Plasma interleukin-6 concentration in Standardbred racehorses determined by means of a novel validated ELISA.
Abstract: To evaluate plasma interleukin 6 (IL-6) concentration in Standardbred racehorses by means of a novel ELISA following validation of the assay for use with equine plasma samples. Methods: Plasma samples obtained from 25 Thoroughbreds for use in assay validation and from 319 Standardbred racehorses at rest 2 to 2.5 hours prior to warm-up and racing. Methods: A sandwich ELISA was developed with equine anti-IL-6 polyclonal antibody and the biotin-streptavidin chemical interaction to enhance sensitivity. The assay was validated for specificity, sensitivity, precision, and accuracy by use of both recombinant and endogenous proteins. Results: For the assay, cross-reactivity with other human and equine cytokines was very low or absent. Serial dilution of plasma samples resulted in proportional decreases in reactivity, indicating high specificity of the method. Partial replacement of detection antibody with capture antibody or pretreatment of samples with capture antibody caused assay signals to significantly decrease by 55%. The inter- and intra-assay precisions were ≤ 13.6% and ≤ 9.3%, respectively; inter- and intra-assay accuracies were within ranges of ± 14.1% and ± 8.6%, respectively, at concentrations from 78 to 5,000 pg/mL, and the sensitivity was 18 pg/mL. Plasma IL-6 concentration varied widely among the 319 Standardbreds at rest (range, 0 to 193,630 pg/mL; mean, 6,153 pg/mL; median, 376 pg/mL). Conclusions: This ELISA method proved suitable for quantification of IL-6 concentration in equine plasma samples. Plasma IL-6 concentration was high (> 10,000 pg/mL) in 9.1% of the Standardbred racehorses, which warrants further investigation.
Publication Date: 2015-12-29 PubMed ID: 26709932DOI: 10.2460/ajvr.77.1.13Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
- Research Support
- U.S. Gov't
- Non-P.H.S.
- Antibodies
- Biochemistry
- Clinical Study
- Cytokines
- Diagnostic Technique
- Disease Diagnosis
- Enzyme-Linked Immunosorbent Assay (ELISA)
- Equine Health
- Horse Breeds
- Horse Racing
- Immune Response
- In Vivo
- Inflammatory Response
- Interleukins
- Laboratory Methods
- Physiology
- Plasma
- Standardbred Horses
- Veterinary Care
- Veterinary Medicine
Summary
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The research involves the development and validation of a new enzyme-linked immunosorbent assay (ELISA) for measuring plasma interleukin-6 (IL-6) concentration in Standardbred racehorses. After validation, this ELISA method was used to determine the plasma IL-6 concentration in a large group of Standardbred racehorses.
Development and Validation of ELISA Method
- The researchers developed a novel ELISA method using equine anti-IL-6 polyclonal antibody and the biotin-streptavidin chemical interaction to boost the assay’s sensitivity.
- The validation process tested the assay’s specificity, sensitivity, precision, and accuracy using both recombinant and endogenous proteins.
- The ELISA method showed minimal cross-reactivity with other human and equine cytokines, indicating high specificity.
- Tests conducted to determine assay precision and accuracy showed inter- and intra-assay precisions of ≤ 13.6% and ≤ 9.3%, respectively. The inter- and intra-assay accuracies were within ranges of ± 14.1% and ± 8.6%, respectively, for concentrations ranging from 78 to 5,000 pg/mL. The assay’s sensitivity was measured to be 18 pg/mL.
Determination of Plasma IL-6 Concentration in Standardbred Racehorses
- After successful validation, the researchers used the newly developed ELISA to measure the plasma IL-6 concentration levels in 319 Standardbred racehorses.
- Test results revealed significant variability in IL-6 concentration among the tested Standardbred racehorses. The range was from 0 to 193,630 pg/mL, with a mean of 6,153 pg/mL and a median of 376 pg/mL.
- A noteworthy finding was that the plasma IL-6 concentration was particularly high (>10,000 pg/mL) in 9.1% of the tested Standardbred racehorses, suggesting an area of study that could benefit from further investigation.
Conclusion
- The novel ELISA, validated in this study, was found to be effective and suitable for the quantification of IL-6 concentration in the plasma samples of equine.
- The research identified potential areas of future study, specifically the group of Standardbred racehorses with notably high plasma IL-6 concentration levels.
Cite This Article
APA
Chen JW, Uboh CE, Robinson MA, Jiang Z, Soma LR.
(2015).
Plasma interleukin-6 concentration in Standardbred racehorses determined by means of a novel validated ELISA.
Am J Vet Res, 77(1), 13-23.
https://doi.org/10.2460/ajvr.77.1.13 Publication
Researcher Affiliations
MeSH Terms
- Animals
- Biomarkers
- Enzyme-Linked Immunosorbent Assay / methods
- Enzyme-Linked Immunosorbent Assay / veterinary
- Horses / blood
- Horses / metabolism
- Interleukin-6 / blood
- Interleukin-6 / metabolism
- Reproducibility of Results
- Sensitivity and Specificity
Citations
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