Magnetic bead enzyme-linked immunosorbent assay (ELISA) detects antigen-specific binding by phage-displayed scFv antibodies that are not detected with conventional ELISA.
An efficient means for the detection of antigen-specific binding by phage-displayed antibodies would facilitate the selection of such phage, especially from libraries with large repertoires of V-genes. We report the development and characterization of a magnetic bead phage ELISA which detects antigen binding phage which could not be detected by conventional ELISA. We were attempting to select phage binding to the oncodevelopmental antigen, heat-stable alkaline phosphatase (HSAP). Although there was an obvious enrichment in the phage titers after successive rounds of selection, we were unable to detect antigen-binding phage by ELISA on a plastic surface. However, ELISA with a suspension of superparamagnetic particles covalently conjugated to HSAP effectively identified antigen-binding phage after the fourth round of selection. This method could also detect antigen-specific binding of individual phage clones. Some of the phage clones bound to either amino- or carboxy-terminal-conjugated HSAP, perhaps reflecting the differences in the exposed epitopes. It is suggested that a sensitive method such as magnetic bead phage ELISA be tried before declaring a phage selection as unsuccessful or concluding that a phage clone does not bind antigen.
Kala
M
M
Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India.
Bajaj
K
K
Sinha
S
S
D000469:Alkaline Phosphatase / Q000378:metabolism; D000937:Antigen-Antibody Reactions; D001287:Attachment Sites, Microbiological; D001435:Bacteriophages / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D018189:Immunomagnetic Separation
D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D000469:Alkaline Phosphatase