Isolation of C-reactive protein by affinity chromatography

Different affinity chromatography methods were compared for the isolation of C-reactive protein (CRP) from pathological fluids. In the presence of calcium ions CRP became bound to agar (Ganrot & Kindmark, 1969), to cyanogen bromide-activated agarose beads (Sepharose 4B) coupled with pneumococcal C substance (Osmand et al ., 1975), to glutaraldehyde-activated polyacrylamide beads (Bio-Gel P300) or agarose-acrylamide beads (Ultrogel AcA 34) coupled with C substance and to `sulphated' polyacrylamide (Bio-Gel P300) beads. After thorough washing, elution with citrate or EDTA yielded CRP of varying degrees of purity. The use of agar provided high yields of heavily contaminated CRP which was substantially purified by passage through a column of insolubilized anti-normal human serum antibodies. Insolubilized C substance provided much purer CRP and the best yields were obtained from Sepharose–CNBr–C substance to which was attached considerably more ligand than was coupled to glutaraldehyde-activated beads. The only significant contaminant of the CRP isolated in this way was a normal serum protein, the P component of amyloid (Clt), which showed calcium-dependent binding to plain unsubstituted Sepharose and Ultrogel, as well as to polyacrylamide beads which had been treated with concentrated sulphuric acid. The use of these easily prepared `sulphated' polyacrylamide beads provides a method for isolating small quantities of CRP contaminated only by P component (Clt), without the need to first prepare C substance. Separation of CRP from P component was readily achieved by absorption of the P component onto agarose beads in the presence of calcium ions. An `electroimmunodiffusion', `rocket' type of assay for pneumococcal C substance was developed, using CRP-rich fluid in the gel instead of antiserum.