Preparation of immunoblot test stripes from a Rubella virus-like particles dye crystal complex as antigen.
Stably transfected Chinese hamster ovary (CHO24S) cells were the source for Rubella virus-like particles (RVLP) containing all structural proteins (E1, E2, C and their dimers). RVLP are secreted from the CHO24S cells into the medium and the time-point for collecting the medium with the highest yield of >100 kDa proteins (with 17 mg protein from 10 ml cell culture supernatant) was after 2 days of incubation. Different methods for RVLP isolation from the cell culture supernatants were assessed by SDS-PAGE and Western blotting (using sera positive or negative for Rubella virus (RV)-specific antibodies or an anti-E1 monoclonal antibody). A combination of membrane filtration with a rapid, novel gradient ultracentrifugation step (using Coomassie brilliant blue G crystals as adsorbens for RVLP that facilitated virus isolation) was the most suitable technique. 132 RV-positive human sera (RV IgG > 20 IU/ml by commercial ELISA) were tested by our "self made" immunoblot test stripes (using RVLP adsorbed to dye crystals as antigen) for the presence or absence of antibodies specific for RV structural proteins. 57.6% of these sera had antibodies against E1, E2 and C, 31% against E1 and C, and 1.5% against E1 only, whereas 3.8% had no RV specific antibodies and only 6.0% were equivocal which demonstrated that these "self made" test stripes can reliably differentiate RV antibody specificities.
Giessauf
A
A
Department of Hygiene, Microbiology and Social Medicine, Innsbruck Medical University, Innsbruck, Austria. adreas.giessauf@uni-graz.at
Flaim
M
M
Walder
G
G
Dierich
M P
MP
Würzner
R
R
D000818:Animals; D000914:Antibodies, Viral / Q000032:analysis; D000918:Antibody Specificity; D000956:Antigens, Viral / Q000235:genetics; D016466:CHO Cells; D004396:Coloring Agents; D006224:Cricetinae; D003460:Crystallization; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D046911:Macromolecular Substances; D012409:Rubella / Q000175:diagnosis / Q000276:immunology; D012412:Rubella virus / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D014162:Transfection; D014771:Virion / Q000276:immunology / Q000302:isolation & purification
D000914:Antibodies, Viral; D000956:Antigens, Viral; D004396:Coloring Agents; D046911:Macromolecular Substances; C040901:rubella antibodies