JP2004117068A - Immunoassay reagent and immunoassay method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an immunoassay method for avoiding a nonspecific reaction and for detecting a target substance quickly and easily. <P>SOLUTION: When a process for adding a blocking agent to an insoluble carrier to avoid the nonspecific reaction in an immunity agglutination reaction, the nonspecific reaction in the immunoassay method is avoided, and immunity can be measured quickly and easily. More specifically, a protein that is specifically combined with a substance to be measured is immobilized to the insoluble carrier along with an inactive protein that is not specifically combined with the substance to be measured. <P>COPYRIGHT: (C)2004,JPO

Description

【００２９】
（比較例２）従来法
各種ＨＴＬＶ−１抗原のラテックス粒子への固定の後にＢＳＡをラテックス粒子へ固定した以外は、実施例２と同じ条件で反応および測定を行い、ＣＯＩ値を求めた。
【００３０】
その結果、従来法では高いＣＯＩ値を示したが、本発明の特異蛋白質（各種ＨＴＬＶ−１抗原）および不活性蛋白質であるＢＳＡを同時に固定する方法ではＣＯＩ値が低下する傾向が認められた。
【００３１】
【発明の効果】
以上説明したように本発明の免疫測定方法は、すなわち特異蛋白質を不活性蛋白質とともに不溶性担体に固定させる工程を含む測定方法は、測定対象物が陽性検体の場合には、より高い測定活性を示し、陰性検体の場合には活性を抑制した値を示すことが判った。このことより、特異蛋白質を不溶性担体に固定したのち、さらに不活性蛋白質を固定して測定する方法に比べ、測定に要する総合時間が短縮され、簡便に感度良く免疫測定を行うことが可能となった。
【図面の簡単な説明】
【図１】ＨＴＬＶ−１陽性検体の場合の測定結果を示す図である。（実施例１および比較例１）
【図２】ＨＴＬＶ−１陰性検体の場合の測定結果を示す図である。（実施例１および比較例１）
【図３】ＨＴＬＶ−１陰性検体の場合の測定結果を示す図である。（実施例１および比較例１）
【図４】ＣＯＩの変化を示す図である。（実施例２および比較例２）
【符号の説明】
１　　ＨＴＬＶ−１陽性検体　１
２　　ＨＴＬＶ−１陽性検体　２
３　　ＨＴＬＶ−１陰性検体　３
４　　ＨＴＬＶ−１陰性検体　４
５　　ＨＴＬＶ−１陰性検体　５
６　　ＨＴＬＶ−１陰性検体　６
７　　ＨＴＬＶ−１陰性検体　７
８　　ＨＴＬＶ−１陰性検体　８
９　　ＨＴＬＶ−１陰性検体　９
１０　　ＨＴＬＶ−１陰性検体１０[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an immunoassay reagent and an immunoassay for measuring a substance to be measured by an immunoagglutination reaction using an insoluble carrier, and more particularly, to an excellent immunoassay in which nonspecific reactions are suppressed and measurement can be performed in a short time. It relates to an assay method and an immunoassay reagent.
[0002]
[Prior art]
As one of the methods for measuring a trace component in a body fluid, an antibody or an antigen corresponding to an antigen or an antibody to be measured is immobilized on an insoluble carrier, and the antigen or antibody is generated by an antigen-antibody reaction between the substance to be measured and the antibody or antigen. BACKGROUND ART Immunoassays for measuring a substance to be measured by detecting the degree of aggregation of an insoluble carrier are widely used. As such immunoassays, those utilizing latex agglutination and those utilizing hemagglutination are known.
[0003]
As a method for detecting the degree of agglutination, a method of determining with the naked eye and a method of irradiating the reaction solution with light and measuring scattered light or transmitted light are used. The latter method, optical measurement, has been used for quantification of antigen or antibody in a sample.
[0004]
In order to obtain higher sensitivity in an immunoagglutination reaction, a method is known in which a water-soluble polymer such as polyethylene glycol or dextran is added to a reaction system as a sensitizer. The addition of the water-soluble polymer promotes the aggregation reaction and allows the reaction to proceed in a shorter time. However, the water-soluble polymer may cause an agglutination reaction even for a negative specimen that does not contain the antigen or the antibody to be measured. Such a reaction is called a non-specific reaction and causes an incorrect diagnosis.
[0005]
In order to reduce non-specific reaction, after immobilizing an antibody or antigen corresponding to the antigen or antibody as the substance to be measured on an insoluble carrier, the blocking agent is specific to the substance to be measured such as serum albumin or gelatin. There is a method of further immobilizing an inactive protein that does not bind to an insoluble carrier.
[0006]
However, if the blocking agent is added after the antibody or antigen corresponding to the antigen or antibody that is the substance to be measured is immobilized on the insoluble carrier, the operation is complicated, and the blocking requires time, and as a result, There was a disadvantage that the time required was long.
[0007]
[Patent Document]
JP 2000-46828 A
[Problems to be solved by the invention]
An object of the present invention is to provide an immunoassay method in which non-specific reactions are suppressed and a target substance can be easily detected in a short time.
[0009]
[Means for Solving the Problems]
The present inventors have conducted intensive studies to achieve the above object, and as a result, if the step of adding a blocking agent to an insoluble carrier can be shortened, a nonspecific reaction in an immunoassay method is suppressed, and in a short time. The present invention was completed by noting that simple immunoassays can be performed.
[0010]
That is, the present invention
1. An immunoassay method comprising a step of immobilizing a protein that specifically binds to the analyte to an insoluble carrier simultaneously with an inactive protein that does not specifically bind to the analyte,
2. An immunoassay method comprising the steps of: mixing a protein that specifically binds to an analyte with an aqueous solution containing an inactive protein that does not specifically bind to the analyte, and immobilizing each protein mixed in an insoluble carrier;
3. The measurement method according to the above item 1 or 2, wherein the insoluble carrier is in the form of particles and the weight of the inactive protein is 1/2000 to 1 when the weight of the insoluble carrier is 1.
4. The measurement method according to any one of the preceding items 1 to 3, wherein the weight ratio of the protein specifically binding to the substance to be measured and the inactive protein to be simultaneously immobilized is 2: 1 to 1:10.
5. The measurement method according to any one of Items 1 to 4, wherein the immunoassay is performed by immunoagglutination.
6. An immunoassay reagent containing a protein that specifically binds to a substance to be measured and / or an inactive protein that does not specifically bind to a substance to be measured, which is used in the measurement method according to any one of the above items 1 to 5.
7. 6. An immunoassay reagent kit used in the measurement method according to any one of the above items 1 to 5.
[0011]
BEST MODE FOR CARRYING OUT THE INVENTION
In the present invention, the protein that specifically binds to the test substance is not particularly limited as long as it is a protein that has a biological or physical binding ability to the test substance. If the test substance is an antigen, the antibody may be used, and if the test substance is an antigen, a protein that binds based on immunological affinity such as the antibody may be used (hereinafter, simply referred to as “specific protein”). There is also.). On the other hand, an inactive protein that does not specifically bind to the analyte is, for example, a protein that does not immunologically bind to the analyte, in other words, a protein that is immunologically inactive for the analyte. (Hereinafter sometimes simply referred to as “inactive protein”).
[0012]
The target substance to be measured according to the present invention is not particularly limited as long as it is a physiologically active substance that can be generally measured using an antigen-antibody reaction, and includes, for example, proteins and lipids, and more specifically, various antigens. , Antibodies, receptors or enzymes. More specifically, various virus (HTLV-1, HIV, HCV, HBs, HBe) antigens or antibodies, CRP, human fibrinogen, FDP, rheumatoid factor, α-fetoprotein (AFP), anti-streptolysin O antibody, treponema pallidum Examples thereof include an antibody, an antibody against a syphilitic lipid antigen, and an antibody.
[0013]
As the insoluble carrier of the present invention, an appropriate insoluble carrier that can carry an antibody or an antigen can be used. Examples of such insoluble carriers include organic polymer powders, inorganic substance powders, microorganisms, blood cells and cell membrane fragments. Examples of the organic polymer powder include insoluble agarose, cellulose, and insoluble dextran, and a latex suspension is preferred. Examples of latex include polystyrene, polystyrene-styrene sulfonic acid copolymer, methacrylic acid polymer, acrylic acid polymer, acrylonitrile-butadiene styrene copolymer, vinyl chloride-acrylate copolymer, and polyvinyl acetate acrylate. And the like. The average particle size of the latex to be used is appropriately selected from the range of 0.05 to 1.0 μm depending on the detection concentration of the object to be measured or the measuring instrument. Examples of the inorganic substance powder include silica, alumina, and metal pieces such as gold, titanium, iron, and nickel.
[0014]
As the measurement method of the present invention, various conventionally known agglutination methods such as a latex agglutination reaction and a blood agglutination reaction can be used.
[0015]
Next, for example, details of an immunoassay by an antigen-antibody reaction using a latex agglutination reagent will be described, but the present invention is not limited thereto. First, when performing the immunoassay, it is necessary to sensitize latex particles to a specific protein. Then, it is necessary to coat the latex particles with an inert protein in order to suppress non-specific reactions. The main feature of the present invention is that sensitization of a specific protein to latex particles and coating of an inactive protein are performed simultaneously. For convenience, "sensitization" and "coating" to an insoluble carrier (here, latex particles) are referred to as "fixation" in the specification of the present application.
[0016]
In order to fix the specific protein and the inactive protein to the latex particles, it is necessary to prepare a solution containing the specific protein and the inactive protein. The method for preparing a mixed solution of both proteins is not particularly limited. For example, a solution containing an inactive protein may be prepared, and then a specific protein may be added to prepare a solution. Alternatively, it can be prepared by preparing a solution containing a specific protein and a solution containing an inactive protein, respectively, and mixing them. A solution containing an active protein and an inactive protein is contacted with latex particles to simultaneously immobilize the active protein and the inactive protein. The fixation can be performed by a physical method or a chemical method according to a known method.
[0017]
The concentration and ratio of the active protein and the inactive protein may vary depending on the molecular weight of the analyte or the protein that specifically binds, the properties of a substance such as a simple substance or a bound substance, and other physical or chemical properties. The optimal conditions can be selected according to the properties of Specifically, for example, the ratio of the specific protein to the inactive protein can be selected from 2: 1 to 1:10, preferably from 1: 1 to 1: 3. In addition, the weight of the inactive protein when the weight of the carrier is 1 can be selected from 1/2000 to 1 and preferably from 1/1000 to 1/100. Conventionally, after fixing a so-called specific protein, when fixing an inactive protein as a blocking agent, it was necessary to add an excessive amount of the inactive protein, but in the method of the present invention, the amount of the inactive protein was reduced. It can be mixed with an active protein and immobilized on an insoluble carrier.
[0018]
When a sample, the above-described fixed corner latex particles and the buffer solution are mixed and placed under appropriate conditions, the analyte specifically binds to a specific protein on the latex particles, and the latex particles aggregate. The substance to be measured can be measured by optically observing or visually observing the degree of aggregation that has occurred. For example, in a method of optically detecting the degree of aggregation of an insoluble carrier, a substance to be measured is measured by measuring scattered light intensity, absorbance or transmitted light intensity with an optical instrument. The measurement wavelength is not particularly limited, and a wavelength of 300 to 2400 nm can be used.
[0019]
Regarding the measurement method, according to a known method, by setting the size or concentration of the insoluble carrier to be used, the reaction time, the scattered light intensity, the absorbance or the increase or decrease of the transmitted light intensity are measured, and these are performed. May be used in combination of two or more.
[0020]
In the present invention, the specific protein and the inactive protein are immobilized on an insoluble carrier such as a reagent for use in the above-described measurement method, specifically, a fixing reagent comprising a solution containing the specific protein and / or an inactive protein, or latex particles. Also included are latex reagents and reagents such as analytes and various lysates of specific proteins and / or inactive proteins. Furthermore, the present invention extends to a reagent kit in which two or more of these reagents are used in combination.
[0021]
【Example】
Hereinafter, the content of the present invention will be specifically described using examples, but the present invention is not limited to these.
[0022]
(Example 1) HTLV- by latex agglutination method for co-sensitization positive samples (samples 1 and 2: commercially available positive panel sera) and negative samples (samples 3 to 10: samples determined to be negative by gelatin agglutination method) It examined using one detection reagent.
[0023]
Various HTLV-1 antigens (p19, gp46 and p21) were mixed in 850 μl of glycine buffer so that the total amount was 9 μg, and 100 μl of bovine serum albumin (BSA) of each concentration was added to adjust the pH to 2.0 to obtain an insoluble carrier Reagent for fixation to
To 50 μl of a latex particle suspension (containing 5 mg of latex particles) having a particle size of 0.8 μm, 950 μl of the reagent for fixing to the insoluble carrier was added, and the mixture was allowed to stand at 37 ° C. for 1 hour. Thereafter, the latex particles were washed with 1% BSA, and various HTLV-1 antigens and BSA were fixed to the latex particles.
[0024]
The measurement was performed using an automatic measuring device (PAMIA-50) manufactured by Sysmex Corporation, and SysmexJournal Vol. 20 No. 1, p77-86 (1997). To the wells of the reaction plate, 80 μl of a buffer for latex agglutination reaction, 10 μl of a sample, and 10 μl of a solution containing the latex particles prepared above were added, and reacted at 45 ° C. About 15 minutes after the start of the reaction, 19 μl of the reaction mixture was added to 950 μl of the sheath liquid in the chamber of the apparatus, and diluted 51 times. The agglutination reaction was stopped by dilution, and then the degree of agglutination was determined using a PAMIA-50 apparatus.
[0025]
(Comparative Example 1)
The reaction was performed under the same conditions as in Example 1 except that BSA was fixed to the latex particles after fixing the various HTLV-1 antigens to the latex particles, and the measurement was performed.
[0026]
The results are shown in FIGS. As shown in FIG. 1, in the case of the HTLV-1 positive sample, the reactivity increased with the amount of BSA added. On the other hand, as shown in FIG. 2, in the case of the HTLV-1 negative sample, the reactivity tended to decrease with the amount of BSA added, suggesting that the non-specific reaction was suppressed. In FIG. 3, the measurement was performed for an HTLV-1 negative sample, and the reactivity decreased most when the BSA concentration was 20 μg / ml.
From the above results, it was found that the measured values tend to be different depending on the difference in the amount of the inactive protein BSA added, so that the optimum addition concentration can be determined depending on the properties of each measurement sample.
[0027]
(Example 2) Simultaneous sensitization method Similar to the method described in Example 1, a study was conducted using a latex particle aggregation method. Various HTLV-1 antigens (p19, gp46 and p21) and 20 μg of BSA were sensitized to latex particles (particle weight: 5 mg) in the same manner as in Example 1, and the reaction and reaction were performed in the same manner as in Example 1 using whole blood as a specimen. A measurement was made. From the measured values, a cut-off index (COI) value was calculated and obtained. The COI value was determined by the calculation formula shown in Equation 1.
[0028]
(Equation 1)

[0029]
(Comparative Example 2) Reaction and measurement were carried out under the same conditions as in Example 2 except that BSA was fixed to latex particles after fixing various HTLV-1 antigens to latex particles, and the COI value was determined.
[0030]
As a result, a high COI value was obtained in the conventional method, but the COI value tended to decrease in the method of simultaneously immobilizing the specific protein (various HTLV-1 antigens) of the present invention and BSA which is an inactive protein.
[0031]
【The invention's effect】
As described above, the immunoassay method of the present invention, that is, the assay method including the step of immobilizing a specific protein on an insoluble carrier together with an inactive protein shows a higher assay activity when the analyte is a positive sample. In the case of a negative sample, it was found that the value showed a suppressed activity. As a result, the total time required for measurement is shorter than that of a method in which a specific protein is immobilized on an insoluble carrier, and then the inactive protein is further immobilized. Was.
[Brief description of the drawings]
FIG. 1 is a diagram showing measurement results in the case of an HTLV-1 positive specimen. (Example 1 and Comparative Example 1)
FIG. 2 is a diagram showing measurement results in the case of an HTLV-1 negative specimen. (Example 1 and Comparative Example 1)
FIG. 3 is a diagram showing measurement results in the case of an HTLV-1 negative specimen. (Example 1 and Comparative Example 1)
FIG. 4 is a diagram showing a change in COI. (Example 2 and Comparative Example 2)
[Explanation of symbols]
1 HTLV-1 positive sample 1
2 HTLV-1 positive specimen 2
3 HTLV-1 negative sample 3
4 HTLV-1 negative sample 4
5 HTLV-1 negative sample 5
6 HTLV-1 negative sample 6
7 HTLV-1 negative sample 7
8 HTLV-1 negative sample 8
9 HTLV-1 negative sample 9
10 HTLV-1 negative sample 10

Claims (7)

An immunoassay method comprising a step of immobilizing a protein that specifically binds to a test substance on an insoluble carrier simultaneously with an inactive protein that does not specifically bind to the test substance. An immunoassay method comprising a step of mixing a protein that specifically binds to an analyte with an aqueous solution containing an inactive protein that does not specifically bind to the analyte, and immobilizing each of the proteins mixed in the insoluble carrier. The method according to claim 1 or 2, wherein the insoluble carrier is in the form of particles, and the weight of the inactive protein is 1/2000 to 1 when the weight of the insoluble carrier is set to 1. The method according to any one of claims 1 to 3, wherein the weight ratio of the protein specifically binding to the substance to be measured and the inactive protein to be simultaneously immobilized is 2: 1 to 1:10. The method according to any one of claims 1 to 4, wherein the immunoassay is performed by immunoagglutination. An immunoassay reagent containing a protein that specifically binds to a substance to be measured and / or an inactive protein that does not specifically bind to a substance to be measured, which is used in the measurement method according to claim 1. . An immunoassay reagent kit for use in the measurement method according to claim 1.

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