Validation of an equine serum amyloid A assay with an unusually broad working range

Abstract

Background Serum amyloid A (SAA) is a major equine acute phase protein and of great value in detection and monitoring of inflammation. A new immunoturbidometric assay based on monoclonal antibodies (VET-SAA, Eiken Chemical Co., Japan) may be useful for SAA measurements in routine diagnostic laboratories. The aim of the study was to validate the VET-SAA immunoturbidometric assay and use it to measure serum SAA concentrations in a variety of clinical cases. Precision was assessed by intra- and interassay coefficients of variation of repeated measurements of serum pools (low, intermediate, high concentrations of SAA). Accuracy was estimated by linearity under dilution. Detection limit was determined by replicate determinations of ionized water. Measurements were compared to measurements performed in a previously validated SAA assay (LZSAA assay, Eiken Chemical Co., Japan). Subsequently, the VET-SAA assay was used for measuring serum SAA concentrations in horses with and without inflammation. Results Detection limit was 1.2 mg/L. Without modifications, the assay measured SAA concentrations with acceptable reliability in a broad concentration range (0 to > 6000 mg/L). In the 0-3000 mg/L range, the assay demonstrated good precision and accuracy, and concentrations correlated well with those obtained in the LZSAA assay, albeit with a slight systematic bias. Concentrations of SAA assessed in horses with and without inflammation followed the expected pattern, with significantly higher concentrations in horses with systemic inflammation than in healthy horses and horses with non-inflammatory disease. Conclusions The assay was unique in its ability to measure SAA concentrations with acceptable reliability over an extreme concentration range. This is relevant in the equine species, where SAA concentrations may reach very high concentrations.

Comparison of a point-of-care serum amyloid A analyzer frequently used in equine practice with 2 turbidimetric immunoassays used in human and veterinary medicine

“…The d obs % between test and control sample was considered acceptable if it was within ±10%, as described for equine SAA assays. 18…”

“…4,5 In addition, its rapid increase over 6–12 h and short half-life of 30–120 min 31 renders SAA a valuable biomarker to closely track disease severity. 7 Different patient-side point-of-care (POC) assays 22,29,30 and turbidimetric immunoassays (TIAs) for laboratory use 17,18 have been validated for equine SAA measurement.…”

“…17,22,29 Although TIA-Hum has proved reliable for equine SAA measurement, 17 its drawback is the combination of monoclonal rat anti-human SAA1 antibody and polyclonal rabbit anti-human SAA1 antibody, which provide a high potential for batch-to-batch variation. 17,18 Furthermore, TIA-Hum is linear only in a limited concentration range, making several dilutions and repeated analyses necessary to obtain a final SAA concentration. 17,18…”

“…17,18 Furthermore, TIA-Hum is linear only in a limited concentration range, making several dilutions and repeated analyses necessary to obtain a final SAA concentration. 17,18…”

“…TIA-Vet is based on the sole use of monoclonal rat anti-human SAA1 antibody, and has an acceptable reliability in measurement of extreme SAA concentrations. 18 Contrary to TIAs, POC assays can be used stall-side, are user-friendly, 22,29 and provide SAA results quickly and without the need for shipment of samples to a reference laboratory. 30 To our knowledge, there are 3 POC assays available for equine SAA measurement 22,29,30 based on the use of anti-human SAA antibody.…”