Development Of A Serological ELISA Kit For Detection Of EV71 Infection Associated With Hand-foot And Mouth Disease And Its Clinical Application

Objective To develop an ELISA(Enzyme-Linked Immunosorbent Assay)method for early rapid detection of serum anti-EV71 antibody and evaluate its clinical application value .Methods To express and purify a recombinant protein VP1 of EV71 as a coated antigen for establishment of an indirect ELISA for detection of serum anti-EV71 IgM and anti-EV71 IgG. Evaluation of anti-EV71 IgM and anti-EV71 IgG serological diagnostic methods in the EV71 disease diagnosis in comparison to RT-PCR(reverse transcription-polymerase chain reaction),isolation of EV71 and micro-neutralizing assays,and clinical evaluation.Results In comparison with RT-PCR, the sensitivity, specificity, positive predictive value and negative predictive value of anti-EV71 IgM antibody were 83%,85%,81% and 87%, respectively. The sensitivity, specificity, positive predictive value and negative predictive value of anti-EV71 IgG antibody were 72%,74%,68% and 77%, respectively. Moreover, compared with viral isolation assay, the sensitivity and specificity of anti-EV71 IgM antibody were 85% and 97%, respectively. The sensitivity and specificity of anti-EV71 IgG antibody were 75% and 77%, respectively. In addition, The titers of anti-EV71 IgG antibody significantly correlated with the titers of neutralizing antibody to EV71 by linear regression analysi(sr =0.72,P<0.001). Finally, the serum titers of anti-IgG from patients with EV71 associated hand food and mouth disease at convalescent stage exhibited significantly higher than that of the same patients at acute stage (P<0.001), but instead of anti-IgM (P=0.287). Coefficient of variation of repeated testing is less than 10%. By 120 clinical samples, anti-EV71 IgM antibody sensitivity and specificity exceeding 85%.。Reagent for 4 weeks, the test results was stabilization.Conclusion Recombinant purified VP1 is used as a coated antigen to develop an indirect ELISA for detection of serum anti-human EV71 IgM and anti-human EV71 IgG antibodies, which has a good diagnostic value and an application prospect.