Isolation of mutants of Aujeszky's disease virus with antigenically altered glycoprotein E by affinity chromatography using monoclonal antibodies.
An efficient method for isolation of virus mutants with antigenically altered proteins is described. The method is based on the separation of viruses with wild-type and antigenically altered proteins by affinity chromatography using monoclonal antibodies (MAbs). A nonessential glycoprotein E (gE) of Aujeszky's disease virus (ADV) was chosen as a model for introducing the antigenic changes. The ADV strain Ka mutagenised with 5-bromo-2'-deoxyuridine was used for the selection of mutants that do not bind to gE-specific MAb conjugated to resin. After three rounds of isolation by affinity chromatography, the resulting viruses that escape the binding to MAb were plaque-purified by plating at limiting dilution, and virus isolates were tested by the gE-specific sandwich ELISA in which the selecting MAb was used as a capture antibody. About 70% of the ADV isolates tested were not recognised by the sandwich gE-ELISA. The analysis of some of virus isolates in indirect ELISA with a panel of 16 gE-specific MAbs revealed that at least several of the generated virus isolates were mutants expressing gE with alterations in the epitope of the selecting MAb 75/7, as well as in the majority of other conformation-dependent epitopes of gE. The method for the production of antigenically altered viruses by affinity chromatography using MAbs is simple and convenient, and can be utilised with MAbs irrespective of their virus-neutralising activity.
Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino. morenkov@ibfk.nifhi.ac.ru
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Morenkov
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Smirnov
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Vrublevskaya
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000956:Antigens, Viral / Q000737:chemistry / Q000276:immunology; D002846:Chromatography, Affinity; D000939:Epitopes; D011558:Herpesvirus 1, Suid / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D016296:Mutagenesis; D011557:Pseudorabies / Q000821:virology*; D014759:Viral Envelope Proteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D000939:Epitopes; D014759:Viral Envelope Proteins