Nanogram quantitation of secreted protein in a recombinant yeast fermentation using an immuno-ligand assay

A liquid-phase immuno-ligand assay has been developed for quantitative determination of recombinant tick anticoagulant protein (rTAP) secreted in yeast fermentations. A polyclonal anti-TAP antibody was labeled with biotin or fluorescein. Labelled antibodies were used in a non-competitive sandwich format to capture rTAP from solution, then reacted with urease-conjugated anti-fluorescein antibody. Detection of the immune complex was by a commercially available silicon-based potentiometric sensor which measures urease activity. Sample throughput was 90 samples per 7 h with a 2 h incubation time. The range of the standard curve was 0.1-10 ng/ml with an assay sensitivity of 0.025 ng/ml. For a mid-range concentration of 1 ng/ml, intraday and interday method precision was determined to be 1.031 +/- 0.061 and 1.077 +/- 0.026 ng/ml, respectively. Typically, spiked samples of 1 microgram rTAP/ml fermentation medium required dilutions of 1/1000 to generate a response in the mid-range of the standard curve. This assay provides a convenient method to quantitate product expression in multiple fermentation samples within 3 h after sampling. In addition, a modified version of the assay was developed which provided accurate results within 1 h of sample acquisition.