[Generation, screening and preliminary identification of specific Fab phage antibody library against Daudi cell strain].

ABSTRACT

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.