Pregnancy-associated Plasma Protein A Purified Monoclonal Antibody Preparation

Background: Pregnancy-associated plasma protein A (PAPP-A) is a macromolecular glucoprotein, mainly produced by the placental synytiotrophoblasts and decidual cells. It was first indentified as a circulating protein in the serum of woman in advanced stages of gestation. PAPP-A concentrations rises throughout gestation. During the first trimester, the decreased PAPP-A levels in matemal blood are concerned with fetal chromosomal abnormalities, fetal anomaly, low birth weight and other abnormal pregnancy outcomes. As decreased circulating levels of PAPP-A antigen correlate with abnormal fetal function, PAPP-A is a valuable index of predicting chromosomal abnormalities and other abnormal pregnancy outcomes during the first trimester.Aims: To develop a method for the purification and identification of human Pregnancy-associated Plasma Protein A, to obtain the hybridoma cells secreting PAPP-A monoclonal antibodies, and to prepare monoclonal antibodies against PAPP-A.Methods: PAPP-A fraction was isolated and purified by DEAE-Sepharose CL-6B ion-exchange and Heparin-Sepharose C1-6B affinity chromatography from later pregnancy serum. The purified PAPP-A was identified by denatured SDS-PAGE and Westem blotting respectively. Balb/c mice were immunized subcutaneously three times with 24μg of high molecular weight PAPP-A in 50% Freund's adjuvant, and titers in immunized mice sera were determined by indirect ELISA. The splenic lymphocytes from the immunized mouse were fused to myeloma Sp2/0 cells and cultured selectivly with HAT and HT. Each supematant was screened by its reactivity to PAPP-A, One of antibody-producing wells was selected for further use through following successive subclonings by limiting dilution method. The selected hybridoma cells were proliferated and injected into Balb/c mice for the generation of ascites. Each ascites was detected for the titers of the PAPP-A monoclonal antibodies. The antibodies in the ascites were purified through protein A column. The obtained PAPP-A monoclonal antibodies were analyzed by SDS-PAGE. The subtype of the antibodies was identified by ELISA. The specificity of the monoclonal antibody was assessed by Western blotting and immunohistochemical staining.Results: The denatured SDS-PAGE showed that PAPP-A dimmer with a relative molecular weight of 200kDa, proMBP subunit migrates as a diffuse spot with molecular mass around 50-90 kDa, Western blotting showed that purified PAPP-A can be indentified specifically by the PAPP-A McAb 3C8.12 hybridoma cell lines were selected. 8 of them are IgG1 and 4 of them are IgG2a subtype. The title of ascite is up to 1:10~5. The denatured SDS-PAGE of PAPP-A McAb showed that heavy and light chain with a relative molecular weight of 51.3 kDa and 28.7 kDa respectively. The purified McAb showed good affinity and specificity against PAPP-A in Western Blotting and immunohistochemical staining.Conclusion: In present study, PAPP-A was successfully isolated from late pregnancy serum. 12 strains hybridoma cells were obtained, which could stably secrete PAPP-A monoclonal antibodies. The antibodies that prepared showed specificity of recognizing PAPP-A, which could provide a potential value for establishing a new method to measure the serum concentrations of PAPP-A.