Preparation Of Monoclonal Antibodies And Genetic Engineering Antibodies Against Blood Group Antigens

Blood group antigens in human erythrocyte surface is quite complex. Up to now 29 blood group systems have been well defined, they are encoded by 38 genes involved in more than 700 blood group antigens. Detection of blood group antigens play crucial roles in many aspects, such as ensuring safe blood transfusion, studying the distrubution of human rares, establishing rare blood group database, as well as serving as diagnostic or therapeutic reagent used clinically. but the essential deteminants to do these is to have adequate blood group antibodies. Unfortunately, they are severely lack and unavailable in china. The goal of the study is to prepare antibodies specifically against different red cell antigens by means of traditional hybridoma technique and antibody engineering technology. These antibodies may be used for idetification of blood group, diagnosis of diseases, and developing international communication, which help accelerate blood group study in china.METHODSThe study is divided into three components in terms of the experimental aims1 Preparation of mAbs against blood group antigens 8 week-old female BALB/c mice were immunized with either type"O"red blood cells or umbilical blood cells. Splenocytes from the immunized mice were fused with Sp2/0 myeloma cells. The clones with the agglutination reaction grade of 4+ were remained for further culture. Specificity of mAbs was identified by panel erythrocytes, prokaryotic expression product of Kell-30c and Western blot analysis. The titer of supernatant and ascitic fluid were 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.2 Construction of ScFv_6D7C9-HPV16 E7 bifunctional moleculeThe variable regions of light and heavy chains were amplified by RT-PCR from 6D7C9, a hybridoma cell line against Glycophorin A and Glycophorin B (GPA/GPB) obtained from the first part of the study. ScFv_6D7C9 was constructed by connecting VH and Vκgene with a linker, it then further transducted into prokaryotic expressive vector pBAD/gIIIA, the positive clone is designated as pBAD-ScFv_6D7C9. Western blot analysis and indirect agglutination were performed to detect its specificity and agglutinating activity. ScFv_6D7C9 - HPV16 E7 bifunctional molecule was contructed by cloning ScFv_6D7C9 genes and the E7 gene of human papollmavirus virus 16 into the same expression vector to form a bifunctional expression vector, named pBAD- ScFv_6D7C9-HPV16 E7. Western blot analysis were performed to detect the expression of bifunctional molecule. Moreover, indirect agglutination was performed to detect its bifunction, that is it has the ability to bind with HPV16 E7 antibody present in patients'serum and RBC, and rapidly detect HPV16 E7 antibody by agglutination response.3 Construction of Fab antibody against Rh(D) antigenThe variable regions of light and heavy chains were amplified by RT-PCR from the PBMCs of volunteers with high titer (1:256-512 by inditect agglutation) antibody to Rh (D) antigen. Meanwhile, the genes of constant regions of light and heavy chains were isolated from pComb3Xtt and pComb3xλphagmid carrying the templates respectively. Vκlight chain and Fd heavy chain were linked by the first splicing overlapping extension PCR (SOE) , and a full-length Fab gene was created by the second SOE. The Fab gene was ligated to phagmid pComb3H-SS and transformed to E. coli XL1-Blue by electroporation. The obtained human Fab phage antibody library was panned using Rh(-)/ Rh(+) RBC four times. the phage antibodies against Rh(D) were highly enriched. Indirect agglutation test, western blot analysis and sequencing analysis were performed to detect the specificity of Fab against Rh (D).RESULTS1 Preparation of mAbs against blood group antigens1.1 mAbs against MNSs blood group antigensEight hybridoma cell lines were obtained. The titer of supernatant was between 1×2^-4–1×2^-8, and the titer of ascitic fluid was between 1×2^-7–1×2^-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.1.2 mAbs against Ii blood group antigenThree hybridoma cell lines against i blood group antigen were obtained. The titer of supernatant was between 1×2^-4–1×2^-8, the ascitic fluid titer of A10G5F9E2E7 was between 1×2^-10–1×2^-11. The immunoglobulin subclasses of the three mAbs were IgG3. Anti-i mAbs were deterimined by agglutination test using panel erythrocytes with the adult i rare blood, adult I blood and fetal i blood.1.3 mAbs against Kell blood group antigenThree hybridoma cell lines, 4H2A8,2F9F5 and 4D10G11, against Kell blood group antigen were obtained. The titer of supernatant was between 1×2^-5–1×2^-10. The immunoglobulin subclasses of all the mAbs were IgG1. Anti-kell mAbs were deterimined by ELASA using prokaryotic expression product of Kell-30c (the 30 amino acids at the C-terminal of kell protein)2 Construction of ScFv_6D7C9-HPV16 E7 bifunctional moleculeSequencing analysis of ScFv_6D7C9 showed that the heavy chain of 6D7C9 belonged to VHII subgroup and the light chain belonged to VκIVsubgroup. Western blot analysis and indirect agglutination proved ScFv was expressed and had agglutinating activity. Based on this, pBAD-ScFv_6D7C9-HPV16 E7 vector was constructed and the bifunctional molecule was expressed in E. coli after being induced by IPTG. Western blot analysis confirmed that the bifunctional molecule was able to bind with HPV16 E7 antibody in serum, but agglutating response failed to be observed when HPV16 E7 antibody in patient's serum was added into the bifunctional protein and then further added fresh human RBC.3 Construction of Fab antibody against Rh(D) antigenThe repertoire of human phage display Fab library was 7.4×106 . After 4 round panning with Rh(-)/ Rh(+) RBC, 7 clones were checked for their binding activities by agglutation test and 2 clones could bind to Rh (+)RBC specifically. Western blot demonstrated that bacterially expressed soluble Fab could recognize Rh (D) antigen. Sequencing analysis showed that the obtained two clones had the identical VH and VK genes. The heavey chain belonged to human VH3-23 subgroup and the light chain was VκⅢsubgroup gene. CONCULSIONS1 mAbs obtained1) Five mAbs against GPA antigen2) Three mAbs against GPA/GPB antigen3) Three mAbs against i antigen4) Three mAbs against kell antigen2 Genetic engineering antibodies1) ScFv_6D7C9 had the ability to agglutate human RBC2) ScFv_6D7C9-HPV(E7) bifunctional molecule could be expressed in fusion, but the HPV16 E7 antibodies in patient's serum failed to be detected by agglutation response.3) Fab antibody, which specifically aggulated Rh(+) RBC, was obtained.