Recombinant polymeric IgG anti-Rh: a novel strategy for development of direct agglutinating reagents.
Anti-Rh alloantibodies are used in research and clinic laboratories to define the Rh antigenic profile of human blood samples. IgM anti-Rh antibodies directly agglutinate Rh-positive RBCs. Anti-Rh antibodies of the IgG isotype bind to Rh antigens with a higher intrinsic affinity than IgM and sensitize RBCs, but do not induce direct hemagglutination. The aim of this work was to produce IgG anti-Rh possessing direct hemagglutinating properties of IgM. To achieve this goal, recombinant antibody technology was used to construct genes encoding Ig light and heavy chains that will form polymers with anti-Rh specificity. Expression vectors and liposome-mediated DNA transfer were used to generate transfectomas secreting human recombinant IgG3 anti-Rh. ELISA, SDS-PAGE, and hemagglutination were used to identify and characterize the recombinant antibody produced. Thus, a recombinant polymeric IgM-like IgG3 anti-Rh antibody was produced that directly agglutinates RBCs with specificity identical to that of the parent non-agglutinating IgG. The results obtained suggest that the technology used here to generate polymeric IgM-like IgG3 anti-Rh antibodies can be applied to produce Rh blood typing reagents. This approach might also be used to develop reagents for which cell surface antigen binding and agglutination or aggregation is required.
Montano
Ramon F
RF
Department of Microbiology, Immunology and Molecular Genetics, and the Molecular Biology Institute, University of California, Los Angeles, CA, USA. rmontano@ivic.ve
Penichet
Manuel L
ML
Blackall
Douglas P
DP
Morrison
Sherie L
SL
Chintalacharuvu
Koteswara R
KR
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006386:Hemagglutination Tests / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007518:Isoantibodies / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D012204:Rh-Hr Blood-Group System / Q000276:immunology*
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007518:Isoantibodies; C033242:RH-antibodies; D011994:Recombinant Proteins; D012204:Rh-Hr Blood-Group System; C044411:polymeric IgG