Development of a colloidal-gold based lateral-flow immunoassay kit for ‘quality-control’ assessment of pyrethroid and endosulfan formulations in a novel single strip format

A simple lateral-flow immuno-chromatographic ‘dipstick’ kit was developed in a novel single strip format to test/verify the label claim of active ingredient in commercial insecticide formulations of endosulfan and the type-II synthetic pyrethroids, cypermethrin, deltamethrin and fenvalerate. Polyclonal antisera were raised in rabbits against immunogens developed by conjugating proteins (bovine serum albumin, BSA and keyhole limpet hemocyanin, KLH) to endosulfan-diol, 4′-hydroxy-cypermethrin and 2′-hydroxy-cypermethrin using an activated ester method through a spacer attachment. Immunoglobulin (IgG) was purified through protein-A affinity chromatography and conjugated to horse radish peroxidase (HRP) and colloidal gold. A competitive ELISA was designed to determine the ability of the test insecticides to compete with the relevant hapten–protein conjugate. A combination of antiserum-4′-OH-Cyper-KLH with the 4′-OH-Cyper-BSA as coating hapten resulted in the highest absorbance (Amax: 1.32) low background value (Amin: 0.05) and good competitive inhibition with cypermethrin (IC99: 2034 μg/L), deltamethrin (IC99: 2297 μg/L) and fenvalerate (IC99: 3546 μg/L). For endosulfan, a combination of antiserum raised against endosulfan-KLH with endosulfan-BSA as the coating hapten gave the highest absorbance (Amax: 1.42), low background value (Amin: 0.05) and good competitive inhibition with endosulfan (IC99: 2378 μg/L). A novel format was designed using the combinations for development of the lateral-flow strip, so as to enable the detection of pyrethroids and endosulfan on a single strip. The strips were designed such that two intensely coloured purple lines appeared when the strip is dipped in a solution devoid of any insecticide. The minimum concentration of the test insecticides, endosulfan (1800 μg/l), cypermethrin (800 μg/l), deltamethrin (1000 μg/l) and fenvalerate (1400 μg/l) was determined, which would be adequate to inhibit the appearance of either of the two lines, when the strip is dipped in a solution containing any one of the diluted insecticides prepared from authentic insecticide formulations. When samples of substandard insecticide formulations are diluted as per the pre-determined dilutions, the aliquots are expected to contain less than the quantity specified above, and would result in the appearance of two purple lines, with one of them at the sample line region of the membrane, the intensity of which would be inversely proportionate to the concentration of the test analyte. The strip can be considered as suitable for on-the-spot analysis of test sample formulations either by farmers, extension workers, quality control officials etc., to generate preliminary prima facie evidence in case of doubtful samples of substandard insecticide formulation.