Prediction of epitope region and preparation of mouse polyclonal antibody of human Shisa-like protein 1(SHISAL1) / 细胞与分子免疫学杂志

Objective To investigate antigen optimization of Shisa like protein 1 (SHISAL1) for preparing mouse anti-human SHISAL1 polyclonal antibody and to identify the specificity of the prepared antibody. Methods Bioinformatics was employed to predict the antigenic epitope region of SHISAL1 protein, and then a polypeptide composed of amino acid residues from the site of 28 to 97 of SHISAL1, termed SHISAL1-N, was selected as the antigen. The coding region of SHISAL1-N was cloned by molecular cloning technique, and then it was inserted into pET-28a to generate pET28a-SHISAL1-N recombinant plasmid. The two recombinant plasmids pET28a-SHISAL1-N and pET28a-SHISAL1 were transformed into BL21 (DE3) bacteria and induced to express by IPTG. The two proteins were purified and immunized to female Kunming mice, respectively. The specificities and sensitivities of the acquired antibodies were detected by Western blot analysis, immunoprecipitation and immunofluorescent cytochemical staining. Results pET28a-SHISAL1-N recombinant plasmid was successfully constructed, and the two fused proteins, SHISAL1 and SHISAL1-N, were induced to express. Moreover, two types of SHISAL1 mouse polyclonal antibodies, derived from SHISAL1-N and SHISAL1 antigens, were obtained. Western blot results showed that the antibody prepared from SHISAL1 antigen was less specific and sensitive compared with the antibody prepared from SHISAL1-N antigen which could specifically identify different endogenous SHISAL1 protein. Immunoprecipitation results showed that SHISAL1-N antibody could specifically pull down SHIISAL1 protein in hepatocellular carcinoma cells and immunofluorescence results demonstrated that SHISAL1-N antibody could specifically bind to SHISAL1 protein in the cytoplasm. Conclusion We have optimized the SHISAL1 antigen and prepared the mouse anti-human SHISAL1 polyclonal antibodies successfully, which can be used for Western blot analysis, immunoprecipitation and immunofluorescence cytochemical staining.

Eukaryotic expression and antigen epitope prediction of the LRRC15 protein in excretory secretory antigens of Taenia solium cysticercus / 中国血吸虫病防治杂志

OBJECTIVE@#To conduct eukaryotic expression of the leucine-rich repeat containing 15 (LRRC15), a differentially expressed protein in excretory secretory antigens of Taenia solium cysticercus, and predict its antigen epitope.@*METHODS@#The molecular weight, stability, amino acid sequence composition, isoelectric point and T lymphocyte epitope of the LRRC15 protein were predicted using the bioinformatics online softwares ExPASy-PortParam and Protean. The full-length splicing primers were designed using PCR-based accurate synthesis, and the LRRC15 gene was synthesized. The recombinant pcDNA3.4-LRRC15 plasmid was constructed and transfected into HEK293 cells to express the LRRC15 protein. In addition, the LRRC15 protein was characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.@*RESULTS@#The recombinant pcDNA3.4-LRRC15 plasmid was successfully constructed, which expressed the target LRRC15 protein with an approximately molecular weight of 70 kDa. Bioinformatics prediction with the ExPASy-PortParam software showed that LRRC15 was a hydrophilic protein, which was consisted of 644 amino acids and had a molecular weight of 69.89 kDa and an isoelectric point of 5.6. The molecular formula of the LRRC15 protein was C3073H4942N846O953S28 and had an instability coefficient is 50.3, indicating that LRRC15 was an instable protein. Bioinformatics prediction with the Protean software showed that the dominant T-cell antigen epitopes were located in 292 to 295, 353 to 361, 521 to 526 and 555 to 564 amino acids of the LRRC15 protein, and the T-cell antigen epitopes with a high hydrophilicity, good flexibility, high surface accessibility and high antigenicity index were found in 122 to 131, 216 to 233, 249 to 254, 333 to 343, 358 to 361, 368 to 372, 384 to 386, 407 to 412, 445 to 450, 469 to 481, 553 to 564, 588 to 594, 607 to 617 and 624 to 639 amino acids. Following transfection of the recombinant pcDNA3.4-LRRC15 plasmid into HEK293 cells, SDS-PAGE and Western blotting identified LRRC15 proteins in cell secretory culture media, cell lysis supernatants and sediments. The LRRC15-His fusion protein was purified from the cell culture medium, and SDS-PAGE identified a remarkable band at approximately 70 kDa, while Western blotting successfully recognized the band of the recombinant LRRC15 protein.@*CONCLUSIONS@#The eukaryotic expression and antigen epitope prediction of the LRRC15 protein in the excretory secretory antigens of T. solium cysticercus have been successfully performed, which provides insights into further understandings of its biological functions.

Identifying protein epitopes recognized by monoclonal antibodies / 生物工程学报

To establish a method for identifying protein epitopes recognized by therapeutic monoclonal antibodies, the programmed death receptor-1 (PD-1) was selected as the target protein. Based on the alanine scanning strategy, a rapid expression method of antigen mutants combining site-directed mutagenesis with mammalian cell expression system was established, the conditions for eukaryotic expression element amplification and cell transfection expression were established. 150 PD-1 protein mutants were co-expressed, and the binding ability of these mutants to anti-PD-1 antibody Pembrolizumab was identified. The epitopes of Pembrolizumab were determined based on the binding ability of protein mutants to antibodies and combined with protein structure analysis, which was highly consistent with the reported crystal structure-based epitopes, indicating that this method is simple and accurate and can be used for epitope mapping of therapeutic monoclonal antibodies.

Mapping of the continuous epitopes displayed on the Clostridium perfringens type D epsilon-toxin

Abstract The epsilon toxin, produced by Clostridium perfringens, is responsible for enterotoxemia in ruminants and is a potential bioterrorism agent. In the present study, 15 regions of the toxin were recognized by antibodies present in the serum, with different immunodominance scales, and may be antigen determinants that can be used to formulate subunit vaccines.

Sequence similarity between thyroid self-protein and hepatitis c virus polyprotein: possible triggering mechanism of autoimmune thyroiditis / Similaridade de sequências entre auto-proteínas da tireoide e poliproteínas do vírus da hepatite C: possível mecanismo de desencadeamento da tireoidite auto-imune

ABSTRACT Background - Exposure to viral antigens that share amino acid sequence similar with self- antigens might trigger autoimmune diseases in genetically predisposed individuals, and the molecular mimicry theory suggests that epitope mimicry between the virus and human proteins can activate autoimmune disease. Objective - The purpose of this study is to explore the possible sequence similarity between the amino acid sequences of thyroid self-protein and hepatitis C virus proteins, using databanks of proteins and immunogenic peptides, to explain autoimmune thyroid disease. Methods - Were performed the comparisons between the amino acid sequence of the hepatitis C virus polyprotein and thyroid self-protein human, available in the database of National Center for Biotechnology Information on Basic Local Alignment Search Tool. Results - The sequence similarity was related each hepatitis C virus genotype to each thyroid antigen. The similarities between the thyroid and the viral peptides ranged from 21.0 % (31 identical residues out of 147 amino acid in the sequence) to 71.0% (5 identical residues out of 7 amino acid in the sequence). Conclusion - Bioinformatics data, suggest a possible pathogenic link between hepatitis C virus and autoimmune thyroid disease. Through of molecular mimicry is observed that sequences similarities between viral polyproteins and self-proteins thyroid could be a mechanism of induction of crossover immune response to self-antigens, with a breakdown of self-tolerance, resulting in autoimmune thyroid disease.

RESUMO Contexto - A exposição a antígenos virais que compartilham sequência de aminoácidos semelhantes a auto-antígenos pode provocar doenças auto-imunes em indivíduos predispostos geneticamente, e a teoria do mimetismo molecular sugere que o mimetismo entre epítopos de vírus e proteínas humanas pode ativar doenças auto-imunes. Objetivo - O objetivo deste estudo foi explorar a possível semelhança entre as sequências de aminoácidos de auto-proteinas da tireóide e proteínas do vírus da hepatite C, utilizando bancos de dados de proteínas e peptídeos imunogênicos, para explicar a doença auto-imune da tireóide. Métodos - Foram realizadas comparações entre as sequências de aminoácidos de poliproteínas do vírus da hepatite C e auto-proteinas da tireóide humana, disponível na base de dados do National Center for Biotechnology Information no Basic Local Alignment Search Tool. Resultados - A semelhança de sequências foi relacionada para cada genótipo de vírus da hepatite C e proteínas da tireóide. As semelhanças entre proteínas da tireóide e os peptídeos virais variaram de 21,0% (31 resíduos idênticos da sequência de 147 aminoácidos) a 71,0% (cinco resíduos idênticos da sequência de 7 aminoácidos). Conclusão - Dados de bioinformática sugerem uma possível ligação entre vírus da hepatite C e doença auto-imune da tireóide. Através de mimetismo molecular observa-se que as semelhanças entre as sequências de poliproteínas virais e auto-proteínas da tireóide pode ser um mecanismo de indução de resposta imune resultando em doença auto-imune da tireóide.

Computational prediction of MHC class I epitopes for most common viral diseases in cattle (Bos taurus).

Viral diseases like foot-and-mouth disease (FMD), calf scour (CS), bovine viral diarrhea (BVD), infectious bovine rhinotracheitis (IBR) etc. affect the growth and milk production of cattle (Bos taurus) causing severe economic loss. Epitope-based vaccine designing have been evolved to provide a new strategy for therapeutic application of pathogen-specific immunity in animals. Therefore, identification of major histocompatibility complex (MHC) binding peptides as potential T-cell epitopes is widely applied in peptide vaccine designing and immunotherapy. In this study, MetaMHCI tool was used with seven different algorithms to predict the potential T-cell epitopes for FMD, BVD, IBR and CS in cattle. A total of 54 protein sequences were filtered out from a total set of 6351 sequences of the pathogens causing the said diseases using bioinformatics approaches. These selected protein sequences were used as the key inputs for MetaMHCI tool to predict the epitopes for the BoLA-A11 MHC class I allele of B. taurus. Further, the epitopes were ranked based on a proposed principal component analysis based epitope score (PbES). The best epitope for each disease based on its predictability through maximum number of predictors and low PbES was modeled in PEP-FOLD server and docked with the BoLA-A11 protein for understanding the MHC-epitope interaction. Finally, a total of 78 epitopes were predicted, out of which 27 were for FMD, 25 for BVD, 12 for CS and 14 for IBR. These epitopes could be artificially synthesized and recommended to vaccinate the cattle for the considered diseases. Besides, the methodology adapted here could also be used to predict and analyze the epitopes for other microbial diseases of important animal species.

Enhancing mucosal immunity in mice by recombinant adenovirus expressing major epitopes of porcine circovirus-2 capsid protein delivered with cytosine-phosphate-guanosine oligodeoxynucleotides

A recombinant replication-defective adenovirus expressing the major epitopes of porcine circovirus-2 (PCV-2) capsid protein (rAd/Cap/518) was previously constructed and shown to induce mucosal immunity in mice following intranasal delivery. In the present study, immune responses induced by intranasal immunization with a combination of rAd/Cap/518 and cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN) were evaluated in mice. The levels of PCV-2-specific IgG in serum and IgA in saliva, lung, and intestinal fluids were significantly higher in the group immunized with rAd/Cap/518 and CpG ODN than animals immunized with rAd/Cap/518 alone. The frequencies of IL-2-secreting CD4+ T cells and IFN-gamma-producing CD8+ T cells were significantly higher in the combined immunization group than mice immunized with rAd/Cap/518 alone. The frequencies of CD3+, CD3+CD4+CD8-, and CD3+CD4-CD8+ T cells in the combined immunization group were similar to that treated with CpG ODN alone, but significantly higher than mice that did not receive CpG ODN. PCV-2 load after challenge in the combined immunization group was significantly lower than that in the phosphate-buffered saline placebo group and approximately 7-fold lower in the group treated with CpG ODN alone. These results indicate that rAd/Cap/518 combined with CpG ODN can enhance systemic and local mucosal immunity in mice, and represent a promising synergetic mucosal vaccine against PCV-2.

Conversion of a murine monoclonal antibody A13 targeting epidermal growth factor receptor to a human monoclonal antibody by guided selection

Epidermal growth factor receptor (EGFR) is an attractive target for tumor therapy because it is overexpressed in the majority of solid tumors and the increase in receptor expression levels has been linked with a poor clinical prognosis. Also it is well established that blocking the interaction of EGFR and the growth factors could lead to the arrest of tumor growth and possibly result in tumor cell death. A13 is a murine monoclonal antibody (mAb) that specifically binds to various sets of EGFR-expressing tumor cells and inhibits EGF-induced EGFR phosphorylation. We isolated human immunoglobulin genes by guided selection based on the mAb A13. Four different human single chain Fvs (scFvs) were isolated from from hybrid scFv libraries containing a human VH repertoire with the VL of mAb A13 and a human VL repertoire with the VH of mAb A13. All the 4 scFvs bound to EGFR-expressing A431 cells. One scFv (SC414) with the highest affinity was converted to IgG1 (ER414). The ER414 exhibited ~17 fold lower affinity compared to the A13 mAb. In addition the ER414 inhibited an EGF-induced tyrosine phosphorylation of EGFR with much lower efficacy compared to the A13 mAb and Cetuximab (Merck KgaA, Germany). We identified that the epitope of A13 mAb is retained in ER414. This approach will provide an efficient way of converting a murine mAb to a human mAb.

A majority of Brazilian patients with rheumatoid arthritis HLA-DRB1 alleles carry both the HLA-DRB1 shared epitope and anti-citrunillated peptide antibodies

The objective of the present study was to evaluate the contribution of the shared epitope (SE), the rheumatoid arthritis (RA) protection model, and the occurrence of anti-cyclic citrullinated peptide (anti-CCP) antibodies in RA patients from a genetically diverse population. One hundred and forty Brazilian RA patients and 161 matched controls were typed for HLA-DRB1 alleles using amplified DNA hybridized with sequence-specific oligonucleotide probes or primers. Patients were stratified according to the presence or absence of SE (DRB1*0401, *0404, *0405, *0101, *1001, and *1402), of the DERAA alleles (DRB1*0103, *0402, *1102, *1103, *1301, *1302, and *1304), and X (all other alleles). Anti-CCP antibodies were measured by ELISA. The combined frequency of SE-positive alleles was significantly greater (76.4 vs 23.6 percent, P < 0.0001) than the controls. The SE/SE and SE/X genotypes were over-represented (P < 0.0001, OR = 6.02) and DERAA/X was under-represented in RA patients (P < 0.001, OR = 0.49), whereas the frequencies of the SE/DERAA, X/X and X/DERAA genotypes were not significantly different from controls. The frequency of anti-CCP antibodies was higher in SE-positive patients than in SE-negative patients (64.6 vs 44.7 percent, P = 0.03; OR = 2.25). Although the Brazilian population is highly miscegenated, the results of this study support the findings observed in most genetically homogeneous populations with RA; however, they are not mutually exclusive but rather complementary. The participation of DRB1-DERAA alleles in protection against RA was also observed (OR = 0.4; 95 percentCI = 0.23-0.68).

Immune responses of selected phagotopes from monoclonal antibodies of Burkholderia pseudomallei.

Random peptide libraries displayed by bacteriophage T7 and M13 were employed to identify mimotopes from 4 monoclonal antibodies (MAbs) specific to Burkholderia pseudomallei. Insert DNA sequences of bound phages selected from four rounds of panning with each MAb revealed peptide sequences corresponding to B. pseudomallei K96243 hypothetical protein BPSL2046, hypothetical protein BpseP_02000035, B. pseudomallei K96243 hypothetical protein BPSS0784, B. pseudomallei 1710b hypothetical protein BURPS1710b_1104, and B. cenocepacia H12424 TonB-dependent siderophore receptor, all located at the outer membrane. The immune responses from all selected phagotopes were significantly higher than that of lipopolysaccharide. The study demonstrates the feasibility of identifying mimotopes through screening of phage-displayed random peptide libraries with B. pseudomallei MAbs.

Use of flow cytometry to develop and characterize a set of monoclonal antibodies specific for rabbit leukocyte differentiation molecules

Flow cytometry was used to identify and characterize monoclonal antibodies (mAbs) that react with rabbit leukocyte differentiation molecules (LDM). Screening sets of mAbs, developed against LDM in other species, for reactivity with rabbit LDM yielded 11 mAbs that recognize conserved epitopes on rabbit LDM orthologues and multiple mAbs that recognize epitopes expressed on the major histocompatibility class I or class II molecules. Screening of mAbs submitted to the Animal Homologues Section of the Eighth Human Leukocyte Differentiation Workshop yielded 7 additional mAbs. Screening of mAbs generated from mice immunized with leukocytes from rabbit thymus or spleen or concanavalin A activated peripheral blood and/or spleen lymphocytes has yielded 42 mAbs that recognize species restricted epitopes expressed on one or more lineages of leukocytes. Screening of the anti-rabbit mAbs against leukocytes from other species yielded one additional mAb. The studies show that screening of existing sets of mAbs for reactivity with rabbit LDM will not be productive and that a direct approach will be needed to develop mAbs for research in rabbits. The flow cytometric approach we developed to screen for mAbs of interest offers a way for individual laboratories to identify and characterize mAbs to LDM in rabbits and other species. A web-based program we developed provides a source of information that will facilitate analysis. It contains a searchable data base on known CD molecules and a data base on mAbs, known to react with LDM in one or more species of artiodactyla, equidae, carnivora, and or lagomorpha.

Identification and characterization of previously described epitopes in HIV-1 subtypes B, C, F and BF in Brazil

Genetic analysis of HIV-1 is essential to improve treatment strategies and select epitopes for vaccine programs. The objective of this study was to determine whether known CD4+ and CD8+ epitopes were present in Brazilian HIV-1 strains. We used previously described CD8+ and CD4+ epitopes from the Los Alamos laboratory to search for these epitopes in the Brazilian sequences using the HIVbase program and we compared the frequency results with the analyses using physical-chemical profile tools from Network Protein Sequence Analysis (NPSA), and the SYFPEITHI program. Furthermore, this analysis was carried out with the Prosite tool using the GeneDoc program and ds/dn analyses using the Synonymous Nonsynonymous Analysis Program (SNAP). The HIVbase epitope mapping demonstrated that 30 CD8+ and 6 CD4+ epitopes were present in the Brazilian sequences at a high frequency. Only two of these epitopes were heavily glycosylated. Interestingly, ds/dn analyses showed evidence of purifying selective pressure. These types of analyses could be useful for the assessment of possible vaccine efficiency in populations.

Characterization of monoclonal antibodies recognizing alpha and beta subunits of human chorionic gonadotropin hormone.

Human chorionic gonadotropin (hCG) hormone is required for maintenance of early pregnancy and is a potential marker in the diagnosis and prognosis of both pregnancy and trophoblastic diseases. Murine hybridomas were generated against purified hCG. Seven hybrid clones secreting antibodies against hCG molecule with IgG1/kappa subclass were established. The indirect ELISA result demonstrated that six MAbs (BEL-1 to BEL-6) recognized hCG in both holo and free beta subunit form with negligible cross-reactivity against a closely related hormone, human luteinizing hormone (hLH). In this fusion, only one MAb (ALC-1) showed a cross-reaction with hLH, which designated an alpha subunit specific. The outcome of Western blot ascertained that ALC-1 recognized the conformational epitope on alpha subunit of hCG at Mr 23 kDa band in nonreducing condition (NR). In contrast, epitopes belonging to all MAbs recognized beta subunit of hCG were in linear peptide structure at Mr 34 kDa band (NR) and Mr 26 kDa band (R). These six MAbs were further characterized by using two beta subunit carboxy-terminal synthetic peptides (beta109-119 and beta109-145). Three of them (BEL-1, BEL-3, and BEL-4) recognized only epitope harboring in beta109-145 fragment, the others recognized both types of the synthetic peptide. In order to select the most suitable MAbs specific to beta subunit of hCG for exploiting with ALC-1 in the sandwich-type immunometric assay, competitive ELISA was employed. Six individual MAbs specific to beta subunit of hCG were used to compete with biotinylated ALC-1 to evaluate the proximity of their epitopes on the holo form of hCG molecule. Of all six MAbs, BEL-5 depicted the lowest percent inhibition result, which indicated the bottom-most steric hindrance effect. Consequently, MAb BEL-5 will be the most appropriate antibody to utilize in concert with ALC-1 in place of devising a sandwich-type immunometric assay for measuring holo-hCG level.

Characterization of a phase 1-d epitope on Salmonella typhi flagellin and its role in the serodiagnosis of typhoid fever.

A monoclonal antibody (MAb) directed against Salmonella typhi 52 kDa flagellin protein has been previously produced by our group. In this study, we have demonstrated that the epitope specific to the MAb is unique to phase 1-d. To map the epitope, plasmids encoding different regions of S. typhi flagellin gene were constructed. Analysis of protein produced from each recombinant plasmid indicated that the epitope specific to the MAb resided within amino acids 171-303 (region IV) of S. typhi flagellin protein. The recombinant region IV flagellin was used to develop an ELISA for the detection of IgM antibody to S. typhi in serum. In the hemoculture-positive typhoid group, the developed ELISA was positive in 77 of 92 cases. In patients with non-typhoidal Salmonella, gram-positive and gram-negative bacteria or dengue virus, the ELISA was negative in all 78 cases. Two from 116 healthy control subjects had positive reactions with the assay. The calculated sensitivity, specificity, positive and negative predictive values of the test were 83.7%, 99.0%, 97.5% and 92.8%, respectively. With such high validity together with the requirement of only a single serum specimen and one day for performing the test, the developed ELISA should become a valuable diagnostic test for typhoid fever.

Cell cycle control protein p34cdc2 homologue and its phase specific accumulation in synchronized onion (Allium cepa L.) root meristem cells.

A key regulator of the cell cycle is a highly conserved protein kinase whose catalytic subunit, p34cdc2, is encoded by cdc2 gene. Immunoblotting with a polyclonal antibody raised against PSTAIRE sequence (found in the N-terminal region of all cdc2 and cdc2 related proteins throughout the phylogenetic scale including higher plants), was used to study the presence of p34cdc2 in onion scale leaves and root tip cells. p34cdc2 homologues are beyond the detection level in scale leaves. PSTAIRE antibody was used to estimate p34cdc2 kinase protein levels during cell cycle in highly synchronous population of Allium cepa L. root meristem cells. p34cdc2 kinase protein showed gradual increase in their levels from S phase to G2 phase boundary. Immunoprecipitation followed by in vitro histone H1 kinase assays also depicted that its kinase activity increased parallel to the increase in p34cdc2 level.

Independent divergences in the CD4 binding site and V3 loop encoded in two seroprevalent Ugandan HIV-1 clinical isolates

"Two major epitopes expressed in HIV-1 have been recently shown to play a central role in virus neutralization. One of these important specificities is a type-specific or group-specific; principal neutralizing determinant (PND) located in the V3 loop of gp120. The other is a more broadly neutralizing determinant associated with the CD4 binding site. Structural and serological studies of the variation in these epitopes have become important in vaccine research. This report describes the analysis of the DNA clones encoding a region of gp120 that overlaps the V3 loop and the putative CD4 recognition site in two new African isolates; UG06c and UG23c. Phylogenetic analyses of the DNA sequences showed that the new African isolates clustered with two very distinct subtypes of HIV-1. UG06c was grouped with U455; D687; and Z321; previously classified as ""HIV-1 subtype A"" in the AIDS and human retroviruses database; and UG23c was grouped with MAL; JY1; NDK; ELI; and Z2Z6 classified as ""HIV-1 subtype D."" Considerable variation was apparent in the V3 loop. The divergence included the presence of the hexapeptides GP-GRSF and GLGQAL at the cap of the loop in UG06c and UG23c; respectively. The GPGR tetrapeptide in UG06c formed a beta-turn configuration similar to that of MN or IIIB. The beta-turn was not found to be a likely conformation for GLGQ. The amino acids previously implicated in CD4 binding and the associated neutralizing activity were relatively conserved. To assess a possible impact of the sequence and conformational variations on serological reactivity; UG06c and UG23c were subjected to neutralization assay.(ABSTRACT TRUNCATED AT 250 WORDS)"

Characterization of a Plasmodium falciparum epitope recognized by a monoclonal antibody with broad isolate and species specificity.

Monoclonal antibody (MAb) 7H8 raised against Plasmodium yoelii reacted with a series of proteins from P. falciparum that range in molecular weight from 46 to 194 kDa. By immunofluorescence assay, this MAb reacted with all isolates of P. falciparum tested. MAb 7H8 was used to screen a genomic expression library of asexual blood stage antigens of P. falciparum, Malayan Camp K+ and 7 independent clones were identified. These 7 clones were sequenced and the epitope recognized by MAb 7H8 in the recombinant protein of one of these clones was mapped. This epitope contained Lys Tyr Pro as core amino acids. However, similar sequences were not found in the other clones, indicating that this MAb binds to a structural epitope formed by different amino acids. The variable composition of the epitope may account for the number of P. falciparum malarial proteins recognized by MAb 7H8.