Purification of rubella virus E1-E2 protein complexes by immunoaffinity chromatography.
A murine monoclonal antibody directed against the E1 membrane glycoprotein of rubella virus was immobilized on an N-hydroxysuccinimide-activated chromatographic support. The antibody was used to purify rubella virus E1-E2 protein complexes from Tween-80/diethyl ether extracts of cell culture supernatants containing virus particles. The adsorption behaviour of immunosorbents with ligand densities of 2.9, 5.4 and 11.1 mg monoclonal antibody per millilitre of gel was investigated using batchwise conditions. Then the immunoaffinity purification process was optimized with regard to adsorption efficiency by adjusting the flow rate, the bed height and the amount of sample loaded onto the column. The optimized immunoaffinity purification process which is reproducible and relatively simple (one-step) had a yield of 73%, a concentration factor of 5-8 and a purification factor of about 2600. No mouse IgG due to ligand leakage could be detected in the immunopurified product using an enzyme immunoassay. High-performance size exclusion chromatography, sodium dodecyl sulphate polyacrylamide gel electrophoresis, immunoblotting and electron microscopy showed that the immunopurified product contained rosette-like structures formed by complexes of E1 and E2 proteins. The product retained its hemagglutinating activity and proved to be suitable for application in a fluorescent enzyme immunoassay for determination of anti-rubella IgG in human serum.
van Sommeren
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AKZO NOBEL, Organon Teknika B.V., Chemistry Research Unit, Boxtel, The Netherlands. tvsommeren@am.Otbc01.Umc.Akzonobel.Nl
Machielsen
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PA
Schielen
W J
WJ
Bloemers
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HP
Gribnau
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TC
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood / Q000276:immunology; D000956:Antigens, Viral / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006386:Hemagglutination Tests; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology; D008024:Ligands; D012412:Rubella virus / Q000737:chemistry* / Q000648:ultrastructure; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*
D000914:Antibodies, Viral; D000956:Antigens, Viral; D007074:Immunoglobulin G; D008024:Ligands; D014759:Viral Envelope Proteins; C061826:E1 envelope protein, Rubella virus; C061827:E2 envelope protein, Rubella virus