Development of an enzyme linked immunosorbent assay and an immunochromatographic assay for detection of organophosphorus pesticides in different agricultural products

Abstract

Objective: Organophosphorus (OP) pesticides are considered hazardous substances because of their high toxicity to nontarget species and their persistence in the environment and agricultural products. Therefore, it is important to develop a rapid, sensitive, and economical method for detecting OP pesticides and their residues in food and the environment.

Methods: A broad, selective monoclonal antibody (MAb) for organophosphorus pesticides was produced. Based on the MAb, an enzyme linked immunosorbent assay (ELISA) and an immunochromatography assay (ICA) for detecting OP pesticides in different agricultural products were developed using a binding inhibition format on microtiter plates and a membrane strip, respectively.

Results: Under the optimized conditions, the IC(50) values of the ELISA ranged from 3.7 to 162.2 ng mL(-1) for the 8 OP pesticides. The matrix interferences of Apple, Chinese cabbage, and greengrocery were removed by 40-fold dilution, the recoveries from spiked samples ranged from 79.1% to 118.1%. The IC(50) values of ICA for the 8 OP pesticides ranged from 11.8 to 470.4 ng mL(-1). The matrix interference was removed from the Chinese cabbage and Apple samples with 5-fold dilution, and the interference was removed from the greengrocery samples with 20-fold dilution. The recoveries from the spiked samples ranged between 70.6 and 131.9%. The established ELISA and ICA were specific selectivity for the 8 OP pesticides.

Conclusions: The established ELISA is a sensitive screening method for the detection of OP pesticides, but the ELISA detection method depends on a laboratory platform and requires a relative long assay time and several steps operation. The established ICA is very useful as a screening method for the quantitative, semi-quantitative or qualitative detection of OP pesticides in agricultural products, and it has advantages over ELISA methods with regard to factors such as the testing procedure, testing time, and matrix interferences, among others.

Figure 1. Molecular structures of the haptens.

Hapten 1 was used as immunizing hapten and homologous coating hapten, hapten 2 to 12 were used as heterogenous coating hapten candidate. ni: no atom.

Figure 2. Schematic diagram of ICA test strip and result judgment.

The ICA test strip was composed of the NC membrane, MAb-gold-conjugated pad, sample pad and absorbent pad, which were pasted onto an adhesive plastic backing. Result judgment was based on the color changed on the T and C line zone.

Figure 3. Flocculation curves.

Each point on the y axis represents the mean D-value of absorbance (528 nm) in two stages (for details, see materials and methods), and the x axis values are the concentration of the mAb (n = 3).

Figure 4. Reading time curves.

Each point on the y axis represents the average value for the G/Peak - ROD, and the x axis values are the time after the addition of the working buffers (n = 3).

Figure 5. Test sensitivity.

A: Concentration range of parathion-methyl standard assayed by the ICA test strip; B: Relative optical density (ROD) curves of parathion-methyl; C: Standard inhibition curves of parathion-methyl; D: The calibration curve from “A”; “B/B0” is binding ratio of antibody/antigen on the test line.

Figure 6. Matrix interference study and correlation of recoveries between the ICA and ELISA.

A: Standard inhibition curves for parathion-methyl in the buffer and different matrices using the multi-analyte ICA, Chinese cabbage and Apple samples were diluted 5-fold, and greengrocery samples were diluted 20-fold; B: Standard inhibition curves for parathion-methyl in the buffer and different matrices using the multi-analyte ELISA, Chinese cabbage, Apple and greengrocery samples were diluted 40-fold; C: The degree of correlation between the multi-analyte ICA and ELISA for analyses of samples spiked with parathion-methyl, parathion, and fenitrothion.

Figure 7. Matrix interference of greengrocery in ICA test strip.

ICA test strip detection of parathion-methyl with 10-, 20-, 40-fold dilutions of greengrocery matrix interference and buffer. Standard solutions of parathion-methyl at each final concentration of 128, 64, 32, 16, 8, 4, 2 and 0 ng mL-1 (numbers across the top of strips from left to right) were tested.