JP2001330611A - Immunoassay reagent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immunoassay reagent, capable of measuring a very small amount of antigen (or antibody) in a living body specimen with high sensitivity, and capable of omitting B/F separating operation. SOLUTION: The immunoassay reagent, for measuring the amount of antigen (or antibody) which is a substance to be measured in the specimen, consists of an insoluble carrier on which the antibody (or antigen) to the antigen (or antibody) and enzyme or an enzyme inhibitor are made to support, the enzyme inhibitor or enzyme and an enzyme substrate.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an immunoassay reagent, and more particularly to an immunoassay reagent capable of measuring an object to be measured with high sensitivity using an insoluble carrier.

[0002]

2. Description of the Related Art In the field of clinical testing, biological samples (blood,
Various diseases are diagnosed using urine and the like, and various measuring methods have been developed and used as methods for diagnosing them. Representative methods of these assays include a biochemical assay utilizing an enzyme reaction and an immunoassay utilizing an antigen-antibody reaction. In recent years, it has been desired to accurately measure components in a biological sample, and an immunoassay utilizing a highly specific antigen-antibody reaction has been actively used.

[0003] As an immunoassay, immunoturbidimetry (TIA) is used.
Method, latex turbidimetry (LIA method), enzyme immunoassay (EIA method), radioimmunoassay method (RIA method), and the like, which are used properly according to the purpose. That is, when the amount of the component contained in the biological sample is relatively large,
The TIA method and the LIA method are used. TIA and LI
In the method A, the components in the biological sample to be measured include, for example, C-reactive protein (CRP), anti-streptolysin-O antibody (ASO), fibrin degradation product (FDP)
And several ng / m 2 as the concentration in the biological sample.
1 or more. On the other hand, when the amount of the component contained in the biological sample is very small, the EIA method or the RIA
The components in the biological sample to be measured include, for example, cancer markers typified by α-fetoprotein (AFP) and hormones typified by insulin, and a concentration of several ng in the biological sample. / Ml or less.

Further, in recent years, importance has been placed on the measurement of trace components in biological samples, and EIA, RIA, and the like have been increasingly used. However, the time required for measurement is short in the TIA method or the LIA method, the operation is simple, and it can be applied to various automatic analyzers (hereinafter, general-purpose automatic analyzers), whereas the reaction time is short in the EIA method and the RIA method. It is long and complicated, and the types of enzymes and radioisotopes used vary widely. Therefore, the EIA method and the RIA method are often used only in a specific automatic analyzer (hereinafter, a dedicated automatic analyzer), and the RIA method requires a specific facility to utilize radioisotopes. There are various problems.

[0005] In recent years, in the measurement of trace components in a biological sample, there has been a demand for a method capable of measuring even a trace amount in order to detect cancer at an early stage or diagnose early stage of infection with an AIDS virus or the like. Means capable of ultra-trace measurement include means for improving the accuracy of the measurement method itself, such as a modification or improvement of the LIA method or EIA method, and LIA method, EIA method, etc., which are used for measuring by conventional methods. It is roughly divided into techniques for improving performance, and some of them have been put to practical use.

As a technique for improving the accuracy of the measuring method itself,
Method of coloring insoluble carrier by LIA method (Japanese Patent Laid-Open No. 1-21)
No. 4760), and a method using a luminescent substance instead of an enzyme as a substance for labeling an antigen or an antibody in the EIA method (Japanese Patent Laid-Open No. 5-34346). Japanese Patent Laid-Open Publication No.
No. 475 discloses a method proposed.

However, none of these methods can be applied to a general-purpose automatic analyzer, and the problem of requiring a dedicated automatic analyzer has not been solved.
The reason why a dedicated automatic analyzer is required is that, as mentioned above,
In a method for measuring a trace component represented by the method A or the RIA method, the reaction time, the operation method, the type of the enzyme or the radioisotope to be used, and the like are variously different depending on the measurement method. Another major reason other than the above is that in a method for measuring trace components that is currently being developed or marketed, an operation called B / F separation (B is a component bound by an immune reaction or the like, and F is an unreacted component) is performed. Since it is always necessary, it cannot be applied to a general-purpose automatic analyzer that cannot perform the B / F separation operation, and a dedicated automatic analyzer that can perform the B / F separation operation is required.

Recently, as disclosed in JP-A-5-249112 and JP-A-7-179495, etc.
Unnecessary measurement methods for / F separation have also been proposed and developed. However, due to problems such as insufficient sensitivity and long measurement time, there are problems such as the necessity of a dedicated automatic analyzer and application to only some general-purpose automatic analyzers.

On the other hand, at the site of clinical examination, an expensive dedicated automatic analyzer is required to perform ultra-trace analysis, and an installation place must be secured. Therefore, measurement of ultra-trace components by a general-purpose automatic analyzer is required. The voice that desires is loud.

[0010] As described above, the measurement of ultra-trace components currently being developed or marketed requires a B / F separation operation despite the strong demands of users, and therefore requires the use of a dedicated automatic analyzer. There is a major problem that measurement is limited.

[0011]

SUMMARY OF THE INVENTION The present invention solves the above-mentioned problems of the prior art, and an object of the present invention is to measure an ultratrace component such as an antigen or an antibody in a sample with high sensitivity. It is another object of the present invention to provide an immunoassay reagent that can perform a measurement easily without requiring B / F separation.

[0012]

Means for Solving the Problems The first invention of the present application is an immunoassay reagent for measuring the amount of an antigen (or an antibody) as a substance to be measured in a sample. A carrier carrying an antibody (antigen) and an enzyme and comprising organic polymer particles or colloidal particles having a particle size of 0.001 to 0.5 μm, an enzyme inhibitor inhibiting the activity of the enzyme, and the enzyme And an immunoassay reagent comprising:

In the immunoassay reagent according to the first invention, the first reagent in which an antibody (or antigen) corresponding to the antigen (or antibody) to be measured and an enzyme are carried on the same insoluble carrier is used. Having an enzyme inhibitor that inhibits the activity of the enzyme and a second reagent containing a substrate of the enzyme, and mixing the first and second reagents with a biological sample containing a measurement target; Three types of reactions proceed. That is, the first
Is an antigen-antibody reaction between the antigen (or antibody) in the biological sample and the antibody (or antigen) carried on the insoluble carrier, and the second reaction is the enzyme on the insoluble carrier and the enzyme in the system. The third reaction is an enzymatic reaction between an enzyme on an insoluble carrier and a substrate in the system.

When the enzyme inhibitor binds to the enzyme, the activity of the enzyme is inactivated or reduced. However, when the antigen (or antibody) is not contained in the biological sample, the insoluble carrier is aggregated by the first reaction. Therefore, the enzyme inhibitor binds to the enzyme on the insoluble carrier, inactivates the enzyme, and does not cause a change in absorbance due to the enzyme. On the other hand, if the biological sample contains an antigen (or an antibody), the first reaction causes aggregation of the insoluble carrier. In this case, the binding of the enzyme and the enzyme inhibitor on the insoluble carrier involved in the aggregation,
It hardly occurs due to steric hindrance of the aggregate, and the enzyme does not deactivate and reacts with the substrate, causing a change in absorbance by the enzyme.

Therefore, by adding an enzyme inhibitor to the reaction system, the third reaction can be made dependent on the amount of the antigen (or antibody) in the biological sample. Therefore, by using the immunoassay reagent of the first invention, the above-mentioned first to third
Are the antigens (or antibodies) in the biological sample
Since the reaction depends on the amount of the compound, the substance to be measured can be measured with higher sensitivity than the LIA method. In addition, unlike the EIA method or the like, a complicated B / F separation operation can be omitted.

[0016] The second invention of the present application is an immunoassay reagent for measuring the amount of an antigen (or antibody) as a substance to be measured in a sample, comprising an antibody (antigen) to the antigen (or antibody); A carrier carrying an enzyme inhibitor and comprising organic polymer particles or colloidal particles having a particle size of 0.001 to 0.5 μm, an enzyme whose activity is inhibited by the enzyme inhibitor, and a substrate of the enzyme And an immunoassay reagent comprising:

In the case where the immunoassay reagent according to the second invention is used, first, an antibody (or antigen) against an antigen (or antibody) which is a substance to be measured in a biological sample and an enzyme inhibitor are carried. When the insoluble carrier and the measurement sample are mixed, the antibody (or antigen) reacts with the antigen (or antibody) in the sample, and an agglutination reaction occurs. As a result of this aggregation reaction, steric hindrance occurs, the enzyme inhibitor on the insoluble carrier is inactivated, and the enzyme inhibitory action is weakened.

That is, the enzyme inhibitory effect is weakened according to the degree of aggregation of the antigen-antibody reaction. Next, as a second reaction, the enzyme inhibitor inhibits the function of the enzyme according to its activity, so that the enzyme present in the system is inactivated according to the activity of the enzyme inhibitor.

Finally, as a third reaction, a color is formed by the reaction between the enzyme and the substrate by adding the substrate. By measuring the degree, the degree of inactivation of the enzyme, and thus the degree of aggregation, That is, it is possible to detect the amount of the antigen (or antibody) that is the substance to be measured.

In the immunoassay reagent according to the second invention, the first to third reactions are all reactions depending on the amount of the antigen (or antibody) in the biological sample. As in the invention, the antigen (or antibody) can be measured with higher sensitivity than the LIA method, and the more complicated B / F
Separation can be omitted.

In the first and second aspects of the present invention, the first to third reactions do not need to be performed in separate steps, and therefore, the measurement reagents need not be divided into three types. By selecting appropriate conditions, a combination of one or two reagents may be used, or a combination of three or more reagents may be used.

In a specific aspect of the present invention (first and second inventions), the amount of change in absorbance when the carrier is aggregated by an immune reaction to increase turbidity is determined by the enzyme reaction between the enzyme and a substrate. Is less than one tenth of the absorbance change caused by the above.

In another specific aspect of the present invention, an enzyme inhibitor which is an antibody against the enzyme is used as the enzyme inhibitor. Hereinafter, details of the present invention will be described.

The substance to be measured according to the present invention includes an antigen or antibody in a biological sample, for example, an antigen or antibody derived from hepatitis B or C: an HIV antigen or antibody; an antigen or antibody derived from syphilis; -Cancer markers represented by fetoprotein; hormones represented by insulin; autakoids, and the like, but are not particularly limited thereto.

The insoluble carrier used in the present invention includes:
Organic polymer particles and colloid particles are used. Examples of the organic polymer particles include natural polymer powders such as insoluble agarose, cellulose, and insoluble dextran; polystyrene, styrene-styrene sulfonic acid (salt) copolymer, styrene-methacrylic acid copolymer, acrylonitrile-butadiene-styrene. Synthetic polymer powders such as a copolymer, a vinyl chloride-acrylate copolymer, and a vinyl acetate-acrylate copolymer are exemplified.
In particular, a latex in which a synthetic polymer powder is uniformly suspended is preferably used.

Examples of the colloidal particles include those composed of elements such as gold, platinum, selenium, tellurium and sulfur. The insoluble carrier varies depending on the purpose of use and intended use, but usually, those produced by chemical synthesis or commercially available can be used. In addition, an insoluble carrier having a sulfonic acid group, a carboxyl group, or the like introduced on the surface can be used as appropriate.

The organic polymer particles or colloid particles preferably have a particle size in the range of 0.001 to 0.50 μm.
0.01 to 0.05 μm is more preferable. The enzyme used in the present invention is not particularly limited as long as it reacts with a substrate to cause a change in absorbance. For example, peroxidase, alkaline phosphatase, β-galactosidase and the like can be exemplified. Enzymes include those obtained from natural products and those obtained by genetic engineering techniques, and any of them can be used.
Usually, an enzyme obtained from a natural product may be used.

When the above enzyme is used for measurement, it is used after being diluted with an appropriate buffer or the like. The buffer is not particularly limited, for example, phosphate buffer, Tris buffer,
Glycine buffer, Good buffer and the like can be mentioned. These buffers may be appropriately selected in consideration of the characteristics of the enzyme and the substrate to be used.

When the above enzyme is diluted in a buffer, the concentration of the enzyme at the time of use is 0.001 to 10 IU / ml.
Is preferred, but depends on the type of enzyme used,
It is not limited to this range.

Next, as the substrate used in the present invention, an appropriate substrate is used as long as it reacts with the enzyme to be used to cause a change in absorbance. For example, when peroxidase is used as the enzyme, a substrate obtained by adding N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline to a hydrogen peroxide solution as a substrate, or Hydrogen peroxide to which o-phenylenediamine or pyrogallol is added.When alkaline phosphatase is used as an enzyme, p-nitrophenylphosphate is used as a substrate, and when β-galactosidase is used as an enzyme, o is used as a substrate. Examples thereof include -nitrophenyl-β-D-galactopyranoside, but are not particularly limited thereto, and may be appropriately selected depending on the purpose and use.

As the above-mentioned substrate, a substrate usually produced by chemical synthesis or a commercially available substrate is used. When used for measurement, the substrate is dissolved or diluted in an appropriate buffer or the like before use. As the buffer, for example, phosphate buffer, Tris buffer, glycine buffer, Good
Examples include a buffer, but are not particularly limited. An appropriate buffer may be selected in consideration of the characteristics of the enzyme and the substrate to be used.

The concentration of the substrate at the time of use is from 0.1 to
1000 mM is preferred, but not particularly limited to this range. Next, the enzyme inhibitor used in the present invention is not particularly limited as long as it binds to the enzyme to be used and deactivates the activity of the enzyme. For example, peptides,
An enzyme inhibitor corresponding to the enzyme to be used, such as an antibody, a fluorine compound or a sulfur compound, may be used as appropriate.

When an antibody against the enzyme (hereinafter referred to as an anti-enzyme antibody) is used as the enzyme inhibitor, any of polyclonal antibody and monoclonal antibody may be used as the antibody species. In addition, as for the method for producing the anti-enzyme antibody, an appropriate known method can be used. In the case of a polyclonal antibody, an anti-enzyme antibody may be produced by immunizing an animal such as a rabbit, goat, or sheep with the enzyme to be used. Monoclonal antibodies can also be obtained using known appropriate methods.

The enzyme inhibitor obtained as described above may be appropriately purified by known chromatography or the like. In some cases, an enzyme inhibitor obtained without special purification may be used. When used for measurement, the enzyme inhibitor is diluted with an appropriate buffer or the like. Examples of the buffer include a phosphate buffer, a Tris buffer, a glycine buffer, a Good buffer, and the like, but are not particularly limited, and may be appropriately selected in consideration of characteristics of an enzyme or a substrate to be used. .

Regarding the concentration at the time of use of the enzyme inhibitor,
The amount is preferably 0.01 to 10 mg / ml, but is not particularly limited to the above range since it varies depending on the type of the enzyme inhibitor used.

In the first invention, the antigen (or antibody)
A method for producing an insoluble carrier carrying an antibody (or antigen) against the enzyme and an enzyme will be described. The method of binding the antibody (or antigen) to the insoluble carrier and the enzyme depends on the type of the antibody (or antigen) and the enzyme used,
Usually, the following method is used. If the solution containing the antibody (or antigen) and the solution containing the enzyme are added simultaneously or sequentially to the suspension of the insoluble carrier and stirred, the antibody (or antigen) and the enzyme are bound to the insoluble carrier by physical adsorption. I do.

For an insoluble carrier having a sulfonic acid group or carboxyl group introduced on its surface, an antibody (or antigen) and an enzyme can be crosslinked to the insoluble carrier by adding an appropriate crosslinking agent. Can be. In this case, it is necessary to modify the antibody (or antigen) and the enzyme so that they can be cross-linked by the cross-linking agent.

Whether the physical adsorption method is used or the cross-linking is used may be appropriately selected in consideration of the physical properties and structure of the antibody (or antigen) to be used and the enzyme. The insoluble carrier carrying an antibody (or antigen) against an antigen (or an antibody) and an enzyme inhibitor used in the second invention can be produced in the same manner as in the first invention. That is, an enzyme inhibitor is used in place of the enzyme used in the production of the insoluble carrier carrying the antibody (or antigen) and the enzyme in the first invention so that the enzyme inhibitor is carried on the insoluble carrier. Thus, an insoluble carrier carrying the antibody (or antigen) and the enzyme inhibitor used in the second invention can be produced in the same manner as in the first invention.

In the first and second inventions, the pH at which the antibody (or antigen) and the enzyme or enzyme inhibitor are bound to the insoluble carrier is preferably in the range of 3 to 10, and the reaction temperature is 2 to 50 ° C. Is preferred. If the pH is out of the above range, the antibody (or antigen) may be denatured because it is a protein. When the reaction temperature is lower than 2 ° C., the reaction rate is low, and it is difficult to obtain an immunoassay reagent having a desired sensitivity. When the reaction temperature is higher than 50 ° C., the antibody (or antigen) may be denatured. .

[0040]

Hereinafter, the present invention will be described in more detail by giving non-limiting examples and comparative examples of the present invention.

Example 1 (1) Preparation of Reagent for Immunoassay 0.02% by weight of polystyrene latex (particle size: 0.05 μm, manufactured by Sekisui Chemical Co., Ltd.) in phosphate buffer (pH 5.
0, 50 mM) dispersion at 4 ° C. in 1 ml,
50 μl of a 1 mg / ml phosphate buffer (pH 5.0, 50 mM) solution of a rabbit antibody and 50 μl of a 1 mg / ml phosphate buffer (pH 5.0, 50 mM) solution of horseradish peroxidase were added to the anti-human HBs antigen, 4 ℃
For 1 hour.

Next, the mixture was centrifuged at 18,000 rpm for 30 minutes, the supernatant was removed, and the precipitate was washed with a phosphate buffer (p
(H5.0, 50 mM) to obtain 1st reagent. Next, 1 ml of a 0.4 mg / ml solution of an anti-horseradish peroxidase monoclonal antibody, N-ethyl-N
-(2-hydroxy-3-sulfopropyl) -3,5-
1 mg / ml solution of dimethoxyaniline 1 ml, 2 mM
1 ml of a 4-aminoantipyrine solution and 1.2 ml of a 10 mM hydrogen peroxide solution were added to a phosphate buffer (pH 7.0).
(5, 50 mM) to obtain a second reagent.

(2) Measurement Using the immunoassay reagent consisting of the first and second reagents, a calibration curve was prepared using standard HBs antigen in the following manner. The HBs antigen titer in was measured.

Standard HBs antigen: HBs antigen is 0, 1
Human sera at concentrations of 0, 25, 50, 75 and 100 IU / ml were provided as standards. Preparation of Calibration Curve An automatic analyzer 7150 manufactured by Hitachi, Ltd. was used. 20 μl of the standard HBs antigen solution prepared in
After mixing with 20 μl and maintaining the mixture at 37 ° C. for 10 minutes, 120 μl of the second reagent was added, and after 1 minute and 10 minutes, the absorbance was measured at a wavelength of 570 nm. The difference between the absorbances after 1 minute and 10 minutes was defined as the absorbance change amount. FIG. 1 shows a calibration curve showing the relationship between the standard HBs antigen titer and the amount of change in absorbance.

In FIG. 1, the vertical axis represents the change in absorbance at 570 nm, and the horizontal axis represents the HBs antigen titer (IU) in serum.
/ Ml). Measurement of HBs antigen-positive serum Standard HBs antigen solution 20 in the section of the above calibration curve
The amount of change in absorbance was determined in the same manner as described above, except that 20 μl of HBs antigen-positive serum was used instead of μl. The obtained change in absorbance was applied to the above calibration curve to determine the HBs antigen titer in HBs antigen-positive serum.

As the HBs antigen-positive serum, six samples (sample names A to F) were prepared, and the measurement was repeated five times for each HBs antigen-positive serum, and the average value and the coefficient of variation (CV) (%) And asked. The results are shown in Table 1 below.

[0047]

[Table 1]

As is clear from Table 1, the use of the immunoassay reagent of this example allows the HBs antigen to have a titer of 10
It can be seen that the titer can be measured with high accuracy even in a trace amount of IU / ml or less.

Example 2 Reagents and Materials Latex: 10% (w / v) polystyrene latex (particle size: 0.05 μm, manufactured by Sekisui Chemical Co.) Latex dilution buffer: 50 mM sodium monophosphate and 50 mM Anti-human HBs antigen rabbit antibody: Purified from rabbit serum to specific immunoglobulin fraction by affinity column Antibody dilution buffer: Latex dilution Anti-horseradish peroxidase polyclonal antibody: Purified from guinea pig serum to immunoglobulin fraction by ammonium sulfate precipitation Enzyme solution (R3 solution): Horseradish peroxidase (25)
7 U / ml, manufactured by Toyobo Co., Ltd.) diluted to 5 U / ml with the above buffer solution for latex dilution. Substrate solution (R4 solution): 1 mM N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3 1,5-Dimethoxyaniline (manufactured by Dojin Chemical Co., Ltd.), 1 mM 4-aminoantipyrine (manufactured by Wako Pure Chemical Industries, Ltd.), and 20 mM hydrogen peroxide solution (manufactured by Santoku Chemical Co., Ltd.). Bovine serum albumin (Fraction V, manufactured by Miles Corp.)
1% (w / v) Sample dilution buffer (R1 solution): polyethylene glycol 6000 (average molecular weight 75
00, manufactured by Wako Pure Chemical Industries, Ltd.) at a concentration of 1% (w / v). Standard HBs antigen: 0, 10, 25, 50, HBs antigen
Human serum containing 75 and 100 IU / ml concentration HBs antigen-positive serum: HBs antigen-positive human serum having a titer around 10 IU / ml

Preparation of Immunoassay Reagent To 2 volumes of latex dilution buffer was added to 1 volume of polystyrene latex (particle size: 0.05 μm, manufactured by Sekisui Chemical Co., Ltd.), and 3.3% (w / v) latex solution was added. Obtained. Anti-human H
For the rabbit antibody and the anti-horseradish peroxidase polyclonal antibody, the Bs antigen was diluted with the above-mentioned phosphate buffer (pH 7.0) to a protein concentration of 100 μg / ml, respectively, to obtain an antigen solution and an antibody solution.
While stirring 300 μl of the above 3.3% latex solution using a magnetic stirrer in an incubator at 25 ° C., the antigen solution and the antibody solution were added to each of 100 μl each.
μl was added quickly and stirred at 25 ° C. for 1 hour.

Next, 0.5 ml of the blocking buffer was added.
The mixture was added and stirred at 25 ° C. for 1 hour. Thereafter, the mixture was centrifuged at 18,000 rpm at 15 ° C. for 20 minutes. To the obtained precipitate, 2 ml of a blocking buffer was added, followed by centrifugation again in the same manner as above, and the precipitate was washed. This washing operation was performed three times. After adding 2 ml of a blocking buffer solution to the precipitate and stirring well, a dispersion treatment was carried out by an ultrasonic crusher to obtain a latex reagent having a solid content of 0.25% (w / v), and stored at 4 ° C.

Preparation of calibration curve Using the latex reagent, the immunoassay reagent of Example 2 consisting of an enzyme solution and a substrate solution, and using an automatic analyzer for biochemistry Model 7170 manufactured by Hitachi, Ltd., standard HBs was prepared in the following manner.
The antigen was measured. In the measurement by the automatic analyzer, the solid content obtained in (2) was 0.25% (w /
The latex reagent of v) was used as R2 solution as it was. The measurement conditions are as follows.

Sample volume 20 μl Sample diluent (R1 solution) 180 μl Latex reagent (R2 solution) 20 μl Enzyme solution (R3 solution) 20 μl Substrate solution (R4 solution) 20 μl Measurement wavelength 570 nm Measurement temperature 37 ° C. The R1 solution was added to the sample placed in the cell of the automatic analyzer, the R2 solution was obtained one minute later, and the R3 solution was obtained four minutes later.
The liquid and the R4 liquid 5 minutes later were charged. Substrate solution (R4 solution)
Was added, and the difference (ΔOD570 nm) between the absorbance approximately 75 seconds later and the absorbance approximately 730 seconds later was measured. The difference in absorbance multiplied by 10,000 was defined as the absorbance change. A calibration curve based on the relationship between the titer of the standard HBs antigen and the amount of change in absorbance was determined and is shown in FIG. In FIG.
The vertical axis shows the change in absorbance at 570 nm, and the horizontal axis shows the HBs antigen titer (IU / ml) in serum.

The standard HBs antigen solution of 20 μm in the section of the calibration curve for the measurement of HBs antigen-positive serum was used.
The amount of change in absorbance was determined in the same manner as in Example 1, except that 20 μl of HBs antigen-positive serum was used instead of 1.
The obtained change in absorbance was applied to the above calibration curve, and HBs
The HBs antigen titer in the antigen-positive serum was determined. Note that HB
Six types of samples (samples G to L) were prepared as s antigen-positive sera, the measurement was repeated five times for each sample, and the average value and coefficient of variation (CV) (%) were determined. The results are shown in Table 2 below.

[0055]

[Table 2]

As is clear from Table 2, it can be seen that the use of the immunoassay reagent of Example 2 enables highly accurate measurement of serum with a very small titer of 10 IU / ml or less.

[0057]

As described above, according to the first and second aspects of the present invention, the above-described first to third reactions are reactions depending on the amount of the antigen (or antibody) in the biological sample, and the LIA A small amount of antigen (or antibody) can be measured with higher sensitivity than the method. Further, since a complicated B / F separation is not required, the workability of the measurement can be improved, and an immunoassay reagent applicable to an automatic analyzer that cannot perform the B / F separation can be provided.

In the present invention, as the insoluble carrier,
Even when the carrier is aggregated by the immune reaction and the turbidity is increased, the amount of change in the absorbance is reduced to 1/10 or less of the amount of change in the absorbance caused by the enzyme reaction. The antigen (or antibody) can be measured with high accuracy.

When an antibody against the enzyme is used as the enzyme inhibitor, the enzyme (specifically) acts on the enzyme, so that the antigen (or antibody) can be measured with higher accuracy.

[Brief description of the drawings]

FIG. 1 is a diagram showing a calibration curve created in Example 1.

FIG. 2 is a diagram showing a calibration curve created in Example 2.

Continued on front page F-term (reference) 4B063 QA01 QA18 QA19 QQ02 QQ79 QQ96 QR03 QR48 QR67 QR83 QS07 QS20 QS39 QX01

Claims (4)

[Claims] 1. An immunoassay reagent for measuring an amount of an antigen (or antibody) as a substance to be measured in a sample, wherein an immunoassay reagent for the antigen (or antibody) and the enzyme are carried. And particle size 0.001-0.5μm
An immunoassay reagent comprising: a carrier comprising organic polymer particles or colloidal particles; an enzyme inhibitor that inhibits the activity of the enzyme; and a substrate for the enzyme. 2. An immunoassay reagent for measuring the amount of an antigen (or antibody) as a measurement target substance in a sample, wherein the antibody (antigen) against the antigen (or antibody) and an enzyme inhibitor are carried. And a particle size of 0.001-0.
An immunoassay reagent comprising: a carrier composed of 5 μm organic polymer particles or colloid particles; an enzyme whose activity is inhibited by the enzyme inhibitor; and a substrate of the enzyme. 3. The amount of change in absorbance when the carrier is aggregated by an immune reaction to increase turbidity is one-tenth or less of the amount of change in absorbance caused by an enzyme reaction between the enzyme and a substrate. The immunoassay reagent according to claim 1 or 2. 4. The immunoassay according to claim 1, wherein the enzyme inhibitor is an antibody against the enzyme.