosphate and 0 3 pl/ml hydrogen peroxide
Correspondence4 Dr R. M. Bernstein, Department of Rheumatology, University of Manchester Medical School, Manchester M 13 9PT, UK.
A. H. Hajeer et al.
for 15 min at room temperature. Absorbance was read at 405 nm using a Multiscan ELISA reader (Flow Laboratories). Detection of monoclonal antibody binding sites by epitope scanning Five hundred and twenty-six peptides representing the complete sequence of the M. tuberculosis hsp65 protein  were synthesised in duplicate. Because of the large number of peptides required to fully represent the sequence of hsp65, two separate syntheses were performed. In the first, 263 peptides representing the amino half of the protein were prepared and in the second the remaining half was synthesised. Peptides, eight amino acids long and overlapping by seven amino acids, were synthesised on polyethylene pins using the Epitope Scanning Kit (Cambridge Research Biochemicals Ltd.). Antibody binding to the hsp65 synthetic peptides, immobilized on polyethylene pins, was measured using a modified ELISA. The pins were blocked with 200 y1 of supercocktail solution (PBS containing 1% BSA, 1% ovalbumin and 01I% Tween 20) for 1 h at room temperature. They were then incubated overnight at 4°C with the appropriate MoAb diluted 1: 1000 in supercocktail solution containing 0 1 % azide. Excess MoAb was washed off using PBS/T. Specific antibody binding to peptides on the pins was detected with peroxidase-conjugated goat anti-mouse immunoglobulins (Sigma) diluted 1:500 in the supercocktail solution. After incubation for 1 h the pins were washed with PBS/T, and then incubated with the substrate solution (ABTS) for 40 min at room temperature. The absorbance of the developed product was read at 405 nm. Pins were used repeatedly with thorough cleaning between assays by sonication for 1 h at 60°C in disruption buffer (1 % (w/ v) SDS, 0 1% (v/v) 2-mercaptoethanol and 0 I M sodium hydrogen phosphate).
Hydrophilicity prediction Hydrophilicity prediction was carried out according to Kyte & Doolittle  over a window of seven amino acids.
Table 1. Binding of the monoclonal antibodies to recombinant hsp65 as detected by ELISA Monoclonal antibody
Raised to Mycobacterium leprae
Antibody binding to hsp65 synthetic peptides Different patterns of antibody binding to the hsp65 8-mer peptides were seen with each of the MoAbs tested (Figs 1 and 2). Binding of the control MoAb to the peptides was low, indicating that high binding activity was not due to non-specific binding of antibody. Monoclonals raised to M. leprae hsp65. Three of these monoclonals (11H9, ML30 and IIC8) each gave a different single major peak of antibody binding (Fig. 1). For each antibody the reactive peptides spanned 4-7 consecutive pins. The amino acid sequences recognized by each of these MoAbs are shown in Table 2, together with their predicted hydrophilicity. Each monoclonal recognized different hsp65 amino acid sequences with different hydrophilicity profiles (Table 2, Fig. 3). IIIE9
1 294+0 152 0281 +0017 1-798 +0-110 0 106 +0004
ML30 IIC8 11HE9 Raised to M. tuberculosis
0 976+0 115
TB78 0m680n +a0025 Control monoclonal antibody
________0_154_0_010 * Mean value (±s.d.) in arbitrary units. t HLA-DR specific control MoAb.
I RESULTS Antibody binding to recombinant M. bovis hsp65 Five out of the six monoclonal anti-mycobacterial hsp65 antibodies, tested at 1: 1000 dilution, bound to the recombinant M. bovis hsp65 in a solid phase ELISA (Table 1), despite some of them having been raised to M. leprae. IIIE9 and the control MoAb (6B6H 1) did not show binding to hsp65.
Absorbance at 405 nm*
Fig. 1. Epitope mapping of Mycobacterium tuberculosis hsp65 using four monoclonal antibodies (IIH9, ML30, IIC8, IIIE9) produced to the M.
leprae protein. Binding of monoclonal antibodies (1/1000) to individual peptides (x-axis) was measured by ELISA and recorded in arbitrary
units (y-axis). MoAb, which gave no binding to recombinant hsp65 in the ELISA, did not bind to the M. tuberculosis hsp65 peptides. Monoclonals raised to M. tuberculosis hsp65. The new MoAb (DC16) bound to a region of 11 amino acids (129-139), with the sequence TLLKGAKEVET in an area of the molecule predicted to be hydrophilic (Figs 2 and 3). The binding site of this MoAb was previously unknown. The MoAb TB78 did not give a peak of binding to the hsp65 peptides although it showed positive reactivity against the recombinant hsp65.
B cell epitopes of mycobacterial hsp65 2800
Lk2_ 1 "--
_____________________ 264 526
Fig. 2. Epitope mapping of Mycobacterium tuberculosis hsp65 for two monoclonal antibodies (DC16, TB78) produced to the M. tuberculosis
protein and for the negative control monoclonal antibody (6B6H 11). Binding of monoclonal antibodies (1/1000) to individual peptides (xaxis) was measured by ELISA and recorded in arbitrary units (y-axis).
The ubiquitous and highly homologous heat-shock proteins, particularly the 65-kD mycobacterial heat-shock protein, have recently generated much interest [1-9]. Many MoAbs have been produced to these proteins and until now the identification of their binding sites has required the expensive and time consuming generation of large peptides and protein fragments [13,14]. The technique of epitope scanning has been used to identify monoclonal  and polyclonal  antibody binding sites on a variety of proteins for which the primary structure is available.
Here, we have described the first application of this technique to the investigation of antibody binding sites on mycobacterial hsp65. We have identified the binding site of a new MoAb DC 16 which was raised against M. tuberculosis hsp65 and does not bind to M. leprae protein (Dr J. Ivanyi, personal communication). This MoAb recognized an 11-amino acid sequence towards the amino terminus of the protein (amino acids 129139). From the hydrophilicity profile of the whole protein this region is predicted to be hydrophilic and thus considered likely to be an antibody binding site. The sequence differs from the same region of M. leprae by the non-conservative substitution of glycine (G) for aspartic acid (D) at position 133. Thus, this single amino acid difference may be responsible for the failure of the antibody to bind to M. leprae hsp65. In a previous study of MoAbs to influenza virus haemagglutinin, the same single substitution of an aspartic acid residue (D) for a glycine (G) also resulted in the loss of antibody binding activity . Monoclonal antibody TB78, also raised to M. tuberculosis failed to recognize any of the peptides by epitope hsp65, s solid-phase The tec ofeitopes maynot solid-phase ELISA The technique of epitope scanning may not be suitable for the detection of discontinuous epitopes, so our result is consistent with the suggestion that TB78 recognizes a discontinuous epitope (Dr J. Ivanyi, personal communication). Four MoAbs raised against M. leprae, for which epitopes have previously been described, were also tested by epitope scanning for binding to peptides representing the M. tuberculosis hsp65. Three of these MoAbs showed binding to equivalent amino acid sequences in M. tuberculosis, although there are some differences in the precise length of the epitopes identified. All three of the epitopes are found in areas of the hsp65 sequence that are highly homologous between M. keprae and M. tuberculosis. The epitope recognized by ML30 is in a region of the sequence predicted to be hydrophobic. Hydrophobicity is normally interpreted as an indication of regions of the protein that are not exposed at the outer 'surface', and therefore not
Table 2. Monoclonal antibody binding sites identified by epitope scanning of the Mycobacterium tuberculosis hsp65 (a) sequence compared with previously identified monoclonal antibody epitopes of M. leprae hsp65 (b). The one letter code is used for each amino acid, and the numbers indicate the position in the amino acid sequence of M. tuberculosis hsp65.
Raised to M. 11H9 ML30
keprae a 113 LGLKRGIEKAVE 124 b AAGANPLGLKRGIEKA a 286 MLQDMAILTGGQVI 299 b KAPGFGDRRKAMLQDMAILTGA
b a b
Raised to M. tuberculosis DC16 a TB78
479 TGVYEDLLAAGVA 491 EYEDLLKAGVAD
Hydrophilic Hydrophobic Amphipathic
not detected KLKLTGDEA
129 TLLKGAKEVET 139 b not published not detected b not published
A. H. Hajeer et al. 4
Fig. 3. Hydrophilicity plot of the Mycobacterium tuberculosis hsp65 according to Kyte & Doolittle  using a window of seven amino acids. Underlined are the positions of the epitopes recognized by four monoclonal antibodies: a, IIH9; b, DC 16; c, ML30; and d, IIC8.
normally accessible to antibodies if the protein is intact. Binding of ML30 antibody to the recombinant hsp65 by ELISA was relatively low compared with other anti-M. keprae MoAbs, whereas binding to peptides in the epitope scanning was strong and similar to the other antibodies tested. These observations are consistent with an epitope that is not on the surface of the protein but becomes accessible to antibody after protein degradation. The fourth monoclonal anti-M. keprae hsp65 antibody, IIIE9, is reported to recognize the M. leprae sequence KLKLTGDEA . In our study, IIIE9 did not bind to any M. tuberculosis peptides. The equivalent region of M. tuberculosis has two substitutions: glutamic acid (E) for lysine (K) at position 426 and glutamic acid (E) for threonine (T) at position 430. These non-conservative substitutions probably cause structural changes which prevent recognition by IIIE9. We have found the technique of epitope scanning to be a useful method for the identification of MoAb binding sites on hsp65, where epitopes are continuous. This approach for identification of epitopes allows many different MoAbs to be tested following a single synthesis of a large number of peptides representing the complete protein sequence. Precise information about antibody binding sites for proteins such as hsp65, from highly homologous families, is important in determining the degree of cross reactivity or species specificity of antibodies in order to ensure correct interpretation of experimental data. Identification of MoAb epitopes by this technique does not depend on the use of predictive algorithms which are known to be unreliable. It is anticipated that further use of the technique and accumulation of more data on the nature of epitopes will enable better predictive algorithms to be formulated.
ACKNOWLEDGMENTS We thank WHO/UNDP, Dr Thomas Gillis (G. W. Long Hansen's Disease Centre, LA, CA) and Dr J. Ivanyi (Tuberculosis Unit, Hammersmith Hospital, London, UK) for supplying monoclonal antibodies, and Dr van der Zee (RIVM, The Netherlands) for supplying recombinant hsp65. This work was supported by an Arthritis and Rheumatism Council project grant and Lupus UK.
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