Possible ways of improving enzymeimmunoassay (EIA) techniques and their application in animal production

To achieve reliable EIA techniques as alternatives to RIA procedures for evaluation of endocrine events in reproduction or for control of residues of anabolic agents, several investigations were performed using the microtitration plate technique. In association with progesterone determination in skimmed milk, kinetic studies were carried out. The speed of the immune reaction is proportional to the concentration of ligands (hormone, tracer, antibody). Therefore, equilibrium of immune reactions is reached later if a progesterone assay is done with skimmed milk instead of final stripping, which has a high but variable fat content and hence a higher progesterone content. A former incubation technique with direct coating of the hormone-specific antibody on to the wells was compared with a new second antibody technique. In the latter technique an excess of affinity purified sheep IgG (anti-rabbit-IgG) is first coated onto the wells, followed by the addition of hormone plus tracer and hormone-specific antibody from rabbits. Variabilities of colour readings were reduced in the new technique (3.2% versus 11.4%) and about 10 to 30 times less antibody was required. The second antibody technique was used for the determination of several hormones (progesterone, trenbolone, diethylstilbestrol, 19-nortestosterone and 13,14-dihydro-15-keto-prostaglandin F2α=PGFM). Comparison of alkaline phosphatase (AP) and horseradish peroxidase (HRP) showed that a similar sensitivity was obtainable with both enzymes. However, the phosphatase system was more practicable as the nitrophenylphosphate solution is stable for several days and phosphatase is insensitive to light and aspirin (stabilizer in prostaglandin assays), whereas peroxidase is inactivated by both and the substrate solution is stable for only a few hours. A direct test for PGFM was established using 2 to 20 μL of plasma per well. Unspecific interferences could be avoided only if 2 μL/well were analysed. A detection limit of ∼ 2 fmol/well (1000 fmol/mL) enables demonstration of PGFM peaks connected with luteolysis. (author)