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Abstract: Objective: : Heat-mediated ELISA (HELISA) technique has been reported to shorten ELISA timings and has major clinical implications in diagnostic field [Bora U, Kannan K, Nahar P. Heat mediated enzyme linked immunosorbent assay. J Immunol Methods 2004; 293: 43–50]. Objective of this study is to find out whether anti-MBL autoantibody can be detected rapidly by HELISA technique. Design and methods: : Activated polystyrene microtiter plate was prepared by a photolinker in a photochemical reaction carried out over a period of 10 min. Lectin was covalently immobilized onto the activated plate at 50 °C in 45 min. Antigenicity of the immobilized lectin was checked by HELISA carried out at elevated temperature (50 °C). The result was further compared with that obtained by conventional ELISA method carried out on an untreated plate over an incubation period of 18 h. Results: : Autoantibody detection carried out by HELISA method (intra- and inter-assay CVs were <9.8%) in 2 h 45 min gives absorbance value comparable to that of conventional ELISA (intra- and inter-assay CVs were <12%) carried out in 18 h in RA patients and healthy controls (n =100). HELISA on a photoactivated surface showed 1.5-fold higher absorbance than those obtained on untreated surface (p =0.00019). The HELISA method is more sensitive (AUC=0.967, 95% CI=0.948–0.987) and can be used to detect autoantibody even at higher dilution than by conventional assay. Excellent correlation was observed when autoantibodies were detected by HELISA and conventional ELISA (R =0.9706). Conclusion: : The present method provides rapid and sensitive detection of autoantibody in rheumatoid arthritis patients. The method is precise and reliable similar to method currently used in diagnostics and could be potentially useful for other immunoassays. [Copyright &y& Elsevier]

Abstract: Occurrence of autoantibodies in patients’ sera is the characteristic feature of autoimmune disorders. We assessed the presence of anti-mannose binding lectin (MBL) autoantibodies in the sera of 107 rheumatoid arthritis (RA) patients and 121 control subjects by enzyme immunoassay. Elevated levels of anti-MBL autoantibodies in the sera of RA patients (P <0.0001) was detected for the first time. The ratios of anti-MBL positive in RA patients and controls were respectively 60.7% and 1.65%. Experiments were then designed to understand the functional relevance of these autoantibodies. An inverse correlation of anti-MBL autoantibodies with serum MBL levels (P =0.001) and MBL complex activity (P =0.02) was observed without genetic association between MBL polymorphisms and anti-MBL autoantibody secretion. A significant increase (P =0.038) in the level of anti-MBL autoantibodies was observed in 23 synovial fluid samples in comparison to the serum samples. Moreover, the anti-MBL autoantibodies were found to be more often present in the sera of RA patients (60.75% sensitivity, 98.35% specificity and 0.913 area under the ROC curve) in comparison to the IgM and IgG isotypes of rheumatoid factors (RF). Anti-MBL autoantibodies were still positive in 25.23% RA patients when both the RF isotypes were negative. Also, in RA patients, at all stages of disease activity and joint deformity, anti-MBL autoantibodies were more often present than both the RF isotypes. Therefore, the significant presence of anti-MBL autoantibodies enunciates that anti-MBL autoantibodies might have a diagnostic value; however, more studies are needed to confirm the role of anti-MBL autoantibodies in the diagnosis of rheumatoid arthritis. [Copyright &y& Elsevier]