Fluorescence fluctuation immunoassay

The homogeneous fluorescent immunoassay described above allows one to measure the brightness of fluorescently tagged carrier particles that are suspended in a background of free, unbound fluorescent sources. We have demonstrated the feasibility of our technique using a gentamicin competitive assay as well as idealized model systems. We have seen that the fluctuation-correlation method is able to discriminate against free background sources because each fluorescing particle in solution contributes to the correlation peak [Eq. (4)] with a weighting equal to the square of its respective intensity. Hence, a few very bright sources contribute disproportionately to the "signal" relative to many weak ones. To take advantage of this property, one would therefore design an assay that uses relatively larger carrier particles, each of which is capable of binding on the order of 10(3) to 10(4) tagged antibodies or antigens. Unfortunately, the nonlinear dependence of the correlation peak on the brightness of the fluorescing species causes the technique to be perturbed by carrier particle aggregation; the apparent bound fluorescence intensity increases with the extent of aggregation. The latter may be an unavoidable consequence of performing assays using raw blood serum, for example. The ultimate usefulness of this method will depend on its sensitivity and speed when applied to "real" assays of clinical significance. These characteristics will be influenced by a number of technical details. Given our limited experience with the method thus far, it would appear that its principal drawback is its relatively slow speed. In order to decrease the time needed for a reliable measurement, one must average the random fluctuations in the fluorescent intensity to zero more quickly. In principle, this can be accomplished by decreasing the shot noise by collecting a larger fraction of the fluorescent light, and increasing the sampling rate. The method requires rather complicated instrumentation; it is by no means clear that this level of complexity is justified given the realistic level of sensitivity that will be obtained by this technique.