Screening And Immunological Identification Of Human ScFv Antibody Against MAGE-A1

MAGE-A1, as one of the MAGE (melanoma associated antigen) family, is often expressed in different types of tumors. With the exception of testis and placenta, the expression of MAGE-A1 is absent in normal tissues. Because it is shared by many tumors and on account of their strict specificity, MAGE-A1 may be a potential target for specific immuno-therapy which is a new method of tumor therapy. Antibodies of murine origin cannot be used in patients because of the likely development of a human anti-mouse antibody response. So the use of mouse antibodies in human has many limitations as the foreign immunoglobulin epitopes. It is, therefore, desirable to have human monoclonal antibodies. One way to obtain these antibodies is through phage display introduced by Winter et al (1991). By this technique, which uses Escherichia coli as a host and filamentous bacteriophages as an expression and display system, antibodies and their encoding DNA are packaged into one single particle. Generation of antibodies by phage display has proved to be a useful tool.Objective1. To screen phage antibodies against MAGE-A1 from a human scFv antibody library by phage display technique and identify their specificity.2. To express the soluble antibody against MAGE-A1, identify its immunological specificity and measure its affinity constant.Methods1. Purified MAGE-A1 was coated on 6-well plate as antigen, the phage antibodies against MAGE-A1 were screened by five times' combining-eluting-amplifying using phage display technique. The positive antibodies were identified by ELISA and dot blotting.2. The phagemid of H9 positive clone was extracted and transformed into E.coli HB2151. The bacteria were induced to express soluble antibody by IPTG. The soluble antibody was identified by dot blotting and immunocytochemical study. The affinity constant of soluble antibody was measured by non-competitive ELISA which used an improved formula of Beatty's.Results1. After five times' combining-eluting-amplifying, the phage antibodies were screened and enriched. The positive phage antibodies identified by ELISA and dot blotting were specific for MAGE-A1.2. The soluble antibody induced by IPTG was also specific forMAGE-A1. Its molecular weight was about 30kD by SDS-PAGE and its affinity constant was about 1.432× 10~6L/mol.Conclusions1. The scFv antibody library is panned successfully against MAGE-A1 using phage display technique. The screened phage antibodies are specific for MAGE-A1.2. The soluble antibody expressed by E.coli HB2151 is also specific for MAGE-A1 with low immunogenicity. It provides a valuable tool for preparing the anti-tumor antibody or immunotoxin for tumor immunotherapy.