Homologous coating antigen AFB1-BSA (OAE) of AFB1 mAb was diluted to a concentration of 2

Homologous coating antigen AFB1-BSA (OAE) of AFB1 mAb was diluted to a concentration of 2.0, 1.0, 0.5, 0.25, 0.125, 0.0625 mg/mL, and coated on the T1 line of the NC membrane. 2A11, 2F6, and 3G2 for AFB1 and 2B6, 4D9 for ZEN were filtered by an indirect non-competitive enzyme-linked immunosorbent assay (inELISA) and an indirect competitive enzyme-linked immunosorbent assay (icELISA), respectively. As AFB1 mAb 2A11 and ZEN mAb 2B6 had the lowest 50% inhibitive concentration (IC50) and cross-reactivity (CR), they were selected for subsequent experiments. By systematically optimizing the preparation condition of gold nanoparticles (AuNPs), AuNPs-labeled mAbs, and detection condition, the visual limit of detection (LOD) of the dual test strip was 1.0 g/L for AFB1 and 5.0 g/L for ZEN, whereas that of the test strip reader was 0.23 g/L for AFB1 and 1.53 g/L for ZEN. The high reproducibility and stability of the dual test were verified using mycotoxin-spiked samples. The dual test strips were highly specific and sensitive for AFB1 and ZEN, which were validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Thus, the proposed AFB1 and ZEN dual test strip is suitable for rapid and simultaneous detection of AFB1 and ZEN contamination in food and feed samples. Keywords: mycotoxins, highly sensitive and specific monoclonal antibodies, dual lateral flow immunochromatographic assay, immunoassay, agro-products 1. Introduction Mycotoxins are low-molecular-weight, toxic secondary metabolites produced under particular environmental conditions by various fungi such as < 0.05) and 128.66% (< 0.05), respectively, and the CRs Rabbit Polyclonal to DNA-PK against AFB1 analogs of 2A11 (0.91C4.35%, mean 2.52%) was lower than that of 2F6 (0.98C4.65%, mean 2.72%) and 3G2 (0.96C14.36%, mean 3.15%) by 7.94% (< 0.05) and 25% (< 0.05), respectively. The lowest IC50 and CR were observed for mAb 2A11. Accordingly, mAb 2A11 was selected for the subsequent studies analyses. Likewise, the IC50 of ZEN mAb 2B6 (10.38 g/L) was lower than that of 4D9 (17.23 g/L) by 65.99% (< 0.05), and the CRs against ZEN analogs of 2B6 (1.28C4.27%, mean 2.308%) was lower than that of 4D9 (1.35C4.88%, mean 2.546%) by 10.31% (< 0.05). Therefore, mAb 2B6 was selected for further experiments (Table 2). Table 1 The IC50 and CRs of the three AFB1 mAbs against AFB1 analogs. for 30 min at 4 C and evaluated using UV. The pH at SB-334867 free base which the highest absorbance occurred was the optimal pH. The optimal amount of AuNPs for labeling mAb was also determined. Briefly, 0.5 mL of SB-334867 free base AuNPs solution at the optimal pH was added to eight Eppendorf (EP) tubes (1.5 mL). AFB1 mAb (or ZEN mAb) (0.1 mL, 1.0 mg/mL) was added to the first tube and then double-diluted to the eighth tube. The mAb SB-334867 free base contents were 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.7825, and 0.391 g, respectively. After thoroughly mixing, the tubes were left to stand for 30 min. NaCl solution (0.1 mL, 10%) was then added, thoroughly mixed, and let to stand for 1 h. The mixtures were centrifuged at 1600 for 30 min at 4 C and analyzed using UV. The minimum amount of mAb required for 1.0 mL of AuNPs to reach the equilibrium point of the scanning curve and an increase of 10% on this basis was the optimal amount for labeling mAb. Finally, AuNPs-labelled mAb was prepared and purified. Briefly, 20 mL of AuNPs solution at the optimal pH and optimal amount of AFB1 SB-334867 free base mAb (or ZEN mAb) were mixed in a centrifuge tube and incubated at room temperature for 30 min, and then 2.0 mL of 10% BSA (at 4 C for 30 min. The supernatant was discarded, and the precipitate was resuspended with AuNPs resuspension (0.05 SB-334867 free base M, pH 7.4 borate buffer solution (BBS) comprising Na2B4O7?10H2O 50 mM) containing BSA 0.15 mM, sucrose 87.72 mM, NaN3 0.05 mM). Centrifugation was repeated once. The resuspended solution was filtered using a 0.45 m membrane and stored at 4C.