Construction Of Single Chain Fv Against Human Erythrocyte And Constitution Of Autologous Red Cell Agglutination Assay

Autologous red cell agglutination assay (AGEN) is a rapid diagnostic method which is a simple, specific and do not require any special equipment. Its sensitivity and specificity is not inferior to ELISA, and can detect both IgG and IgM. The method is safe, fast, accurate and inexpensive. Without separation of serum and equipments, the illness is diagnosed with a drop of whole blood within two minute. It is very suitable for the poor condition of the grass-roots hospitals, voluntary blood donation and the emergency transfusion, and therefore it has a good application prospects. This method relies on a bifunctional reagent that combined human red blood cells and antigen or antibody. When the blood of patients exist the relevant antigen or antibody, the red blood cells begin to agglutinate due to intermolecular cross-linking. The purpose of this study was to prepare the non-agglutinating human RBC monoclonal antibody and thereby form bispecific antibody reagent used for clinical diagnosis.In this study, the non-agglutinating human RBC monoclonal antibodies were prepared through the BALB/c mice were immunized with three kinds of immunogen. The antibodies were engineered bispecific antibody by chemical conjunction. Furthermore, the reagent was made to become whole-blood agglutinating reagent and hemagglutination test methods were studied preliminarily. Main research contents are as follows:1. Human O-type RBC, RBC whole membrane and RBC membrane protein were injected intraperitoneally and intravenously to the BALB/C mice. Then the spleen cells which were collected from immunized mice were fused with the SP2/0 myeloma cells with the ratio of 5:1. By detection of indirect ELISA, red blood cell direct agglutination test and indirect agglutination test and cloning of limited dilution method, a hybridoma cell was obtained and secreted non-agglutinating human RBC monoclonal antibodies after cloning of three times. The cell was named 1C3. After repeated passage, cryopreservation and recovery, the hybridoma cell could secret antibody capacity stably, and its subtype belonged to IgG1. Ascites induced with conventional methods were purified by the methods of octanoic acid-ammonium sulfate precipitation and DEAE cellulose chromatography. The ELISA titers of immunity and ascites can reach between 1:10⁴ and 1:10⁶. The McAbs had no cross-reaction with other species and a high affinity.2. Non-agglutinating human RBC monoclonal antibodies and hepatitis B surface antibodies were digested with pepsin. Further, the monoclonal antibody and hepatitis B surface antibodyF(ab^')₂ fragments were obtained by purify. Through this method of and two-step,F(ab^')₂ segments of human RBC McAbs and hepatitis B surface antibodies are coupled into bispecific antibody. By SDS-PAGE analysis, bispecific antibodies were prepared successfully with the method of glutaraldehyde' one-step. The bispecific antibodies were made to form whole-blood agglutinating reagent that is used to diagnose HBV surface antigen. The activity of the reagent was made a preliminary identification and a method of detecting hepatitis B surface antigen in the blood was established.