Preparation Of Monoclonal Antibodies (McAb) Against Cysticercus Cellulosae And Identification Of Linear Epitode Recognized By The McAb

Cysticercosis is a major zoonosis caused by the metacestode of the parasitic worm Taenia solium. Which is endemic in Latin America, India, Asia, and Africa and is highly detrimental to human health and pig-breeding. Diagnosis plays an important role in preventing and curing cysticercosis. Methods of immunological diagnosis, especially ELISA and EITB(or Western-blot), are currently the hot spot researched at home and abroad.Which can be devided into antibody-detecting and antigen-detecting methods according to objects detected. Screening of antigens specific to cysticercus cellulosae ,especially specific antigens which can be expressed or synthesized in vitro, and establishment of hybridoma cell strains which secrect monoclonal antibodies specific to cysticercus cellulosae are bases on which methods of immunological diagnosis are established.In this study, technique of monoclonal antibody production by cell hybridization was used to establish hybridoma cell strains which can secrect monoclonal antibodies specific to cysticercus cellulosae.Techniques of solid-phase peptide synthesis and epitope screening by overlapping peptides were used to find the epitope specific to cysticercus cellulosae.All the study will provide bases on which rapid immunodiagnostic approaches can be established.Cystic fluid antigens are of the most specific in the structure antigens of Cysticercus cellulosae.Firstly,the components of cystic fluid antigens were characterized in this study.The result shows there are about thirteen protein components in the cystic fluid ,in which three main proteins show strong immuno-reactivity with serum from human cysticercosis.Relative molecular mass of the three proteins are 97-,18-and 10-KD by SDS-PAGE.For further study,proteins were isolated by SDS-PAGE individually.And then the isolated proteins 18-and 10-KD were immunized to two group of BALB/c mice respectively.Spleen was removed aseptically to isolated spleen cells for fusing with myeloma cells after super-immunization.Spleen cells were fused with Murine myeloma NSO cells for hybridoma. Hybridoma were screened by ELISA and single cell cloning by limited dilution method to produce monoclonal antibody specific to cysticercus cellulosae. Two strains of hybridoma cell ,TSCF-18₁1H12B8 and TSCF-10₁2G5B5,which secret antibodies specific to cysticercus cellulosae were gained eventually.Monoclonal antibodies TSCF-18₁1H12B8 and TSCF-10₁2G5B5 showed no cross-reactivity with any antigens from Trichinella spiralis Cysticercus tenuicollis Sarcocystis miescheriana and Ascaris suum by ELISA.Western-blot with TSCF showed monoclonal antibody TSCF-18₁1H12B8 recognize 18KD and 10KD protein bands and only 10KD protein was recognized by monoclonal antibody TSCF-10₁2G5B5. In order to identify antigenic epitope recognized by McAb TSCF-181H12B8,a series of overlapping peptides,based on the published sequence for Taenia solium 18-kD varl cystic fluid antigen, were synthesised.A 9-aa sequence epitope, TS443: QIAQLAKNW,was identified by epitope mapping with overlapping peptides. Blast in NCBI protein database with the sequence TsM18-KDa varl protein shows similar epitope is shared with TsM14-,18-and 21-KDa glycoprotein antigens. TsM14-,18-and 21-KDa glycoproteins are classified as the family of TsM 8-KDa diagnostic proteins by some researchers. Nine further peptides were synthesised based on this epitope replacing each residue sequentially by Ala or Glu. Four critical amino acids(I52, A56, K57 and W59),which play important roles in the binding of the McAb,were identified in the epitope with Dot-ELISA.The epitope was evaluated in the serodiagnosis of cysticercosis.The result showed the epitope was not sensitive to serologically diagnose human cysticercosis.Tt's value in serodiagnosis of porcine cysticercosis was not determined for nonspecific factors in the assay. In addition,a sandwich ELISA,based on the McAb TSCF-10₁2G5B5,was established to diagnose porcine cysticercosis.which showed high specifity in serological cross-reactivity with other porcine parasites.A maximal dilution of 1:12800 was determined in the detecting of cystic fluid by the sandwish ELISA.Sensitivity varyed by muscle sample from different tissues of cysticercosis-infection pig in the sandwish ELISA.The maximal dilution was 1:800 for tongue muscle,followed by cardiac muscle and diaphragm muscle,a maximal dilution of 1:100 was determined for griskin. Detection for normal control muscle showed negtive at different dilutions.Application in diagnosing serum circulating antigens of cysticercosis for the sandwish ELISA shows a low sensitivity for porcine serodiagnosis,the maximal dilution of porcine serum was 1:10,and a much dissatisfing result for human serodiagnosis.