SU1464089A1 - Method of conducting immunoanalysis - Google Patents

Abstract

The invention relates to immunoassay methods and can be used in medicine, agriculture, industry for the determination of biologically active compounds. The aim of the invention is to increase the sensitivity of the immunoassay. Compounds of the class of metalloporphirins, which are capable of intensive phosphorescence, were used as a label for immunoassay. Conducting an immunoassay involves obtaining reagents covalently labeled with metal porphyrins, conducting immunological reactions, including with the participation of labeled compounds, and detecting specific binding on phosphorescence of the label. Detection is carried out with microsecond time resolution in solution at room temperature or in liquid nitrogen. The sensitivity of the method is 0.05 nmol / L. 4 il. i (L

Description

one

The invention relates to immunoassay methods and can be used in medicine, agriculture, industry for the determination of biologically active compounds.

The purpose of the invention is to increase the sensitivity of the method — by using methadloporphyrins as markers,

FIG. Figure 1 shows the absorption spectrum of Pd-coproporphyrin I in an aqueous solution (absorption maxima of 396.514 and 548 nm); in fig. 2 - corrected emission spectrum of Pd-propropphyrin 1; in fig. 3 — Dependence of the luminescence intensity on concentration: 1) P6-coproporphyrin I (excitation at 395 nm, recorded at 665 nm); 2) europium (excited at 337 nm, registered at 616 nm); in fig. 4 - calibration curve for the determination of monoclonal antibodies by the solid phase method (concentration of labeled antibodies in the second stage is 30 nmol / l).

Example 1. Preparation of Pd (2 +) - coproporphyrin I. To a solution of 72.7 kg of coproporphyrin I dihydrochloride in 40 ml of glacial acetic acid, do-. Add a prepared solution of 75 mg of PdCl in 15 ml of freshly purified dimethylformalin and heat the mixture for 1 hour at 120 ° C (spectrophotometric control at a wavelength of 620 nm until the absorption bands of nonmetal porphyrin in dimethylformamide solution disappear). K go VI h

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| To this solution, 1.5 ml of water was added to the solution and the ophthalmic solution was gradually taken up to room temperature. The precipitated crystalline precipitate is filtered off, washed with 10 ml of acetic acid, then with water and ethanol and dried in a vacuum desiccator. The output of Pd (2 +) - | Coproporphrin I 65 mg (85.7%). The absorption and phosphorescence spectra are shown in FIG. 1 and 2. Vre: and the lifetime of phosphorus in an oxygen-free aqueous solution at 0.67 ms. The phosphorescence quantum yield is 0.20. EXAMPLE 2 Antibody Production Labeled with Pd (2 +) -Coproporphyrin I To 1 mg Pd (2 +) -Copropofirin I in 1 MP of water is added 1.5 mg 1-cyclo-. Hex-1-3-(2-morpholinoethyl) carbodiimide-p-toluenesulfonate and incubated for 15 min, maintaining the pH 5.8-6.2. The resulting solution is added to 20 mg of monoclonal antibodies dissolved in 2 ml of 0.1 M carbonate buffer,

IpH 9.0, and incubating for 3 hours in the dark. The resulting solution was chromatographed on a column with a carrier. Touoreag 1 HW-55, collecting fractions of the protein peak. The content of Pd (2 +) - coropopherphin in the labeled protein is determined by the absorption at 280 and 390 nm. The sensitivity of detec1-5I on a LS-5 pulsed luminescence spectrometer (Perkin Elmer) with a time-resolution of antibodies labeled with Pd-coproporphyrin I (composition 1; 3) is not inferior to the sensitivity of a euro-nebula in the definition on the same instrument and is 0.01 nMol / L (Fig. 3).

Froze Conducting solid phase immunoassay. On. The surface of polystyrene tubes adsorb rabbit antibodies against globulins from 1 MP of 0.1 M carbonate buffer at a concentration of 10 μg / mp for 3 hours at room temperature. Then the solution is poured and the tubes are washed 3 times with phosphate buffered saline.

14640894

ferry with Triton X-100 (K-phosphate

0.01 M, pH 7.4, NaCl 0.15 M, 0.05% Triton X 100; FSBT).

In tubes with adsorbed antibodies pour 1 ml of standard solutions of monoclonal antibodies in PBS and incubate i h at. After that, the solutions are sewed, the tubes are washed 2 times with PBS, poured into them with 1 MP PBS containing 10 µg of monoclonal antibodies labeled with Pd-coproporphyrin I, and incubated for 1 hour at dark.

15 Then the solutions are drained, the tubes are washed 3 times with FSBT, and they are poured into them with 1 MP of desorbing solution (0.1 M NsiOH). After 20 minutes, 0.1 ml of a 1 M solution of sodium bisulfite is added and the intensity of phosphorescence is measured.

Example 4: Recording of phosphorescence. The excitation is carried out with a nitrogen laser (337 nm) with a frequency of 50 Hz.

25 and a half-width of the 10 not. Filters are used in registration, which are cut off by quanta with wavelengths less than 620 nm. The delay time is 0.3 ms, the counting is 0.7 ms, the counting time is 1 s. The calibration curve 30 is shown in FIG. 4. The sensitivity of the analysis is 0.01 nmol / l.

35

Claims (1)

Invention Formula The method of carrying out an immunoassay by obtaining antibodies or antige-. new, labeled with luminescent compounds, carrying out immunochemical reactions, followed by detection of specific binding by luminescence intensity, which is the case with luminescence. In order to increase the sensitivity of the method, phosphorescent metal porphyrins are used as the luminescent compound 45, and the detection of a specific bond is based on the intensity of phosphorescence. 40 Invention Formula The method of carrying out an immunoassay by obtaining antibodies or antige-. new, labeled with luminescent compounds, carrying out immunochemical reactions, followed by detection of specific binding by luminescence intensity, which is the case with luminescence. in order to increase the sensitivity of the method, phosphorescent metal porphyrins are used as the luminescent compound, and detection of a specific bond is based on the intensity of phosphorescence. 1.0 0.8 0.6 0. 0.2. 600 650 700  -G  9 800 (ffff) 850 TO .0 V.5 3.0 5 to s 1.Q 0.5 LOfffl) -eleven -7 / -eleven -YU -9-8 LQGlCjJ l FIG. in- / o-f -W -9 L06 (C) M -eight Fig.Z t -7  - 2 7