Development and application of a rapid IgG antibody ELISA kit for Chikungunya virus
Objective To develop an ELISA kit to detect IgG antibodies of Chikungunya virus(CHIKV),providing a new method for epidemiological investigation and detection in the field for CHIKV infection.Methods Using the CHIKV-specific recombinant protein pMal-chik23 as diagnostic antigen,HRP-labeled anti-IgG antibody as color-developing antibody,and the working concentration of diagnostic antigen,serum to be tested and second antibody were optimized using orthogonal.The reaction conditions of ELISA reaction,such as coating,blocking,incubation,and color-developing were systematically optimized.The cut-off value for ELISA detection was established based on the assessment of a large clinical sample set.On this basis,the specificity,sensitivity,and stability of the ELISA response were evaluated to develop and assemble a rapid ELISA kit for the detection of Chikungunya fever IgG antibodies.Results On the basis of systematic conditions optimization,an indirect ELISA kit for the detection of IgG antibodies against CHIKV was developed and assembled.The optimal reaction conditions were identified as 1.0 μg/mL antigen was coated using carbonate buffer at 4 ℃ for 24 hours.Then the microplate was blocked using HBV blocking solution at 37 ℃ for 4 hours.100 μL/well samples to be tested were diluted at 1∶101,reacted at 37 ℃ for 40 minutes,and washed 4 times with PBST.Thus,HRP-labeled rabbit anti-human IgG was diluted at 1∶20 000,HRP-labeled sheep anti-mouse IgG was diluted at 1∶10 000,reaction at 37 ℃ for 30 minutes,and washed 5 times with PBST.Finally,100 μL/well TMB solution was added and incubated at 37 ℃ for 10 minutes.Then terminate the reaction with 50 μL of 20％H2SO4 and measure the A450 value at dual wavelengths of 450/630 nm(A450).The evaluation results showed that ELISA A450 of Chikungunya fever-positive samples were more than 0.43,while the ELISA A450 of negative samples was less than 0.04,and the S/N ratio＞10.Specificity test showed that the developed kit had no cross-reaction with 9 other similar arbovirus species such as Sindbis,Geta,Ross River,and Dengue virus.The stability evaluation of the reagent kit indicated that it had high stability,with a coefficient of variation(CV)within the microplate ranging from 0.76％to 2.12％,the coefficient of variation between the microplate ranged from 0.64％to 1.85％,and the coefficient of variation between batches ranged from 0.83％to 2.31％,all of which were less than 3％.The sensitivity of the kit did not decrease significantly after being stored at 4° C for 1 year.Conclusions A rapid indirect ELISA kit for the detection of Chikungunya fever IgG antibodies was successfully developed,exhibiting good sensitivity,specificity,and stability.
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