Development of canine C-reactive protein assays

Abstract

C-reactive protein (CRP), which is released during tissue damage and inflammation, is a useful nonspecific inflammatory marker in both human and veterinary clinical practice. Veterinarians have often used human CRP assays to analyze samples from canine patients, but cross-reactivities between the species affect assay sensitivity and reliability, leading to inaccurate inflammation assessment. To improve the efficiency of inflammation assessment, we developed a canine CRP detection enzyme-linked immunosorbent assay (ELISA) for quantitative analysis and an immunochromatography assay (ICA) for semiquantitative point-of-care (POC) analysis. The ELISA demonstrated an assay detection limit of 0.5 ng/mL, quantitative linear assay range of 1.6–100 ng/mL, and intra- and inter-assay coefficient of variations of 0.7 to 10.0% and 6.0 to 9.0%, respectively; the recovery rates of samples spiked with purified canine CRP were 105 to 109%, and the parallelism assessments were 82.7 to 104.4%. The correlation between the CRP level results obtained with the ELISA and those of a currently available quantitative POC assay was 0.907 with the regression formula of y = 0.55x + 0.05. In addition, the ICA requires only 5 μL samples and a 10-min assay time, and clearly distinguished positive, weak positive, and negative samples (P < 0.001) at an approximately 5–10 µg/mL cut-off value. The developed canine CRP ELISA and ICA showed reliable assay results and a high correlation with a commercially available POC assay in clinical use. The ICA can be a useful canine CRP screening test for diagnostic purposes in veterinary clinics.

Measurement and clinical applications of C‐reactive protein in gastrointestinal diseases of dogs

“… 29 , 32 The ELISA developed by Waritani et al was shown to have an intraassay CV between 0.7% and 10.0% and an interassay CV between 6.0% and 9.0%. 38 When spiked with purified canine CRP, this assay also demonstrated a recovery between 105% and 109%. 38 Given these performance parameters and the assay's good correlation with the previously validated Laser CRP‐2, 34 it appears sufficiently accurate for clinical use.…”

“… 38 When spiked with purified canine CRP, this assay also demonstrated a recovery between 105% and 109%. 38 Given these performance parameters and the assay's good correlation with the previously validated Laser CRP‐2, 34 it appears sufficiently accurate for clinical use. To the authors’ knowledge, clinical validation of this assay has not yet been reported in the primary literature.…”

“…Commercial laboratory assays allow for the evaluation of CRP at a much greater scale than that of POCTs. Multiple canine‐specific ELISAs are commercially available for the measurement of CRP, including the Phase Range canine CRP ELISA 37 and one recently developed by Waritani et al, which is, to the authors’ knowledge, not yet commercially available 38 . The Phase Range canine CRP ELISA (Tridelta; Tridelta Development Ltd, Kildare, UK) was shown in one study to have an intraassay CV between 6.9% and 10.1% and an interassay CV between 7.5% and 29.0% 37 .…”

“…The Tridelta assay has been used in a number of studies 29,32 . The ELISA developed by Waritani et al was shown to have an intraassay CV between 0.7% and 10.0% and an interassay CV between 6.0% and 9.0% 38 . When spiked with purified canine CRP, this assay also demonstrated a recovery between 105% and 109% 38 .…”

“…Multiple canine‐specific ELISAs are commercially available for the measurement of CRP, including the Phase Range canine CRP ELISA 37 and one recently developed by Waritani et al, which is, to the authors’ knowledge, not yet commercially available. 38 The Phase Range canine CRP ELISA (Tridelta; Tridelta Development Ltd, Kildare, UK) was shown in one study to have an intraassay CV between 6.9% and 10.1% and an interassay CV between 7.5% and 29.0%. 37 While this interassay CV is considered unacceptable, this is not unusual for assays when a sample is evaluated that is close to the detection limit of the assay (mean result: 54.9 mg/L; working range: 3.7‐60 mg/L when sample diluted 1:500).…”