An application of protein microarray in the screening of monoclonal antibodies against the oyster mushroom spherical virus.
The oyster mushroom spherical virus (OMSV) is a causative agent of dieback disease in the oyster mushroom, Pleurotus ostreatus. Outbreaks of this virus occasionally result in serious disease that is associated with hefty economic losses. Thus, the detection and removal of OMSV-infected spawn is considered to be a crucial step for the stable production of P. ostreatus. For the detection of OMSV, we attempted to generate monoclonal antibodies (mAbs) against an RNA polymerase domain (RPD) of an OMSV protein. In an effort to simplify the laborious multistep mAb screening process, we developed a protein microarray on a slide glass that is chemically modified with the RPD protein. The culture supernatants of 87 hybridoma cells, which were prepared from the fusion of RPD-immunized mouse spleen cells with myeloma cells, were spotted onto the RPD-coated microarray. The binding of mAb to RPD was detected via Alexa 488 dye-labeled anti-mouse immunoglobulin G (IgG) as a secondary antibody. Of 87 samples, 13 evidenced a significant level of fluorescence signal intensity. Subsequent immunoblot analysis revealed that the specificity of each mAb against RPD coincided with the corresponding fluorescence signal intensity, thereby indicating the effectiveness of the protein microarray in mAb screening.
Kim
Sang-Woo
SW
Deparment of Microbiology and Research Institute of Life Science, Gyeongsang National University, Chinju 660-701, Korea.
Kim
Min-Gon
MG
Jung
Hyo-Am
HA
Lee
Kyung-Hee
KH
Lee
Hyun-Sook
HS
Ro
Hyeon-Su
HS
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology*; D005260:Female; D006825:Hybridomas / Q000276:immunology / Q000378:metabolism; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D020076:Pleurotus / Q000821:virology*; D040081:Protein Array Analysis; D017434:Protein Structure, Tertiary; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D014780:Viruses / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D014764:Viral Proteins