Epitope mapping of anti-glomerular basement membrane (GBM) antibodies with synthetic peptides

Autoantibodies to the non-collagenous (NC1) domain of the α3(IV)-chain of type IV collagen are found in sera from patients with anti-GBM nephritis. These antibodies have been shown to be pathogenic. In this study the antibody specificity has been investigated in patients with Goodpasture’s syndrome and from a patient with atypical anti-GBM antibodies, recognizing the α1(IV)-chain only. Overlapping synthetic peptides, covering the complete NC1 domains of the α1(IV)- and α3(IV)-chains were used in sandwich ELISA and competitive ELISA. None of the Goodpasture sera showed reactivity to the synthetic peptides. However, antibodies from the patient with atypical anti-GBM antibodies recognized a 20 amino acid peptide from the α1(IV)-chain. The reactive peptide was further narrowed down with glycine substitution of the different amino acids. We have localized the epitope to the four last C-terminal amino acids of the α1(IV)-chain, with the sequence 1754-MRRT. The two arginine residues were found to be essential for antibody binding. Threonine is important, while methionine is of less importance. These four amino acids are also determined to be the smallest peptide that could inhibit the binding of the autoantibodies to the native α1(IV)-chain. This study shows that overlapping peptides can be used to map linear epitopes. However, for conformational epitopes such as the Goodpasture epitope, other methods must be used. It would be prognostically important to know the fine specificity of anti-GBM antibodies, since the patient with anti-α1(IV) antibodies had a mild disease, while the Goodpasture patients with anti-α3(IV) antibodies had a rapidly progressive disease.     

Conformation dependence of antigenic determinants on the collagen molecule

Three different types of antigenic determinants were demonstrated in soluble collagen with the aid of rat, rabbit and chicken antisera to native collagen. Helical antigenic determinants which require an intact triple-helical structure of the molecule are mainly recognized by rat antisera. Renaturation of the serologically inactive unfolded polypeptide chains (denatured collagen) is accompanied by a significant recovery of serological activity. Central antigenic determinants which are probably located in the same regions of amino acid sequence are less accessible in the native antigen and become exposed upon denaturation. Equal titres for both types of determinants are found in chicken antisera. Immunization with denatured collagen, however, revealed a response restricted to the central type. Isolated antibodies specific for terminal non-helical antigenic determinants, as yet only known to occur in rabbit antisera, reacted equally well with native collagen, the unfolded polypeptide chains and with small cyanogen bromide peptides. Independence of conformation is therefore suggested for these antigenic structures.     

Involvement of more than a single polypeptide chain in the helical antigenic determinants of collagen

Rat antibodies to native triple helical calf collagen were used to study the structural requirements of helical antigenic determinants revealed in this particular model. Renaturation of random-coiled chains of collagen in the original proportion of two α1-chains and one α2-chain to triple-helical structures is accompanied by complete recovery of antigenic activity. Although physically similar to native collagen, triple-helical structures prepared from individual chains alone were inactive or exhibited a strongly reduced activity. These results were interpreted as to the involvement of parts of the α1 and α2-chains in several dinstinct helical antigenic determinants of collagen. Further studies with collagenase-derived, triple helical fragments of variable length revealed successive loss of antigenic activity upon reduction in size. However, the smallest fragment, having a length of 78 nm, still contained some of the original antigenic determinants. The antibodies to helical antigenic determinants also showed rather limited interspecies cross-reactions as opposed to the very strong cross-reactions found previously between antigenic determinations of sequential nature in random-coiled α-chains. Helix formation of two different types of α-chains essentially masks these cross-reacting structures.     

Mapping of antigenic determinants within peptides of a variant surface glycoprotein of Trypanosoma brucei

The location of antigenic determinants in the primary amino acid sequence of the variant surface glycoprotein of Trypanosoma brucei MITat 1.6 was investigated using monoclonal antibodies in conjunction with the known cyanogen bromide and tryptic cleavage patterns of this antigen. The cyanogen bromide digestion fragments of the antigen were purified and used to raise polyclonal antisera, which were specific for the appropriate cyanogen bromide fragment and partial digestion products, as well as recognising the intact variant surface glycoprotein. Competition radioimmunoassays were carried out between these antisera and nine monoclonal antibodies specific for MITat 1.6 variant surface glycoprotein, which have previously been characterised and shown to recognise five antigenic determinants of which only one is exposed on the surface of the living trypanosome. The binding of the monoclonal antibodies to the major tryptic peptide of MITat 1.6 variant surface glycoprotein was investigated by immunoblotting and by competition radioimmunoassay, and revealed that the five antigenic determinants recognised by the nine monoclonal antibodies are all located in the N-terminal two thirds of the MITat 1.6 variant surface glycoprotein molecule. Three of the determinants are located in an immunodominant region apparently formed by the folding together of two of the cyanogen bromide peptides. The other two determinants appear to be more conformationally labile; one of these is the determinant which is exposed on the surface of the living trypanosome, which is located in the N-terminal one third of the molecule.     

Allergens in Hymenoptera venom. XVI: Studies of the structures and cross-reactivities of vespid venom phospholipases

Phospholipases (PLs) isolated from the venoms of three species of yellow jackets, white-faced hornets, European hornets, and paper wasps were studied by peptide mapping after limited enzyme hydrolyses and cyanogen bromide cleavage. Significant differences in the primary structures were observed. Cross-reactivities of vespid PLs were studied by precipitation reactions in gel with rabbit antibodies, immunoblots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels with rabbit antisera, and RAST inhibition with individual sera from vespid-reactive patients. The cross-reactivities of both human IgE antibodies and rabbit IgG antibodies were variable and not reciprocal between antigen and antibody, suggesting that there are multiple antigenic determinants on the PL molecules and that individuals respond to different determinants. No general patterns of cross-reactivity could be observed.     

Demonstration of a unique antigenic specificity for the collagen alpha1 (II) chain from cartilaginous tissue.

The rabbit antibody response to native collagen (chain composition [alpha1(II)3) from cartilaginous tissue, has been examined by agglutination assays, gel diffusion, haemagglutination-inhibition studies, and immunoadsorption. The results show that the rabbit antibody response to the cartilage-type collagen is characterized by considerable reactivity to both helical [alpha1(II)]3 as well as alpha1(II) chains. This is in contrast to the rat antibody response to the same antigens where titres are generated to largely helical antigenic determinants. Similarly to the rat response, rabbit antibodies to [alpha(II)]3 exhibit no strong cross-reaction with the genetically distinct [alpha(I)]2ALPHA2 collagen or its component chains. Strong cross-reactions were, however, observed between bovine and chick alpha1(II) chains. One of the major antigenic sites on [alpha1(II)]3 collagen appears to reside in the sequence represented by CB-11, a peptide derived from the helical portion of the [alpha1(II)]3 molecule after cyanogen bromide cleavage. The data, however, are compatible with the presence of other antigenic determinants which are probably located in the amino- and carbocy-terminal portions of the molecule.     

Epitope analysis of the Goodpasture antigen using a resonant mirror biosensor

We have used a new technique for studying molecular interactions—a resonant mirror biosensor—to identify B cell epitopes within the Goodpasture antigen, which has recently been identified as the non-collagenous domain of the α3-chain of type IV collagen (α3(IV)NC1). Recombinant antigen (r-α3) was immobilized onto the sensing surface of a sample cuvette, and the binding of patients’ autoantibodies or a MoAb to the Goodpasture antigen was followed in real time. All patients’ sera bound r-α3 in this system, while control sera did not bind. A MoAb inhibited the binding of all patients’ autoantibodies to r-α3, from 27% to 90% (mean inhibition 60%), and patients’ sera cross-inhibited the binding of each other to the antigen. Binding was inhibited by pre-incubation of autoantibody with both native sheep α3(IV)NC1 and purified human α3(IV)NC1 monomers. Inhibition experiments using soluble overlapping peptides from human α3(IV)NC1 identified putative B cell epitopes. These results suggest that there is a major immunodominant epitope on the Goodpasture antigen, and that there is very limited heterogeneity in the autoantibody response in Goodpasture’s disease. The resonant mirror biosensor can be successfully used to monitor antibody–antigen binding using polyclonal sera, and to map epitopes on autoantigens.     

Induction of neutralizing antibodies and immunity in vaccinated guinea pigs by cyanogen bromide-peptides of VP3 of foot-and-mouth disease virus.

The specificity of guinea pig antisera against large cyanogen bromide-cleaved peptides of the virus capsid protein VP3 of foot-and-mouth disease virus type O1, strain Kaufbeuren has been characterized by double immunodiffusion, virus neutralization and protection tests. Antibodies to purified 146S particles and the cleavage peptides of VP3 showed an incomplete cross-section against VP3 peptide antigen when reacted in immunodiffusion tests, indicating that new antigenic determinants are exhibited by the peptides which are not recognized by the antiserum against the native virus proteins. The immune response against the reduced, unfolded chain constituents of VP3 was lower in comparison to that of native virus particles but still some immunological determinants remained actively capable of inducing virus-neutralizing antibodies in immunized guinea pigs.     

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.     

天花粉蛋白裂解片段的抗原性研究

本文采用21株抗天花粉蛋白单抗对4个经溴化氰裂解的天花粉蛋白裂解片段进行抗原决定基分析。7株单抗特异结合天花粉蛋白裂解片段CBTf_1,2株单抗特异结合CBTf_2,2株单抗特异结合CBTf_4,7株单抗呈交叉反应,3株单抗与片段无结合反应。采用纯化的CBTf_1,CBTf_2对4株抗天花粉蛋白单抗的竞争结合实验显示:这两个片段对单抗的百分结合率可达到或接近50%,证明经溴化氰裂解的天花花粉蛋白片段仍保持原有的抗原性,这为天花粉蛋白结构和功能关系的研究提供重要资料。     

Pattern of humoral reactivity to type II collagen in rheumatoid arthritis

Humoral immunity directed against type II collagen (CII) is a common although not specific feature of rheumatoid arthritis (RA). We have shown that 10 to 15% of the sera either from RA patients (n = 88) or from healthy controls (n = 149) reacted with native human CII. Conversely, autoantibodies to the alpha-1 (II) chains were significantly more frequent in the RA group (26.1% versus 6.0%, P<0.001), suggestingthatdenaturedCII may bean autoantigenin RA. Thus, human CII was cleaved with cyanogen bromide (CB), and immunoblotting techniques were performed on 19 RA and 21 normal sera. Among the four major CB peptides, CB10 and CB11 were recognized by most of the sera tested without distinction between normal or RA sera. Inhibition experiments using an ELISA have shown that: (i) antibodies to the native CII molecule did not cross-react with those recognizing the CB peptides, and vice-versa; (ii) the binding of the sera to native CII was partially inhibited by pre-incubation with alpha-1 (II) chains, and vice-versa; (iii) pre-incubation of the sera with CB peptides partially blocked the binding to alpha-1 (II) chains, whereas pre-incubation of the sera with alpha-1 (II) chains totally inhibited the reactivity against CB peptides; and (iv) a substantial proportion of the epitopes recognized by anti-CII autoantibodies was neither species specific nor type specific. Taken together, these findings reveal the existence of several populations of anti-CII autoantibodies: some antibodies react exclusively with conformational determinants of the CII molecule, and others are directed towards linear structures of alpha-1 (II) chains.     

Characterization of human helper and suppressor factors with special reference to HLA-DR determinants

Human helper and suppressor cells can be induced entirely in vitro, by culturing lymphocytes with a small or large dose of antigen for 4 days in a Marbrook—Diener system. The corresponding helper or suppressor factor (vitro) can then be released by a second pulse of antigen for 1 day. However, if sensitization has taken place in vivo by a naturally encountered antigen, then helper or suppressor factor (vivo/vitro) can be released by a single pulse of antigen for 1 day from putative in vivo-induced helper or suppressor cells. Helper factor_vitro was compared with helper factor_vivo/vitro, stimulated by a streptococcal protein antigen derived from Streptococcus mutans. The results of affinity chromatography suggest that both helper factors have an antigen-specific binding site and bind to monoclonal antibodies directed against HLA-DR, MT, β₂M and μ chain and a `constant' part on helper factor (HF) determinants. The HF<sub>vivo/vitro</sub> unlike HF<sub>vitro</sub> may reflect in vivo-sensitization and thus assay putative in vivo-sensitized antigen specific helper cells. Similarly, suppressor factor <sub>vivo/vitro</sub> might be a measure of in vivo-induced suppressor cells. Like HF, suppressor factor (SF) has an antigen-specific binding site, HLA-DR, MT and β<sub>2</sub>M determinants and a `constant' portion of SF. The DR determinants were further characterized by immunoadsorption, using monoclonal antibodies to the α- and β-chain of DR antigen. The results suggest that helper and suppressor factor_vivo/vitro express similar Ia determinants. Both expressed an α-chain determinant, recognized by one of the two anti-α-chain antibodies (TAL/1B5), and a β-chain non-polymorphic determinant recognized by one of the four anti-β chain antibodies used (DA6.231). However, both HF and SF derived from DRw6- but not from DR4-typed lymphocytes expressed MT1 (DRw6,1,2) or MT2 (DRw6,3,5,8) determinants. As DR and MT determinants can be expressed on the same molecule, it is possible that both are involved in helper and suppressor activities.     

Antigenic determinants of influenza virus hemagglutinin. I. Cyanogen bromide peptides derived from A/MEMPHIS/72 hemagglutinin possess antigenic activity.

Immunoglobulin G prepared from rabbit antisera raised against three strains of influenza virus was adsorbed with suitable recombinant viruses to yield IgG directed only against the polypeptide moiety of the hemagglutinin. Immune complexes formed by incubation of these antibodies with purified hemagglutinin heavy chain, or its derivatives, were detected by affinity chromatography using protein A-Sepharose. It was found that reduction and alkylation (carboxymethylation) of hemagglutinin heavy chain did not affect the antigenicity of the molecule. Peptides derived from the heavy chain by cyanogen bromide cleavage still possessed substantial antigenic activity. Carboxymethylation of these peptides, however, caused a more dramatic decrease in the binding of antibody.     

Studies on the specificity of antibodies to ovalbumin in normal human serum: technical considerations in the use of ELISA methods.

As part of a study designed to reveal information about molecular features of allergenic food proteins after absorption from the gut the specificity of antibodies in normal human serum to hen's egg ovalbumin was investigated using ELISA techniques. Preliminary investigations with monoclonal antibodies and hyperimmune rabbit antiserum specific for ovalbumin in its native and denatured form established that the molecule underwent an extensive conformational change on adsorption to polyvinyl chloride microtitre plates. The native conformation could be retained by using antibodies to couple the protein to the surface. Serum from 90% of healthy adult human donors contained IgG antibodies to ovalbumin. In nearly all cases the antibodies were specific predominantly for the native molecule and could not be absorbed with denatured ovalbumin or peptides prepared from it by cleavage with cyanogen bromide or trypsin. Antibodies to denatured ovalbumin were detected in most sera but at very low levels and were preferentially absorbed by the homologous antigen; peptides and native ovalbumin showing variable absorptive activity. Thus, although ovalbumin is ingested largely in a denatured form, the serum antibody response is stimulated mainly by topographic epitopes of the native molecule.     

A study of the alpha2-macroglobulin homologues of various species

The antigenic relationships between the α₂-macroglobulin homologues of a large number of species (predominantly mammalian) have been studied by the gel diffusion precipitin technique using a specific antiserum to human α₂-macroglobulin raised in a rabbit.

This antiserum has been absorbed with eleven different mammalian sera and the ability of the absorbed samples to cross-react with all these sera has again been tested.

From these results an attempt has been made to calculate the minimum number of antigenic determinants on each α₂-macroglobulin homologue recognized by this rabbit antiserum.

     

Immunogenicity and specificity of collagen: VI. Separation of antibody fractions with restricted specificity from anticollagen sera using an immunoadsorbent technique

Rabbit antisera to calf collagen were investigated using columns of denatured rabbit collagen insolubilized by coupling to diazotized p-aminobenzyl cellulose. Only antibodies with general collagen specificity were bound onto the immunoadsorbent. Species specific antibodies passed through the column and were isolated in the effluent. Column-bound antibodies could be desorbed by elution with collagen peptides. The separated antibody fractions exhibited in haemagglutination—inhibition experiments with calf and rabbit collagen and collagen peptides a characteristic behaviour allowing the differentiation between them. The results support the assumption drawn from serological experiments that most antisera contain mixtures of antibodies with different specificities.     

Immunogenicity and specificity of collagen: V. Demonstration of three different antigenic determinants on calf collagen

Eighty rabbits were immunized with native or denatured acid-soluble calf skin collagen. Fifty-five antisera showed haemagglutination titres higher than 1:64. These sera were investigated with calf skin collagen, pepsin-treated calf skin collagen and rabbit skin collagen, used as coating antigens and inhibitors. Three serologically distinct collagen-antibody fractions could be demonstrated: a general, non-species specific fraction reacting with all three antigens, a species specific fraction active with the two calf preparations, and an antibody fraction to pepsin-labile structures of collagen reacting only with untreated calf collagen. The antibody fractions occur in the antisera either alone or as mixtures. The titres of distinct antibody fractions present in the mixed sera were determined by haemagglutination—inhibition experiments.

Antibodies to pepsin-labile structures could be equally well inhibited by collagen peptides obtained after trypsin or collagenase treatment. A ten-fold higher amount of peptides as compared with undergraded antigen was necessary to obtain the same inhibition effect. The results were compared with previously reported peptide inhibition experiments performed with the other two types of antibodies.

     

Radioimmunoassay for the aminoterminal peptide of procollagen pα1(I)-chain

Peptides derived from the aminoterminal portion of the pα1(I)-chain of calf and sheep procollagen were labeled with iodine-125. Despite changes in electrophoretic homogeneity after labeling, reaction of the labeled peptide with antisera to unlabeled peptide was retained. Antisera to procollagen or the isolated procollagen peptide showed high titers for the native peptide, a much weaker binding with the reduced and alkylated peptide and little or no reaction with collagen. Antisera to collagen showed strong binding with collagen and a weaker but distinct reaction with the procollagen peptide. Evidence was obtained that a minor contaminant of procollagen peptide was present in the acid-extracted collagen and that there were no shared antigenic determinants. Bovine serum and amniotic fluid contained 1–10 μg/ml reactive antigen. These results indicate that the labeled peptides can be used as a specific, sensitive and accurate assay for the amino-terminal portion of procollagen in biological samples.     

Detection of immunologically active zones in equine growth hormone.

Peptide fragments, obtained from equine growth hormone by cyanogen bromide cleavage and further chemical treatment, were isolated and identified.Their immunological reactivities were tested by hemagglutination and complement fixation methods using rabbit antisera against native hormone. Antigenic determinants were detected in the fragments comprising amino acid sequences 5-72 and 73-123, this last one being predominant.Fragment 124-178 had very low reactivity.Nitration of peptide 73-123 did not modify its immunological properties,but oxidation diminished them.Comparison of the antigenicity of equine growth hormone fragment 73-123 with that of the homologous ovine growth hormone fragment 86-123 lent support to the hypothesis that at least one antigenic determinant area in the horse hormone fragment is dependent on sequence 86-123.     

Structural features of antigenic determinants on variant surface glycoproteins from Trypanosoma brucei

The immunochemical structure of two variant surface glycoproteins (VSGs) from Trypanosoma brucei has been studied using monoclonal and polyclonal antibodies. These two VSGs, WaTat 1.1 and WaTat 1.12 have been shown to possess cross-reactive surface-exposed antigenic determinants [Barbet et al., Nature 300, 53-57 (1982)] and similar N-terminal amino acid sequences [Olafson et al., Molec. Biochem. Parasit. 12, 287-298 (1984)]. Monoclonal and polyclonal antibodies were raised against the soluble forms of the two VSGs and against their reduced, alkylated and cyanogen bromide (CNBr) cleaved forms. None of the monoclonal antibodies which bound to the surface of living trypanosomes bound to CNBr fragments of the VSGs nor to denatured VSGs. Polyclonal antibodies raised against denatured and cleaved VSG did not bind to the surface of the living trypanosomes. These results suggest that the variable surface exposed antigenic determinants of VSG are topographically assembled structures. It was also shown that the conserved amino terminal peptides of WaTat 1.1 and WaTat 1.12 do not contain antigenic determinants.