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

Abstract

Abstract. Haptoglobin is a positive moderate acute phase protein (APP) in cats. Measurement of haptoglobin can be used in the diagnosis, prognosis, and monitoring of systemic inflammatory disease, especially by creating profiles with major APPs. The aim of our study was to validate a sandwich enzyme-linked immunosorbent assay (ELISA) for measurement of feline haptoglobin. The validation included an assessment of precision, accuracy, detection limit, method comparison with a spectrophotometric assay, and evaluation of the overlap performance. The concentration of haptoglobin was measured in serum from 27 healthy and 23 sick cats. The coefficients of variation were 2.5-4.7% for intra-assay variability and 7.1-11.6% for interassay variability. The ratio of observed to expected dilutional parallelism of 4 serum samples was 108.1-118.4%. The ratio of observed to expected spike recovery of 4 serum samples was 90.8-94.0%. The lower detection limit was 0.19 g/L. Method comparison revealed a positive correlation (r s = 0.949, P < 0.0001) and a proportional bias between the methods of −38.9%. Agreement between the methods was not clinically acceptable. Overlap performance of the ELISA was deemed satisfactory. The sandwich ELISA measures feline haptoglobin with an analytical and overlap performance acceptable for clinical purposes. Given the observed bias, the ELISA cannot be used interchangeably with the spectrophotometric assay.

Serum haptoglobin concentrations in feline inflammatory bowel disease and small-cell alimentary lymphoma: a potential biomarker for feline chronic enteropathies

“…The use of the ELISA method described has only been validated in cats by a single study. 18 This could be viewed as a limitation of its potential accuracy. Also, samples obtained for this study were performed via endoscopy and were not full-thickness surgical biopsies.…”

“…The assay is a feline-specific sandwich ELISA for the quantitative determination of HPT in feline serum and has been previously validated by others for use in cats. 18 The assay has low intra- and inter-assay precisions of 3.3% and 8.6%, respectively. Dilution linearity ranged from 90.4% to 102.9%, and spike recovery ranged from 98.4% to 103.8%.…”

“…Haptoglobin (HPT) is a moderate, positive acute-phase protein with immunomodulating properties found in a variety of species, including humans and cats. 17–23 Its concentration decreases slowly in serum, compared with major acute-phase proteins, 16,19 making it a more accurate predictor of chronic or ongoing inflammatory disease processes. In humans, HPT has been found to be increased in a variety of inflammatory processes, including IBD, 23 and it has also been found to be elevated in a variety of malignancies, including lymphomas and leukemias.…”

“…22,24,25 Stiller et al validated the use of the sandwich ELISA for the measurement of HPT in cats for clinical use. 18 Several studies have confirmed that serum HPT (sHPT) is elevated in ‘sick’ cats with a variety of inflammatory disease processes; 19–21,26–29 however, none has directly evaluated sHPT in cats with chronic enteropathies or neoplasia.…”

Serum Insulin-like Growth Factor-1 Is a Biomarker of Testosterone Production and Intact Acrosome in Asian Elephants (Elephas maximus)

“…The intra-assay CV was evaluated from the CV of the same samples three times in duplicates. The total imprecision assay CV(CV total ) was calculated: CV total [ 38 ]. Acceptable intra- assay and inter-assay CV were less than 10% and 15%, respectively.…”