JPS62298761A - Fluorescent immunoassay - Google Patents

Abstract

PURPOSE:To measure a plurality of antigens in the same sample at a high sensitivity simultaneously, by using a labelled antibody produced by bonding an antibody to a phycobiliprotein as fluorochrome protein. CONSTITUTION:The bonding of an antibody to a phycobiliprotein is executed by an ordinary method. A bonding is performed between R-phycoerythrin and an anti-human albumin antibody, C-phycocyanine and an anti-human immunoglobulin G antibody, allophycocyanine and an anti-humam immunoglobulin A antibody. On the other hand, three kind of antibodies not labelled are immobilized on respective carboxylated polyacryl amide gel beads. These three kind of bead suspensions are taken into equivalent test tubes and a mixed reference liquid of an antibody to be inspected is added to each of the test tubes and the supernatant is thrown away after a reaction. Liquids of antibodies labelled of said three kinds are added to the beads to react, after a washing and separation are conducted, the beads are suspended in a buffer. The intensity of fluorescence is measured using excitation wavelength and emission wavelength suitable for individual phycobiliproteins.

Description

Detailed Description of the Invention 3. Detailed Description of the Invention [Field of Industrial Application] The present invention relates to an immunoassay method, and particularly to an immunoassay method suitable for detecting trace components in biological samples.

[Conventional technology]

Conventionally, immunoassay methods using phycobiliproteins have been described in Clinical Chemistry, Vol. 29 (1983).
) pages 1582 to 1586 (CIin, Ch.
ew,, 29, (1983) Pρ, 1582-
1586) discusses an immunoassay method using phycoerythrin, but it uses one type of protein, and there is no known example of using multiple types of phycobiliproteins in an assay by binding them to different antibodies.

[Problem that the invention seeks to solve]

The above-mentioned conventional technology discusses the use of one type of phycobiliprotein and does not take into consideration the detection of multiple measurement targets from the same sample. Not enough information could be obtained quickly to make a decision. Another option is to label different types of low-molecular organic fluorescent substances, but these labeling reagents generally have low solubility in water, are difficult to bind to antibodies, and have low quantum yield and molar absorption. Due to problems such as low coefficients except for a few reagents (fluorescein and rhodamine B), it has been extremely difficult to measure multiple antigens simultaneously with high sensitivity.

An object of the present invention is to measure antigens with high sensitivity from the same sample containing multiple antigens.

[Means for solving problems]

In order to achieve the above purpose, C
- Phycobili proteins consisting of phycoerythrin, B-phycoerythrin, R-phycoerythrin, C-phycocyanin, R-phycocyanin, allophycocyanin, etc. were used, combined with antibodies, and applied to immunoassays.

Here, the term "antibody" includes the Fab' component (active cleavage fragment) of the antibody. Binding of antibodies and phycobiliproteins is carried out by a conventional method ([Chemical modification of proteins (2, 2)].

Written by Ohno, Kanaoka, Sakiyama, and Maeda, published by Gakkai Publishing Center, 198.
1 year, etc.), the amino group or carboxyl group of the antibody and the carboxyl group or amino group of the phycobiliprotein are bonded with a cross-linking reagent, or the Fab' of the antibody is
This can be carried out by bonding the thiol group of the component and the amino group of the phycobiliprotein in a similar manner.

[Effect]

Phycobiliproteins are a group of fluorescent pigment proteins, and both absorption and fluorescence wavelengths are in the long wavelength region of 480 nm or more, which is less susceptible to interference by coexisting components.

In addition, the quantum yield is extremely high at 0.51-0.98, -
T-/L/extinction coefficient is also 2.3-24. IX]05M
Very large at ``cm''.Low molecular fluorescent substance fluorescein (molar extinction coefficient 9 x 10'M'c
m'.

A fluorescence intensity several to several tens of times higher than the quantum yield (0.65) can be obtained. The main phycobiliproteins are B-, b-, and R-
These include phycoerythrin, C- and R-phycocyanin, and allophycocyanin. Since it is a protein, it is water-soluble, and functional groups for binding to antibodies are easily available.

〔Example〕

Examples of the present invention will be described below.

Example I A thiol group is introduced into R-phycoerythrin and an anti-human albumin antibody using N-succinimidyl-3-(2-pyridyldithio)propionic acid (clinical method).
Chemistry (CHn.

Chem, ) 29.1582 (1983)). Mix thiolated antibody and R-phycoerythrin,
Both are combined by H, -8-8- exchange reaction. Excess thiol groups are rendered inactive with N-ethylmaleimide.

On the other hand, thiol groups were introduced into C-phycocyanin and anti-human immunoglobulin G antibody in the same manner, and C-phycocyanin and anti-human immunoglobulin G antibodies were introduced in the same manner.
- Prepare phycocyanin-labeled antibodies. Furthermore, allophycocyanin is bound to anti-human immunoglobulin A in the same manner.

Albumin and immunoglobulin G in human serum were detected using the above three types of phycobiliprotein-labeled antibodies.

Immunoglobulin A was measured. First, three mixed standard solutions (physiological saline containing AO.5g/roomQ, immunoglobulin AO.5g/roomQ)
Dilute it. On the other hand, three types of unlabeled antibodies were each placed on carboxylated polyacrylamide gel beads (
(diameter 3 μm to 10 μm). This 3
Take an equal amount of the seed bead suspension into a test tube, add the mixed standard solution to it, react at 37°C for about 1 hour, centrifuge,
The supernatant was discarded. Next, add the three types of labeled antibody solutions,
The reaction was carried out at 37°C for about 1 hour. Add washing buffer (P
H7.4.

After stirring well, centrifuge and discard the supernatant. The precipitated polyacrylamide beads were suspended in a measurement buffer (pH 7.4, 0.05M phosphate buffer) and immediately transferred to a fluorescence measurement cell.
Fluorescence intensity was measured. R-phycoerythrin has an excitation wavelength of 480 nm.

Emission wavelength 580 nm, C-phycocyanin excitation wavelength 6
Allophycocyanin was measured sequentially at an excitation wavelength of 650 nm and an emission wavelength of 665 nm. A calibration curve obtained by this measurement procedure is shown in FIG. Approximately good calibration curves were obtained for each measurement target. The results of assaying 20 human serum samples according to the above procedure using this standard curve were compared with the measured values using a nephelometer, and the correlation coefficient was 0.97 to 0.98, indicating good agreement. It was observed.

Example 2 An antibody against a measurement target is immobilized on a polystyrene test tube. In this example, an anti-human α-fetoprotein antibody and an anti-ferritin antibody were adsorbed. A sample containing the antigen was placed in this test tube and reacted with the antibody for about 2 hours. Next, after washing the test tube with washing buffer,
Phosphate buffered saline (pH 7.4) containing about 1 mg/mfl of anti-human α-fetoprotein antibody labeled with phycocyanin and anti-ferritin antibody labeled with R-phycoerythrin was added, and the mixture was heated at 37°C to approx. I kept it for an hour. The solution in the test tube was aspirated and introduced into a flow cell of a fluorometer to measure the fluorescence intensity, and the antigen concentration was determined from the degree of decrease in the intensity. Both antigens could be measured separately using 20 standard samples in which both antigens were mixed arbitrarily.

〔Effect of the invention〕

The present invention makes it possible to measure multiple antigens from the same sample with high sensitivity by utilizing multiple phycobiliproteins with strong fluorescence intensity and different excitation and emission spectra for the purpose of highly sensitive multi-item analysis.

Furthermore, since multiple antigens can be measured in the same sample, the total measurement time can be shortened and the amount of sample can be reduced.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing a calibration curve used in Example 1 of the present invention.

Claims (1)

[Claims] 1. A fluorescence immunoassay characterized by comprising the steps of preparing an antibody bound to phycobiliproteins having different fluorescence emission wavelengths for each antigen to be measured, and reacting the antibody with a sample containing a plurality of antigens to be measured. Law.