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Immunological screening of a cDNA library using avidin-biotin-peroxidase method

Authors
Journal
Genetic Analysis Techniques
0735-0651
Publisher
Elsevier
Publication Date
Volume
2
Issue
2
Identifiers
DOI: 10.1016/0735-0651(85)90002-0
Disciplines
  • Biology

Abstract

Abstract The antigen-expressing cDNA clones were identified through the use of a highly sensitive immunoenzymatic technique. Bacterial colonies grown on nitrocellulose filters were lysed in situ. The prints of bacterial colonies were reacted sequentially with 1) primary antibody, 2) biotin-labeled secondary antibody, 3) avidin-biotin-peroxidase complex, and 4) the chromogenic substrate of peroxidase, diaminobenzidine (DAB). Compared to the widely used 125I-protein A or 125I-secondary antibody method, the current procedure has the following advantages: 1) it allows rapid detection of antigen-positive colonies within 4 hr after cell lysis; 2) it is highly sensitive due to the amplification of antigen-antibody reaction by the coupled biotin-avitin reaction; 3) positive colonies can be easily located and correctly picked, even at a high plating density, as each individual colony has a clearly defined edge and the color reaction has excellent signal-to-noise ratio; 4) the reagents do not decay, and no handling of radioactive isotope is required; 5) the original screening filter is stable for at least 6 months without significant color changes.

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