Epitopes of Escherichia coli ribosomal protein S13

To analyze the immunochemical structure ofEscherichia coli ribosomal protein S13 and its organizationin situ, we have generated and characterized 22 S13-specific monoclonal antibodies. We used a competitive enzyme-linked immunosorbent assay to divide them into groups based on their ability to inhibit binding of one another. The discovery of five groups with distinct binding properties suggested that a minimum of five distinct determinants on S13 are recognized by our monoclonal antibodies. The locations of the epitopes detected by these monoclonal antibodies have been mapped on S13 peptides. Three monoclonal antibodies bind a S13 C-terminal 34-residue segment. All the other 19 monoclonal antibodies bind a S13N-terminal segment of about 80 residues. The binding sites of these 19 monoclonal antibodies have been further mapped to subfragments of peptides. Two monoclonal antibodies recognized S13_1–22; three monoclonal antibodies bound to S13_1–40; the binding sites of three other antibodies have been located in S13_23–80, with epitopes possibly associated with residues 40–80. The remaining 11 monoclonal antibodies did not bind to these subfragments. These data provide molecular basis to the structure of S13 epitopes, whosein situ accessibility may reveal the S13 organization on the ribosome.     

Antigenic specificity of monoclonal antibodies to human myoglobin

Two monoclonal antibodies directed against different sites of the human myoglobin molecule have been tested for their cross-reactivities against several myoglobins including seven from mammalian species. The relation between their cross-reactivities and their amino acid sequences had led to a possible localization of two antigenic domains in human myoglobin. Each domain includes residues previously considered not to be directly involved in the antigenic structure of myoglobin. Unlike polyclonal serum antibodies, monoclonal hybridoma antibodies directed to a native protein often fail to bind to supposedly antigenic protein fragments. This is explicable in terms of the concept of antigenic domains. Such domains are numerous and overlapping, each comprising a number of contributory amino acid side chains which need not necessarily include continuous sequences of amino acids and which need not exhibit measurable antigenicity in isolation from the rest of the domain.     

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.     

Multiple overlapping epitopes in the three antigenic regions of horse cytochrome c1

To gain a better understanding of the diversity of epitopes on a protein, the specificities of 103 monoclonal antibodies to a model antigen, horse cytochrome c(cyt c), were analyzed. The antibodies were generated in in vitro monoclonal, secondary antibody responses against horse cyt c coupled to hemocyanin in splenic fragment cultures. For this assay, horse cyt c-primed murine B lymphocytes were transferred to irradiated, hemocyanin-primed recipients. A panel of seven mammalian cyts c differing at one to six residues out of 104 and cyanogen bromide-cleaved fragments of horse cyt c containing residues 1-65, 1-80, and 66-104 was used to examine the specificities of the antibodies. Twenty-two distinct reactivity patterns were observed, even though the majority of the monoclonal antibodies were found to bind in the three previously identified antigenic regions of the molecule about residues 44-47, 60-62, and 89-92. The results indicate that each of the three antigenic regions consists of multiple overlapping epitopes. Few of the antibodies directed to any given antigenic region bound polypeptide fragments inclusive of the epitope sequences, demonstrating that some antibodies were more conformationally dependent than others. Only 13% of the antibodies bound to cyanogen bromide-cleaved polypeptide fragments that together encompassed the entire length of the protein. Considering the large number of antibodies analyzed and the reoccurrence of 13 of the 22 clonotypes in different lymphocyte donors, it is likely that the antibody specificities tabulated herein approach yet do not completely enumerate the total inventory of the horse cyt c-specific B cell repertoire. The remarkable diversity for epitope recognition within antigenic regions observed here is likely to pertain to protein antigens in general, and strongly supports the widely held notion that the entire surface of a protein is potentially antigenic. The restriction of the epitopes of horse cyt c to three antigenic regions where the amino acid sequences of the mammalian cyts c differ probably results from tolerance of the mice to their own cyt c.     

Antigenic structure of histone H2B

Antigenic determinants of histone H2B were localized using a series of 23 overlapping fragments of H2B obtained either by chemical and enzymatic cleavage of the histone or by solid-phase peptide synthesis. The ability of peptides to bind H2B antibodies was measured in an enzyme-linked immunosorbent assay, using antisera directed against calf thymus and chicken erythrocyte H2B as well as four anti H2B monoclonal antibodies obtained from autoimmune mice. Seven antigenic determinants were localized in the H2B molecule in the vicinity of residues 1-11, 6-18, 15-25, 26-35, 50-65, 94-113 and 114-125. Two of these determinants (residues 6-18 and 26-35) were revealed only through the binding properties of antibodies isolated from autoimmune mice. The usual correlation between hydrophilicity and antigenicity was found to hold for four of the epitopes, and the N- and C-termini of H2B were both antigenically active.     

Comprehensive Mapping of Common Immunodominant Epitopes in the Eastern Equine Encephalitis Virus E2 Protein Recognized by Avian Antibody Responses

Eastern equine encephalitis virus (EEEV) is a mosquito-borne virus that can cause both human and equine encephalitis with high case fatality rates. EEEV can also be widespread among birds, including pheasants, ostriches, emu, turkeys, whooping cranes and chickens. The E2 protein of EEEV and other Alphaviruses is an important immunogenic protein that elicits antibodies of diagnostic value. While many therapeutic and diagnostic applications of E2 protein-specific antibodies have been reported, the specific epitopes on E2 protein recognized by the antibody responses of different susceptible hosts, including avian species, remain poorly defined. In the present study, the avian E2-reactive polyclonal antibody (PAb) response was mapped to linear peptide epitopes using PAbs elicited in chickens and ducks following immunization with recombinant EEEV E2 protein and a series of 42 partially overlapping peptides covering the entire EEEV E2 protein. We identified 12 and 13 peptides recognized by the chicken and duck PAb response, respectively. Six of these linear peptides were commonly recognized by PAbs elicited in both avian species. Among them five epitopes recognized by both avian, the epitopes located at amino acids 211–226 and 331–352 were conserved among the EEEV antigenic complex, but not other associated alphaviruses, whereas the epitopes at amino acids 11–26, 30–45 and 151–166 were specific to EEEV subtype I. The five common peptide epitopes were not recognized by avian PAbs against Avian Influenza Virus (AIV) and Duck Plague Virus (DPV). The identification and characterization of EEEV E2 antibody epitopes may be aid the development of diagnostic tools and facilitate the design of epitope-based vaccines for EEEV. These results also offer information with which to study the structure of EEEV E2 protein.     

Epitope mapping of monoclonal antibodies toEscherichia coli ribosomal protein S3

The antigenic structure ofEscherichia coli ribosomal protein S3 has been investigated by use of monoclonal antibodies. Six S3-specific monoclonal antibodies secreted by mouse hybridomas have been identified by immunoblotting of two-dimensional ribosomal protein separation gels. By using a competitive enzyme-linked immunosorbent assay, we have divided these monoclonal antibodies into three mutual inhibition groups, members of which are directed to three distinct regions of the S3 molecule. The independence of these monoclonal antibody-defined regions was confirmed by the failure of pairs of monoclonal antibodies from two inhibition groups to block the binding of biotinylated monoclonal antibodies of the third group. To determine the regions recognized by these monoclonal antibodies, chemically cleaved S3 peptides were fractionated by gel filtration and reverse-phase high-performance liquid chromatography. The fractionated peptides were coated on plates and examined for specific interaction with monoclonal antibody by enzyme immunoassay. In this manner, two epitopes have been mapped at the ends of the S3 molecule: one, in the last 22 residues, is recognized by three monoclonal antibodies; and the second, in the first 21 residues, is defined by two monoclonal antibodies. The third S3 epitope, recognized by a single monoclonal antibody, has been localized in a central segment of about 90 residues by gel electrophoresis and immunoblotting. These epitope-mapped monoclonal antibodies are valuable probes for studying S3 structurein situ.     

Epitope mapping of monoclonal antibodies toEscherichia coli ribosomal protein S3

The antigenic structure ofEscherichia coli ribosomal protein S3 has been investigated by use of monoclonal antibodies. Six S3-specific monoclonal antibodies secreted by mouse hybridomas have been identified by immunoblotting of two-dimensional ribosomal protein separation gels. By using a competitive enzyme-linked immunosorbent assay, we have divided these monoclonal antibodies into three mutual inhibition groups, members of which are directed to three distinct regions of the S3 molecule. The independence of these monoclonal antibody-defined regions was confirmed by the failure of pairs of monoclonal antibodies from two inhibition groups to block the binding of biotinylated monoclonal antibodies of the third group. To determine the regions recognized by these monoclonal antibodies, chemically cleaved S3 peptides were fractionated by gel filtration and reverse-phase high-performance liquid chromatography. The fractionated peptides were coated on plates and examined for specific interaction with monoclonal antibody by enzyme immunoassay. In this manner, two epitopes have been mapped at the ends of the S3 molecule: one, in the last 22 residues, is recognized by three monoclonal antibodies; and the second, in the first 21 residues, is defined by two monoclonal antibodies. The third S3 epitope, recognized by a single monoclonal antibody, has been localized in a central segment of about 90 residues by gel electrophoresis and immunoblotting. These epitope-mapped monoclonal antibodies are valuable probes for studying S3 structurein situ.     

Human Monoclonal Antibodies to a Novel Cluster of Conformational Epitopes on HCV E2 with Resistance to Neutralization Escape in a Genotype 2a Isolate

The majority of broadly neutralizing antibodies to hepatitis C virus (HCV) are against conformational epitopes on the E2 glycoprotein. Many of them recognize overlapping epitopes in a cluster, designated as antigenic domain B, that contains residues G530 and D535. To gain information on other regions that will be relevant for vaccine design, we employed yeast surface display of antibodies that bound to genotype 1a H77C E2 mutant proteins containing a substitution either at Y632A (to avoid selecting non-neutralizing antibodies) or D535A. A panel of nine human monoclonal antibodies (HMAbs) was isolated and designated as HC-84-related antibodies. Each HMAb neutralized cell culture infectious HCV (HCVcc) with genotypes 1–6 envelope proteins with varying profiles, and each inhibited E2 binding to the viral receptor CD81. Five of these antibodies neutralized representative genotypes 1–6 HCVcc. Epitope mapping identified a cluster of overlapping epitopes that included nine contact residues in two E2 regions encompassing aa418–446 and aa611–616. Effect on virus entry was measured using H77C HCV retroviral pseudoparticles, HCVpp, bearing an alanine substitution at each of the contact residues. Seven of ten mutant HCVpp showed over 90% reduction compared to wild-type HCVpp and two others showed approximately 80% reduction. Interestingly, four of these antibodies bound to a linear E2 synthetic peptide encompassing aa434–446. This region on E2 has been proposed to elicit non-neutralizing antibodies in humans that interfere with neutralizing antibodies directed at an adjacent E2 region from aa410–425. The isolation of four HC-84 HMAbs binding to the peptide, aa434–446, proves that some antibodies to this region are to highly conserved epitopes mediating broad virus neutralization. Indeed, when HCVcc were passaged in the presence of each of these antibodies, virus escape was not observed. Thus, the cluster of HC-84 epitopes, designated as antigenic domain D, is relevant for vaccine design for this highly diverse virus.     

Genetic control of immune response to staphylococcal nuclease. XII: Analysis of nuclease antigenic determinants using anti-nuclease monoclonal antibodies

Summary SJL mice, which are high responders to Staphylococcal nuclease (nuclease), were immunized and used to produce hybridoma cell lines secreting anti-nuclease monoclonal antibodies (mAb). Ten stable clones were derived from a single fusion. Seven of these produced antibodies of the IgG_1, isotype and were more precisely characterized for antigenic specificity. Only one hybridoma cell line (54-10-4) produced anti-nuclease antibodies capable of inhibiting enzymatic activity of nuclease. Binding inhibition analyses strongly suggest that the other monoclonal antibodies, which failed to inhibit nuclease activity detect two different antigenic regions, or epitopes, of the molecule: epitope cluster 1 domain is defined by hybridomas 54-2-7, 54-5-2, 54-9-8, and 54-10-8; epitope cluster 2 by 54-5-1 and 54-1-9. Because of its capacity to inhibit nuclease enzymatic activity mAb 54-10-4 was considered specific for a third epitope of the nuclease molecule called epitope 3. Binding studies of these monoclonal antibodies were extended to peptide fragments of the nuclease molecule in order to examine possible cross-reactions with such fragments, as has previously been reported for antibodies purified from polyclonal antisera. Monoclonal antibodies specific for epitope cluster 1 on the native molecule also bound to the fragments 1–126 and 49–149 but failed to bind to fragment 99–149, suggesting that the corresponding epitope(s) is determined by amino acids localized between residues 49 and 99. The epitope clusters 2 and 3 appeared to be expressed only on the native molecule. Monoclonal antibodies of different clusters exhibited very different migration patterns on isoelectric focusing while monoclonal antibodies of the same cluster were indistinguishable, which suggests that they may have originated from the same B cell precursor. Taken together these data suggest that this panel of monoclonal antibodies detects at least three distinct epitopes of the nuclease molecule, one of which could be involved in the determination of the enzymatic site.     

Characterization of antigenic properties of horseradish peroxidase with monoclonal antibodies

Monoclonal antibodies (mcAbs) specific to alkaline isoenzymes of horseradish peroxidase were used to characterize the antigenic properties of horseradish peroxidase. The results of a competitive binding assay indicated that monoclonal antibodies can be divided into three groups directed against distinct parts of the protein. The interaction of monoclonal antibodies with native and modified horseradish peroxidase showed also three different patterns of reactivity. Antibodies from groups I and II are directed against epitopes which are conformational and formed by tertiary structure elements. Epitopes recognized by these antibodies are sensitive to heme removal or partial denaturation of peroxidase. Antibodies from group III bind specifically with epitopes consisting of primary or secondary structure elements. The antigenic determinants recognized by antibodies from group III PO ₁ and 36F ₉ were shown to be linear (continuous) and formed by amino acid residues 261-267 and 271-277, respectively, as determined by the peptide scanning method (PEPSCAN). The location of revealed linear antigenic determinants in the molecular structure of peroxidase is analyzed.     

The concept and operational definition of protein epitopes

The antigenic determinants or epitopes of a protein correspond to those parts of the molecule that are specifically recognized by the binding sites or paratopes of certain immunoglobulin molecules. Epitopes are thus relational entities that require complementary paratopes for their operational recognition. Some authors consider that the concept of epitope necessarily involves the two properties of antigenic reactivity (ability to bind to a paratope) and immunogenicity (ability to induce an immune response). Such a view creates difficulties because it makes the existence of epitopes in a protein depend on immunogenetic and regulatory mechanisms of the immunized host. The delineation of epitopes can be achieved by antigenic cross-reactivity studies or by X-ray crystallography. Both approaches require specific criteria for deciding which residues of the antigen are in contact with the paratope and are functionally part of the epitope. The relative contribution of static accessibility, segmental mobility and induced fit to immune recognition remains controversial. Each of the methods used for analysing antigenic specificity is subject to various operational constraints originating from the type of experimental probe and from the format sensitivity and specificity of the immunoassay used. If a protein is assumed to contain as many epitopes as the number of different monoclonal antibodies that can be raised against it, the delineation of epitopes corresponds to the summation in various hosts of the immune repertoire specific for the antigen. Neutralization epitopes are a special subclass of the epitopes of infectious agents and toxins that are specifically recognized by antibody molecules able to neutralize the biological activity of the antigen. The identification of neutralization epitopes is important for the development of synthetic vaccines because it is this type of epitope that should be mimicked by synthesis and used as a vaccine for eliciting protective immunity. The first demonstration that synthetic peptides could elicit antibodies that neutralized viral infectivity was made by Anderer and his colleagues in the 1960s in their work with tobacco mosaic virus. Nearly 20 years passed before it was shown that antibodies to synthetic peptides were also able to neutralize the infectivity of other viruses such as foot-and-mouth disease, polio and hepatitis B viruses.     

Antigenic determinants on chicken riboflavin carrier protein. A study with monoclonal antibodies

Monoclonal antibodies raised against chicken egg white riboflavin carrier protein were classified into seven categories each recognizing a distinct epitope. Of these, six were directed against conformation dependent epitopes and one to a sequential epitope. The roles of lysine residues and the post-translationally attached phosphate and oligosaccharide moieties in the antigenicity of riboflavin carrier protein recognized by the monoclonal antibodies were investigated. The binding region of three monoclonal antibodies could be located within the 87–219 amino acid sequence of the protein and one antibody among these recognized a sequence of 182–204 amino acid residues. All the monoclonal antibodies were able to recognize riboflavin carrier proteins present in the sera of pregnant rats, cows and humans indicating that the epitopes to which they are directed are conserved through evolution from chicken to the human.     

Epitopes ofEscherichia coli ribosomal protein S13

To analyze the immunochemical structure ofEscherichia coli ribosomal protein S13 and its organizationin situ, we have generated and characterized 22 S13-specific monoclonal antibodies. We used a competitive enzyme-linked immunosorbent assay to divide them into groups based on their ability to inhibit binding of one another. The discovery of five groups with distinct binding properties suggested that a minimum of five distinct determinants on S13 are recognized by our monoclonal antibodies. The locations of the epitopes detected by these monoclonal antibodies have been mapped on S13 peptides. Three monoclonal antibodies bind a S13 C-terminal 34-residue segment. All the other 19 monoclonal antibodies bind a S13N-terminal segment of about 80 residues. The binding sites of these 19 monoclonal antibodies have been further mapped to subfragments of peptides. Two monoclonal antibodies recognized S13_1–22; three monoclonal antibodies bound to S13_1–40; the binding sites of three other antibodies have been located in S13_23–80, with epitopes possibly associated with residues 40–80. The remaining 11 monoclonal antibodies did not bind to these subfragments. These data provide molecular basis to the structure of S13 epitopes, whosein situ accessibility may reveal the S13 organization on the ribosome.     

Discovery of Immunodominant B Cell Epitopes within Surface Pneumococcal Virulence Proteins in Pediatric Patients with Invasive Pneumococcal Disease

The identification of immunodominant B cell epitopes within surface pneumococcal virulence proteins in pediatric patients with invasive pneumococcal disease (IPD) is a valuable approach to define novel vaccine candidates. To this aim, we evaluated sera from children with IPD and age-matched controls against 141 20-mer synthetic peptides covering the entire sequence of major antigenic fragments within pneumococcal virulence proteins; namely, choline-binding protein D (CbpD), pneumococcal histidine triad proteins (PhtD and PhtE), pneumococcal surface protein A (PspA), plasminogen and fibronectin binding protein B (PfbB), and zinc metalloproteinase B (ZmpB). Ten immunodominant B cell epitopes were identified: CbpD-pep4 (amino acids (aa) 291–310), PhtD-pep11 (aa 88–107), PhtD-pep17 (aa 172–191), PhtD-pep19 (aa 200–219), PhtE-pep32 (aa 300–319), PhtE-pep40 (aa 79–98), PfbB-pep76 (aa 180–199), PfbB-pep79 (aa 222–241), PfbB-pep90 (aa 484–503), and ZmpB-pep125 (aa 431–450). All epitopes were highly conserved among different pneumococcal serotypes, and four of them were located within the functional zinc-binding domain of the histidine triad proteins PhtD and PhtE. Peptides CbpD-pep4, PhtD-pep19, and PhtE-pep40 were broadly recognized by IPD patient sera with prevalences of 96.4%, 92.9%, and 71.4%, respectively, whereas control sera exhibited only minor reactivities (<10.7%). Their specificities for IPD were 93.3%, 95%, and 96.7%; their sensitivities were 96.4%, 92.9%, and 71.4% and their positivity likelihood ratios for IPD were 14.5, 18.6, and 21.4, respectively. Furthermore, purified antibodies against CbpD-pep4, PhtD-pep19, and PhtE-pep40 readily bound on the surfaces of different pneumococcal serotypes, as assessed by FACS and immunofluorescence analysis. The identified immunodominant B cell epitopes provide a better understanding of immune response in IPD and are worth evaluation in additional studies as potential vaccine candidates.     

UreB蛋白B细胞抗原表位快速筛选与鉴定

以幽门螺旋杆菌(Helicobacterpylori,Hp)主要抗原蛋白尿素酶B(ureaseB,UreB)为靶蛋白,建立一种新的B细胞抗原表位筛选与鉴定方法.运用Fmoc固相肽合成法合成11条HpUreB蛋白的单表位抗原肽片段,在其氨基端标记FITC荧光素,应用荧光偏振方法(fluorescencepolarization,FP)快速鉴定这些肽片段的抗原性,并通过FP法在大规模样品中快速筛选相应抗体滴度高、分布人群广的优势抗原表位肽.结果表明,合成的11条UreB蛋白线性抗原肽中,10条具有较强的抗原性,其中No.2、No.5和No.11抗原肽相应的特异性抗体在感染Hp的人群中分布较广,抗体滴度较高,为UreB的优势抗原表位肽.对抗原表位进行多参数综合分析与设计,通过FP技术快速鉴定抗原肽,并筛选优势抗原表位肽,对于疾病的抗原表位谱研究具有重要的意义,同时在疾病的诊断、分型及治疗中具有重要的应用前景.     

Monoclonal antibodies to the collagen binding domain of human plasma fibronectin

Five independent hybrids producing monoclonal antibodies to human plasma fibronectin have been obtained by fusing P3/X63-Ag8 myeloma cells with immune mouse splenocytes. The specificity of these monoclonal antibodies (MABs) for fibronectin was demonstrated by three independent tests: binding to the purified soluble molecule, immunofluorescence staining of insoluble extracellular matrices produced by endothelial cells in vitro, immunostaining of fibronectin tryptic peptides after separation on SDS-PAGE and transfer to nitrocellulose sheets. Two antibodies (MAB 29 and 52) recognized selectively human fibronectin while the others (MAB 5, 30 and 59) reacted also with plasma fibronectin from calf, hamster and chicken. Four distinct epitopes were recognized by the MABs studied. MAB 5, 30, 52 and 59 reacted with distinct antigenic sites, while MAB 29 and 52 bind to the same site. Antigenic fragments were identified by immunostaining of fibronectin tryptic peptides. MAB 5 reacted with a collagen binding fragment with a molecular weight of 120 K. In addition, each of the MAB 29, 30, 52 and 59 reacted with peptides with a molecular weight of 40 K that bind to gelatin. Since these antibodies do not inhibit fibronectin-collagen interaction, it is concluded that their corresponding epitopes are clustered in a region close, but not coincident, to the collagen binding site of fibronectin.     

Localization of epitopes of herpes simplex virus type 1 glycoprotein D.

We previously defined eight groups of monoclonal antibodies which react with distinct epitopes of herpes simplex virus glycoprotein D (gD). One of these, group VII antibody, was shown to react with a type-common continuous epitope within residues 11 to 19 of the mature glycoprotein (residues 36 to 44 of the predicted sequence of gD). In the current investigation, we have localized the sites of binding of two additional antibody groups which recognize continuous epitopes of gD. The use of truncated forms of gD as well as computer predictions of secondary structure and hydrophilicity were instrumental in locating these epitopes and choosing synthetic peptides to mimic their reactivity. Group II antibodies, which are type common, react with an epitope within residues 268 to 287 of the mature glycoprotein (residues 293 to 312 of the predicted sequence). Group V antibodies, which are gD-1 specific, react with an epitope within residues 340 to 356 of the mature protein (residues 365 to 381 of the predicted sequence). Four additional groups of monoclonal antibodies appear to react with discontinuous epitopes of gD-1, since the reactivity of these antibodies was lost when the glycoprotein was denatured by reduction and alkylation. Truncated forms of gD were used to localize these four epitopes to the first 260 amino acids of the mature protein. Competition experiments were used to assess the relative positions of binding of various pairs of monoclonal antibodies. In several cases, when one antibody was bound, there was no interference with the binding of an antibody from another group, indicating that the epitopes were distinct. However, in other cases, there was competition, indicating that these epitopes might share some common amino acids.     

Continuous B-epitope maps of cytochrome P450cam (CYP101) obtained by peptide scanning: correlation to spatial structure

Protein continuous B-epitopes can be revealed using short synthetic peptides that overlap a known protein sequence. Since the whole protein surface is considered to possess antigenic properties, a question that arises is whether a set of linear B-epitopes determined by peptide scanning correlates with a protein spatial structure. We have chosen cytochrome P450cam (CYP101) of Pseudomonas putida, with known 3D structure, as a template. Sera of two rabbits and antibody egg yolk preparations from three chickens were produced against the P450cam molecule. These polyclonals were analyzed separately in ELISA with 409 overlapping P450cam hexapeptides. The whole set of continuous antigenic sites of P450cam covered about 45% of the P450cam sequence. However, immunodominant sites (those revealed with more than 50% antibody preparations), the so-called "antigenic core," represent only 9% of the protein sequence. While the amount of water-accessible residues in the total antigenic map (42%) was close to that in the whole native P450cam molecule (39%), the amount of water-accessible residues in the antigenic core was significantly higher (64%). These results led to the conclusion that antigenic core epitopes can be associated to the molecular surface, whereas epitopes with low detection frequency may partly correspond to unfolded regions of the protein molecule.     

A simple method of determination of antigenic determinants in proteins with known primary structure

A simple and rapid method is proposed for the localization of antigenic determinants in proteins of known primary structure exemplified by human myoglobin. The polypeptide chain of myoglobin was cleaved with BrCN (at Met residues) or with bromosuccinimide (at Trp and Tyr residues) under conditions which on average gave less than one scission per myoglobin molecule. The "single-hit" cleavage products were separated by gel electrophoresis and transferred to nitrocellulose by electroblotting. The peptides containing intact antigenic determinants were vizualized by immuno-peroxidase staining with four monoclonal anti-myoglobin antibodies. Comparison of the lengths of the immuno-reactive peptides with the known positions of methionine, tryptophan and tyrosine residues suggested that the four monoclonal antibodies were bound by myoglobin over the region Trp-14 to Met-55. As compared with other methods of localization, the method proposed is much faster and takes much lesser amount of protein.