Objective: To establish and validate an ELISA method to measure the titer of botulinum antitoxin type A. Methods: Botulinum toxoid type A was used as coated antigen coated enzyme label plate, and diluted serum samples were added after plate blocking. The horseradish peroxidase labeled rabbit anti-horse IgG (IgG-HRP) was subsequently used as the enzyme-labeled antibody. Tetramethyl benzidine(TMB) was added for 10-15 min for color development, and the reaction was terminated by 2 mol·L-1 of sulfuric acid. Finally, the A450 nm value was read on the enzyme marker. The logarithm of concentration(X) of the sample concentration of standard curve was taken as the horizontal coordinate and the A450 nm value(Y) was taken as the vertical coordinate to draw the standard curve. The serum sample concentration could be obtained by substituting A450 nm value of serum samples to be tested into the curve equation, and the titer of the serum samples could be obtained by multiplying the dilution ratio. Results: Under the optimized conditions, the specificity of the method was high, there was no cross reaction with B, C, D, E and F type of antitoxin serum except botulinum antitoxin type A. In the linear range of 50-400 mIU·mL-1, the linear relationship was high with r＞0.990 0. The accuracy was between 80% and 120% and the RSD of precision (repeatability and intermediate precision) was less than 20%. The titers for 20 batches of botulinum antitoxin type A serum were determined by ELISA in the range of 525-2 450 IU·mL-1, and the neutralization test was in the range of 675-2 400 IU·mL-1. The coefficient of correlation r=0.906 0(P＜0.001), which was significantly positive correlated between the two methods. Conclusion: The established ELISA method could acquire stable results under laboratory conditions, which provides a simple detection method for botulinum antitoxin type A in vitro.
ZHANG Ling-ling, DUAN Li-juan, LIU Jian-guang, LI Yu-he, XIE Xiao-rong, SU Hui, LIANG Ling-Yu, GAO Jian-jun
. Establishment and validation of an ELISA method for detection of botulinum antitoxin type A[J]. Chinese Journal of Pharmaceutical Analysis, 2023
, 43(3)
: 525
-530
.
DOI: 10.16155/j.0254-1793.2023.03.20
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