[Preparation and identification of monoclonal antibodies against Type M/N and Glycophorin A/B]

Abstract

Aim: To prepare monoclonal antibodies(mAbs) against Type M/N and monoclonal antibodies against Glycophorin A and Glycophorin B (GPA/GPB) and identify rare blood group MkMk.

Methods: 8 week-old female BALB/c mice were immunized with type "O" red blood cells, Splenocytes from the immunized mice were fused with Sp2/0 myeloma cells, and positive hybridoma clones were screened by panel erythrocytes. The titer of supernatant and ascitic fluid was tested by direct and indirect agglutination. The subclasses and isotypes were identified by monoclonal antibody isotyping kit. The complemental structures of the combining sites of their antibody antigen were determined by enzyme-treated red cells. The specificity of the GPA/GPB mAbs was identified by Western blot analysis.

Results: Eight hybridoma cell lines against Type M/N and GPA/GPB were obtained. The titer of supernatant was between 1 x 2(-4) - 1 x 2(-8), and the titer of ascitic fluid was between 1 x 2(-7) - 1 x 2(-12). The immunoglobulin subclasses of all the mAbs were IgG except 1C1C9C4, which was IgM. Four anti-M mAbs and one anti-N mAb were deterimined by agglutination test using panel erythrocytes and the test of the combining sites in antiody antigen. Western blot analysis proved that three mAbs recognized GPA/GPB protein.

Conclusion: Eight hybridoma cell lines against Type M/N and GPA/GPB have been obtained successfully. Among them, four mAbs recognize M, one recognizes N and three recognize GPA/GPB. Our study may be useful to research into MN blood group and for serological diagnosis. The anti-GPA/GPB mAbs can be used to prepare diabodies for the treatment and diagnosis of some of diseases.