Immunoaffinity purification of the NS1 protein of Murray Valley encephalitis virus: selection of the appropriate ligand and optimal conditions for elution.
A novel approach was used to select the most suitable antiviral monoclonal antibody (mAb) and elution conditions for immunoaffinity purification of the NS1 protein of Murray Valley encephalitis virus (MVE). Crude NS1 protein was subjected to a variety of chemical conditions produced by common elution buffers, and tested with a panel of NS1-specific mAbs by ELISA to determine which buffers denatured antigenic epitopes. Buffers that caused least structural damage to NS1 epitopes were tested by ELISA for dissociation of NS1-mAb complexes adsorbed to the solid phase. For each mAb analysed, the conditions required to break the mAb-NS1 complex on the solid phase were similar to those required to release antigen bound to mAb-sepharose beads. From these results we selected an appropriate antibody for affinity purification of the non-structural viral protein NS1 on CNBr-activated sepharose columns. Elution at pH 11.5 yielded good recoveries of highly pure and antigenically intact NS1 dimer. The results demonstrate that the appropriate ligand and optimal elution conditions can be rapidly determined generally by immunoassay in microtitre plates.
Hall
R A
RA
Department of Microbiology, QE II Medical Centre, University of Western Australia, Nedlands.
Coelen
R J
RJ
Mackenzie
J S
JS
D000911:Antibodies, Monoclonal; D002213:Capsid / Q000276:immunology / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D004048:Diethylamines; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005416:Flavivirus / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D015636:Magnesium Chloride; D014758:Viral Core Proteins / Q000276:immunology / Q000302:isolation & purification*; D017361:Viral Nonstructural Proteins
D000911:Antibodies, Monoclonal; D004048:Diethylamines; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; D015636:Magnesium Chloride; C034281:diethylamine