Localization of key amino acid residues in the dominant conformational epitopes on thyroid peroxidase recognized by mouse monoclonal antibodies

Autoantibodies to thyroid peroxidase (TPO), the major target autoantigen in autoimmune thyroid diseases, recognize conformational epitopes limited to two immunodominant regions (IDRs) termed IDR-A and -B. The apparent restricted heterogeneity of TPO autoantibodies was discovered using TPO-specific mouse monoclonal antibodies (mAbs) and later confirmed by human recombinant Fabs. In earlier studies we identified key amino acids crucial for the interaction of human autoantibodies with TPO. Here we show the critical residues that participate in binding of five mAbs to the conformational epitopes on the TPO surface. Using ELISA we tested the reactivity of single and multiple TPO mutants expressed in CHO cells with a panel of mAbs specifically recognizing IDR-A (mAb 2 and 9) and IDR-B (mAb 15, 18, 64). We show that antibodies recognizing very similar regions on the TPO surface may interact with different sets of residues. We found that residues K713 and E716 contribute to the interaction between mAb 2 and TPO. The epitope for mAb 9 is critically dependent on residues R646 and E716. Moreover, we demonstrate that amino acids E604 and D630 are part of the functional epitope for mAb 15, and amino acids D624 and K627 for mAb 18. Finally, residues E604, D620, D624, K627, and D630 constitute the epitope for mAb 64. This is the first detailed study identifying the key resides for binding of mAbs 2, 9, 15, 18, and 64. Better understanding of those antibodies' specificity will be helpful in elucidating the properties of TPO as an antigen in autoimmune disorders.     

The effect of dithiotreitol on thyroid peroxidase and microsomal antigen epitopes recognized by auto and monoclonal antibodies.

The effect of disulphide bridges reduction of the microsomal antigen (Mic-Ag) and thyroid peroxidase (TPO) by dithiotreitol (DTT) has been investigated. The reaction of all 67 tested sera from untreated hyperthyroid Graves' and from 22 Hashimoto's patients with high microsomal antibodies (aAb) titer was diminished by 90-95% by DTT, at pH 9.6. The remaining 5-10% of the activity was not destroyed by DTT. The residual Mic-Ag after DTT reduction was able to inhibit the binding of all 45 Graves' and 22 Hashimoto's tested aAb's to the native microsomal antigen by 100% at high concentration. Reaction of affinity purified TPO with two monoclonal antibodies (mAb) were diminished by 80% to 95% by DTT pretreatment, while the reaction of one mAb with TPO was only slightly affected. The reaction of TPO and Mic-Ag with rabbit polyclonal anti-TPO serum (rabbit a TPO) was diminished by 60% by DTT pretreatment. The immunological reactivity of TPO with aAb's was diminished by 65% after DTT pretreatment. The microsomal antigen-aAb's complex was not destroyed by DTT. Results presented in this paper suggest conformational epitope structure of the Mic-Ag recognized by aAb's in patients with thyroid autoimmune disease (AITD).     

Simultaneous binding of two monoclonal antibodies to epitopes separated in sequence by only three amino acid residues

Two monoclonal antibodies recognizing distinct epitopes the outer boundaries of which are separated by only three amino acid residues, a maximum of 10A, were demonstrated to bind simultaneously to a short synthetic peptide. The affinity of binding of the two monoclonal antibodies and of Fab' fragments derived from them was determined. The stoichiometry of the interaction was analysed by velocity sedimentation and by gel permeation chromatography experiments. The results indicate that the immune complexes formed are composed of two antibody molecules in association with one or two peptide molecules.     

Dendritic cells of the mouse recognized by two monoclonal antibodies

A new monoclonal antibody, MIDC-8, is described which shows a comparable tissue distribution as the recently described NLDC-145 antibody. It recognizes interdigitating cells, veiled cells and Langerhans cells in lymphoid organs and the skin of the mouse. In contrast to NLDC-145 it recognizes a cytoplasmic component of these cell types. Its distribution is more restricted to the T cell-dependent areas than NLDC-145. Isolation of dendritic cells from lymph nodes, spleen and thymus revealed that both antibodies react with the in vitro isolated dendritic cells. The results show that these antibodies can be used to study dendritic cells in vitro and emphasize the relationship between the in vivo interdigitating cells of the T cell areas and the in vitro isolated dendritic cells.     

Evaluation of conformational epitopes on thyroid peroxidase by antipeptide antibody binding and mutagenesis

Autoantibodies to thyroid peroxidase (TPO) recognize predominantly conformational epitopes, which are restricted to two distinct determinants, termed immunodominant domain region (IDR) A and B. These dominant determinants reside in the region with structural homology to myeloperoxidase (MPO)-like domain and may extend into the adjacent complement control protein (CCP) domain. We have explored the location of these determinants on the MPO-like domain of the structural model of TPO, by identifying exposed hydrophilic loops that are potential candidates for the autoantigenic sites, generating rabbit antipeptide antisera, and competing with well characterized murine monoclonal antibodies (mabs) specific for these two IDRs. We recently defined the location of IDR-B, and here report our findings on the location of IDR-A and its relationship to IDR-B, defined with a new panel of 15 antipeptide antisera. Moreover, in combination with single amino acid replacements by in vitro mutagenesis, we have defined the limits of the IDR-B region on the TPO model. The combination of antisera to peptides P12 (aa 549-563), P14 (aa 599-617) and P18 (aa 210-225) inhibited the binding of the mab specific for IDR-A (mab 2) by 75%. The same combination inhibited the binding of autoantibodies to native TPO from 67 to 94% (mean 81.5%) at autoantibody levels of 5 IU. Fabs prepared from the antipeptide IgG and pooled in this combination were also effective in competition assays, thus defining the epitopes more precisely. IDR-A was found to lie immediately adjacent to IDR-B and thus the two immunodominant epitopes form an extended patch on the surface of TPO. Finally, by single amino acid mutagenesis, we show that IDR-B extends to residue N642, thus further localizing the boundary of this autoantigenic region on the structural model.     

Structurally defined epitopes of Haemophilus ducreyi lipooligosaccharides recognized by monoclonal antibodies.

By use of enzyme-linked immunosorbent assay and immunoblotting techniques, the migration patterns and binding epitopes of lipooligosaccharides (LOS) from 10 Haemophilus ducreyi strains were investigated with two monoclonal antibodies (MAbs), MAHD6 and MAHD7, raised against LOS from H. ducreyi ITM 2665. Closely related LOS, with defined structures, from Haemophilus influenzae, Bordetella pertussis, Aeromonas spp., and synthetic glycoproteins were also included in the analyses. The MAbs bound to conserved epitopes of LOS exposed on the surface of H. ducreyi. The MAb MAHD6 reacted with 8 of the 10 LOS from H. ducreyi but with none of the other Haemophilus or Bordetella spp. with structurally defined LOS. It is suggested that MAb MAHD6 binds to a LOS epitope (-DD-Hepp-1-->6-beta-D-Glcp-). This LOS epitope is not present in the hexasaccharide structure of LOS from H. ducreyi ITM 4747 (E. K. H. Schweda, A. C. Sundström, L. M. Eriksson, J. A. Jonasson, and A. A. Lindberg, J. Biol. Chem. 269:12040-12048, 1994). Because MAb MAHD6 reacts with the epitope mentioned above, it also discriminates between the two LOS structures, the hexasaccharide group and the nonasaccharide group, of H. ducreyi strains. MAb MAHD7 recognizes the common conserved inner core region of the LOS because it reacts with all H. ducreyi strains and with LOS with minor components in the inner core epitope structure. Rabbit polyclonal sera raised against the LOS from strains CCUG 4438 and CCUG 7470 were tested with the 10 LOS from the H. ducreyi strains. The antiserum to CCUG 7470 reacted with all H. ducreyi strains as did MAb MAHD7, whereas the antiserum to CCUG 4438 reacted with only its homologous strain and strain ITM 4747. Also, the LOSs of our reference strains CCUG 4438 and CCUG 7470 were structurally analyzed by use of sugar analyses and electrospray ionization-mass spectrometry. The hexasaccharide and nonasaccharide structures obtained from LOS of strains CCUG 4438 and CCUG 7470 were identical to the described LOS structures from H. ducreyi ITM 4747 and ITM 2665, respectively. In conclusion, the MAb MAHD6 recognizes an epitope present in the nonasaccharide LOS group, whereas the MAb MAHD7 recognizes a conserved epitope on LOS of H. ducreyi, which is present in all strains of H. ducreyi tested. Two major groups of oligosaccharides were distinguished by their LOS structures and the reactivity of monoclonal as well as polyclonal antibodies. The majority of H. ducreyi strains possess a nonasaccharide structure of LOS.     

Common and specific epitopes of Campylobacter flagellin recognized by monoclonal antibodies.

Murine monoclonal antibodies to Campylobacter jejuni recognized a flagellin epitope common to most Campylobacter species and an epitope restricted to C. jejuni and C. coli. These epitopes are distinct from the serotype-specific epitope recently detected on the flagellin and have not been described previously.     

Identification of the amino acid residues contributing to monoclonal antibody-defined DQw1 epitopes.

The reactivity of monoclonal antibodies (mAbs) R1, S1, and S5, shown previously to recognize polymorphic epitopes on HLA-DQ molecules, have been found to correlate with the presence of certain DQB1 alleles. mAb S5 reacts with cells expressing DQB1*0503, 0601, 0602, 0603, or 0604 alleles while R1 and S1 react with all DQB1 alleles except *0201 and 0301. In the case of R1 and S1, sequence comparison of these chains suggests the involvement of residues 45-47 (GVY) in formation of the epitopes. This prediction has been confirmed by showing that a G----E mutation in position 45 of the DQB1*0302 gene eliminates binding of both mAbs.     

Functionally different subpopulations of mouse macrophages recognized by monoclonal antibodies

Four rat anti-mouse macrophage monoclonal antibodies are described. Three of them are highly specific for macrophages, and one cross-reacts with granulocytes. All 4 antibodies do not react with membrane antigens shared by all macrophages, but with antigens present only on subpopulations of 20-50% of the cells. All antibodies are directly or indirectly cytotoxic for macrophages. The subpopulations defined by these antibodies can be correlated with certain macrophage functions. Thus, antibody M43 eliminates macrophages that are activated by lymphokine to cytotoxicity. Antibodies M43 and M57 eliminate macrophages that kill antibody-coated tumor targets, and clone 102 (strictly macrophage-specific) eliminates natural killer cells. Only M143, reacting with 10-30% of macrophages, has not yet been correlated with any function. With the use of these antibodies, cells of the macrophage lineage with specific functions can be recognized and eliminated from a given population.     

Localization of two epitopes recognized by monoclonal antibody PCG-4 on Clostridium difficile toxin A.

The toxin A gene of Clostridium difficile contains a 2.5-kb region encoding a series of contiguous repeating units located at the COOH terminus of the molecule. We previously showed that the monoclonal antibody (MAb) PCG-4, which neutralizes the enterotoxic activity of toxin A, binds to epitopes located within these repeating units. In the present study, we subcloned a series of fragments from this portion of the gene. The recombinant peptides expressed from the gene fragments were examined for reactivity with MAb PCG-4 to identify the epitopes involved in binding. Our results showed that MAb PCG-4 recognizes epitopes in amino acid residues 2097 through 2141 and amino acid residues 2355 through 2398.     

Identification of epitopes recognized by a panel of six anti-human IgG2 monoclonal antibodies.

Human IgG2 contains several subclass specific amino acid residues or deletions in the CH1 and CH2 domains and also in the hinge region. These substituted residues are the structural correlates for IgG2 specific epitopes. Since human IgG2 has different biological properties from other subclasses, some IgG2 epitopes may be located in regions correlating with sites determining the biological functions. Previously, we produced three anti-IgG2 monoclonal antibodies (mAbs) with highly specific and interesting reactivities using improved immunization protocols. However, it has been almost impossible to identify epitopes conventionally, because human IgG2 is so resistant to proteolysis that various proteolytic fragments could not be isolated. In this study, we identified the epitopes recognized by anti-IgG2 mAbs by SDS-PAGE, Western blotting, amino acid sequence analysis and peptide/mAb binding ELISA, thus overcoming the need for fragment isolation. A panel of six anti-human IgG2 mAbs, including the current WHO/IUIS specificity standards (HP6002, HP6008, HP6014) and our own (HG2-6A, HG2-30F, HG2-56F), reacted with distinct epitopes. The residues essential to expression of the epitopes recognized by the mAbs were: Pro234, Val235 and Val309 for HG2-56F, HG2-30F and HP6008, respectively. HP6014 reacted with the epitope expressed by Thr214 and its neighboring residues. HG2-6A was reactive with the hinge region, and HP6002 was assumed to be directed against discontinuous epitopes requiring intact Fc for expression. Through these studies, the pepsin and papain cleavage sites of human IgG2 were also clarified.     

Neutralization of CRPV infectivity by monoclonal antibodies that identify conformational epitopes on intact virions.

Monoclonal antibodies were generated against cottontail rabbit papillomavirus (CRPV) and tested for neutralization of CRPV-induced papillomas on domestic NZW rabbits. Intact CRPV was semi-purified on CsCl gradients and used to immunize BALB/c mice. Hybridomas were prepared from a fusion with lymph node cells, and supernatants from growing hybridomas were analyzed by enzyme-linked immunosorbent assay (ELISA) for reactivity to both intact and disrupted CRPV virion antigen. Supernatants from 22 cultures were initially selected that were responsive to CRPV. Ten were reactive to intact CRPV alone, 4 were reactive only to disrupted CRPV, and 8 were reactive to both intact and disrupted CRPV virion antigen. None of these supernatants contained antibodies which recognized epitopes on CRPV capsid proteins (L1 and L2) that were separated on Western blots. Five hybridomas which produced antibodies that bound to intact CRPV, and did not react to intact HPV-11 or BPV-1 were selected and tested for antibody-mediated neutralization of CRPV infectivity. All five monoclonal antibodies were neutralizing, and identified epitopes on intact CRPV virions which were non-linear and conformational in nature. The five neutralizing monoclonal antibodies appeared to recognize a similar epitope or epitope cluster on the intact CRPV virion as determined by competition ELISA.     

Repeated helical epitopes of defined amino acid sequence in human type III collagen identified by monoclonal antibodies.

Two monoclonal antibodies, designated 1F8 (IgG1) and 5B10 (IgG1), have been produced in mice against native human type III collagen. These antibodies were highly type and species specific, recognizing the triple helical domain of type III as tested by ELISA. Immunofluorescence studies using each of these antibodies resulted in a fibrous staining pattern in human skin dermis. Immunogold electron microscopy resulted in a periodic distribution of gold particulates along banded collagen fibrils. Assuming that the total contour length of pepsin digested type III collagen is 300 nm, measurements of antibody-antigen complexes visualized by rotary shadowing revealed that each antibody bound at the same two sites: one approximately at the middle of the helix (153 nm from the N-terminus), the other at a site one-quarter the triple helical length from the N-terminus (75 nm). That the one-quarter binding site was closest to the N-terminus was determined by antibody incubation following tadpole collagenase treatment, which results in a larger, N-terminus containing fragment (binding antibody) and a smaller C-terminus containing fragment (not binding antibody). Located at each antibody binding epitope is a sequence of 10 amino acids: Gly-Ala-Hyp-Gly-Leu-Arg-Gly-Gly-Ala-Gly. Renatured cyanogen bromide-cleaved(CB)-peptides, CB4 and CB8, containing these repeated sequences reacted with each antibody, whereas other renatured type III CB-peptides were unreactive as determined by Western blotting analysis and ELISA. This was further confirmed by inhibition tests using a 10 residue synthetic peptide of identical sequence, which yielded 20-30% inhibition of antibody binding to native type III collagen at 4 degrees C. However, no inhibition was noted at higher temperature. These results indicate that both monoclonal antibodies recognize a specific helical conformation of 10 or slightly fewer residues in the three identical polypeptide chains comprising type III collagen.     

Rapid characterization of protein epitopes recognized by monoclonal antibodies using direct probing on thin-layer and paper chromatograms

A simple method for the comparison and identification of protein epitopes recognized by monoclonal antibodies directly on thin-layer plates and 3MM paper chromatograms is described. Enzyme digests of myelin basic protein were separated on thin-layer plates and 3MM paper, fixed with glutaraldehyde and probed directly with affinity-purified mouse monoclonal antibodies. Detection of the immunoreactive peptides was enhanced using a second rabbit anti-mouse immunoglobulin and finally located using an alkaline phosphatase-conjugated anti-rabbit immunoglobulin. By probing the same enzyme digests of MBP with various monoclonal antibodies raised against MBP, a different binding 'pattern' of reactive peptides is rapidly obtained for monoclonal antibodies of differing specificities. This procedure was extended to the identification of the antigenic determinant using synthetic peptides. The major advantages of this procedure are its simplicity, non-radioactive nature and speed. Furthermore, there is the possibility of sequencing immunoreactive peptides eluted from the 3MM paper.     

Detection of the major epitopes of human protamine P1 recognized by rabbit and mouse antibodies

The characterization of the major antigenic determinants present in human protamine P1 has been carried out by the use of specific rabbit polyclonal and mouse monoclonal antisera raised against protamine P1. This basic protein, the full amino acid sequence of which has been determined here, has been cleaved by cyanogen bromide and/or by pepsin to generate a discrete number of peptides. These have been purified, characterized by partial amino acid sequencing and used for the determination of their antigenic reactivities with antisera to native protamine P1. Both rabbit polyclonal and mouse monoclonal antibodies were able to recognize the NH2-terminal CNBr peptide encompassing residues 1-36 to the same extent as the intact protamine. A minor epitope present on the COOH-terminal peptide 37-50 could be detected only with the polyclonal rabbit antisera. Attempts to further cleave the P1 molecule in order to isolate peptides shorter than fragments 1-36 whilst retaining full antigenic reactivities, were unsuccessful. This suggests that the epitopes in P1 are conformation-dependent and located for the most part on the amino-terminal half of the molecule, which comprises the characteristic central arginine cluster. The implication of these findings for the studies of the specificities of autoantibodies in sera from infertile and vasectomized individuals is discussed.     

Determination of antigenic epitopes recognized by four monoclonal antibodies to glutathione S-transferase pi (GST-pi).

Four independent antibodies (6A, 5F, 2H and 2F) to Glutathione S-transferase pi (GST-pi) were selected to characterize their epitopes. Amino acid analysis of 5.6 K and 7.4 K tryptic peptides appeared to suggest that the epitope recognized by antibodies 2H and 2F is located in the N-terminal 44 peptides of GST-pi, and that of 6A and 5F is located in the C-terminal 69 peptides. Reactivities of antibodies 6A and 5F with two synthetic peptides indicated that 6A recognized an epitope in the C-terminal hydrophilic fragment 176Leu-209Gln, and could be distinguished from 5F which recognized an epitope in the 141Thr-176Leu hydrophobic fragment. The differential immuno-reactivity of antibodies 6A and 5F with GST-pi itself, was characterized by the particularly high reactivity of 6A and almost no reactivity of 5F with the natural conformation of GST-pi in solution. This may be explained by differences in the hydropathic natures of their epitopes. The 6A antibody was useful for immunodetection of GST-pi in circulation, while 5F was found to be most suitable for histochemical staining of tumor tissue.     

A rhoptry antigen of Plasmodium falciparum contains conserved and variable epitopes recognized by inhibitory monoclonal antibodies

Four monoclonal antibodies produced against Plasmodium falciparum recognize an antigen in merozoites that is localized in rhoptries, as judged by a punctate, double dot fluorescence pattern. All four antibodies bound to the same affinity purified antigen in a two site immunoradiometric assay. Immunoprecipitation of antigen by monoclonal antibody followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis yielded protein bands of 80, 66 and 42 kDa. Western blotting gave bands of 80 and 66 kDa only with three of the antibodies: the fourth did not blot. Based on protease inhibitor data the 66 kDa band is considered to be a cleavage product of the 80 kDa band, but the 42 kDa band does not appear to derive from the latter and may be a coprecipitation product. This group of antigens labels with both [35S]methionine and [3H]histidine. Two of the monoclonal antibodies inhibited merozoite invasion of erythrocytes. One of these inhibitors recognizes a variable epitope, whereas the second recognizes a highly conserved epitope present in all 106 primary isolates of P. falciparum tested from Brazil, Thailand and Papua New Guinea.     

New polymorphic HLA-DR epitopes recognized by three monoclonal antibodies produced against DR103 transfected L cells

Production of monoclonal antibodies directed against polymorphic epitopes of HLA class II molecules using whole human cells as immunogen has often proved ineffective, because most of the antibodies produced are directed against non-MHC human cell surface molecules. One approach to overcome this problem is the use of transfected mouse L cells expressing a single HLA class II allele as immunogen. By immunizing C3H mice with DR103-transfected L cells, we obtained 3 mAb, OHA TM901, OHA TM902, and OHA TM903, that recognize different polymorphic epitopes of the HLA-DR molecule. The molecular specificities of the 3 mAb were determined on a large panel of B-lymphoblastoid cell lines (B-LCL), peripheral blood cells and HLA class II transfectants from the XIth International Histocompatibility Workshop. Interestingly, the 3 polymorphic mAb detect new HLA-DR epitopes shared by several specificities: OHA TM901 reacts with DR1 (DR101, DR103), DR9 (DR901) and DR10 (DR1001) molecules; OHA TM902 recognizes the same molecules but also DR8 (DR801, 802, 803); OHA TM903 reacts with all DR types except DR3 (DR301, 302), DR7 (DR701, 702) and DR52. Surprisingly, OHA TM901 reacts with DR9 transfectants and B-LCL but not with DR9 peripheral blood lymphocytes. Biochemical analyses indicate that the 3 mAb immunoprecipitate HLA-DR products and react in western blots with DR alpha/beta-dimer but not with free alpha- or beta-chains. This study shows that transfected L cells are very useful tools for the production and the fine characterization of mAb recognizing polymorphic epitopes of HLA class II molecules.     

A unique human IgE-binding epitope on the Bermuda grass pollen recognized by mouse lambda-type monoclonal antibodies

A group of six mouse monoclonal antibodies (MoAbs) with the unusual lambda-type light chain were generated by fusion of NS-1 cells with splenic cells derived from BALB/c mice immunized with crude extracts of Bermuda grass pollen (BGP). Four of them were IgG1, one was IgG2b, and one was IgG3. Binding inhibition assay showed that they recognized the same (or very similar) epitope. Using sera from BGP-allergic patients, it was found that the specific binding between the IgE antibodies and the MoAb 26-11-fixed antigen could be blocked by MoAb 26-11 itself and another MoAb 9-13 in a dose-dependent manner. It appears that the epitope recognized by the lambda-type MoAbs is a human IgE-binding antigenic determinant. Further physico-chemical analyses showed that this epitope was stable under heat but sensitive to treatments of sodium periodate and proteinase K. Results from these studies indicate that this unique epitope which leads to the generation of lambda-type MoAbs is part of a glycoprotein.