A time-resolved immunofluorometric assay for quantification of the bovine collectin conglutinin
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Published on: December 2, 2010
T Krogh-Meibom1, U Holmskov, P Løvendahl
1Department of Animal Health and Welfare, Danish Institute of Agricultural Sciences, Research Centre Foulum, P.O. Box 50, DK-8830 Tjele, Denmark.
Insights
A new time-resolved immunofluorometric assay (TRIFMA) accurately measures bovine conglutinin. This reliable assay is suitable for various plasma concentrations and sample conditions.
Area of Science:
- Veterinary immunology
- Biochemical assay development
Background:
- Conglutinin is a bovine plasma protein crucial for innate immunity.
- Accurate quantification of conglutinin is essential for understanding bovine immune responses.
Purpose of the Study:
- To develop and validate a high-capacity, time-resolved immunofluorometric assay (TRIFMA) for quantifying bovine conglutinin.
- To establish the assay's performance characteristics, including sensitivity, specificity, and reliability.
Main Methods:
- A non-competitive sandwich assay format was employed using polyclonal capture antibodies and a monoclonal detection antibody.
- The assay utilized biotin-labeled secondary antibodies and streptavidin-Eu(3+) for enhanced fluorescence detection on an automated system.
- Plasma samples were analyzed after overnight incubation with immobilized antibodies.
Main Results:
- The developed TRIFMA demonstrated a low detection limit (0.34 ng/ml) and a broad working range (0.80 ng/ml–0.20 µg/ml).
- Excellent assay precision was observed with intra-plate and inter-plate coefficients of variation ranging from 5.0–8.3%.
- High recovery rates (90.9–98.8%) and no observed circadian rhythm in plasma conglutinin levels were noted.
Conclusions:
- The developed TRIFMA is a specific, reliable, and sensitive method for measuring bovine conglutinin in plasma.
- The assay's robustness allows for sample storage at -20°C and tolerance to repeated freeze-thaw cycles.
- This assay provides a valuable tool for immunological research and diagnostics in cattle.
Abstract:
A high capacity time-resolved immunofluorometric assay (TRIFMA) for the bovine collectin conglutinin was developed. The TRIFMA was constructed as a non-competitive sandwich assay based on polyclonal antibodies as the capture reagent and a novel monoclonal antibody raised against conglutinin as the detection reagent and was set up to run on an automatic analyzer designed for the TRIFMA detection system. Polyclonal antibodies immobilized on microtiter plate wells were incubated overnight at 4 degrees C with diluted plasma samples, including quality controls (QC) and dilutions of a plasma with known conglutinin concentration. Conglutinin was sandwiched between the capture antibodies and the monoclonal antibody and the detection 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 0.34 ng/ml and the working range 0.80 ng/ml-0.20 microg/ml. Intra-plate and inter-plate coefficients of variation (CV) were in the range of 5.0-8.3% and 6.2-7.2%, respectively, at concentrations of 3.4 and 150 ng/ml. Recovery was 90.9+/-2.4% and 98.8+/-2.5% when samples were spiked with 20 ng/ml and 100 ng/ml purified bovine conglutinin (BK). No circadian rhythm (24-h variation) in conglutinin plasma levels was observed across animals, indicating that the plasma levels were not influenced by, e.g. feeding. Samples could be stored at -20 degrees Celsius and were not sensitive to repeated freezing and thawing. In conclusion, the developed TRIFMA for bovine conglutinin is specific and reliable over a measurement range covering most situations.
