A monoclonal antibody-based competitive antibody-coated enzyme-linked immunosorbent assay (ELISA) was developed and optimized for determining chlorpyrifos residue in agricultural products. The IC 50 and IC 10 of this ELISA were 3.3 ng/mL and 0.1 ng/mL respectively. The average recoveries in six agricultural products were between 79.5% and 118.0%, with the intra-assay coefficient of variation being less than 8 %. The limit of detection for all tested products was 30 ng/g. To the best of our knowledge, this assay has the best specificity among all the published research on ELISAs for chlorpyrifos.

The mechanism of specific recognition in pesticide immunochemistry was investigated by computer-based strategy, and a rapid method for the identification of antibody specificity was developed. Based on the previously produced anti-parathion monoclonal antibody (mAb), the DNA sequence was analyzed by polymerase chain reaction (PCR). From the translated amino acid sequences, a three-dimensional structure of the antibody was constructed by homology modeling method, and then it was coordinated by 1 ns molecular dynamics under the explicit solvent condition. The stereochemical property and folding quality were further assessed by Procheck and Profile-3D. The self-compatibility score for the antibody model was 98.7, which was greater than the low score 46.2 and close to the top score 102.6. In addition, parathion and several structural analogues were docked to the constructed antibody structure. The docking results showed that the interaction energy (-40.54 kcal/mol) of antibody-parathion complex was the lowest among all the tested pesticides, which accounted for the high specificity of the antibody to parathion and perfectly matched with the experimental data. Moreover, three residues, Phe165, Asp107 and Thr100 were recognized as the most important residues for antibody reacting with parathion. The interaction energy negatively correlated with the antibody specificity.

A new method for specific antibody production was developed using antibody (Ab)-pesticide complex as a unique immunogen. Parathion (PA) was the targeted pesticide, and rabbit polyclonal antibody (Pab) and mouse monoclonal antibody (Mab) were used as carrier proteins. The Ab-PA complexes were generated by conjugating the two antibodies with an excessive dosage of PA. It was shown that the sensitivity of homologous enzyme-linked immunosorbent assay (ELISA) using the new antibodies was similar to that using original antibodies. However, the new mouse Pab had not only similar positive recognizing spectrum as the original Mab, but also a significantly improved sensitivity in heterologous ELISA when some recognizable competitors were applied. IC(50) value of ELISA based on a combination of the new mouse Pab and hapten 9 was 0.24 ng/mL, which was 445.54 times as that of the homologous ELISA. An Ab-pesticide complex may be a suitable alternative immunogen for producing highly specific antibody to improve the immunoassay sensitivity.

Myeloid differentiation factor 88 (Myd88) plays an important role in both innate and adaptive immune response. In this study, the full-length complementary DNA (cDNA) of myd88 from Misgurnus anguillicaudatus was characterized. The myd88 cDNA is 1333 bp in length and contains an 855 bp open reading frame encoding a predicted protein of 284 amino acids. The predicted protein possesses typical Myd88 domain structural features including a death domain in the N-terminus, and box 1, 2, and 3 motifs of the Toll/IL-1 receptor domain in the C-terminus. Quantitative real-time PCR (qRT-PCR) revealed that myd88 messenger RNA (mRNA) was ubiquitously expressed in all examined tissues, especially highly in brain, kidney, blood, intestines and liver. qRT-PCR and western blotting were used to determine the mRNA and protein levels of Myd88 after Aeromonas veronii challenge, respectively. The Myd88 was remarkably upregulated in response to infection of A. veronii. These results suggested that Myd88 may play a vital role during the immune response of M. anguillicaudatus against bacterial infection.

A heterologous indirect enzyme-linked immunosorbent assay (ELISA) was developed, which was based on monoclonal antibody (Mab) to determine parathion residue in agricultural and environmental samples. Eight Mabs were produced by introducing the heterologous indirect ELISA into the screening procedure. It was shown that these Mabs had more broad-reactivity with twenty competitors than that of 5H7 (Mab produced by homologous screening). So it became much easier using these new Mabs to develop heterologous immunoassays. In addition, all the developed heterologous ELISAs could be used to determine parathion residue in semiquantitative or quantitative level, and their detection limitation (LOD) was around 2 ng/mL. Compared with the previous heterologous ELISA (hapten 11/5H7) with IC(50) of 13.3 ng/mL, a better heterologous ELISA (hapten 11/1E1) was obtained with IC(50) of 3.8 ng/mL, which improved the sensitivity 3.5 times. Finally, the latter was applied to parathion residue determination in spiked agricultural and environmental samples without extraction or cleanup. The average recoveries of parathion added to water, soil, cucumber, Chinese cabbage and carrot were between 80.4 % and 111.8 %. The LOD for water and soil samples was 5 ng/mL, and the LOD for cucumber, rice and corn samples was 10 ng/mL.