Bioaffinity assay methods are commonly used in the analytics of various biologically active molecules. Different applications of these methods are widely used in routine diagnostics and research laboratories. The most commonly used bioaffinity assay methods are immunoassays in which antigens serve as biospecific reagents.
The method of this invention refers to fluorometric bioaffinity assays in which the first biospecific reagent Ab (for example, a protein, antibody, nucleotide) is bound to a solid reaction matrix, which may be, for example, the inner wall of a reaction cuvette or microparticle suspension. Components bound to the reaction matrix are later referred to as solid phase. Another biospecific reagent, which is later referred to as ligand (biologically active molecule such as a steroid or other hormone, drug or oligonucleotide), is labelled with a fluorescent label. This reagent is later simply referred to as label. The assay method of this invention is a so called compentitive assay in which a reagent bound to the solid matrix has affinity both to an analyte and labelled ligand.
The competitive assay method of this invention is particularly suitable for small analyte molecules Ag. Characteristic to the competitive method is that the concentrations of the reaction components are adjusted so that the concentration of the biospecific reagent Ab is typically lower than that of the analyte and labelled ligand and the type of the assay is also called as limited reagent assay. Labelled ligand Ag* may be the same molecule as analyte molecule Ag, or it may be another molecule containing a corresponding affinity determinant such as Ag. Since the concentration of reagent Ab is lower than that of other components in the reaction solution, components Ag and Ag* bind to reagent Ab of the solid phase in their proportional concentrations. This is therefore called competitive binding. A standard curve of this assay is non-linear (sigmoidal). The standard curve refers to a graph obtained from calibration measurements. The graph presents dependence of signal response (fluorescence intensity) on analyte concentration.
A problem with conventional assay and research methods lies in their complexity. For example, the determination of hormons from blood with a competitive fluorometric immunoassay method requires several steps as follows: separation of cells from the serum, dispensing and dilution of the serum sample, addition of the labelled reagent, incubation, separation of the free fraction by washing, addition of the measurement solution and measurement of the signal. The majority of known measuring devices require at least 10-100 microliter reaction volumes. The separation step is very difficult to perform reliably if the reaction volume is less than 10 microliter.
There is a constant need for simpler microvolume and more cost-effective assays for routine diagnostics. In practice, this means a single step assay method with liquid volumes only a fraction of volumes of current assays. The new fluorometric biospecific assay method of this invention is a single step method, it does not require separation of the label and suits well for assaying very small samples, which may be either solutions or cell suspensions. In the method according to this invention, the signal strength is also independent of a sample size, and the smallest possible sample size depends only on the available liquid handling techniques.
An example of computer simulation of a typical assay has been presented in FIG. 1 and FIG. 2. The method according to this invention can be performed for example as follows:
A solid phase is prepared in advance. The solid phase is coated with biospecific reagent Ab which binds said analyte and ligand. The solid phase may be located either on the inner wall of the reaction cuvette, or on the surface of the microparticles added to the reaction volume.
A ligand is labelled with a fluorescent molecule.
The sample to be determined and the labelled ligand Ag* is added into the reaction cuvette as such or with the microparticle suspension; and due to bioaffinity, analyte molecules Ag and labelled ligands Ag* start to bind competitively to reagent molecules residing on the surface of the solid phase.
A laser beam is focused through an objective lens into the liquid in the cuvette. The parameters of the laser beam and the numerical aperture of the lens are selected so that the focal volume of the laser beam is significantly smaller than the volume of the cuvette; and in practice limited by diffraction. Moreover, the laser wavelength and the intensity is selected to generate two-photon excitation in the fluorescent molecules of the solution. The excited states of the fluorescent molecules will relax with a specific fluorescence decay rate, emitting photons which are detected with a appropriate photon detector. The signal strength obtained from the detector or the single photon count rate is directly proportional to the concentration of fluorescent labels in the solution.
The less analyte there is in the solution the more fluorescent label is bound to the solid phase. It is possible, therefore, on the basis of the calibration and the signal obtained from the label, to determine the concentration of the analyte in the solution.