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Development of Canine C-Reactive Protein Assays

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Development of Canine C-Reactive Protein Assays Waritani et al. Acta Vet Scand (2020) 62:50 https://doi.org/10.1186/s13028-020-00549-9 Acta Veterinaria Scandinavica BRIEF COMMUNICATION Open Access Development of canine C-reactive protein assays Takaki Waritani* , Dawn Cutler and Jessica Chang Abstract C-reactive protein (CRP), which is released during tissue damage and infammation, is a useful nonspecifc infam- matory 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 afect assay sensitivity and reliability, leading to inaccurate infammation assessment. To improve the efciency of infammation assessment, we developed a canine CRP detection enzyme-linked immunosorbent assay (ELISA) for quantitative analysis and an immunochro- matography 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 coefcient of variations of 0.7 to 10.0% and 6.0 to 9.0%, respectively; the recovery rates of samples spiked with purifed 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-of value. The devel- oped 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. Keywords: C-reactive protein, Dogs, Enzyme-linked immunosorbent assay, Immunochromatography assay, Infammation Findings immunosorbent assays (ELISAs) [6, 7] and latex aggluti- Te level of canine serum C-reactive protein (CRP), an nation tests [3, 8]. However, due to the poor cross-reac- acute-phase protein released during tissue damage and tivity of anti-human CRP antibodies with canine CRP, infammation, is elevated 100- to 1000-fold in surgical some of these human CRP assays demonstrate relatively trauma, irritation, and infammatory diseases, including unreliable assay results and mislead infammation assess- pyometra, panniculitis, acute pancreatitis, polyarthritis, ment. To achieve more reliable results, several commer- septic arthritis, and hemangiosarcoma [1–3]. Te serum cial canine CRP-specifc assays have been introduced level dynamics indicate that canine CRP is a useful, non- to the market. However, technical improvements are specifc, sensitive infammatory marker [1, 2, 4]. needed to decrease between-run imprecision [9, 10]. Te canine serum CRP level is generally measured Canine CRP ELISAs can be useful for routine checkups with human diagnostic CRP immunoassays, including but are not appropriate as a rapid test for daily clinical use automated biochemical analyzers [5], enzyme-linked because of the time and labor required. Recently, com- mercially available canine CRP point-of-care (POC) assay *Correspondence: [email protected] kits, including quantitative immunoassays using analyz- Chondrex, Inc., 2607 151st Place North East Redmond, Washington 98052, ers [11, 12] and semiqualitative immunochromatography USA assays (ICAs) [10], have been developed. Unfortunately, © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Waritani et al. Acta Vet Scand (2020) 62:50 Page 2 of 5 these kits may still require investigation as they tend to a backing sheet (DCN Diagnostics, Carlsbad, CA, USA), yield imprecise results [13]. and a device case (DCN Diagnostics). Te assay was per- Te aim of this study was to develop a highly reliable formed by loading 5 μL of undiluted canine serum fol- canine CRP ELISA and ICA using anti-canine CRP mon- lowed by 100 μL of BSA/PBS containing 0.1% Tween 20 oclonal antibodies (mAbs). Tese assays were validated in the sample well of the device. If both the red sample using clinical samples and compared with a commercially line and control line appeared in the test window of the available POC assay (Laser CRP-2 Analyzer from Arrows device after 10 min, the assay was deemed to have a posi- Co., Ltd, Osaka, Japan) (Laser). tive result, which was further distinguished as positive MAbs against canine CRP were developed by cell with a dark sample line and weak positive with a light hybridization of SP2/0-Ag4 cells and splenocytes from sample line. If the sample line did not appear, the assay BALB/c mice (Envigo, Indianapolis, IN, USA) immunized was deemed to have a negative result (Fig. 1). with purifed canine CRP (LEE Biosolutions, Maryland Te developed ELISA demonstrated an assay detection Heights, MO, USA). Hybridomas producing anti-CRP limit of 0.5 ng/mL, and a quantitative linear assay range mAbs were screened using an indirect canine CRP ELISA of 1.6–100 ng/mL for the standard curve (R = 0.996) and then cloned by the limiting dilution method. Te (Additional fle 1). Te intra-assay and inter-assay coef- specifcity of the anti-canine CRP mAbs was confrmed fcient of variations were 0.7 to 10.0% and 6.0 to 9.0%, by western blot analysis (data not shown). Te mAbs pro- respectively, with a range of 3.2 to 50 ng/mL of CRP in duced by hybridomas were purifed by Protein G afnity serum samples diluted with BSA/PBS (n = 3) (Addi- chromatography (GE Healthcare Systems, Chicago, IL, tional fle 2). Te spike tests in which three serum sam- USA) from culture medium collected with a miniPERM ples were spiked with known amounts of purifed CRP culture system (Sarstedt, Newton, SC, USA). demonstrated recovery rates ranging from 105 to 109% We developed a canine CRP ELISA employing a (Additional fle 2). Te parallelism assessments using four one-step sandwich assay system using purifed canine samples demonstrated recovery rates ranging from 82.7 CRP and two anti-canine CRP mAbs. Briefy, the mAb to 104.4% in several sample dilutions (Additional fle 3). 4D3C1 was diluted in 100 μL of 0.05 M phosphate-buf- Te canine serum test samples consisted of 16 sam- ered saline bufer, pH 7.4 (PBS), added to each well of a ples from healthy blood donors as healthy control dogs 96-well ELISA plate and incubated at 4 °C overnight. (control group) and 38 samples from client-owned dogs After washing the plates with PBS containing 0.05% presented to the Research Institute of Biosciences, Azabu Tween 20 (PBST), 50 μL of the mAb 3B4D3 conjugated University, Sagamihara, Japan as diseased dogs (patient with horseradish peroxidase in PBS containing 1% group). All experimental protocols were approved by the bovine serum albumin (BSA) (BSA/PBS) and 50 μL of Animal Experiment Committee of Azabu University. canine CRP standards or diluted canine serum samples Te CRP levels of the patient group were evaluated (1:1000 or higher dilution) in BSA/PBS were added to using both our ELISA and the Laser commercial POC the wells. Te plate was incubated for 1 h at room tem- assay at Azabu University (Additional fle 4). Undiluted perature (i.e. around 25 °C). After washing the plates serum samples were applied to the Laser assay, but serum with PBST, color was developed at room temperature samples diluted to 1:1000 or higher with BSA/PBS were for 25 min by adding 100 μL of 3,3′,5,5′-tetramethylb- used for our ELISA because of its high assay sensitivity. enzidine solution (MP Biomedicals, Santa Ana, CA, Tree of the 38 samples assayed were omitted from this USA). After adding 50 μL of 2 N sulfuric acid to stop analysis because their serum CRP levels were out of the the enzymatic reaction, absorbance values were meas- Laser’s quantitative assay range. Te correlation between ured at 450 nm. Te CRP levels in samples were calcu- the CRP levels obtained with our ELISA and those lated using linear regression based on a standard curve obtained with the Laser assay was 0.907 with the regres- in Microsoft Excel and multiplied by the corresponding sion formula of y = 0.55x + 0.05 (Fig. 2). Te slope of 0.55 dilution factor to obtain true values (μg/mL). suggests that our ELISA obtained approximately 55% A canine CRP ICA was developed by modifying a lower CRP levels than the Laser assay. Since the Laser previously described technique [14]. Briefy, the kit was assay employs a CRP positive cut-of value of 10 µg/mL, composed of a high-fow nitrocellulose membrane strip our ELISA would have an equivalent positive cut-of (MilliporeSigma, Burlington, MS, USA) prebound with value of 5.5 µg/mL.
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