Purification of measles virus by affinity chromatography and by ultracentrifugation: a comparative study.
The purification of viruses by ultracentrifugation in regions with limited resources is often hampered by problems of instrument maintenance. Viral antigens must therefore be imported, which results in delays in obtaining epidemiological data. To address this problem, we undertook the development of purification procedures by affinity chromatography using measles virus (strain Y15) and monkey IgG (anti-measles) coupled covalently via their carbohydrate residues to glass spheres as a model system. The measles virus prepared in this way was compared with measles virus purified by ultracentrifugation using three criteria: the degree of purification, yield and the sensitivity in HA and ELISA. The purification of the antigen by ultracentrifugation (AgU) was undertaken using 10(9) infected cells, and by affinity chromatography (AgAc) using 10(7) infected cells. The total concentration of proteins before purification was 5000 mg for AgU and 11.3 mg for AgAc. After purification by AgU 21.5 mg protein was obtained which was assessed by specific hemagglutinating units (HAU/mg protein) and found to be 179 for AgU and 372-744 for AgAc. Based on total HAU, the yield by centrifugation was 2.4%, whereas the affinity chromatography produced essentially 100%. By ELISA, using reference sera to calibrate the antigens, maximum OD was obtained with 0.5 microgram/well of AgAc and with 2 micrograms/well of AgU. From the relatively small amount of starting material, good yields were obtained with increased specific activity economy in coating by ELISA. These results favour the adoption of affinity chromatography for purifying viral antigens.
Njayou
M
M
Laboratoire de Biochimie Microbienne, Faculté des Sciences, Yaounde, Cameroun.
Quash
G
G
D000818:Animals; D000956:Antigens, Viral / Q000276:immunology / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D006386:Hemagglutination Tests; D007074:Immunoglobulin G / Q000378:metabolism; D008459:Measles virus / Q000276:immunology / Q000302:isolation & purification*; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000378:metabolism; D014461:Ultracentrifugation; D014709:Vero Cells
D000956:Antigens, Viral; D007074:Immunoglobulin G; D009195:Peroxidase