JPH07333225A - Immunoassay enhanced in sensitivity - Google Patents

Abstract

PURPOSE:To enhance the measuring sensitivity of immunoreaction due to multistage reaction by repeatedly performing the formation of an immune composite containing an object to be measured held to the same solid phase and the separation (B/F separation) operation of a bound label and a free label to cumulatively increase the amt. of the composite. CONSTITUTION:An immune composite consisting of an insolubilized first immune substance and a specific substance is formed by performing incubation along with a solid phase to which a first immune substance specifically reacting with the antibody or antigen (specific substance) to be measured in a fluid sample is bonded and the solid phase having the immune composite bonded thereto is separated from the reaction mixture. An immune composite composed of an insolubillzed immune substance is newly formed by incubating a newly prepared fluid sample along with a solid phase and separated from the reaction mixture having the immune composite held thereto. By repeating this process, the immune composite containing a substance to be measured is cumulatively increased on the solid phase. By this constitution, the amt. of the immune composite held on the solid phase, that is, the absolute amt. of the substance to be measured is cumalatively increased and detection sensitivity is enhanced.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a heterogeneous immunoassay method, and more particularly to an immunoassay method capable of highly sensitively quantifying specific components in body fluids, particularly blood sperm, plasma and urine. Specific substances in the body fluid include all substances such as tumor markers, hormones, drugs, antibodies, and pathogen-derived antigens.

[0002]

2. Description of the Related Art Immunoassay methods are roughly classified into a homogeneous method and a heterogeneous method. In the heterogeneous method, a bound label and a free label are separated (B / F separation)
For that purpose, a solid phase in which an antigen or an antibody or a ligand specifically reacting with them is insolubilized is used. As the solid phase, beads, test tubes, microplates, microparticles, glass fiber aggregates, cellulose fiber aggregates, membrane filters and the like are used.

[0003]

However, in the conventional heterogeneous immunoassay method, the operation for producing the immune complex containing the substance to be measured, which is carried out on the solid phase, is a one-time operation, and the immunoreactivity is appropriate. In order to maintain
Since it was necessary to use a considerably diluted sample, the amount of the immune complex as a reaction product, and hence the amount of the substance to be measured contained therein, was small, and the detection sensitivity had to be low.

As a result of various studies, the inventor of the present invention has developed a method for significantly improving the measurement sensitivity based on the conventional method.

[0005]

Means for Solving the Problems The present invention is a heterogeneous multi-step reaction in which the production of an immune complex retained on the same solid phase and the operation of B / F separation are repeated a plurality of times to progressively increase the amount of the complex. A genius immunoassay method is presented.
Various known methods or processes can be applied to the method for producing the immune complex each time used in the present invention.

The present invention can be implemented in response to all of these. Typical aspects are listed below. Here, the "specific substance" is an antibody or antigen to be measured,
The “immunity substance” refers to an antibody or an antigen that specifically reacts with the specific substance.

In the first embodiment, the insolubilized first immunological substance is obtained by incubating the fluid sample with a solid phase bound with a first immunological substance which reacts specifically with the specific substance in the fluid sample. Forming an immune complex consisting of a specific substance and separating the solid phase carrying the immune complex from the reaction mixture. By incubating the newly prepared fluid sample with the solid phase, an immune complex composed of the insolubilized first immunological substance-specific substance is newly formed, and the solid phase carrying the immunological complex is separated from the reaction mixture. To do. By repeating this step, the immune complex containing the substance to be measured is accumulated on the solid phase.

In the second embodiment, the fluid sample is specifically bound to a solid phase having a first immunological substance bound thereto which reacts specifically to the particular substance in the fluid sample, and to the particular substance. By incubating with a second antibody labeled with a reacting detectable labeling substance
An immune complex composed of the specific substance-labeled second immunological substance is formed, and the solid phase carrying the immune complex is separated from the reaction mixture. By incubating the newly prepared fluid sample and the labeled second immunity substance with the solid phase, an immune complex composed of the insolubilized first immunity substance-specific substance-labeled second immunity substance is newly formed, and the immunization is performed. The solid phase holding the complex is separated from the reaction mixture. By repeating this step, the immune complex containing the substance to be measured is accumulated on the solid phase.

In a third aspect, the fluid sample is incubated with a first immunological agent that specifically reacts with the particular agent in the fluid sample, thereby immunizing the first immunological agent-specific agent. The immune complex is captured on the solid phase by forming a complex and incubating the immune complex with a solid phase bound with a ligand that specifically reacts with the first immunological substance, The solid phase holding the complex is separated from the reaction mixture. By incubating the newly prepared fluid sample with a first immunological substance, an immune complex composed of the first immunological substance-specific substance is newly formed, and the immune complex is captured on the solid phase, The solid phase bearing the immune complex is separated from the reaction mixture. By repeating this step, the immune complex containing the substance to be measured is accumulated on the solid phase.

In a fourth embodiment, the fluid sample is a first immunological substance that specifically reacts with the specific substance in the fluid sample, and a detectable substance that specifically reacts with the specific substance. By incubating with a second antibody labeled with a labeling substance, the first immunological substance-specific substance-labeled second
Forming an immune complex composed of an immunological substance, capturing the immune complex on a solid phase bound with a ligand that specifically reacts with a first immunological substance, and reacting the solid phase holding the immune complex Separate from the mixture. The newly prepared fluid sample,
By incubating with the first immunity substance and the labeled second immunity substance, an immunocomplex composed of the first immunity substance-specific substance-labeled second immunity substance is newly formed, and the immune complex is formed on the solid phase. The solid phase that is captured and retains the immune complex is separated from the reaction mixture. By repeating this step, the immune complex containing the substance to be measured is accumulated on the solid phase.

In a fifth aspect, the fluid sample is incubated with a first immunological substance that specifically reacts with the specific substance in the fluid sample, and the specific substance labeled with a detectable labeling substance. Thereby forming an immune complex composed of the first immunological substance-specific substance (or labeled specific substance), and capturing the immune complex on a solid phase bound with a ligand that specifically reacts with the first immunological substance. Then, the solid phase carrying the immune complex is separated from the reaction mixture. By incubating the newly prepared fluid sample with the first immunological substance and the labeled specific substance, an immune complex composed of the first immunological substance-specific substance (or labeled specific substance) is newly formed, and the immunocomplex is formed. The body is captured on the solid phase and the solid phase carrying the immune complex is separated from the reaction mixture. By repeating this step, the immune complex containing the substance to be measured is accumulated on the solid phase.

According to such a method, the amount of immune complex retained on the solid phase, that is, the absolute amount of the substance to be measured is cumulatively increased, and the detection sensitivity is improved.

Further, in the present invention, the number of times of repeating the operation of producing the immune complex retained on the solid phase and the B / F separation is preferably 2 to 5 times. The sensitivity will not increase.

Further, in the present invention, among the heterogeneous immunoassay methods, the method using a fiber aggregate such as glass fiber filter paper as the solid phase is particularly useful.

In general, the fiber assembly has a large surface area and can trap a larger number of immune complexes as compared with an ordinary planar solid phase, and thus has a remarkably wide dynamic range. Further, due to the network structure, the diffusion distance between the solid phase and the liquid phase becomes short, and the reaction rate is fast.

Since the solid phase has a large surface area, the dynamic range can be secured even if the addition of the reaction solution and the like is repeated according to the present invention. Since the reaction distance is fast because the diffusion distance between the solid phase and the liquid phase is short, the reaction in which the immune complex is repeatedly added and solidified also proceeds extremely quickly.

Further, if the "repeated addition / removal" operation of the present invention is automated, it becomes more practical.

[0018]

According to the present invention, it is possible to measure up to a low concentration range which cannot be detected by the conventional measuring method. In clinical tests, it is possible to measure up to such concentration range.
The present invention has great significance because it may lead to the discovery of clinical significance and a new measurement target item. Examples will be shown below, but the specific method is not limited thereto.

[0019]

【Example】

1. Preparation of labeled antibody 1) Acridinium ester labeled antibody Acridinium-1 (manufactured by Dojinka Chemical Co., Ltd.) was labeled with anti-AFP monoclonal antibody A (manufactured by Medics) and purified by gel filtration. 2) A biotin-labeled antibody NHS-LC-BIOTIN (manufactured by PIERCE) is labeled with anti-AFP monoclonal antibody B (manufactured by Kyoto Daiichi Kagaku) and purified by dialysis.

2. Preparation of solid phase (Avidin-bonded glass fiber filter paper) 3 glass fiber filter paper (Toyo filter paper GA-100φ9mm)
-Aminopropyltriethoxysilane (manufactured by Nacalai Tesque) and introduced NHS-LC-B to the introduced amino groups.
A product obtained by reacting IOTIN (manufactured by PIERCE), then binding avidin to the biotin introduced, and blocking with casein (Block Ace (trade name) manufactured by Snow Brand).

3. Measurement 1) Antigen-antibody reaction-Acridinium-labeled antibody 10 μg / ml, 10 μl-Biotin-labeled antibody 10 μg / ml, 10 μl-Sample: AFP 0 ng / ml and AFP 1 ng
/ Ml of blood sperm diluted 10 times with phosphorus buffered saline 80 μl. The above three components are mixed and incubated at room temperature for 10 minutes. 2) Immobilization reaction Take 65 μl of the antigen-antibody reaction solution, spot it on the solid phase, and incubate at room temperature for 5 minutes. 3) B / F separation Phosphate-buffered saline is added to the solid phase on a Buchner funnel and washed by suction. 4) Multistep reaction The operations of 1) to 3) are repeated a predetermined number of times. 5) Measurement of luminescence The luminescence on the solid phase in which the antigen-antibody complex is captured is measured by a luminescence measuring device by a homemade photon counting method.

4. Results Table 1 shows the luminescence amount of AFP 0 ng / ml, and Table 2 shows AFP.
The amount of luminescence of 1 ng / ml and the SN ratio are shown in Table 3. Incidentally, S
The N ratio is calculated by the following formula. SN ratio = (AFP 1 ng / ml luminescence amount-AFP
0 ng / ml luminescence amount) / AFP 0 ng / ml luminescence amount Further, Table 4 shows the relationship between each luminescence amount and SN ratio and the number of multistage reactions.

As can be seen from the results, by repeating the multi-step reaction, the signal component was clearly increased proportionally,
SN improves.

[0024]

[Table 1] AFP 0 ng / ml

[0025]

[Table 2] AFP 1 ng / ml

[0026]

[Table 3] SN ratio

[0027]

[Table 4]

Claims (5)

[Claims] 1. In a heterogeneous immunoassay method,
Highly sensitive immunoassay by multi-step reaction characterized in that the production of an immunocomplex containing a substance to be measured held on the same solid phase and the operation of B / F separation are repeated a plurality of times to gradually increase the amount of the complex. Method 2. The highly sensitive immunoassay method according to claim 1, wherein the number of repetitions is 2 to 5 times. 3. The highly sensitive immunoassay method according to claim 1 or 2, wherein the solid phase is a solid phase using a glass fiber aggregate as a carrier. 4. The highly sensitive immunoassay method according to claim 3, wherein an acridinium ester is used as the labeling substance for the immune complex. 5. The highly sensitive immunoassay method according to claim 1, wherein the antibody forming the immune complex is labeled with biotin, and avidin is used as a ligand that binds to the solid phase.