[Construction and clinical applications of a humanized phage library of platelet GPIIb/IIIa single chain Fv antibody]

Abstract

Objective: To screen out the anti-platelet GPIIb/IIIa autoantibody which can inhibit with aggregation function of platelet and construct a humanized anti-platelet GPIIb/IIIa single chain Fv library by phage surface display technology.

Methods: Idiopathic thrombocytopenic purpura (ITP) patients whose plasma contains anti-platelet GPIIb/IIIa autoantibodies were screened out. The antibodies can inhibit the aggregation function of platelet by using MAIPA assay and platelet aggregation test. The heavy chain and light chain variable region genes of human immunoglobulin were amplified by RT-PCR from peripheral blood lymphocytes mRNA of the patients screened out and randomly combined through a DNA linker encoding the peptide (Gly(4)Ser)(3) to construct single chain Fv gene. ScFv gene was digested with SfiI/NotI restricted digest enzyme to ligate the pHEN2 cloning vector, then were electrically transformed to E.coli TG1. The TG1 containing ScFv-pHEN2 was rescued by helper phage M13K07 to produce ScFv phage antibody.

Results: Of 95 chronic ITP patients, 41 (43.2%) were found positive for anti-platelet GPIIb/IIIa autoantibody, 5 (5.3%) were markedly positive. 2 (2.1%) patients plasma significantly inhibited the aggregation function of platelet. The lengths of VH and VL were about 380 to 400 bp. They were successfully linked by DNA linker to form ScFv fragment of about 780 bp. After cloning ScFv to phagemid pHEN2 and transforming ScFv-pHEN2 to TG1, 2.1 x 10(7) clones formed. After M13K07 rescue, 1.62 x 10(10) cfu/ml ScFv phage antibodies were produced.

Conclusion: Few anti-platelet GPIIb/IIIa antibodies can inhibit the aggregation function of platelet. A phage antibody library has been constructed by phage surface display technology. Humanized anti-platelet GPIIb/IIIa ScFv phage antibody can be screened from this library.