| Natural bispecific antibody (nBsAb) is a new antibody entity characterized by two distinct antigen-binding sites and natural production in vivo. Accumulating studies have revealed that natural bispecific antibody can be detected in long-term passive immunization and some diseases. However, nBsAb in human diseases has been poorly investigated except for rheumatoid arthritis, and no data have been reported regarding the possible role of naturally occurring nBsAb in vivo. Hashimoto thyroiditis (HT) is a common autoimmune disease of thyroid. Anti-thyroid peroxidase antibodies (anti-TPO) and anti-thyroglobulin antibodies (anti-Tg) are a hallmark of HT, high titers of these can be detected several years before apparent symptoms appear. It is an ideal model by which to investigate nBsAb in diseases:1) it is a chronic disease;2) there is a consistent presence of higher titer autoantibodies in serum, including anti-TPO and anti-Tg antibodies;3) IgG4is one predominant subclass among anti-TPO and anti-Tg on the basis of currently available data. As described above, HT fulfills the conditions for nBsAb production.This study aims to establish a double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) for anti-TPO/Tg nBsAb detection. Then determine the prevalence of anti-TPO/Tg nBsAb in HT patients and preliminary explore its potential role by investigating the associations of this nBsAb with clinical and laboratory indicators. First, a double-antigen sandwich ELISA for anti-TPO/Tg nBsAb detection was developed, in which the plates were coated with Tg and the peroxidase labeled TPO was used as detection antibody by a three-step development optimization and validation strategy. Serum samples were obtained from136HT patients,92diseased controls and99healthy controls for anti-TPO/Tg nBsAb detection. The prevalence of anti-TPO/Tg nBsAb in HT was44.9%(61/136), significantly higher than that of diseased controls (2.2%,2/92)(p<0.0001) and healthy controls (0%,0/99)(p<0.0001). Then the authenticity of anti-TPO/Tg nBsAb was verified by performing several assays, including a competitive inhibition assay to exclude a cross-reaction between TPO and Tg, RF test to exclude its interference and size-exclusion chromatography to demonstrate the monomeric IgG nature of anti-TPO/Tg nBsAb. A series of studies regarding the producing mechanism of anti-TPO/Tg nBsAb were carried out. It showed that the IgG subclass of anti-TPO/Tg nBsAb was IgG4which determined by a new magnetic separation technique; Anti-TPO/Tg nBsAb could be generated in vitro and the denaturing condition (GSH) was a necessary requisite for the reaction. However, no correlation was observed between anti-TPO/Tg nBsAb and serum GSH/serum total antioxidant capacity; There was a unexpected negative correlation between anti-TPO/Tg nBsAb and serum total IgG4(r=-0.697, p=0.025) in IgG4thyroiditis patients. The highlight of the research is the preliminary study regarding the possible role of nBsAb in vivo. HT patients who were nBsAb-positive were prone to have significantly lower levels of serum C-reactive protein and tumor necrosis factor alpha compared to the nBsAb-negative individuals (p<0.05). The serum amyloid A and interferon gamma levels also showed a similar trend in the two groups. The results provide importance evidences to suggest that nBsAb may be a protective factor exerting a potent anti-inflammatory effect on autoimmunity. We successfully identified a new type of nBsAb against TPO and Tg in HT patients for the first time, enriching the context of the research for nBsAb in human diseases. Also we preliminarily discussed its potential role based on inflammatory markers analysis. The results from this study indicate a protective effect of anti-TPO/Tg nBsAb in pathogenesis of HT and extend prior knowledge about nBsAb in diseases. |
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