Validation of a Commercially Available Human Immunoturbidimetric Assay for Haptoglobin Determination in Canine Serum Samples

Abstract

Haptoglobin is a positive acute-phase protein with a valuable role as a marker of inflammation in both human and veterinary medicine. The aim of this study was to validate a commercially available immunoturbidimetric method designed for human haptoglobin determination (Izasa SA, Barcelona, Spain) for its use in canine samples. Cross-reactivity between anti-human haptoglobin antiserum and canine haptoglobin was found when agarose gel immunodiffusion and ELISA tests were performed. The use of canine pooled serum with haptoglobin concentration of 6.3 g/L as standard provided higher analytical range than commercially available standards. Intra-assay and inter-assay coefficients of variation were 2.49% and 4.60%, respectively. A linear regression model between immunoturbidimetric results and a previously validated spectrophotometric method (Tridelta Development Limited, Ireland) yielded a slope at 95% confidence interval of 0.94 (0.86, 1.02) and y-intercept at 95% confidence interval of 0.11 (-0.59, 0.82). No significant differences were produced by anticoagulants, lipaemia and bilirubinaemia, although haemolysis significantly decreased haptoglobin. A significant increase of haptoglobin concentration was detected in inflammatory conditions such as pyometra and leishmaniasis, in neoplastic conditions, and after glucocorticoid administration. Canine serum haptoglobin concentration can be reliably measured using the commercially available Izasa immunoturbidimetric method developed for human haptoglobin determination. This method is precise and accurate, provides a wider analytical range than previous reported methods, and can be easily automated and used for routine haptoglobin determination in canine samples.

Validation of an enzyme-linked immunosorbent assay for measurement of feline haptoglobin

“…Based on haptoglobin concentrations measured in the ELISA, cats with marked acute phase reactions could be differentiated from healthy cats and diseased cats without systemic inflammation as already demonstrated in dogs. 11,41 As expected, the observed increases were of lesser magnitude than others reported in positive major APPs, such as serum amyloid A in cats. 14,39 However, measuring haptoglobin as a positive moderate APP together with a major APP is recommended to create APP profiles.…”

“…3,4,9 Assays for the detection of haptoglobin in serum or plasma can be classified into 2 main groups: 1) spectrophotometric assays, which are based on the ability of haptoglobin to bind hemoglobin 11,17 ; and 2) immunoassays. 3,41 A commercially available multispecies-specific kit based on the spectrophotometric method has been used to measure feline haptoglobin. 13,23,24,28 This kit has only been validated independently in canine serum, demonstrating reliability for detecting canine haptoglobin.…”

Acute Phase Protein Response in the Capybara (Hydrochoerus Hydrochaeris)

“…The basal concentration of these proteins in healthy animals can vary, depending on the animal species. For example, in sheep and cattle, levels of Hp in serum from healthy animals are not detectable, whereas in other species, such as pigs and dogs, Hp is quantifiable in healthy animals, with values of up to 3 g/l Tecles et al, 2007a, c). Conversely, no significant differences were found between male and female basal values (data not shown), suggesting that sex has no influence on the concentrations of Hp, ASG, or albumin in healthy capybaras.…”

“…A second pool of plasmas with expected high APP concentrations was obtained from animals with induced experimental inflammation after administration of turpentine oil. Precision, linearity, and the limit of detection were calculated for each protein following previously published protocols (Tecles et al, 2007a).…”

Validation of an Automated Method for Salivary Alpha-Amylase Measurements in Pigs (Sus Scrofa Domesticus) and its Application as a Stress Biomarker

“…For the validation of the method, previously described protocols were used, 23,24 and the following parameters were evaluated: 1) precision, 2) linearity under dilution, and 3) detection limit. For precision, 4 pools of saliva, 2 with high and 2 with low alpha-amylase activity were used for the precision studies.…”