An Automated Biochemical Assay for Haptoglobin: Prevention of Interference from Albumin

Abstract

Abstract. Measurement of the acute phase serum protein, haptoglobin (Hp), is performed by biochemical methods based on haemoglobin binding, in many veterinary diagnostic laboratories. During attempts to develop a robust biochemical assay for serum Hp it was discovered that serum albumin interfered with the assay system increasing results by as much as 0.28 mg/ml, which could affect interpretation of results especially in species with low normal Hp concentrations. A reagent cocktail (SB-7) was devised which inhibited the interfering effect of albumin. An automated assay for Hp utilising SB-7 was developed for production as a biochemical assay kit and was evaluated for use in veterinary diagnosis. The intra-assay coefficients of variation were of 0.9%, 0.9% and 1.3% for Hp concentrations of 2.0, 1.0 and 0.23 mg/ml, respectively and interassay coefficients of variation of 1.7% and 4.5% for Hp of 2.08 mg/ml and 0.24 mg/ml, respectively. The lower limit of detection of was 0.02 mg/ml, linearity extended to 8 mg/ml and recovery was 101±7% (mean ± SD). The assay had correlation coefficients (R 2 ) of 0.96 and 0.90 when compared with immunodiffusion assays of canine Hp and bovine Hp, respectively. Lipaemia and bilirubinaemia caused no interference. Haemolysis did not affect measurement of low levels of Hp, but at serum Hp concentrations of 0.4 and 1.8 mg/ml the apparent Hp concentration was decreased. Elevated concentrations of Hp were measured in cattle with mastitis, dogs with polyarthritis and rats experimentally infected with Bordetella pertussis. The automated assay is precise and has negligible interference from albumin.

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

“…Intra-assay and inter-assay CVs were lower than 5% and similar to those reported by other authors (Wiedmeyer and Solter, 1996;Eckersall et al, 1999), except for inter-assay CV for samples with Hp concentration lower than 3 g/L. The higher between-run CV observed with samples with low Hp concentration does not have any clinical relevance since these variations only provide values within the canine reference range.…”

“…Hp values for healthy dogs range between 0.0 and 3.0 g/L (Eckersall et al, 1999). Therefore, canine samples from healthy animals could be assayed without previous dilution using the immunoturbidimetric method described here, and even serum samples from diseased animals if Hp is 2-fold above the upper normal range.…”

“…A human Hp immunoturbidimetric method using a polyclonal goat antihuman Hp antiserum (Izasa SA, Barcelona, Spain), and a commercially available spectrophotometric kit (Tridelta Development Limited, Ireland) previously validated for dogs (Eckersall et al, 1999) were used for the determination of Hp in canine serum samples. The analyses were performed using a Cobas Mira Plus multiparametric autoanalyser (ABX Diagnostics, Montpellier, France).…”

“…Spectrophotometric methods are based on the ability of Hp to bind to haemoglobin (Hb). After Hp-Hb complex formation, Hp concentration can be measured by two different ways: (a) Hp-Hb complex formation produces changes in absorbance characteristics of Hb in proportion to the concentration of Hp (Harvey, 1986); (b) use of the innate peroxidase activity of Hp-Hb, which can be quantified at a mildly acidic pH by addition to hydrogen peroxide in the presence of a chromogen at 600 nm wavelength (Makimura and Suzuki, 1982;Eckersall et al, 1999). Immunological methods use specific antibodies against Hp, and the formation of antigen-antibody complexes can be detected and quantified by: (a) addition of a secondary enzyme-labelled antibody against the primary specific anti-Hp antibody (ELISA methods) (Katnik et al, 1998;Yee and Brown, 1999); (b) appearance of spectrophotometrically detectable turbidity in the reaction cuvette due to a precipitate that appear when antigen and antibody are mixed in the correct proportions (immunoturbidimetric methods) (Moseley, 1980).…”

Erythrogram, leukogram, and acute phase protein reference intervals for healthy newborn Murrah buffalo calves (Bubalus bubalis) within the first month of life

“…An ABX Pentra 400 (Horiba ABX SAS, Montpellier, France) was used to analyze serum concentrations of haptoglobin with the methods described in Eckersall et al (1999) ( Table 1). ELISA methods were used to analyze serum concentrations of SAA (Tridelta Development Limited, Maynooth, Co. Kildare, Ireland) and CRP (Life Diagnostics, Inc., West Chester, Pennsylvania, USA) (Table 1).…”