Identification Of The Polyreactivity Of Natural Antikeratin MAb 3B4 And Primary Analysis Of Its Molecular Basis

Natural autoantibodies are immunoglobulins that exist in the serum of healthy people and animals in the absence of external antigenic stimulation. In about the middle age of the 20th century , the found of NAA challenged some theories of traditional immunology, especially "clonal selection theory" .Many studies demonstrated that most NAA can bind a variety of totally unrelated self and foreign antigens, i. e polyreactive. As its existence does , the polyreactivity of NAA does' t abide by the ' lock and key' traditional hypothesis of antigen - antibody interaction, which has long dominated immunological thinking. So the questions emerge:Is polyreactivity an immunoreaction or just a nonspecific reaction? Does polyreactive NAA has some special feature? What' s the molecular mechanism of polyreactivity?Many studies showed polyreactive antibodies recognize and bind antigens through HCDR3, indicating that polyreactivity isan immunoreaction but not a nonspecific reaction. Polyreactive antibodies are coded by gene of germline configuration. They are more flexible and hydrophilic, so they can easily to change their confomation to bind antigens inferred by researches. But there haven' t been direct studies explaining correctly the molecular mechanism of polyreactivity.Anti-keratin auto antibody (AK auto Ab) is a very important member of NAA, which was first found by Krogh inl970. Since then, a lot of work has been made about its biolpgy speficity and function. Studies indict that AK auto Ab generally exists in the sera of normal people and animals, which has important function of modulation on physiology and pathology in the body to maintain the balance of human immune system. But the polyclonal components and difficulty of purification has limited the studies of AK auto Ab. There has been few research on polyreactivity of AK auto Ab.Recently our research group has got three hybridomas secreting monoclonal IgM AK auto Ab by infusing splenocytes of mice bred in specific pathogen free conditions with Sp2/0 cells directly, which provide very good materials for us to further the study of AK auto Ab about it' s specificity and biology function.The present reseach would like to choose monoclonal antibody (mAb)3B4, the one has the higher affinity, to study it' s most important specificity, ie polyreactive binding reaction and answer some of the questions about polyreactivity.Methods1. Identification of polyreactivity of mAb3B4.ELISA and immunohistochemistry were applied to test the binding reactivity of 3B4 against some different antigens and tissues. In order to eliraate false-positive reaction, every research step are contoled well. Absorption and inhibit-competitive ELISA were applied to differ polyreactivity from nonspecificity.2. The construction and expression of phage-display scFv of mAb3B4.The antigen-binding site of antibody is in the variable region. In order to prove the polyreactivity is through the conventional antigen-binding site, we express and test the antigenic specificity of scFv of 3B4. Phage antibody technology was acted.The VH and VLgenes of 3B4 were amplified by PCR from plasraids of pMDT- VHand pMDT- Vu and then cloned into the phagemid pscMH. E. coli was transfected by recombined pscFv and super infected by the helper phage VCSM13.The antigenic specificity of the scFv concentrated from the supernatant of the transfected E. coli was detected by indirect ELISA.3. Site-directed mutagenesis analysis on polyreactivity of scFv.After analysing the gene and amino acid seqence of the variable region of 3B4, site-directed mutagenesis based on PCRwas applied to the possible critical site responsible for polyreactivity. Mutagenesis was performed by fast PCR based on pscFv. Then the pscFv with mutation was expressed, the reactivity chaging profile tested by ELISA may tell us some molecular basis of polyreactivity. Results1. 3B4 is highly polyreactive.Indirect-ELISA and immunohistochemisty showed that 3B4 supernants were polyreactive with poor species-specific and relatively restriced Ag-binding repertoire. Absorption ELISA and competitive ELISA indirected that 3B4 recognizes epitopes of antigens through CDRs. So we conclude that polyreactivity is an immunoreaction but not a non-specific reaction.2. the scFv phage antibody was polyreactive as mAb3B4.We successfully constructed and expressed phage-display scFv of mAb3B4, whose polyreactivity indicated that the recognizing region of 3B4 is in variable chain but not Fc.3. Site-directed mutagenesis analysis on polyreactive of scFv.DNA sequence analysis showed that the VH and VL genes of 3B4 were highly homologous with germline genes. They are very hydrophilic and flexible shown by amino acids sequence analysis. The variable region of 3B4 differs from many other high homologous immunoglobulin genes or amino acids just by HCDR3, indicating HCDR3 may be critical for the specificity of 3B4. We...