人卵透明带蛋白ZP3a和ZP3b肽段的免疫原性及其抗血清体外抑制人精子-半透明带结合

分析大肠杆菌表达的重组人卵透明带-3（r-huZP3）蛋白两个肽段（r-huZP3a^22-176及r-huZP3b^177-348）的免疫原性,比较两者抗血清体外抑制人精子-半透明带结合的能力。以制备性聚丙烯酰胺凝胶电泳（PAGE）纯化的大肠杆菌表达蛋白为抗原,主动免疫新西兰兔产生抗huZP3a^22-176及huZP3b^177-348抗血清：通过ELISA测定比较两者的抗体应答水平;以蛋白印迹和免疫组化方法测定两者抗血清同重组表达蛋白、大然人卵ZP以及卵巢组织的反应性;通过竞争性半透明带结合试验（hemizona assay,HZA）观察两者抗血清体外抑制人精子-ZP结合能力。结果显示：未与人分子蛋白载体耦联的r-huZP3a^22-176和r-huZP3b^177-348抗原都在免疫兔中产生了较高的抗体滴度,而且它们的抗血清可识别人肠杆菌表达的各自重组ZP3肽以及人卵细胞表面天然ZP,两者抗血清也都能在体外抑制人精子-ZP结合。由此可见,r-huZP3^22-176及r-huZP3b^177-348蛋白具有良好免疫原性,所产生抗血清也都显示出细胞和组织特异性。因此,单一或合并两个huZP3肽段均可作为抗原研制检测不明原因性不孕妇女中是否存在抗自身ZP抗体的诊断试剂盒,另外它们的抗血清也能用于鉴定已知huZP3表位肽段的最小B-细胞表位基序。     

Delineation and conformational analysis of two synthetic peptide models of antigenic sites on rodent cytochrome c

Two regions of rodent cytochrome c, one within the first four residues of the molecule, which is N-acetylated, and one at a beta bend around residue 44, are known to be immunogenic and antigenic in rabbits. Using sequential peptide synthesis, we have determined the residues required for linear synthetic peptides within these sequences to bind to antibody raised in rabbits to intact rat cytochrome c. The residues that were important in binding the N-terminal peptides were N-acetylglycine at position 1 and valine at position 3. The smallest peptide sequence around residue 44 that would bind to antibodies was Gln-Ala-Ala-Gly-Phe. A theoretical conformational analysis of these peptides showed that the amino-terminal tetrapeptide adopts a wide statistical ensemble of conformational states and that the addition of residues beyond 41 and 45 in the other sequence does not appear to stabilize longer peptides in the native beta-bend conformation. Thus, the antigenicity conferred by Phe-46 and Gln-42 in this peptide is most likely due to the direct interaction of the side chains of these residues with the antibody binding site. The demonstration here that native conformation is not essential for antigenic peptides to bind to antibodies raised against the whole protein indicates that the association energy between antigen and antibody can be sufficient to induce conformation in conformationally flexible peptides. This supports the concept that anti-protein and anti-peptide antibodies may invoke conformational changes in cross-reactive protein antigens and may explain why longer peptides, which may adopt stable nonnative secondary structure, often do not bind to antibodies raised to the whole molecule.     

Antigen-antibody interaction. Synthetic peptides define linear antigenic determinants recognized by monoclonal antibodies directed to the cytoplasmic carboxyl terminus of rhodopsin

The specificities of four monoclonal antibodies rho 1D4, 1C5, 3A6, and 3D6 prepared by immunization of rod outer segments containing rhodopsin have been defined using synthetic peptides. All of these antibodies interact within the 18 residues at the COOH terminus of rhodopsin and recognize linear antigenic determinants of 4-11 residues. Twenty-seven synthetic peptide analogs of varying lengths of native sequence or containing single amino acid substitutions at each position of the COOH-terminal 18 residues have provided some insight into the mechanism of antigen-antibody binding. Our results clearly demonstrate that antibodies can be highly specific at key positions as shown by the loss of binding on single amino acid substitutions in the binding site. In contrast single amino acid substitutions at other positions in the binding site only affect affinity for some antibodies. Ionic interactions can dominate immunogenic determinants. Immunogenic determinants are not restricted to highly charged hydrophilic regions on the surface of a protein and may be dominated by hydrophobic interactions. Although certain side chains can dominate the interaction of the antigen with antibody, our results are in agreement with the interpretation that the free energies of all the contact points are additive and a certain free energy must be present to achieve binding. Antibodies with different specificities directed to the same region of the protein antigen can be produced in an immune response. Peptide antigens representing regions of a protein antigen bind best to the anti-protein antibody when the sequence is shortened to contain only those residues binding to the specificity site in the antibody. Cross-reactivity between protein antigens can be explained by conservation of the critical residues in the combining site.     

Preparation of Antibodies to β Subunits of γ-Aminobutyric AcidA Receptors

Antisera were produced in rabbits against synthetic peptides based on two regions of the cDNA sequence of the beta 1 subunit of bovine gamma-aminobutyric acidA (GABAA) receptors. The deduced amino acid sequences were similar in other beta subunits of bovine, rat, and chick receptors, predicting cross-reactability with all beta subunits. One antiserum (anti-beta e) was raised against an extracellular moiety near the invariant disulfide loop thought to be located near the neurotransmitter binding domain; the other (anti-beta c) was raised against an intracellular moiety containing a consensus sequence for cyclic AMP-dependent protein kinase phosphorylation of a serine residue. Predicted secondary structures suggested high potential immunogenicity for the chosen antigen peptides. Both antisera at high dilutions recognized the same polypeptide bands on western blots of GABAA receptors purified from three regions of bovine brain (four bands at 57, 54, 53, and 52 kDa in cerebral cortex) but fewer bands (57, 54, and 52 kDa) in hippocampus and cerebellum (one major band at 54 kDa, traces at 57 and 53 kDa). This is consistent with the presence of multiple beta subunits whose expression varies with brain region, as shown by molecular cloning. The anti-beta c antibody was able to immunoprecipitate purified GABAA receptor [3H]-muscimol binding, 87% in bovine cortex and 75% in total rat brain; the anti-beta e was unable to immunoprecipitate any antigen. These antibodies indicate a region-dependent heterogeneity of beta subunits and should be useful for analyzing structure, function, and localization of GABAA receptor subtypes in brain.     

Antibodies Raised Against Synthetic Peptides React with Choline Acetyltransferase in Various Immunoassays and in Immunohistochemistry

Abstract: Antisera were raised in rabbits against five synthetic peptides. These peptides have been identified as potentially antigenic epitopes from the sequence of porcine choline acetyltransferase (ChAT) using primary and secondary structure analysis. All five antisera recognized immunoaffinity-purified antigen from porcine brain in an ELISA and on western blots. Four antisera recognized ChAT on dot blots, and another four antisera reacted with native and degraded enzyme in a sandwich ELISA using monoclonal antibodies as the capture antibody. One peptide antiserum was of similar avidity in this sandwich ELISA as a polyclonal antibody raised against immunoaffinity-purified ChAT. The same antiserum reacted with the enzyme from human placenta in an ELISA and on western and dot blots and recognized ChAT in rat, primate, and human neurons. Thus, a single peptide (amino acids 168- 189) provides the means for easy, reliable, and reproducible generation of antibodies against ChAT suitable for replacing conventional polyclonal and monoclonal antibodies.     

Exploring antibody recognition of sequence space through random-sequence peptide microarrays

A universal platform for efficiently mapping antibody epitopes would be of great use for many applications, ranging from antibody therapeutic development to vaccine design. Here we tested the feasibility of using a random peptide microarray to map antibody epitopes. Although peptide microarrays are physically constrained to ～10(4) peptides per array, compared with 10(8) permitted in library panning approaches such as phage display, they enable a much more high though put and direct measure of binding. Long (20 mer) random sequence peptides were chosen for this study to look at an unbiased sampling of sequence space. This sampling of sequence space is sparse, as an exact epitope sequence is unlikely to appear. Commercial monoclonal antibodies with known linear epitopes or polyclonal antibodies raised against engineered 20-mer peptides were used to evaluate this array as an epitope mapping platform. Remarkably, peptides with the most sequence similarity to known epitopes were only slightly more likely to be recognized by the antibody than other random peptides. We explored the ability of two methods singly and in combination to predict the actual epitope from the random sequence peptides bound. Though the epitopes were not directly evident, subtle motifs were found among the top binding peptides for each antibody. These motifs did have some predictive ability in searching for the known epitopes among a set of decoy sequences. The second approach using a windowing alignment strategy, was able to score known epitopes of monoclonal antibodies well within the test dataset, but did not perform as well on polyclonals. Random peptide microarrays of even limited diversity may serve as a useful tool to prioritize candidates for epitope mapping or antigen identification.     

Analysis of immune complexes in rheumatoid arthritis for Epstein-Barr virus antigens reveals cross-reactivity of viral capsid antigen and human IgG

We recently defined the immunochemical characteristics of immune complexes (IC) isolated from synovial fluid (SF) of patients with rheumatoid arthritis with the use of Western blot analysis. In the present study, we probe for exogenous antigens in the IC by examining the specificity of antisera raised against the IC. Anti-IC antisera demonstrated strong reactivity against the viral capsid antigen (VCA) of Epstein Barr virus (EBV), which was not explained by preimmune reactivity, polyclonal B cell activation, or Fc-mediated binding in the immunofluorescence or ELISA systems used to measure antibody titers. However, comparable anti-VCA reactivity was detected in antisera raised against non-rheumatoid SF. This phenomenon was not due to antigen since monoclonal anti-VCA antibody probing the IC by Western blot detected only IgG, nor to idiotype/anti-idiotype interaction since normal IgG absorbed out the anti-VCA reactivity. A monoclonal anti-VCA antibody competitively inhibited the binding of anti-IgG to IgG, and Fc fragment of IgG competitively inhibited the monoclonal antibody binding to VCA. No relationship between IgG anti-VCA antibody and IgG rheumatoid factor could be demonstrated. These data demonstrate an unexpected cross-reactivity of Fc fragment of IgG and VCA of EBV through the analysis of SF IC.     

An antigenic determinant common to human chorionic somatomammotropin and human growth hormone revealed by limited proteolysis of immune complexes.

An epitope of human chorionic somatomammotropin for one of the monoclonal antibodies raised against the whole antigen has been identified. We compared the release of peptides from limited proteolysis of the antigen in the presence and absence of the related antibody. Using enzymes of different specificity, we could determine the amino acid sequence that can be considered at least inclusive of the epitope. The monoclonal antibody selected is 100% cross-reactive with human growth hormone, so the antigenic determinant identified is shared by the two protein hormones.     

The immunological activity of some of the chymotryptic peptides of sperm-whale myoglobin

1. Sperm-whale apomyoglobin was digested with chymotrypsin in a dialysis sac. The ultrafiltrate contained incompletely hydrolysed fragments which partially inhibited the precipitation of metmyoglobin and apomyoglobin by some antisera produced against metmyoglobin. The inhibitory activity was stable to heating at 100 degrees and depended on the peptide structure. 2. The fragments were fractionated according to molecular size and were purified by ion-exchange chromatography. Six pure peptides and two peptides which contained a minor impurity were isolated. Their amino acid compositions and N-terminal amino acid sequences were determined and their entire amino acid sequences deduced from the known amino acid sequence of sperm-whale myoglobin. 3. The peptides formed no detectable precipitates with the antisera. Five of the eight peptides partially inhibited the precipitation of apomyoglobin and/or metmyoglobin by one antiserum. Six of the peptides inhibited the precipitation of apomyoglobin by one or other of two antisera; at least two of these peptides inhibited both antisera. One peptide failed to inhibit the precipitation of either antigen by either antiserum. Two of the peptides possessed the same serological specificity. 4. The molar ratios of inhibitors to antigen for 50% of the maximum inhibition decreased as the molecular size of the inhibitor increased. With one antiserum and with apomyoglobin as the antigen, molar ratios 12 and 80 were obtained for peptides with molecular weights 2051 and 793 respectively. 5. The size and structure of an antigenic site is discussed in relation to the known steric configuration of myoglobin.     

Identification of immunodominant epitope of F1 antigen of Yersinia pestis

The F1 antigen of Yersinia pestis has been identified as one of the major protective antigens of this bacterium. The present study aims to delineate major and minor antigenic sites of F1 antigen. Using algorithmic predictions, five peptide sequences (P1, P2, P3, P4 and P5) spanning the C-terminal region were identified and synthesized. Antibodies were generated in mice against the peptides, native F1 protein and polymerized F1 antigen using liposomes as mode of immunization. Cross-reactivity between F1 antigen and peptides was tested using both solid and solution phase assays. Similar assays were done with rabbit anti-F1 sera. Competitive inhibition assays using a different combination of antisera and competing antigen identified P2 peptide FFVRSIGSKGGKLAAGKYTDAVTV (142-165) as the immunodominant sequence. The results indicate that this sequence appears to be exposed on the surface of F1 molecule. In a solid phase binding assay, P2 peptide was recognized even at high F1 antisera dilution. However, when antisera raised to different peptides were tested for binding to F1 antigen, antisera to P4 peptide showed maximal immunoreactivity. This implies more accessibility of this region during immobilization on solid surface. There was consistency in the results obtained for different strains of mice as well as for the rabbit antisera. Such a sequence of F1 antigen, which is recognized widely in animals of different genetic background, would be useful for diagnosis and subunit vaccine.     

Comparison between different rabbit antisera against the glucocorticoid receptor

Seven antisera against the glucocorticoid receptor (GR), raised in different rabbits immunized with highly purified (in case of five rabbits apparently homogeneous) preparation of GR from rat liver cytosol, were compared concerning titer and cross-reactivity. The titers of protein A-purified antisera (10 mg/ml) were in the range 1:100-1:320 as measured by enzyme-linked immunosorbent assay, ELISA, (defined as the dilution giving 50% of maximum absorbance). All seven antisera bound to the rat GR with a Stokes radius of 6.1 nm, but no antiserum reacted with the proteolytically induced steroid binding domain with Stokes radius 3 nm. However, the antigenic determinant(s) of the non-ligand-binding domain(s), split off from the steroid binding domain, is preserved following digestion with alpha-chymotrypsin or trypsin, respectively, since immunoactivity is still detectable by ELISA. Only two of four antisera tested cross-reacted with the GR from human lymphocytes. The same two antisera cross-reacted with chick embryo liver GR. Four out of four antisera tested cross-reacted with mouse liver GR as well as with rabbit lung GR. For these antisera, antibody binding to the GR prior to steroid- or DNA-binding did not influence the ability of the GR to interact with the ligand or DNA-cellulose, respectively. No difference regarding avidity of the antisera for activated or non-activated GR was observed. Furthermore, none of the antisera tested cross-reacted with the estrogen, progestin, androgen or mineralocorticoid receptors in rat. These findings indicate that the antisera from different rabbits raised against the same antigen all react with a certain domain of the rat GR, but show species differences as well as receptor class specificity.     

Evolutionary transition pathways for changing peptide ligand specificity and structure

We identified evolutionary pathways for the inter- conversion of three sequentially and structurally unrelated peptides, GATPEDLNQKL, GLYEWGGARI and FDKEWNLIEQN, binding to the same site of the hypervariable region of the anti-p24 (HIV-1) monoclonal antibody CB4-1. Conversion of these peptides into each other could be achieved in nine or 10 single amino acid substitution steps without loss of antibody binding. Such pathways were identified by analyzing all 7 620 480 pathways connecting 2560 different peptides, and testing them for CB4-1 binding. The binding modes of intermediate peptides of selected optimal pathways were characterized using complete sets of substitution analogs, revealing that a number of sequential substitutions accumulated without changing the pattern of key interacting residues. At a distinct step, however, one single amino acid exchange induces a sudden change in the binding mode, indicating a flip in specificity and conformation. Our data represent a model of how different specificities, structures and functions might evolve in protein-protein recognition.     

Protection against Pseudomonas aeruginosa infection by passive transfer of anti-flagellar serum

The specificity of adsorbed flagellar antisera for H-antigen was demonstrated in vitro by cross-agglutination assays, motility inhibition, and an ELISA. The specific flagellar antibody was determined to be an IgG. Complete protection against burn wound sepsis was achieved with flagellar antisera. Cross-protection experiments revealed that protection was not only H-antigen dependent, but specific for the flagella antigen type. Antiserum raised against b-type flagella would only protect against homologous bacterial challenge and not against a-type flagellated strains. Results using a-type antisera were consistent, giving protection only against the homologous strain. In contrast, protective capacity was selectively removed from antisera by adsorbing with Fla+ cells. Bacteria colonized the burn wounds of passively protected mice to similar levels as seen in nonprotected animals, but the colonization remained localized and did not result in systemic infection, a pattern similar to infections with motility mutants observed in other studies. Animals rendered neutropenic prior to burning were not protected with flagellar antisera. These data suggested a role for phagocytic cells in protection. Immobilization by flagellar antiserum was observed both by microscopic studies and by inhibition of colony spreading. Antiflagellar antibody is hypothesized as exerting its protective capacity possibly in two ways; first by inhibiting the motility of invading bacteria by binding to the flagellum and immobilizing the bacteria, and secondly by acting as an opsonin, targeting either immobilized or mobile cells for phagocytosis.     

Characterization of immune response to synthetic peptides derived from the hemagglutinin of measles virus

We describe here four synthetic peptides derived from the hemagglutinin of measles virus. The peptides were predicted by a computer program combining hydrophilicity, flexibility, surface probability, secondary structure and antigenic index parameters of the amino acid sequence of measles virus hemagglutinin. Rabbits were immunized with the synthesized peptides conjugated to purified protein derivative using immunostimulating complex as adjuvant. Anti-peptide antisera raised in rabbits against the peptide conjugates reacted well with the homologous peptides and with measles virus antigen as tested with plate ELISA. None of these sera had either neutralizing or hemagglutination inhibiting antibody or reacted with measles hemagglutinin protein in Western blot and reacted weakly in immunofluorescence. Human sera positive for measles virus antibody reacted with the synthesized peptides indicating that the selected locations function as partial antigenic sites.     

Influence of carbohydrate moieties on the immunogenicity of human immunodeficiency virus type 1 recombinant gp160.

The role of carbohydrates in the immunogenicity of human immunodeficiency virus type 1 (HIV-1) glycoproteins (gp160 and gp120) remains poorly understood. We have analyzed the specificity and neutralizing capacity of antibodies raised against native gp160 or against gp160 deglycosylated by either endo F-N glycanase, neuraminidase, or alpha-mannosidase. Rabbits immunized with these immunogens produced antibodies that recognized recombinant gp160 (rgp160) from HIV-1 in a radioimmunoassay and in an enzyme-linked immunosorbent assay. Antibodies elicited by the different forms of deglycosylated gp160 were analyzed for their reactivity against a panel of synthetic peptides. Compared with anti-native gp160 antisera, serum reactivity to most peptides remained unchanged, or it could increase (peptide P41) or decrease. Only antibodies raised against mannosidase-treated gp160 failed to react with a synthetic peptide (peptide P29) within the V3 loop of gp120. Rabbits immunized with desialylated rgp160 generated antibodies which recognized not only rgp160 from HIV-1 but also rgp140 from HIV-2 at high titers. Although all antisera produced against glycosylated or deglycosylated rgp160 could prevent HIV-1 binding to CD4-positive cells in vitro, only antibodies raised against native or desialylated gp160 neutralized HIV-1 infectivity and inhibited syncytium formation between HIV-1-infected cells and noninfected CD4-positive cells, whereas antibodies raised against alpha-mannosidase-treated gp160 inhibited neither virus replication nor syncytium formation. These findings indicate that the carbohydrate moieties of gp160 can modulate the specificity and the protective efficiency of the antibody response to the molecule.     

Antibody to a synthetic peptide detects a conserved region of retrovirus transmembrane proteins

N-terminal amino acid sequence analysis of the transmembrane protein of baboon endogenous virus revealed an internal 13 residue identity with the transmembrane homolog of murine leukemia virus. A tridecapeptide Glu-Val-Val-Leu-Gln-Asn-Arg-Arg-Gly-Leu-Asp-Leu-Leu corresponding to this region was chemically synthesized and antibody to the peptide was raised in rabbits. The rabbit antisera recognized the protein in Western blots. The specificity of the antisera was tested against a panel of retroviruses. Transmembrane proteins of type C retroviruses as well as type D were identified.     

Mapping antibody binding sites on cytochrome c with synthetic peptides: Are results representative of the antigenic structure of proteins?

Crystallographic work on antigen-antibody complexes has revealed that extensive surface areas of proteins may interact with antibodies. On the other hand, most experimental approaches to locate and define antigenic determinants of protein antigens rely on the linear sequence of the polypeptide chain. Hence the question arises whether mapping of antibody binding sites by analysis of the reactivity of anti-protein antibodies with synthetic peptides can provide a representative picture of the antigenic structure of a protein antigen. We have addressed this question using yeast iso-1 cytochrome c as a protein antigen against which antisera were raised in rabbits. The reaction of the antisera with 103 synthetic hexapeptides covering the entire sequence of cytochrome c was tested by the pepscan procedure in which peptides are coupled to polyethylene rods and tested by ELISA. For the assay, anti-cytochrome c antibodies were fractionated by affinity chromatography on native yeast iso-1 cytochrome c and on apo-cytochrome c; the latter is a random coil. It was found that only antibodies retained by the apo-cytochrome c affinity column react with synthetic peptides. These antibodies comprise a small fraction, probably less than 2%, of all cytochrome c-specific antibodies. The majority of antigenic determinants, which seem to consist of strongly conformation-dependent topographic epitopes, could not be uncovered by the peptide approach. Epitope mapping with short peptides seems of limited usefulness in the case of small, globular, and conformationally stable proteins like cytochrome c.     

Cross-reactive immunodeterminants on Streptococcus sanguis and collagen. Predicting a structural motif of platelet-interactive domains.

Cross-reactive immunodeterminants on a fibril-associated surface antigen of Streptococcus sanguis and types I and III collagen participate in the induction of aggregation of human platelets. To further understand the basis for this apparent molecular mimicry, antitype-specific collagen antibodies, anti-KPGEPGPK (an analogue of platelet-interactive domains on collagen) and a panel of KPGEPGPK-like synthetic peptides were used as probes. When collagen or S. sanguis cells were pretreated with the anti-collagen antisera, the induction of aggregation of platelet-rich plasma was greatly delayed or abrogated. These anti-collagen antibodies also neutralized KPGEPGPK and purified S. sanguis platelet-interactive antigens as inhibitors of S. sanguis or collagen-induced aggregation of platelets in plasma. In immunoblot analyses, these anti-collagen antibodies reacted with S. sanguis platelet-interactive antigens. Additionally, antisera against the platelet-interactive antigen of S. sanguis selectively reacted with undigested type I collagen and with fragments CB3 and CB6 of cyanogen bromide-treated type I collagen. Finally, when platelets were pretreated with synthetic peptides containing specific amino acid substitutions within the KPGEPGPK sequence, the time to onset of platelet-rich plasma aggregation by both agonists was altered. The hierarchical pattern of responses of platelets to these peptides and predictions of the structural changes produced by simulated insertions of each peptide into the CB4 sequence of type III collagen suggested conformational requirements for interactions with platelets. Thus, these data show that cross-reactive immunodeterminants of S. sanguis and collagen induce platelet aggregation. The platelet-interactive domains are predicted to be characterized by a structural motif with the consensus sequence X-P-G-E-P/Q-G-P-X.     

Epitope mapping employing antibodies raised against short synthetic peptides: a study of the nicotinic acetylcholine receptor

Antibodies were raised against eight synthetic peptides matching preselected portions of the amino acid sequence of nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata. To increase the probability of obtaining antibodies specific for the exact sequence of the immunizing peptide, peptides of only five to seven amino acids in length were employed. Even under these limiting conditions some of the polyclonal rabbit immune sera showed cross-reactivity with other peptides and/or other sequence regions of the receptor. Further studies with polyclonal and monoclonal sera suggested that conformation and charge pattern rather than linear sequence are the essential determinants of antibody epitopes. Application of antibodies for topological studies therefore requires that the antibody specificity for a particular region of the antigen has been firmly established. Epitope mapping with the eight anti-peptide immune sera provides information on the accessibility to antibody of matching sequences within the receptor molecule. We find the sequence portions alpha 81-85, alpha 127-132, and alpha 190-195 to be freely accessible both at membrane-bound and at purified receptor. Binding of anti-alpha 387-392 serum does not prove accessibility of this region as the serum cross-reacts strongly with peptide fragments corresponding to the regions alpha 165-200 and beta 190-200 of nAChR from Torpedo californica. To permit binding of anti-alpha 137-142 immune serum, treatment of the receptor with endoglycosidase is required, showing that Asn-141 indeed is glycosylated in native nAChR. The homologous sequence of the other subunits differing only in one sequence position from alpha 137-142 is not accessible in native nAChR to antibody, indicating clear differences in folding of the receptor polypeptides. Sequence portions alpha 395-401 and alpha 161-166 must first be exposed by appropriate treatment to permit binding of respective serum. These results and previous epitope mapping studies by other laboratories are discussed with respect to the limited sequence specificity of antibodies.