Purification and immunoactivity identification of rabbit-derived polyclonal antibody against peanut allergen Ara h 3

The peanut allergen protein Ara h 3 is widely present in various peanut products, and even a small intake can trigger allergic reactions, posing a serious threat to the health of peanut allergy patients. To enable accurate detection of Ara h 3 for immunological analysis, it is essential to prepare antibodies with high purity and specificity. Using defatted peanut powder as the raw material, centrifugation was performed at a solid-to-liquid ratio of 1∶10 to eliminate insoluble proteins, followed by dialysis for 48 hours. Anion exchange chromatography using a HiTrap Q HP column was subsequently employed to separate peanut allergens with isoelectric points differing markedly from that of Ara h 3. During gradient elution with varying sodium chloride concentrations, three distinct peaks were observed in the separation profile. Following electrophoretic analysis, fractions containing high levels of Ara h 3 in the eluate were pooled. The pooled eluate was filtered through a 0.45 μm membrane and injected into a HiLoad Superdex 200 pg preparative Size Exclusion Chromatography column equilibrated with Tris-HCl buffer (10 mmol/L, pH 7.9). Elution was continued with the same buffer, and multiple distinct peaks were observed in the separation profile. Fractions corresponding to these peaks were analyzed by electrophoresis, and those with high purity were pooled. The pooled fractions were concentrated using polyethylene glycol (PEG, 20,000) to obtain highly purified peanut allergen Ara h 3. This purified Ara h 3 was conjugated to CNBr-activated Sepharose^TM 4B, which had been activated overnight with HCl (2 mmol/L). Laboratory-prepared serum underwent ammonium sulfate fractionation to remove impurities. The resulting ammonium sulfate precipitate was injected into the prepared immunoaffinity column. Different elution buffers were used to selectively elute non-specific and specific components, yielding high-purity, highly specific anti-Ara h 3 polyclonal antibodies. The protein mass concentration eluted by Gly-HCl was 0.6 mg/mL. The purified protein was concentrated using polyethylene glycol (PEG, 20,000) and stored at -20 ℃ for subsequent use. SDS-PAGE analysis confirmed that most non-specific substances remaining after ammonium sulfate precipitation were effectively removed through immunoaffinity purification, significantly enhancing antibody purity. Western blot and ELISA test results demonstrated that the purified antibodies maintained high specificity following immunoaffinity purification, with a titer reaching 1∶51 200. Compared with the titer achieved by ammonium sulfate precipitation, the titer of the antibody obtained via immunoaffinity chromatography was markedly improved. The successful purification of the antibody provides a foundation for the development of a test strip for detecting the peanut allergen Ara h 3.