Evaluation of an enzyme-linked immunosorbent assay for determination of porcine haptoglobin.
An enzyme-linked immunosorbent assay for quantification of haptoglobin in porcine serum was evaluated. The detection limit when expressed as the estimated concentration of a blank sample was 0.0003 mg/ml. The precision of the assay was acceptable with intra-assay coefficients of variation below 4% and inter-assay coefficient of variation below 5% for serum concentrations ranging from 1.0 mg/ml and above. For samples with a concentration below 0.8 mg/ml, the inter-assay coefficient of variation was above 10% The assay maintained linearity under dilution. Recovery was proportional. Haemolysis significantly decreased the measured concentration of haptoglobin in paired serum samples (one-sided t-test, P < 0.0001, degrees of freedom = 29). No significant effect on the concentration due to repeated freezing and thawing of serum was observed. The biological variation in individual pigs with no clinical signs of disease was estimated by nested analysis of variance. Between-pig variation was 86.0% within-pig variation was 13.3% and analytical variation was 0.7%. The one-sided critical difference was 12.3% and the two-sided critical difference was 14.6%. The index of individuality was 0.2. The maximum allowable analytical imprecision was 2.6% and the maximum analytical inaccuracy was 9.9%. The number of samples required to determine the true haptoglobin value in an individual pig when accounting for the day-to-day fluctuation was 5. In conclusion, the haptoglobin assay was found to be suitable for quantification of haptoglobin in non-haemolysed porcine serum samples with a haptoglobin concentration above 1.0 mg/ml. The analytical variance was found to be low. The haptoglobin concentration in serum was found to characterize individual animals. A large inter-animal variation in haptoglobin level was found.
Petersen
H H
HH
Royal Veterinary and Agricultural University, Dep of Clinical Studies, Large Animal Medicine, Frederiksberg, Denmark. hhp@kvl.dk
Nielsen
J P
JP
Jensen
A L
AL
Heegaard
P M
PM
D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary*; D005260:Female; D006242:Haptoglobins / Q000032:analysis* / Q000276:immunology; D006461:Hemolysis; D008297:Male; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013552:Swine / Q000097:blood*
D006242:Haptoglobins