Abstract
Ultrasensitive sandwich immunoassays for detecting the small molecule semicarbazide (SEM) were developed based on derivatization. Several SEM derivatizing agents were synthesized by linking o-nitrobenzaldehyde (NBA) and biotin with dihydroxyalkanes (different lengths), which were then used to evaluate the distance effect of two epitopes. Sandwich ELISA for SEM derivatives was developed using an anti-SEM-NBA antibody and horseradish peroxidase-labeled avidin or anti-biotin antibody as a secondary conjugate. The advantageous distances of the two epitopes under the double-antibody sandwich and antibody-avidin sandwich modes were ≥12 and ≥13Å, respectively. Under the distances, the sensitivities of the sandwich ELISA were no lower than those of competitive ELISA. The obtained optimal EC50 values were 11.2pg/mL (double-antibody sandwich with the epitope distance ≥16Å) and 7.3pg/mL (antibody-avidin sandwich with the epitope distance ≥17Å). Compared with competitive ELISA, the developed method achieved a 30-fold improvement in sensitivity, with simpler aquatic product pretreatment.
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