An immunoturbidimetric assay for bovine haptoglobin

Abstract

In cattle, the serum protein haptoglobin (Hp) is a major acute phase protein (APP) that rises in concentration over a thousand fold following stimulation by pro-inflammatory cytokines. As such, this APP is a valuable biomarker for infection, inflammation and trauma in cattle. The assay for bovine Hp is becoming more commonplace in clinical pathology and in experimental studies when a biomarker of innate immunity is required. The most widely used assay for Hp utilises its binding to haemoglobin (Hp-Hb binding assay), which at low pH enables the preservation of the native peroxidase activity in the haemoglobin. This assay is used for all species, including species such as dog, cat and pig where the level of Hp is higher in healthy animals of these species than in healthy cattle, and therefore a bovine-specific immunoassay that can be automated would be desirable. Thus, a novel-automated species-specific immunoturbidimetric (IT) assay has been developed. Validation studies showed intra- and inter-assay CVs of below 5% and 9% respectively and a recovery of 99% from samples spiked with bovine Hp and a limit of quantification of 0.033 g/L. The assay is not affected by icterus or lipaemia but had moderate interference from haemoglobin and showed a significant correlation with the Hp-Hb binding assay. This novel IT assay for bovine Hp will allow automated analysis of this important bovine APP to identify changes in the Hp concentration not detectable by current Hp-Hb binding assays. It will enable the incorporation of this assay into herd health assessments, animal welfare analysis and for bovine medicine and research.

Biomarkers of sepsis in pigs, horses and cattle: from acute phase proteins to procalcitonin

“…Among these three species, Hp concentration can also rise in non-infectious pathologies such as local aseptic inflammation, fatty liver, and after transport or surgeries (Murata and Miyamoto, 1993; Nakagawa et al ., 1997; Parra et al ., 2006; Guzelbektes et al ., 2010; Heinonen et al ., 2010; Zabrecky et al ., 2015), as well as in infectious pathologies caused by viruses and parasites (Heegaard et al ., 2000; Pomorska-Mól et al ., 2014; El-Deeb et al ., 2018). In addition to immunoturbidimetric methods, the existence of spectrophotometric assays that can be automated allows the use of Hp in routine diagnostics (Brady et al ., 2019). Hp as well as other APPs can also be measured in saliva (Cerón et al , 2019).…”

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

“…Our assay is a particle‐enhanced method that results in a lower LoD (0.005 mg/mL) and LoQ (0.007 mg/mL) than that reported by Brady et al, using direct immunoturbidimetry (LoQ 0.03 mg/mL) 37 . The LoD and LoQ of our assay were low enough to measure Hp in all the serum samples used for reference value establishment.…”

“…To the authors' knowledge, the present assay and the one developed by Brady et al 37 are the only turbidimetric immunoassays for bovine Hp that have been developed and validated and that are specific for the bovine species. It has been proposed that turbidimetric immunoassays should eventually replace the Hp‐Hb binding method for Hp quantification in animals 37 …”

“…An interference of around 25% was reported for hemoglobin at 1.5 g/L with the in‐house Hp‐Hb assay 38 . The TI developed by Brady et al 37 also shows slight hemoglobin interference at 4 g/L (10%).…”

Changes in Acute Phase Response Biomarkers in Racing Endurance Horses

“…The concentrations of total proteins, albumin, urea, cholesterol, triglycerides, Fe and the enzyme activities of aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) were analysed by Abbott commercial kits using an automated biochemistry analyser Architect c4000 (Abbott, Abbott Park, IL, USA). The concentration of Hp was determined by an automated spectrophotometric haemoglobin-HP binding assay [ 22 , 23 ]. The Cp concentration was determined by an automated spectrophotometric assay using a biochemistry analyser Architect c4000 (Abbott, USA) [ 24 ].…”