Direct antigen capture by soluble scFv antibodies. A method for detection, characterization, and determination of affinity.
For ease of detection, soluble forms of phage-displayed scFv antibodies are usually expressed with a tag, e.g., c-myc or His (Histidine). The binding is then assayed by a monoclonal antibody to the tag. In this article, we describe the use of biotinylated antigen for detecting soluble scFv antibodies without utilizing the peptide tag detection system. The scFv antibodies were against the oncoplacental antigen heat-stable alkaline phosphatase (HSAP). The method essentially consisted of either reverse Western or antigen capture enzyme-linked immunosorbent assay (ELISA). In the reverse Western, periplasmic extract was electrophoresed, and binding to biotinylated antigen was detected by the detection system based on streptavidin-horseradish peroxidase. The antigen capture ELISA utilized the binding of periplasmic extract to a polystyrene plate. We have also demonstrated the use of antigen capture ELISA for studying specificity and affinity of the selected clones. Although these techniques have been developed for antibodies to HSAP, they have general utility for phage expression systems without a peptide tag.
Kala
M
M
Department of Biochemistry, All India Institute of Medical Sciences, New Delhi-110029, India.
Bajaj
K
K
Sinha
S
S
D000469:Alkaline Phosphatase / Q000276:immunology*; D000915:Antibody Affinity; D000918:Antibody Specificity; D019921:Biotinylation; D015153:Blotting, Western; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D007527:Isoenzymes / Q000276:immunology; D019151:Peptide Library; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D012995:Solubility
D007135:Immunoglobulin Variable Region; D007527:Isoenzymes; D019151:Peptide Library; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase