engleski

Assay of haptoglobin, the plasma acute‐phase protein, by hemoglobin binding: Optimization of a novel chromogen

Background: Haptoglobin (Hp) can be assayed by a biochemical assay based on its binding to hemoglobin (Hb) and preservation of the Hb peroxidase activity with the peroxidase‐ catalyzed reaction of 4‐aminoantipyrine and 8‐anilino‐1‐ naphthalone sulfonic acid as chromogen. The resulting product has low stability potentially lead‐ ing to poor precision. New peroxidase substrates are available that may improve the Hp assay. Objective: Assess an alternative peroxidase substrate for the assay of Hp. Method: The substrate, N, N’‐bis(2‐hydroxy‐3‐ sulfopropyl)‐ tolidine (SAT‐3, Dojindo, Japan) in buffer was optimized for use on an Architect c4000 Analyzer (Abbot, USA) so that 3 ml of sample was mixed with 157 mL of Hb reagent, 80 mL of SAT‐ 3 solution including H2O2 was added and absorbance at 660 nm determined. Validation was by determination of precision, limit of quantifica‐ tion, interference and detection of clinical changes by monitoring plasma Hp in healthy dairy cows (n = 10) and those with clinical mastitis (n = 10). Results: The SAT‐ 3chromogen product was stable for several min‐ utes post‐reaction with intra‐assay and inter‐ assay CVs below 6.5%. The limit of quantification assay was 0.05 g/L. Lipemia had no effect on Hp results to 0.16 g/L while hemolysis increased the apparent Hp concentration above 0.07 g/L of Hb. Haptoglobin in plasma from healthy cows (n = 10) was <0.05 g/L in all samples, while the median (range) in plasma from cows with clinical mastitis (n = 10) was signifi‐ cantly greater (P = .0011) at 0.15 (<0.05‐0.84) g/L. Conclusion: The use of SAT3 is effective as a peroxidase substrate for the assay of Hp in plasma.

acute phase protein, haptoglobin, SAT3

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