Isolation of human C-reactive protein and serum amyloid P component.
Procedures are described for the isolation in high yield of consistent, highly purified preparations of human C-reactive protein (CRP) and serum amyloid P component (SAP). CRP was obtained from malignant ascitic and pleural fluids by calcium-dependent affinity chromatography on pneumococcal C-polysaccharide covalently coupled to cyanogen bromide-activated Sepharose. It was then gel filtered on Ultrogel AcA44 (acrylamide-agarose beads) in the presence of calcium ions, combining molecular sieve chromatography with removal of contaminating SAP by its affinity of agarose. Residual trace contaminants were removed by immunoabsorption with anti-normal human serum and anti-SAP antibodies insolubilised on Sepharose and/or by absorption with Sepharose-Con A to remove glycoproteins and Blue-Sepharose to remove albumin. After a final gel filtration step on Sephacryl S-300 35-45% of the initial CRP was recovered in pure form according to biochemical and immunochemical criteria. SAP was isolated from normal serum by calcium-dependent affinity chromatography on unsubstituted Sepharose beads, followed by solid-phase immunoabsorption of contaminants and finally gel filtration on Sephacryl S-300. At least 50% of the SAP in the starting material was recovered in pure form according to biochemical and immunochemical criteria. Ready availability of such preparations facilitates biochemical, biophysical and particularly biological studies of these plasma proteins.
D000682:Amyloid / Q000276:immunology / Q000302:isolation & purification*; D000818:Animals; D001202:Ascitic Fluid / Q000032:analysis; D002097:C-Reactive Protein / Q000276:immunology / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D006801:Humans; D007106:Immune Sera / Q000494:pharmacology; D010996:Pleural Effusion / Q000032:analysis; D011817:Rabbits; D000683:Serum Amyloid P-Component; D012756:Sheep
D000682:Amyloid; D007106:Immune Sera; D000683:Serum Amyloid P-Component; D002097:C-Reactive Protein