FI70330C - FOERFARANDE FOER UTFOERING AV IMMUNOBESTAEMNINGAR - Google Patents

Description

70330

Method for performing immunoassays

The invention relates to a fluorometric or phosphorimetric immunoassay method in which an antibody or antigen 5 labeled with a fluorescent or phosphorescent label is attached to the inner wall of a measuring vessel.

In fluorometric immunoassays (FIA), the antigen or antibody is adsorbed onto, for example, cuvettes or microtiter plates made of polystyrene or polyacrylic. This so-called the solid phase-bound antigen or antibody is reacted with an antibody, or antigen, respectively, in the test sample, and an antibody or antigen, respectively, labeled with a fluorescent molecule. The less antibody or antigen in the sample, the more labeled α1-15 will adhere to the solid phase. When the labeled antibody or antigen remaining in the liquid phase is separated, and the amount of labeled substance adhering to the solid phase is measured with a fluorometer, the concentration of antibody or antigen in the sample is determined.

In known methods, excess labeled antibody or antigen must be washed away prior to fluorometric measurement.

It is an object of the present invention to provide a fluorometric solid phase immunoassay method in which 25 additional tracers do not need to be removed from the measuring vessel prior to fluorometric measurement.

In the method of the invention, the antigen or antibody is attached to the wall or bottom of the measuring vessel and the fluorescence radiation is measured through the wall or bottom. After the reaction of the 30 samples and the added, labeled antibody or antigen with the solid phase antigen, or antibody, respectively, a denser liquid than the sample is added to the measuring vessel, displacing the sample basin, 70330 leave out. Thus, all fluid transfers associated with the assay are fluid additions. This speeds up the configuration and makes it crucial 5 to automate it.

When a liquid denser than the sample is added to the sample in the method, the liquid is more suitably colored so that it strongly absorbs light at either the excitation or emission wavelength of the fluorescence or both, i.e. being 10 black in color. In this way, fluorescence can also be measured directly from below without interfering with excess tracer or background radiation. Preferably, the method is implemented in such a way that the solid phase is at the bottom of the measuring vessel and both the excitation light is conducted to the measuring vessel and the fluorescent light is collected on the detector through the bottom of the measuring vessel.

The liquid and dye added to the method should, of course, be such that they do not interfere with the measurement between the antigen-antibody reaction and prevent the marker from fluorescing.

Any staining should be such that the fluorescence of the tracer in the free liquid of the sample is not significantly visible in the measurement channel. If the colored liquid mixes with the sample, the absorption coefficient 25 must be very high. If the colored liquid does not mix with the sample and is heavier, an absorption coefficient of the order of 1 / mm is sufficient if the thickness of the colored liquid layer is several millimeters.

Fluorocarbons which are liquid at room temperature, such as, for example, trichlorotrifluoroethane (Freon-113) and dibromotetrafluoroethane (Freon-11 ^ B2), are well suited as denser, immiscible liquids. These can be colored black, eg with fine carbon powder (soot).

According to one embodiment, a liquid denser than the sample is added to the measuring vessel, which is allowed to solidify before the measurement.

Of course, the method can also be applied to a method that utilizes phosphorescence.

Claims (5)

A method for performing immunoassays, wherein 5 antibodies or antigens labeled with a fluorescent or phosphorescent label and attached to the bottom or side walls of a measuring vessel are excited by appropriate radiation and a liquid that displaces i0 from the sample basin, and that both excitation radiation is introduced into the sample and fluorescence or phosphorescence radiation is collected on the detector through the wall or bottom of the measuring vessel. A method according to claim 1, characterized in that a liquid denser than the sample is dispensed into the measuring vessel, which strongly absorbs the excitation radiation at the wavelength. Method according to Claim 1 or 2, characterized in that a liquid denser than the sample is dispensed into the measuring vessel, which strongly absorbs the emission radiation at the wavelength. Method according to Claim 2 or 3, characterized in that the liquid denser than the sample is black. Method according to one of Claims 2 to 4, characterized in that both the excitation radiation is introduced into the sample and the fluorescence or phosphorescence radiation is collected on the detector through the bottom of the measuring vessel. Method according to one of Claims 1 to 5, characterized in that a liquid denser than the sample is dispensed into the measuring vessel, which is allowed to solidify in the measuring vessel.