Inactivated EV71 virus were used as the coating antigen to titrate anti-EV71 IgG levels in the serum samples by sandwich enzyme-linked immunosorbent assay (ELISA) as described previously [3 (link)]. Briefly, 96-well plates were coated with polyclonal anti-EV71 antibody overnight at 4 °C and blocked with the buffer containing 2 % (w/v) bovine serum albumin for 2 h at 37 °C. Inactivated EV71 virus was added to the well and incubated for 2 h after washing thrice with the buffer (0.05 % (v/v) Tween 20 in PBS). The sera were analyzed at twofold serial dilutions by starting at 1:100. The plates were incubated at 37 °C for 1 h and washed thrice with buffer. HRP conjugated goat anti-mouse IgG (CWBIO, China) was then added into each well in a 1:2000 dilution, and incubated at 37 °C for 1 h. The plates developed with TMB solution (Tiangen Biotech, China) in a dark room for 15 min after washing three times with buffer, and the reaction was stopped by adding 2 M H2SO4. The absorbance at 450 nm was evaluated using a microplatereader (Bio-Rad, USA).
Tmb solution
Manufactured by Tiangen Biotech
Sourced in China
TMB solution is a laboratory reagent used as a substrate in colorimetric enzyme-linked immunosorbent assay (ELISA) applications. It provides a chromogenic reaction that can be measured spectrophotometrically to quantify the presence and concentration of target analytes.
Automatically generated - may contain errors
2 protocols using tmb solution
1
Quantification of Anti-EV71 IgG by ELISA
2
ELISA for Recombinant VP27 Protein
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Recombinant VP27 protein was diluted in carbonate-bicarbonate buffer (pH 9.5) to a final concentration of 2 µg/mL and coated on 96-well plates (100 μL per well). Following adsorption of the antigen after incubation at 4°C overnight, the plates were washed with PBST 3 times and then blocked with 5% non-fat milk at 37°C for 1.5 h. Next, 100 μL of serum from immunized mice was incubated in the plate at a 1:1,000 dilution at 37°C for 1 h, and subsequently washed 3 times with PBST. Then, 100 μL of HRP-conjugated goat anti-mouse IgG (1:10,000; Solarbio, Beijing, China) was added into the plates and incubated at 37°C for 1 h. After washing with PBST 3 times, 100-μL TMB solution (TIANGEN, Beijing, China) was added into the plates at 37°C for 15 min. The reaction was stopped with 50-μL sulfuric acid (2 mol/L; Sangon Biotech Co., Ltd, Shanghai, China), and absorbance was measured at 450 nm using a microplate reader (Model550; Bio-Rad, Hercules, CA).
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