A simple immunological detection method for the direct screening of genes from clone banks.
A simple immunoassay has been developed which can be used in the isolation of particular gene(s) from a clone bank of recombinant plasmids. A clone bank of the DNA is constructed with a plasmid vector and maintained in Escherichia coli. The recombinant clones were filtered onto a hydrophobic grid membrane and grown up into individual colonies, and a replica was made onto nitrocellulose paper. The bacterial cells were then lysed with chloroform and the proteins were immobilized onto the nitrocellulose paper. The nitrocellulose paper is then reacted with a rabbit antibody preparation made against the particular antigenic product to detect the recombinant clone which carries the corresponding gene. The bound antibodies can be detected easily by a colorimetric assay using goat anti-rabbit antibodies conjugated to horseradish peroxidase. Positively reacting clones can be recovered from the master hydrophobic grid membrane filter for further characterization. We proposed to call this method "colony ELISA blot" and described the isolation of the genes coding for the soluble antigens of Pasteurella haemolytica using this method.
D003001:Cloning, Molecular / Q000379:methods*; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000032:analysis; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D007118:Immunoassay / Q000379:methods; D010325:Pasteurella / Q000235:genetics / Q000276:immunology; D010957:Plasmids; D014170:Transformation, Genetic
D004269:DNA, Bacterial; D004274:DNA, Recombinant; D004262:DNA Restriction Enzymes