Validation of new automated turbidimetric immunoassays for the measurement of haptoglobin and inter‐α‐trypsin inhibitor heavy chain H4 specific for the bovine species

Abstract

Background Good strategical programs are required for the early detection of disease even in the absence of evident clinical signs, which is crucial in satisfying animal welfare. Haptoglobin (Hp) and inter‐α‐trypsin inhibitor heavy chain H4 (ITIH4) are acute phase proteins and good biomarkers of early inflammation in cattle, with plasma levels that significantly increase after injury or infection. Objectives We aimed to develop and validate two new immunoturbidimetric methods for Hp and ITIH4. Methods Species‐specific antibodies were obtained and used to develop the immunoassays. For the Hp assay, antibodies were fixed to latex microparticles to enhance detection. The immunoassays were set up in an automated analyzer to carry out validation studies. Reference intervals were calculated using Reference Value Advisor. Results The Hp immunoturbidimetric method had a linear analytical range up to 0.40 mg/mL. The limit of detection (LoD) was 0.005 mg/mL, and the limit of quantification (LoQ) was 0.007 mg/mL. Total imprecision was less than 7%. Comparison with ELISA and single radial immunodiffusion (SRID) showed good correlation, whereas the comparison with the colorimetric method showed constant and proportional differences. The ITIH4 immunoassay showed linearity up to 5 mg/mL, and the LoD was 0.002 mg/mL. Total imprecision was less than 6%. Method comparison showed a good correlation with single radial immunodiffusion, both methods being equivalent. Bilirubin, triglycerides, and hemoglobin presented no interference in any of the assays. Reference intervals were 0.007‐0.017 mg/mL for Hp and 0.2‐0.7 mg/mL for ITIH4 in dairy cows 10 days before parturition. Conclusions Immunoturbidimetric methods developed for Hp and ITIH4 can measure basal and increased levels of these proteins, showing adequate precision, accuracy, and robustness.

Haptoglobin Electrochemical Diagnostic Method for Subclinical Mastitis Detection in Bovine Milk

“…The main features (assay format, detection system, bioreceptor, detection range, LOD, and matrix) of each strategy are detailed in Table 1. The reported methods for Hp detection include different readouts from turbimetric [25,49], colorimetric [50,51] and (chemi)luminescence [27,52] detection, among others. Moreover, the LOD reported are in accordance with the complexity of these strategies.…”

“…This assay showed low sensitivity for Hp and α1-acid glycoprotein and was performed on bovine blood, not milk matrices [24]. More recently, Bassols et al have designed a immunoturbidimetric assay for bovine Hp and α-trypsin inhibitor heavy chain H4, also intended for serum analysis [25]. An SPR sensor for the detection of Hp using haemoglobin as a bioreceptor molecule was developed by Akerstedt et al This sensor presented a limit of detection (LOD) of 1.1 µg mL −1 .…”

Introducing the special issue on acute phase proteins in veterinary medicine

“…A good example of validation that illustrates the problems inherent in the use of different methodologies is presented by Bassols et al 7 This is the first description of newly developed immunoturbidometric assays for haptoglobin and inter‐α‐trypsin inhibitor heavy chain 4 (ITIH4), implemented on an automated wet chemistry analyzer. Haptoglobin and ITIH4 are both positive acute phase proteins in cattle.…”