Single-step purification of rat C-reactive protein and generation of monospecific C-reactive protein antibody.
Although Sepharose-phosphorylcholine affinity chromatography has been used extensively to purify some acute phase proteins, the operation has usually been a laborious multi-step procedure. By modifying previously described multi-step protein purification assays, centigram quantities of pure rat C-reactive protein (CRP) could be obtained in a single chromatographic step using affinity chromatography. Rat serum was passed over a column of p-aminophenylphosphorylcholine and extraneous proteins eluted with Tris-saline-Ca2+ buffer. Similar to other purification procedures, CRP was eluted with phosphorylcholine in a Tris-saline-Ca2+ buffer. The technical detail which distinguished this procedure from others, was the use of a phosphorylcholine gradient shallow enough (0.95 mM-2.5 mM) to resolve the eluent into two peaks; the first peak was composed largely of the contaminant, serum amyloid protein (SAP), and the second was composed of CRP. Although there was some overlap between the first and second peak, pure CRP could be obtained by pooling fractions from the trailing shoulder of the second peak. Using this single step procedure, a greater than 25% yield of SAP-free, purified CRP could be obtained. The purified CRP was free of SAP contamination as measured by sodium dodecyl sulfate gradient polyacrylamide gel electrophoresis. Purified CRP was determined to be free of rat albumin, IgG and the C3 component of complement using immunoelectrophoresis. This one-step affinity column chromatography procedure provides a simple, efficient method for collecting large quantities of rat CRP pure enough to be used to obtain a monospecific goat, anti-rat CRP antibody.
D000818:Animals; D000917:Antibody Formation; D002021:Buffers; D002097:C-Reactive Protein / Q000276:immunology / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006041:Goats / Q000276:immunology; D005779:Immunodiffusion; D007122:Immunoelectrophoresis; D008297:Male; D051381:Rats; D011917:Rats, Inbred Lew; D011919:Rats, Inbred Strains
D002021:Buffers; D002097:C-Reactive Protein