| Enterovirus 71 (EV71), a member of the family Picornaviridae, is known as one of the major causative agents of hand, foot and mouth disease(HFMD), and sometimes associated with a series of central nervous system diseases in some infected children so much as lead to severe cases and death. Accordingly, developing a quick homogeneous alternative approach to human IgM detection looks so crucial for early diagnosis of HFMD.According to the characteristic of HFMD, we established a method for EV71 IgM detection which is called AlphaLISA technology with high sensitivity, specificity, homogeneous, low background, wide dynamic ranges, no-wash steps and low sample volumes.In this study, we successfully purified and verified the recombinant protein EV71 VP1 1-297 and protein EV71 VP1 1-177. After proteins were biotinylated, the optimal quantities of the antigen was determinated by ELISA, and the lowest protein quantity finally is 37.5ng per well. Adjusting acceptor beads, dornor beads, Bio-protein and volume of serum, AlphaLISA reaction system was optimized in total assay volume 20 ul containing serum 2μl.Subsequently, we detected the serum samples in 10 healthy subjects with commercial ELISA IgM kits and 30 samples from the patients of HFMD with AlphaLISA, respectively, for verifying the protocol newly established. The results revealed IgM negative for the total 10 healthy subjects by ELISA as well as AlphaLISA. of the 30 HFMD patients, positive serum IgM was detected in 16 with respectively ELISA and AlphaLISA, and yet, additional detection of the negative samples among the 10 healthy subjects by ELISA revealed another 6 IgM positive with AlphaLISA. The results of the AlphaLISA assay are coincident with ELISA kits, suggesting that AlphaLISA can be competent for EV71 IgM serum detection in patients of HFMD. |
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