Abstract
The use of heterologous immunoassays containing antibodies raised against a different biological species for quantification of serum proteins is studied and discussed, taking as example the case of the use of a commercially available heterologous assay containing antibodies against human C-reactive protein (hCRP) for quantification of CRP in serum of dogs. This assay was adapted and validated for measurements of canine CRP (cCRP) and compared with three different homologous assays containing species-specific canine antibodies, which are currently commercially available for cCRP determination. Serum samples from healthy and diseased dogs (n = 44) were used. Analytical evaluation included precision, accuracy, limit of detection and lower limit of quantification for all assays. In the case of the heterologous assay also cross-reactivity of the antibody of the heterologous assay with cCRP was evaluated by a Western-Blot analysis giving a positive result. The heterologous assay showed similar results than the homologous assays in all the tests of the analytical evaluation that indicated that the assay was precise and accurate. Method comparison showed a high correlation between all assays (r≥0.9). The Bland-Altman test revealed that the heterologous assay showed a proportional error when compared with the homologous automated assays and a random error when compared with the point-of-care assay. All four CRP assays were able to detect higher CRP values in dogs with inflammatory conditions compared with healthy dogs. It is concluded that heterologous immunoassays could be used for quantification of serum proteins in different species, provided that the antibody has cross-reactivity with the protein to be measured and the assay give satisfactory results in the analytical validation tests. In addition, use of species-specific calibrators and an appropriate batch validation are recommended in these cases.
Highlights
C-reactive protein (CRP) is a major positive acute phase protein (APP) in dogs, produced mainly in the liver in response to increased concentrations of pro-inflammatory cytokines as part of the innate immune response [1,2,3]
standard deviation (SD) and coefficient of variation (CV) were calculated from 10 replicate measurements of the different concentrations
Measurements of the control material consisting in pure canine CRP (cCRP) having a mean value of 42 mg/L showed an inaccuracy of 2.2, 4.5, 3.8 and 9.8% for the Olympus, Avacta, Gentian and Fujifilm assays, respectively
Summary
C-reactive protein (CRP) is a major positive acute phase protein (APP) in dogs, produced mainly in the liver in response to increased concentrations of pro-inflammatory cytokines as part of the innate immune response [1,2,3]. PLOS ONE | DOI:10.1371/journal.pone.0172188 February 21, 2017
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