Concentrations of haptoglobin in bovine plasma determined by ELISA or a colorimetric method based on peroxidase activity.
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abstract
The objective was to compare different procedures for determination of haptoglobin in bovine plasma. Nine Angus steers were vaccinated against Mannheimia haemolytica to stimulate an acute-phase response. Blood samples were collected immediately prior to vaccination (day0), and on days1, 3, 5, 7 and 10. Plasma samples were frozen in duplicates at -80C. One set of the duplicates was analysed for haptoglobin concentrations using a commercial ELISA kit. A day effect was detected (p<0.01) because haptoglobin peaked on day3 and returned to baseline on day7 relative to vaccination. The second duplicate was analysed using a procedure that measures haptoglobin-haemoglobin complexing by estimating differences in peroxidase activity (CPPA) with results expressed as optical density. Further, based on the ELISA results, the plasma sample with the greatest haptoglobin concentration was also serially diluted into a plasma sample with negligible haptoglobin concentration from the same steer (1:1 through 1:1024 dilution). These dilutions were used within the CPPA method to generate a standard curve and estimate plasma haptoglobin concentrations (CPPA+STD). A linear standard curve was generated (r(2) =0.99). A day effect similar to the ELISA method was detected for the CPPA and CPPA+STD methods (p<0.01). Results obtained from CPPA and ELISA methods were positively correlated (r=0.97; p<0.01). The values generated by the CPPA+STD procedure were similar (p=0.38) compared to the values generated by the ELISA method. In conclusion, assessing concentrations of haptoglobin in bovine plasma using the CPPA and CPPA+STD methods generate highly correlated or similar results, respectively, compared to ELISA. Therefore, the CPPA+STD and CPPA methods can be used as a less expensive alternative to ELISA to determine concentrations or monitor changes in plasma haptoglobin in bovine samples.
