Development of an ultrasensitive sandwich immunoassay for detecting small molecule semicarbazide.

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 ≥13 Å, respectively. Under the distances, the sensitivities of the sandwich ELISA were no lower than those of competitive ELISA. The obtained optimal EC50 values were 11.2 pg/mL (double-antibody sandwich with the epitope distance ≥16 Å) and 7.3 pg/mL (antibody-avidin sandwich with the epitope distance ≥17 Å). Compared with competitive ELISA, the developed method achieved a 30-fold improvement in sensitivity, with simpler aquatic product pretreatment.

Upconversion luminescence nanoparticles-based immunochromatographic assay for quantitative detection of triamcinolone acetonide in cosmetics.

Triamcinolone acetonide (TCA) abuse in cosmetics is a common phenomenon. A rapid lateral flow immunochromatographic assay (ICA) was developed for the quantitative detection of TCA using a probe based on upconversion luminescence nanoparticles. Lanthanide-doped upconversion nanoparticles (UCNPs) were synthesized in a system comprising water and ethylene glycol, and a silicon dioxide layer was covered at the carboxyl site. A binding site protection strategy was used to decrease the background signal of UCNPs-ICA. Using dexamethasone derivative as a coating antigen, the optimal UCNPs-ICA exhibits good dynamic linear detection for TCA in the range 1.0-100 ng mL-1 with a median inhibitory concentration of 9.8 ng mL-1. The detection limits for TCA in a cosmetic sample are 20 µg kg-1. The pretreatment of samples only needs dilution with water, suggesting the assay can quantitate TCA on-site using a portable upconversion luminescence reader with a cumulative analysis time of only 10 min.

High-specificity quantification method for almond-by-products, based on differential proteomic analysis.

A highly specific competitive enzyme-linked immunosorbent assay (ELISA) protocol has been developed to identify and classify almond products based on differential proteomic analysis. We applied two-dimensional electrophoresis to compare the differences between almond and apricot kernels to search for almond-specific proteins. The amino acid of apricot Pru-1 was sequenced and aligned to almond Pru-1. One peptide, RQGRQQGRQQQEEGR, which exists in almond but not in apricot, was used as hapten to prepare monoclonal antibody against almond Pru-1. An optimized ELISA method was established using this antibody. The assay did not exhibit cross-reactivity with the tested apricot kernels and other edible plant seeds. The limit of detection (LOD) was 2.5-100µg/g based on different food samples. The recoveries of fortified samples at levels of twofold and eightfold LOD ranged from 82% to 96%. The coefficients of variation were less than 13.0%. Using 7M urea as extracting solution, the heat-treated protein loss ratios were 2%, 5% and 15% under pasteurization (65°C for 30min), baking (150°C for 30min) and autoclaved sterilization (120°C for 15min), respectively.