A time-resolved immunofluorometric assay for quantification of collectin-43

Abstract

CL-43 is a serum collectin involved in the innate immunity of cattle and variability of serum CL-43 may relate to disease in cows. A high capacity time-resolved immunofluorometric assay (TRIFMA) for the bovine collectin-43 (CL-43) was developed. The TRIFMA was constructed as a noncompetitive sandwich based on polyclonal antibodies and a novel monoclonal antibody (mab) raised against CL-43 and was set up to run on an automatic analyser designed for the TRIFMA detection system. The polyclonal antibodies were immobilized on microtiter plate wells and incubated with diluted plasma samples, including quality controls (QC) and dilutions of a plasma with known CL-43 concentration. CL-43 was sandwiched between the capture antibodies and the monoclonal antibody and the detection was optimised with biotin-labelled secondary antibodies and streptavidin-Eu 3+. Plates were washed four times between each step and finally incubated with enhancement solution before measuring the fluorescence. The assay detection limit was 0.24 ng/ml and the working range was 0.54–22 ng/ml. Recovery was 92.3% when samples were spiked with 2.0 ng/ml of CL-43. Intraplate and interplate coefficients of variation were in the range of 1.11–2.36% and 0.70–1.35%, respectively. No circadian rhythm (24-h variation) in CL-43 plasma levels was observed, indicating that plasma levels were not influenced by e.g. feeding. Samples could be stored at −20 °C and were not sensitive to repeated freezing and thawing. In conclusion, the developed TRIFMA for CL-43 is specific and reliable over a measurement range covering most situations.