JPS592346B2 - Two-component simultaneous quantitative enzyme immunoassay - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an enzyme immunoassay method that can simultaneously quantify two components (two types of antigens) contained in a test liquid.

Enzyme immunoassay is a method that has recently become widely used as a method for quantifying trace components derived from living organisms.
An example is as follows.

A fixed amount of antibody is attached to a suitable carrier (solid phase) such as the inner wall of a glass test tube or the surface of plastic beads to make it insoluble (solid phase), and a fixed amount of enzyme-labeled antigen is measured against this. When the target antigen is added, the enzyme-labeled antigen and the antigen to be measured competitively bind to the immobilized antibody through an antigen-antibody reaction, and at this time, the amount of the enzyme-labeled antigen that binds to the antibody increases. can be measured by measuring the enzyme activity of the enzyme-labeled antigen bound to the antibody, which changes depending on the amount of antigen to be measured, or conversely by measuring the activity of the enzyme not bound to the antibody. The amount of antigen required can be determined. When measuring trace components using the enzyme immunoassay method described above, for example in mass screening of newborns, the sample that can be collected is extremely small, and it is difficult to quantify many components. Measurement of the item is required.

Therefore, simultaneous quantitative determination of two components has the advantage of requiring less sample and shortening measurement time. The present invention was developed to meet this need, and will be described below. A method for simultaneously quantifying two components, such as antigens A and B, contained in a test liquid will be described.

As shown in Figure 1, anti-A antibody (antibody A) was immobilized on the inner wall of test tube 1, while anti-B antibody (antibody B) was immobilized on beads 2
After placing the beads in a test tube, test solution 3 is added, and then enzyme-labeled antigen B is added. Antigen A in the test solution is absorbed by antibody A on the inner wall of the test tube. Antigen B in the test liquid and enzyme-labeled antigen B competitively bind to antibody B on the surface of the piece. In this case, the amount of enzyme-labeled antigen B that binds to antibody B is determined depending on the amount of antigen B to be measured. After the above binding is completed, the beads are removed from the test tube, washed, and the activity of the enzyme bound to antibody B on the beads is measured. f) The amount of antigen B can be determined. On the other hand, the amount of antibody A can be determined by washing the test tube from which the beads were taken out, adding enzyme-labeled antigen A to it and binding it to antibody A, and then measuring the activity of the enzyme bound to antibody A. be able to. The present invention will be explained below by exemplifying a method for simultaneously measuring thyroxine T4 and thyroid stimulating hormone TSH.

. Use the reagents shown below. (1) Buffer: (A) 0.05M phosphate buffer PH7. O(0.1(Fll bovine serum albumin B
SAIO. Contains 9% sodium chloride) (8) 0.05
M phosphate buffer pH7. Ol] (0.9 (contains fl sodium chloride)) (1) Anti-T antibody immobilized test tube: Boehringer T4 kit test tube (3) T: Miles product (4) T4 standard solution: T4O ~ 200 mm /TIIJI, aqueous 1 solution (5) Peroxidase standard T4: Standard T4 of T4 kit manufactured by Boehringer (6) TSH: Product of Carpiochem (7) TSH standard solution: TSH 100μU/m2
Solution (8) Peroxidase Lysigma product HOrSeradiShperOxidaSeType
32OU/3f1 (9) anti-TSH antiserum: LTB Chemical Co. product 2 (11 peroxidase-labeled TSH: peroxidase was oxidized using NaIO, then bound to THS in a basic water bath solution, and reduced using NaBH. Thereafter, it was purified by gel chromatography using Sephadex G-100, and the fractions with high enzyme activity and immunoactivity were used.

(11) Anti-rabbit 1gG antibody-immobilized beads (second antibody-immobilized beads): Diluted solution of anti-rabbit 1g (/f different antibody (goat)) purified by affinity chromatography. (0D280nm 0.15-0.2) and left at 4° C. for 1 day or more. When used, wash with buffer (B) and then immerse in buffer (4) for 1 hour or more.

Using the reagents prepared as described above, measurements are performed by the method shown below. anti-T4
In an antibody-immobilized test tube, add 1001 tons of anti-TSH serum diluted approximately 100,000 times as 100 μm T standard solution and 20 μm TSH standard solution.
Contains peroxidase standard TSHlOOtL buffer (A
) and one anti-rabbit 1gG antibody-immobilized bead, and left at 4°C overnight.The beads were then transferred to another test tube, washed with buffer B, and then added with buffer BO. 65Trl
J! / and p-hydroxyphenylpionic acid 571f
50 μm of aqueous solution and 5.0 μm of a 0.01% H2O2 aqueous solution were added, and the mixture was allowed to stand at room temperature for 1 hour.

Next, 50μ of a 1.25% potassium cyanide aqueous solution and 50μ of an IN sodium hydroxide aqueous solution were added to stop the enzyme reaction, and the fluorescence intensity was determined by Ex32OnrrkEm4.
Measurement was performed using O5 nm light. On the other hand, 1 m of peroxidase standard T4 solution was added to the anti-T4 antibody-immobilized test tube from which the beads had been removed, and after leaving it at room temperature for 2 hours, the solution in the test tube was discarded, and buffer solution [F]) was added. After washing the test tube, add 0.9 μm of buffer (8), 50 μm of p-hydroxyphenylpropionic acid 51 Vm aqueous solution, and 0.0 μm of p-hydroxyphenylpropionic acid.
Add 50μ of 1% H2O2 aqueous solution and leave at room temperature for 1 hour.

Next, 50μ of a 1.25% potassium cyanide solution and 50μ of an IN aqueous sodium hydroxide solution were added to stop the enzyme reaction, and the fluorescence intensity was adjusted to Ex32Onm in the same manner as above.
、． Measured using Em4O 5 nm light 〇 When measuring T4 and TSH by the above method, T4 is 0n.
9/Tn). 25n9Δπmu 50n9/Mj. lOOn
gr No Yamaichiyo and 200n9Z Nawate? Using an aqueous solution, TSH
As 0IjXJ/i and 5μJd, 10μV/NLj
! Using an aqueous solution of /
The fluorescence intensity was measured using 100 μJTSH and 100 μJTSH, and the calibration curve shown in FIG. 2 was obtained.

According to the method shown above, T standard solution 20μ and TS
Instead of using 100μ of the H standard solution, repeat the above method using 120μ of the test solution to measure the fluorescence intensity of each, and calculate the T4 and TSH contained in the test solution from the above calibration curve. can be measured.

Furthermore, T4 and TSH were measured using the combinations shown below, and the results shown in FIG. 3 were obtained.

This result shows that the presence of the second antibody beads has no effect on the measurement of T4.
According to the method of the present invention, even when only a small amount of test liquid can be obtained.

Two types of components can be measured simultaneously using the sample.

[Brief Description of the Drawings] Fig. 1 is an explanatory diagram showing the method of the present invention, Fig. 2 is an explanatory diagram showing the method of the present invention,
The figure shows a calibration curve obtained by the method of the present invention.

Claims (1)

[Claims] 1 Two types of antigens A and B contained in the test liquid
In the method of simultaneously quantifying by enzyme immunoassay, a test tube with anti-A antibody (antibody A) immobilized on its inner wall, beads with anti-B antibody (antibody B) immobilized on the surface thereof,
After adding a test solution containing antigens A and B and enzyme-labeled antigen B to perform an antigen-antibody reaction, the beads are removed and the activity of the enzyme bound to antibody B on the beads is measured. On the other hand, the feature includes the step of adding enzyme-labeled antigen A to the test tube from which the beads have been taken out, causing an antigen-antibody reaction, and then measuring the activity of the enzyme bound to antibody A on the inner wall of the test tube. A two-component simultaneous quantitative enzyme immunoassay method.