Hrp labeling kit

Monoacetylmorphine (MAM), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), sulfo N-hydroxysuccinimide (sulfo-NHS), bovine serum albumin (BSA), Ovalbumin (OVA), complete Freund’s adjuvant (CFA), incomplete Freund’s adjuvant (IFA), horseradish peroxidase (HRP), goat anti-rabbit IgG-HRP, 3,3',5,5'-Tetramethylbenzidine (TMB) substrate were purchased from Sigma Chemical Co. Chandigarh, India. HRP labeling kit was purchased from (Thermofisher Scientific, USA). Standard heroin, morphine, and codeine samples were provided by the Central Forensic Science Laboratory (CFSL), Chandigarh, India. Q-Sepharose, Protein-A Sepharose, and Sepharose CL 6B were procured from Amersham Biosciences, Chandigarh, India. Super signal west picochemiluminescence substrate was purchased from Pierce Biotechnology, Chandigarh, India. Polystyrene ELISA plates were procured from NUNC, Chandigarh, India.

To produce the ascites fluid, hybridoma cells in medium were intraperitoneally injected into a mature female BALB/c mouse that had previously been injected with 0.5 mL of pristine. After 14 days, the ascites fluid was collected, and monoclonal IgG from ascites was purified using Protein G column chromatography (GE Healthcare). The isotype of the purified antibody was determined using a mouse mAb isotyping kit (Roche) according to the manufacturer's instructions. The ENR mAb was conjugated to horseradish peroxidase (HRP) using an HRP labeling kit (Thermo Scientific), then stored at -20℃ until use.