Protein quantification from complex protein mixtures using a proteomics methodology with single-cell resolution

We have developed an extremely sensitive technique, termed immune-detection amplified by T7 RNA polymerase (IDAT) that is capable of monitoring protei ns, lipids, and metabolites and their modifications at the single-cell leve l. A double-stranded oligonucleotide containing the T7 promoter is conjugat ed to an antibody (Ab), and then T7 RNA polymerase is used to amplify RNA f rom the double-stranded oligonucleotides coupled to the Ab in the Ab-antige n complex. By using this technique, we are able to detect the p185(her2/neu ) receptor from the crude lysate of T6-17 cells at 10(-13) dilution, which is 10(9)-fold more sensitive than the conventional ELISA method. Single-cha in Fv fragments or complementarity determining region peptides of the Ab al so can be substituted for the Ab in IDAT, In a modified protocol, the oligo nucleotide has been coupled to an Ab against a common epitope to create a u niversal detector species. With the linear amplification ability of T7 RNA polymerase, IDAT represents a significant improvement over immuno-PCR in te rms of sensitivity and has the potential to provide a robotic platform for proteomics.