Detection of biotin in individual sea urchin oocytes using a bioluminescence binding assay

The ability to detect biomolecules in single cells is important in order to fully understand the processes by which many biochemical events occur. To that end, we have developed a bioluminescence binding assay capable of meas uring the intracellular biotin content of individual cells. The assay depen ds on competition between an aequorin-biotin conjugate (AEQ-biotin) and fre e biotin within the oocytes for binding sites on the protein avidin. The as say is performed by microinjecting each component into the oocytes and foll owing the resulting bioluminescence within the oocyte upon triggering of ae quorin. Results obtained using sea urchin oocytes show that the assay perfo rmed within the cells behaves in a manner consistent with assay theory. Usi ng the assay, the individual biotin content of the oocytes is an average of similar to 20 amol. To our knowledge, this is the first reported multicomp onent binding assay to be performed inside an intact single cell.