Preparation Of Monoclonal Antibody Against Human B-cell Lymphoma

Objective To prepare monoclonal antibody(McAb) against human B-cell lymphoma or construct anti-human B-cell lymphoma phage display from mouse immuned with Raji cell, which helps establish a base for lymphoma radioimmunotherapy. Methods (1) McAbs were screened by indirect ELISA and tissue specificity of McAbs was identified by flow cytometry and immunochemical SP staining.(2)The heavy-chain and light-chain variable region gene(VH and VL) repertoire of immunoglobulin were amplified from the spleen cell mRNA by RT-PCR and joined by a DNA linker encoding peptide as a single-chain Fv (ScFv) fragment with overlap extension. These fragments were cloned into the phagemid pHEN-1 and the phage display library was constructed. Results (1)A murine McAb SZ-138 was obtained and its titer in ascites was 3.2?0"4.McAb was typed by reaction with subclass-specific rabbit anti-mouse antibodies and were found to be IgGl.Flow cytometry showed that McAb SZ138 was positive with Raji cell and Daudi cell,while did not react with red blood cell,white blood cell , inactive platelets, ECV304 and K562 cell.Immunohistochemical SP staining confirmed that the antigen identified by McAb SZ-138 was expressed on the gastroenteric glands and liver,but very little or not on heart,brain, tonsil.spleen etc. Western blotting demonstrated that McAb reacted with the antigen which molecular weight was about 170kd under reducing condition.(2)An anti-human B-cell lymphoma phage display was successfully constructed. Conclusions The McAb which has a high immuno-activity and tissue specificity gained by hybridoma technique and phage libray for repertoire single chain antibody of anti-human B-cell lymphoma may be useful for diagnosis and treatment of malignant lymphoma.