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

Abstract

BackgroundSerum 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.ResultsDetection 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.ConclusionsThe 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.

Reference intervals of acute phase proteins in healthy Andalusian donkeys and response to experimentally induced endotoxemia

“…Species‐specific validation of any APP test is mandatory to obtain reliable data 13 . Our validation results show that every immunoassay performed reliably in donkeys, with good repeatability and accuracy (linearity under dilution) in our ranges of measurements, in agreement with requirements for this type of analysis 24,26 …”

“…13 Our validation results show that every immunoassay performed reliably in donkeys, with good repeatability and accuracy (linearity under dilution) in our ranges of measurements, in agreement with requirements for this type of analysis. 24,26 One limitation of our study is more prolonged sampling post-LPS. Including donkeys with naturally occurring disease could clarify mid-and long-term variations in the concentrations of the APPs studied in this species.…”

“…Tests were validated for donkeys following a modified protocol previously reported in horses and using published guidelines for immunoassay validation 24‐26 . Specific aliquots from the tested donkeys were used for these validations.…”

“…Tests were validated for donkeys following a modified protocol previously reported in horses and using published guidelines for immunoassay validation. [24][25][26] Specific aliquots from the tested donkeys were used for these validations. Precision was evaluated by calculating the intra-assay (same day) and interassay ( Reported concentrations in horses (22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37) .08…”

Bullous amyloidosis in a horse: first description in veterinary medicine

“…4 Serum AA also was elevated in this horse [1,436 µg/mL (0-8 mg/µL)]. 5 These clinicopathological findings confirmed the diagnosis of AAamyloidosis with a unique phenotype called bullous amyloidosis.…”