| Natural IgG antibody molecule is made up of two identical heavy chains and two identical light chains held together by interchain disulfide bonds between different chains. It is a protein complex composed of four peptide chains arranged in a Y-shape structure. It is well accepted that the two antigen binding sites of antibody molecule are both structurally and functionally identical. Thus, one antibody recognizes one particular antigen, which is known as monospecificity. However, recent studies have revealed that human IgG4antibodies are dynamic molecules with unique structural properties and undergo’Fab-arm exchange’in vivo, which results in bispecific antibodies (BsAb) Natural bispecific antibody is able to bind two different antigens simultaneously, which challenges the commonly accepted’one antibody-one antigen’paradigm. Natural bispecific antibodies recognizing2unrelated antigens can be raised from rabbits by a specific immunization procedure and an animal model of producing natural bispecific antibodies has been established. Later, a group of bispecific auto-antibodies against IgG and CCP in sera from RA patients were identified. We speculate that in the circumstances of distinct antigens’chronic and simultaneous stimulation (i.e. Autoimmune disease, chronic parasitic, bacterial and viral infections, tumor growth and other chronic processes), the vertebrate immune system is prone to producing bispecific antibodies against these antigens. Chronic hepatitis C infection is a persistent infection with virus replication and antibody levels against the HCV proteins (Core, NS3, NS4, NS5) were found to be relatively constant, which leads us to speculate that some natural bispecific antibody against HCV proteins might exist. This study aims to identify natural bispecific antibodies against HCV proteins during chronic HCV infection and explore the relationship of the finding BsAb and hepatitis C infection. Then purify the identified bispecific antibody and investigate the underlying producing mechanism. First, double antigen sandwich biotin-streptavidin ELISA methods were established to detect HCV NS3/NS4, NS3/NS5and NS3/Core BsAbs in91Hepatitis C patients. Unexpectedly, bispecific antibodies against NS3and NS5were observed. To determine the prevalence of the NS3/NS5natural BsAbs in a larger population, the cut-off value was set at mean OD+8SD of100health controls and a sample was considered positive if the OD value was above the set cut-off and at least0.090OD. NS3/NS5natural BsAbs were detected in41%(145/354) patients with HCV infection. The association of NS3/NS5BsAb OD values and HCV RNA levels in115patients were analysed and no correlation were observed (Spearman r=-0.1210, p=0.1977). The specificity of the NS3/NS5natural BsAb was confirmed by competition inhibition assay and cross-reactive exclusion assay. The observed dual reactivity was due to NS3Ab and NS5Ab aggregation was excluded by high resolution gel filtration chromatography. The purification of NS3/NS5natural BsAb by affinity chromatography was failure. However, the isotype of the NS3/NS5BsAb was determined to be IgGl but not IgG4by Protein G affinity chromatography and magnetic bead isolation method. This natural BsAb can not be obtained by half-molecule exchange experiment in vitro, which indicates the producing mechanism of NS3/NS5natural BsAb is different from the producing mechanism of IgG4BsAb. We propose a hypothesis that this naturally producing IgG1BsAb is caused by the failure of allelic exclusion of B cells. In this study we first identified a novel naturally occurring bispecific IgG1antibody against HCV NS3and NS5in sera from hepatitis C patients. Further studies regarding the pathological/biological implications and the production mechanism of NS3/NS5natural BsAb in HCV are necessary. |
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