Enhanced specificity in immunoscreening of expression cDNA clones using radiolabeled antigen overlay.
A highly sensitive and specific method has been developed for immunoscreening clones from an expression cDNA library. The procedures utilize a radiolabeled antigen detection method described originally for the immunoblotting of plasma proteins (5). Screening of rat alpha 1-antitrypsin clones was used. Comparison between Western blots of alpha 1-antitrypsin using both labeled antigen and protein A detection methods showed that the former yielded lower background and greater sensitivity than the latter. Further, this technique was shown to have a lower detection limit of less than 20 ng through Western blot analysis of varying concentrations of alpha 1-antitrypsin. The procedures are based on the expression of the protein by cDNA clones containing the DNA inserts in the correct reading frame. Following the transfer of phage proteins to nitrocellulose membranes, the bivalent antibodies bind monovalently to both nitrocellulose-bound-antigen in the phage lysates and radiolabeled antigen. The radiolabeled antigen overlay method is superior to the protein A detection method in sensitivity, specificity and reproducibility. This improved method can be applied in general for screening expression cDNA libraries, provided that the specific antiserum and radiolabeled antigen are available.
Chao
S
S
Dept. of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston 29425-2251.
Chao
L
L
Chao
J
J
D000818:Animals; D000941:Antigens; D001709:Biotechnology; D015153:Blotting, Western; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000276:immunology; D015723:Gene Library; D007457:Iodine Radioisotopes; D051381:Rats; D000515:alpha 1-Antitrypsin / Q000235:genetics / Q000276:immunology
D000941:Antigens; D007457:Iodine Radioisotopes; D000515:alpha 1-Antitrypsin; D004247:DNA