Immuno affinity purification of foot and mouth disease virus type specific antibodies using recombinant protein adsorbed to polystyrene wells.
The specificity of foot and mouth disease virus (FMDV) serological tests depends largely on the quality and purity of the antibodies used. Such type specific antibodies can be generated by hybridoma technology. Alternatively, the specific antibodies can be selected from polyclonal serum by immunoaffinity chromatography using recombinant protein/peptide bound affinity matrices. Based on this approach, we purified selectively antibodies against the major epitopes of VP 1 of FMDV serotype Asia 1 using recombinant protein adsorbed to polystyrene wells. Optimum buffer conditions were standardised for efficient elution. Buffer consisting of 4 M MgCl2 with 75 mM HEPES pH 6.5 was found to be optimum with respect to elution efficiency of bound antibodies and integrity of antigen. The specific reactivity of eluted antibodies was confirmed by dot-enzyme linked immunosorbent assay (dot-ELISA) and antigen capture reverse transcription polymerase chain reaction (Ag/RT-PCR). The effect of temperature and repeated elution on the stability of coated protein were studied.
Bayry
J
J
Indian Veterinary Research Institute, Bangalore.
Prabhudas
K
K
Bist
P
P
Reddy
G R
GR
Suryanarayana
V V
VV
D000818:Animals; D000914:Antibodies, Viral / Q000235:genetics / Q000302:isolation & purification*; D000918:Antibody Specificity; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology*; D005537:Aphthovirus / Q000145:classification / Q000276:immunology*; D002213:Capsid / Q000235:genetics / Q000276:immunology*; D036022:Capsid Proteins; D004356:Drug Storage; D007163:Immunosorbent Techniques / Q000295:instrumentation; D011137:Polystyrenes / Q000378:metabolism*; D011994:Recombinant Proteins / Q000276:immunology*; D012680:Sensitivity and Specificity; D012703:Serotyping
D000914:Antibodies, Viral; D000956:Antigens, Viral; D036022:Capsid Proteins; D011137:Polystyrenes; D011994:Recombinant Proteins; C442501:VP1 protein, Foot-and-mouth disease virus