Development of a sensitive heterologous ELISA method for analysis of acetylgestagen residues in animal fat

A new heterologous ELISA method for detecting acetylgestagen multi-residues in animal fat was developed. An antibody generated has high cross-reactivity with relative progestogens (up to 50%) and no cross-reactivity with other steroids (<0.1%) in homologous ELISA. Three heterologous immunoassay were developed and one of them improved not only sensitivity but also the class-selectivity compared with the homologous assay against these progestogens. The IC₅₀ for four acetylgestagens, chlormadinone acetate (CMA), 17α-hydroxyprogesterone acetate (HPA), megestrol acetate (MEGA) and medroxyprogesterone acetate (MPA) were, 4.5, 2.5, 2.9 and 1.8 μg/L, respectively. The cross-reactivity for CMA, HPA and MEGA were 40%, 72% and 62% when they were compared with MPA. Progestogens recoveries from spiked swine fat averaged between 61% and 78%. Results obtained from LC/MS/MS method showed the heterologous ELISA method developed was reliable and suitable for rapid screening the four progestogens residues in fat tissues.