A modified screening method for pcDNA-1 expression libraries which is applicable to both surface and intracellular antigens. Cloning of a colon carcinoma antigen.

Abstract:

:A modification of the COS cell transient expression system for isolating cDNAs encoding antibody defined molecules was used to clone the carcinoma antigen defined by monoclonal antibody CORC-6. Bacteria were transformed with cDNA prepared from colon carcinoma cells and immediately divided into aliquots containing approximately 500 independent clones. The aliquots were grown for 8 h before frozen. Screening was performed by isolating plasmid from each aliquot, transfecting COS-7 cells, and staining the fixed cells with monoclonal antibodies. The aliquot giving rise to antigen expressing cells was then subdivided until a single antigen encoding colony was obtained. This approach allows a library to be rescreened and is equally applicable to cytoplasmic and surface localized antigens. Using this approach, the antigen defined by CORC-6 was identified as carcinoembryonic antigen.

journal_name

J Immunol Methods

authors

Grimm T,Johnson JP

doi

10.1016/0022-1759(95)00155-4

subject

Has Abstract

pub_date

1995-10-26 00:00:00

pages

305-12

issue

2

eissn

0022-1759

issn

1872-7905

pii

0022175995001554

journal_volume

186

pub_type

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