Analytical Validation of a Point-of-Care Assay and an Enzyme-linked Immunosorbent Assay for the Quantification of Equine Serum Amyloid A in Comparison with a Turbidimetric Immunoassay
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Beschreibung
In this prospective study, carried out from January 2021 to February 2022, we evaluated the key characteristics of VMRD-POCA (Veterinary Medical Research and Development, Pullman, WA, USA, Point-of-Care Assay), VMRD-ELISA (Enzyme-Linked Immunosorbent Assay), and VET-SAA (Veterinary-Specific Immunoturbidometric Assay, Eiken Chemical Co., Tokyo, Japan) evaluating linearity, precision, interferences, total observed error, recovery rates, hook effect, and method comparison across different concentration ranges.
The aims of this prospective study were as follows:
- To validate the stall-side VMRD-POCA for quantifying equine SAA in serum samples, using VET-SAA based on monoclonal rat anti-human SAA1 antibodies as the reference method.
- To perform an analytical validation of the VMRD-ELISA for quantifying equine SAA in serum samples in a low concentration range (<300 mg/L). Focusing on inter- and intra-assay precision, linearity, total observed error, recovery rates, resistance to interference, and correlation in comparison to the VET-SAA and VMRD-POCA assays.
Ethical approval of the study was given by the Regierungspraesidium Giessen (ethics committee number JLU_kTV_03_2021). Three different concentration levels and interferences were used to assess analytical performance.
A total of 77 equine serum samples were analyzed to determine the analytical performance of the VMRD-POCA, VMRD-ELISA, and VET-SAA. Method comparison revealed a strong correlation between VMRD-POCA and VMRD-ELISA (rs = 0.93), a good correlation between VMRD-POCA and VET-SAA (rs = 0.89), and a moderate correlation between VMRD-ELISA and VET-SAA (rs = 0.62).
Bias analysis showed variations, with VMRD-POCA showing a proportional bias of -81.3% vs. VET-SAA and VMRD-ELISA showing a bias of -89.4% vs. VET-SAA. In contrast, VMRD-POCA and VMRD-ELISA showed a lower bias (-3.3%).
The VMRD-POCA had intra-assay CVs ranging from 14.9% to 31.4% and inter-assay CVs of up to 44%, surpassing the 12.5% quality threshold in all concentration groups. The VMRD-ELISA demonstrated high precision at low SAA concentrations (<300 mg/L), with an intra-assay CV of 13.1%. The VET-SAA had lower intra-assay CVs (3.3%-6.5%) but higher inter-assay variability at SAA concentrations greater than 1000 mg/L.
Total observed error (TEobs) values exceeded the 37% threshold for VMRD-POCA vs. VMRD-ELISA (60% and 63%, respectively), whereas VMRD-POCA vs. VET-SAA remained below 37% in the SAALow (19%) and SAAMed (20%) groups but exceeded this limit in the SAAHigh range (52%).
The VMRD-POCA demonstrated strong linearity up to 1,841 mg/L (R² = 0.94). However, recovery rates differed from the acceptable 80-120% range, especially at high SAA concentrations.
Interference analysis revealed that intralipid caused the most bias (66% in VMRD-POCA and 36% in VET-SAA), while bilirubin caused deviations of -30% (VMRD-POCA), -26% (VET-SAA), and -24% (VMRD-ELISA). VMRD-ELISA had the lowest susceptibility to interferences (bias < 10%). Unlike previous studies that found hook effects in other POCAs at SAA concentrations >3000 mg/L, the VMRD-POCA showed no hook effect even at 15,300 mg/L.
In conclusion, the VMRD-POCA and VMRD-ELISA demonstrated strong correlation with VET-SAA (rs = 0.89 and rs = 0.62, respectively), confirming their potential for equine SAA measurement. However, the VMRD-POCA exhibited high intra- and inter-assay CVs (up to 31.4% and 44%, respectively), which may impact its reliability in clinical settings. The VMRD-ELISA showed good precision at low SAA concentrations (<300 mg/L) but was limited by its narrow working range.
Interference effects were substantial, particularly with intralipid (bias of 66% in VMRD-POCA and 36% in VET-SAA), whereas VMRD-ELISA exhibited the lowest susceptibility to interferences (bias <10%).
The absence of a hook effect in VMRD-POCA up to 15,300 mg/L represents a key advantage over other POCAs, where hook effects have been observed at SAA concentrations >3000 mg/L.
Veterinarians should consider potential interferences, high variability, and assay-specific biases when interpreting SAA results. While VMRD-POCA and VET-SAA provide reliable tools for clinical inflammation monitoring, careful sample handling, dilution accuracy, and method selection remain crucial to ensure precise and meaningful SAA measurements.
Autor*in
Emre Aner
Themen in »Analytical Validation of a Point-of-Care Assay and an Enzyme-linked Immunosorbent Assay for the Quantification of Equine Serum Amyloid A in Comparison with a Turbidimetric Immunoassay«
Equines Serum-Amyloid A (SAA) Point-of-Care-Test (POCT) Analytische Validierung Interferenzen und Präzision Methodenvergleich und Korrelation Hook-Effekt in SAA-Messungen