The Preparation and Identification of a Monoclonal Antibody against Citrinin and the Development of Detection via Indirect Competitive ELISA

• Main Authors: Shimuye Kalayu Yirga, Sumei Ling, Yanling Yang, Jun Yuan, Shihua Wang
• Format: Article
• Language: English
• Published: MDPI AG 2017-03-01
• Series: Toxins
• Subjects: citrinin; conjugation; cell fusion; monoclonal antibody; ic-ELISA
• Online Access: http://www.mdpi.com/2072-6651/9/3/110

Citrinin (CTN) is a hepato-nephrotoxic mycotoxin produced by fungi genera of Aspergillus, Monauscus, and Penicillium. CTN contaminates grains, fruits, juices and vegetables, and causes various toxic effects to humans and animals. It has small molecular weight, which is non-immunogenic to animals. Thus, CTN was conjugated to bovine serum albumin (BSA) and ovalbumin (OVA), respectively, by amide bonds using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Mice were immunized with CTN-BSA conjugates, and spleen cells of the immunized mice were fused with Sp2/0 myeloma cells to obtain 21H27 hybriodoma cell. Ascitic fluid of hybridoma cell was produced in mice abdomen, and purified using caprylic/ammonium sulfate precipitation method. The 21H27 anti-CTN mcAb was the IgG2a antibody subclass, and cross-reactivity results indicated that anti-CTN mcAb is specific to CTN with high affinity (2.0 × 108 L/mol). Indirect competitive ELISA (ic-ELISA) results showed that the linear range of detection was 0.01–5.96 ng/mL and the IC50 was 0.28 ng/mL with a lower detection limit (LOD) of 0.01 ng/mL. The average recovery was 93.8% ± 1.6% with a coefficient variation of 1.0%–4.3%. Hence, anti-CTN mcAb secreted by 21H27 hybridoma cell was successfully produced and can be used to detect CTN contaminated feed and foodstuffs.