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.

Homogeneous fluorescent immunoassay.

A fluorescent immunoassay based on the correlation of fluctuations in particle number measures the amount of tagged species bound to micrometer-sized beads and is insensitive to background fluorescence. Without separation steps, a competitive assay can resolve I nanogram of gentamicin per milliliter from a total sample volume of only 10 microliters.

Diffusion coefficients of hemoglobin by intensity fluctuation spectroscopy: effects of varying pH and ionic strength.

Measurements of the mutual diffusion coefficients (D) of the liganded human hemoglobins (Hb) oxy-HbA and oxy-HbS were performed as a function of Hb concentration (CHb), pH, and ionic strength (tau) by intensity fluctuation spectroscopy (IFS). Average diffusion coefficients, (D), and normalized variances, ((D/(D) - 1)2), were recorded. Results are reported and select features are discussed quantitatively. (a) for tau = 0.15 M, the shape of the (d) vs. CHb curve is found to vary with pH. We developed a precise description of this effect in the form of an algebraic relationship between (D), CHb, and Z, the titration charge. (b) only slight differences between the (D) values of oxy-HbS and oxy-HbA are observed, at tau = 0.15 M, for CHb Less Than or Equal To 10 g%. These differences are explained by the theory of part a. (c) No evidence of aggregation is found in solutions of oxy-HbA or oxy-HbS, at tau = 0.15 M, for CHb Less Than or Equal To 10 g%. (d) Indications of aggregation appear in oxy-HbA solutions at very low concentrations of salt. An estimate is made of the extent of aggregation, and the average radius of a cluster is determined.

Fluorescence immunoassay based on long time correlations of number fluctuations.

We report the development of a fluorescence-based immunoassay technique relying on the physical phenomena of random number fluctuations and diffusion, which we review. By determining the autocorrelation of the fluctuations in the fluorescent intensity, this methid is able to measure the amount of labeled antigen or antibody that is bound to micrometer-sized carrier particles in solution. The principal advantage of this technique is its insensitivity to small, fast-diffusing sources. It also discriminates against weakly fluorescent contaminants of size comparable to the carrier particles. We demonstrate these attributes by using two model systems: a human IgG assay and an idealized system consisting of polystyrene fluorescent spheres and rhodamine dye.

Quantitative verification of the existence of high molecular weight protein aggregates in the intact normal human lens by light-scattering spectroscopy.

The method of quasi-elastic light-scattering spectroscopy was used to establish quantitatively the concentration of high mmolecular weight (HMW) aggregates present in the normal human intact lens as a function of age. The concentration of HMW proteins increases monotonically with age. HMW proteins are absent in the infant lens, but represent 3% of the total soluble lens protein at age 60 years. The percent concentration of HMW proteins measured in intact lenses of various ages by quasi-elastic light scattering is in striking agreement with values determined biochemically.