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Aim: To generate and screen the specific Fab phage antibody library against human Daudi cell strain in B-lymphoma and identify the positive clones., Methods: BALB/c mice were immunized with Daudi cells, and antisera were titrated by ELISA. Following the demonstration of sufficient antibody titer, total RNA was extracted from splenic lymphocytes of the immunized mice and RT-PCR was used to amplify kappa light chain and Fd fragments of heavy chain. After restrictive digestion with Sac I/Xba I and Xho I/Spe I, the kappa light chain and the Fd fragments were successively inserted into the phagemid vector pComb3H-SS and then electroporated into E.coli XL1-Blue. The specific Fab phage antibody library against Daudi cell strain in human B-lymphoma was constructed by infection of helper phage VCSM13. Following six rounds of biopanning with Daudi cells, the antigen binding activities of random clones were tested by ELISA to select the positive clones, which were further DNA sequenced, expressed in E.coli XL1-Blue and identified by Western blot., Results: The Fab phage antibody library with 3.13x10(7) size was constructed and four positive clones which specifically recognized Daudi cell strain were isolated. In amino acid sequences, the variable heavy domains (V(H)) were found to be 80%-02.394% and variable light domains (V(L)) 88%-95% homologous with respective murine germline genes in GenBank. Furthermore, soluble Fab antibodies of the positive clones were successfully expressed in E.coli XL1-Blue and the reactivity with the membrane proteins of Daudi cells was demonstrated by Western blot., Conclusion: Fab phage antibody library is successfully constructed and specific antibodies against membrane antigens in Daudi cells are obtained, which provides an experimental foundation for the further investigation of B-lymphoma immunotherapy.

Aim: To generation of human Fab fragment against GP GPIIb/IIIa from phage display library and to study its effect on platelet aggregation., Methods: An antibody phage display library was constructed from spleen cells from a donor with idiopathic thrombocytopenic purpura(ITP) whose anti-GPIIb/IIIa antibody was positive. High-affinity human mAbs was selected by panning against GPIIb/IIIa expressed on CHO123 cells. The binding specificity of phage antibody to GPIIb/IIIa was detected by ELISA and Western blot. And the effect of antibody on platelet aggregation was analyzed., Results: After three rounds of panning with CHO123, phage antibodies against GPIIb/IIIa were enriched specifically. 2 positive phage antibodies with high specific for GP IIb/IIIa were verified, and the purified antibody can inhibited ADP induced platelet aggregation., Conclusion: Human antibodies against GPIIb/IIIa are obtained from phage display library.

Aim: To construct and screen the specific Fab phage antibody library against human Raji cell strain in B-lymphoma., Methods: BALB/c mice were immunized with Raji cells, and the antibody light chain kappa genes and heavy chain genes Fd from the spleen cells were amplified by RT-PCR. After restrictive digestion with Sac I/Xba I and Xho I/Spe I, the light chain kappa genes and heavy chain genes Fd were inserted into the phagemid vector pComb3H-SS successively and then electroporated into E.coli XL1-Blue. The specific Fab phage antibody library against Raji cell strain in human B-lymphoma was constructed by infection of helper phage VCSM13. The specific antibodies against Raji cells were obtained after selected with Raji cells. The binding activity with antigens was identified by ELISA and the positive clones were sequenced., Results: The Fab phage antibody library with 2.18 x 10(7) volume was constructed and eight positive clones which specifically recognized Raji cell strain were isolated. Sequence analysis of the two positive clones showed that the variable heavy domains (VH) and variable light domains (VL) were highly homologous with the registered murine Ig heavy chain V region sequences and kappa light chain sequences, respectively., Conclusion: Fab phage antibody library was successfully constructed and specific antibodies against membrane antigens in Raji cells were obtained, which will provide an experimental foundation for the further investigation of B-lymphoma immunotherapy.