Technical note: time-resolved fluoro-immunometric assay for intact insulin in livestock species

Insulin levels in ruminants are often very low and hence are difficult to measure with commercially available RIA kits designed for use with human serum or plasma samples. Those assays may also have high cross-reactivity with nonintact insulin. An assay originally invented for human insulin and based on a pair of monoclonal antibodies binding to specific parts of the insulin molecule was further developed and validated for use with bovine or porcine plasma or serum. The assay is of the sandwich type, with the catching antibody coated to the solid phase of microtiter plate wells and with the detecting antibody labeled with europium, and measured as time-delayed fluorescence. The assay protocol includes an incubation step in which plasma samples of 50 microL are incubated with buffer and detecting antibody for 3 h in coated wells, followed by an enhancement step in which the fluorescence from the europium label is stabilized before measurement. This gives a sensitivity of 3 pmol/L and a possible working range up 16,700 pmol/L. There is no cross-reactivity with pro-insulin or IGF-I. Calibrators are prepared in heat-inactivated serum from the relevant species. Porcine and bovine insulin have different calibration curves; porcine insulin is more reactive and has a higher background than bovine insulin. Validation results show low CV values, parallel dilution of samples, and a recovery ratio close to unity. Comparison with a commercial RIA shows good agreement, except at low concentrations, at which the RIA determinations are inaccurate. Plasma samples from other domestic species (horse, sheep, goat, and mink) have also been assayed, but it is emphasized that calibrators should be prepared in heat-inactivated serum from the appropriate species, and preferably insulin from that species should be used for calibration.