Performance assessment and validation of a sensitive immunoassay for detection of ruminant and porcine heat stable proteins in compound animal feedstuffs

Sandwich ELISAs have been produced to detect ruminant and porcine heat stable proteins which have been heated to 130 degreesC, 2.7 bar (absolute pressure), for 30 mins and incorporated into compound animal feedstuffs (CAFs). Sample preparation involves heat treatment, removal of CAF proteins and analyte concentration. The test system incorporates a separate ELISA to detect non-specific binding of certain plant derived CAF components, each of which are identified. Both ruminant and porcine heat stable antigens are detected at a concentration of 1250 ppm. %CVs obtained within assay were 6.97% and between assay 9.07%. The maximum calculated false negative rate over 3 years of routine operation of the ELISAs was < 1%. Each assay plate incorporates 12 negative controls permitting an additional 3SD calculation to overcome any in-plate variation.