Description
Principle of the Assay: The principle of the double antibody sandwich ELISA is represented in Figure 1. In this assay the CRP present in samples reacts with the anti-CRP antibodies which have been adsorbed to the surface of polystyrene microtitre wells. After the removal of unbound proteins by washing, anti-CRP antibodies conjugated with horseradish peroxidase (HRP), are added. These enzyme-labeled antibodies form complexes with the previously bound CRP. Following another washing step, the enzyme bound to the immunosorbent is assayed by the addition of a chromogenic substrate, 3,3',5,5'-tetramethylbenzidine (TMB). The quantity of bound enzyme varies directly with the concentration of CRP in the sample tested; thus, the absorbance, at 450 nm, is a measure of the concentration of CRP in the test sample. The quantity of CRP in the test sample can be interpolated from the standard curve constructed from the standards, and corrected for sample dilution.
Background: Acute phase proteins are plasma proteins which increase in concentration following infection, inflammation or trauma. The first acute phase protein to be recognized was discovered in humans by Tillet and Frances in 19301. This C-reactive protein (CRP) is so named because it is able to effect precipitation of somatic C-polysaccharide of Streptococcus pneumonia. CRP is an alpha globulin with a mass of 110,000 to 140,000 daltons, and composed of five identical subunits, which are non-covalently assembled as a cyclic pentamer. It is synthesized in the liver and, in humans, is normally present as a trace constituent of serum at levels less that 0.3 mG/dL. The levels in serum rise quickly following acute tissue damage and also falls very rapidly once the stimulus is removed. It has been proposed that the function of CRP is to aid in complement activation, influence phagocytic cell function, and augment cell mediated cytotoxicity. Investigations over the past few years have shown that quantification of these in plasma or serum can provide valuable diagnostic information in the detection, prognosis, and monitoring of disease not only in humans, but in companion animals and farm herds as well2