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Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes
Published on: December 20, 2014
A time-resolved immunofluorometric assay for quantification of collectin-43
T Krogh-Meibom1, U Holmskov, P Løvendahl
1Department of Animal Health and Welfare, Danish Institute of Agricultural Sciences, Research Centre Foulum, DK-8830 Tjele, Denmark.
Insights
A new assay accurately measures bovine collectin-43 (CL-43), a key protein in cattle innate immunity. This reliable method aids in understanding CL-43
Area of Science:
- Veterinary Immunology
- Biochemical Assays
- Innate Immunity
Background:
- Bovine collectin-43 (CL-43) is crucial for cattle innate immunity.
- Variations in CL-43 levels may correlate with bovine diseases.
- A reliable method to quantify CL-43 is needed for disease research.
Purpose of the Study:
- To develop a high-capacity, reliable assay for quantifying bovine CL-43.
- To validate the assay's performance characteristics for clinical and research applications.
Main Methods:
- Developed a noncompetitive sandwich time-resolved immunofluorometric assay (TRIFMA) using polyclonal and monoclonal antibodies.
- Utilized an automated analyzer for high-throughput detection.
- Optimized detection using biotin-labeled antibodies and streptavidin-Eu3+.
Main Results:
- The assay demonstrated a detection limit of 0.24 ng/ml and a working range of 0.54-22 ng/ml.
- High recovery (92.3%) and low coefficients of variation (1.11-2.36% intraplate, 0.70-1.35% interplate) were achieved.
- CL-43 plasma levels showed no circadian variation and were stable during storage and freeze-thaw cycles.
Conclusions:
- The developed TRIFMA is a specific, reliable, and robust method for measuring bovine CL-43.
- This assay provides a valuable tool for investigating the role of CL-43 in cattle health and disease.
- The assay's performance characteristics support its use in diverse research and diagnostic settings.
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-Eu3+. 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 degrees 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.
