Design and synthesis of immunoconjugates and development of competition inhibition enzyme-linked immunosorbent assay (CIEIA) for the detection of O-isopropyl methylphosphonofluoridate (sarin): An organophosphorous toxicant

• Published in: Journal of hazardous materials 2011-09, Vol.192 (3), p.1720-1728
• Main Authors: Sathe, Manisha, Merwyn, S., Ghorpade, R., Agarwal, G.S., Rao, M.K., Rai, G.P., Kaushik, M.P.
• Format: Article
• Language: English
• Subjects: Animals; Antibodies; Antibodies - chemistry; Applied sciences; Bioconjugation; bovine serum albumin; Cattle; Chemistry Techniques, Analytical; cross reaction; detection limit; enzyme-linked immunosorbent assay; Enzyme-Linked Immunosorbent Assay - methods; Exact sciences and technology; Female; Gas Chromatography-Mass Spectrometry - methods; Hapten; haptens; Haptens - chemistry; Immunoconjugates - chemistry; Immunoconjugates - immunology; Inhibitory Concentration 50; Models, Chemical; Organophosphorus Compounds - analysis; ovalbumin; Ovalbumin - chemistry; pesticides; Pollution; Polyclonal antibody; Protein; Rabbits; Reproducibility of Results; Sarin; Sarin - analysis; Serum Albumin - chemistry; Spectrophotometry, Ultraviolet - methods; Temperature; toxic substances; Toxicant
• ISSN: 0304-3894; 1873-3336
• DOI: 10.1016/j.jhazmat.2011.07.011

[Display omitted] ► Production of polyclonal antibodies against organophosphorous toxicant sarin (GB). ► The haptens used in the present studies have not been studied so far. ► Antibodies showed negligible cross reactivity (CR) with other OP toxicants. ► CIEIA to distinguish GB agent in the presence of other OP toxicants or pesticide. ► Polyclonal antibodies for the selective detection of sarin. Three haptens of the organophosphorus (OP) toxicant ‘sarin’ having different spacer arm were designed and synthesized. Haptens were conjugated with BSA (bovine serum albumin) and ovalbumin (OVA) for raising antibody and coating antigen. High antibody titer with higher specificity was obtained from 4-(4-(isopropoxy(methyl)phosphoryloxy)phenylamino)-4-oxobutanoic acid (hapten B) having reasonable long spacer arm. For the standard curve, an IC 50 (inhibitory concentration) of free antigen was found to be 0.415 μg mL −1 on the basis of indirect competitive ELISA. The study revealed that heterology in competition inhibition enzyme immunoassay (CIEIA) produced remarkable improvement in the sensitivity and specificity of the assay. Under the optimized conditions, the quantitative working range was found to be 0.19–1.56 μg mL −1 with a limit of detection (LOD) of 0.05 μg mL −1. The antibodies showed negligible cross reactivity (CR) with other OP toxicants and pesticides, which makes the assay suitable for the selective detection of sarin.