Purification and determination of C-reactive protein and inter-α-trypsin inhibitor heavy chain 4 in dogs after major surgery through generation of specific antibodies

A new automated turbidimetric immunoassay for the measurement of canine C‐reactive protein

“…The use of specific reagents is fundamental for the development of robust and accurate species‐specific turbidimetric immunoassays. Our antibodies are very specific because the antiserum is immunoadsorbed with canine serum deprived of cCRP . Accordingly, the dose–response curve obtained with canine serum was similar to that obtained with purified cCRP, and no matrix effect was revealed in the linearity under dilution study.…”

“…Assay calibration was performed with a multipoint calibration curve using a cCRP calibrator consisting of purified cCRP (150 mg/L) in a matrix based on a 0.1 M Tris Chloride, 0.15M NaCl, 3 mM CaCl 2 buffer at pH 7.4 and containing 10 mg/mL bovine serum albumin (BSA) and 0.09% sodium azide as a stabilizer. The development and establishment of the cCRP value in the calibration material by single radial immunodiffusion (SRID) using a cCRP standard serum has been previously described …”

Preparation of canine C‐reactive protein serum reference material: A feasibility study

“…To prepare the calibrator, purified cCRP was diluted in a matrix containing 0.1 mol/L Tris buffer pH 7.5, 0.2 mol/L NaCl, 3.0 mmol/L CaCl 2 , 10 g/L BSA, and 0.09% sodium azide. The concentration of cCRP in the calibrator was determined by single radial immunodiffusion using a previously developed anti‐cCRP‐specific antiserum and a cCRP standard serum . The cCRP calibrator was analyzed in duplicate at 3 different dilutions (1:1.5, 1:2, and 1:3) in 3 different assays, and the mean value was assigned (146 mg/L).…”

“…BlueStar Plus Prestained Protein Markers (Nippon Genetics Europe, Dueren, Germany) were used as molecular weight markers. Western blot was performed using a previously developed specific anti‐cCRP antiserum as the primary antibody (diluted 1:2000) . Horseradish peroxidase labeled with anti‐rabbit IgG (Sigma, diluted 1:20,000) was used as the secondary antibody.…”

“…International reference materials with values assigned from purified and highly characterized proteins are desirable . We previously purified cCRP and the molecular characteristics of this protein (molecular weight, reactivity with specific anti‐cCRP antibodies, and identity) were established using electrophoretic and immunochemical techniques, and mass spectrometry . In the present work, we describe the preparation of reference material for cCRP intended to be used for transfer of values to secondary or tertiary materials (calibrators and controls) and not for direct use in laboratory assays.…”