JPH0763758A - Immunological measuring method - Google Patents

Abstract

PURPOSE:To provide a method by which a component to be measured can be accurately, quickly, and easily measured with high reproducibility even when the concentration of the component is such a low level that the component cannot be measured by the conventional method and a reagent composition used for the method. CONSTITUTION:In an immunological measuring method for trace components in which measurement is performed based on variation in turbidity or intensity of scattered light caused by an antigen-antibody reaction, an antigen (or antibody) modified in advance with biotin is used as the antigen (or antibody) against an antibody (or antigen) to be measured and the measurement is performed under the presence of avidin or streptoavidin. This invention also aims at providing a reagent composition used for the measuring method.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunological assay method capable of specifically, rapidly and accurately quantifying a trace component in a body fluid such as serum, plasma or urine, and a reagent composition used therefor.

[0002]

BACKGROUND OF THE INVENTION In recent years, automatic analyzers capable of simultaneous analysis of a large number of specimens and multiple items have become widespread, and attempts have been made to apply the immunoassay method for trace components utilizing antigen-antibody reaction to automatic analyzers. .

As a method applicable to an automatic analyzer,
So-called immunoturbidimetric method (TIA) for measuring a target component by measuring a change in turbidity caused by an antigen-antibody reaction
As a typical example, a so-called immuno ratio method, which measures a target component by measuring a change in scattered light intensity caused by an antigen-antibody reaction, and the like.

However, in these methods,
When the amount of the component to be measured in the sample is small (that is, in the low concentration range), the measured value becomes lower than the theoretical value, which causes a problem that the target component cannot be accurately measured.

In order to overcome such problems, in these methods, a method of adding a large amount of an antigen-antibody reaction promoter such as polyethylene glycol (PEG) and chondroitin sulfate to the assay system, Methods for increasing the absolute amount are being studied. However, any of the methods has a problem that a measurement error caused by a coexisting substance in a sample, a measurement error caused by an insoluble matter generated by a nonspecific reaction, and the like occur, which is further problematic. There is a situation where improvement is desired.

[0006]

SUMMARY OF THE INVENTION The present invention has been made in view of the above situation, and it is possible to accurately and reproducibly measure a component to be measured even in a low concentration range, and to provide an automatic analyzer. It is an object of the present invention to provide an applicable immunoassay method and a reagent composition used therefor.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to an immunoassay for a trace component, which is carried out based on a change in turbidity or a change in scattered light intensity caused by an antigen-antibody reaction, and an antibody (or The invention of the above-mentioned measuring method, characterized in that an antigen (or antibody) previously modified with biotin is used as an antigen (or antibody) against (antigen), and the measurement is carried out in the presence of avidin or streptavidin.

The present invention also combines a reagent containing an antigen (or antibody), which is pre-modified with biotin, with respect to the antibody (or antigen) to be measured, and a reagent containing avidin or streptavidin. It is an invention of a reagent composition for immunological measurement, which is formed together.

That is, the inventors of the present invention have earnestly studied to find a method for increasing the measurement sensitivity of a component to be measured in a low concentration range without causing a measurement error due to the influence of a coexisting substance or a nonspecific reaction. As a result, the antigen (or antibody) to be added to the measurement reagent for the antibody (or antigen) to be measured
In advance with biotin, and by performing the desired measurement in the presence of avidin or streptavidin,
The inventors have found that a calibration curve with high reaction sensitivity and good linearity can be obtained even in a low concentration range, and have completed the present invention.

The avidin-biotin reaction has high specificity,
Since the binding force is strong, it is used in the enzyme immunoassay or the radioimmunoassay when binding the solid phase to the antigen-antibody reaction product or the binding of the antibody or the like to the labeled product, or In the latex turbidimetric method and carrier agglutination method, it is used for binding a carrier and an antigen (or antibody). However, there is no example in which an avidin / biotin reaction is used in combination when performing an immunoturbidimetric method or an immunonephelometric method using an antigen-antibody reaction,
It was quite surprising that the combined use of the avidin / biotin reaction markedly increased the measurement sensitivity in the case where the amount of the component to be measured in the sample was small (that is, in the low concentration range).

As the avidin or streptavidin used in the present invention, commercially available products can be used as they are, and the quality and the degree of purification are not particularly limited. Further, the amount to be used varies depending on the amount of biotin modified to the antigen (or antibody) to the measurement target and the measurement item, and is not particularly limited, but usually 0.01 to 1000 μg / ml as the concentration in the reaction solution, preferably Is 0.1 to 100 μg / m
l, more preferably 5-100 μg / ml.

As a method for binding biotin to an antigen or an antibody, for example, a commercially available biotinylation reagent, more specifically, for example, biotin having a succinimide group introduced (eg, NHS-biotin) or N-hydroxysuccinimide ( NHS) and biotin bound via a spacer, etc., to react with the amino group of the antibody or antigen protein [eg J. Biol. Chem., Vol. 264, 272-279 (1989)],
For example, commercially available N- [6- (Biotinamide) hexyl] -3 '-(2'-pyrid
yldithio) propionamide (biotin-HPDP) and N-Iod
A method of reacting oacetyl-N-biotinylhexylenediamin with a thiol group of an antigen or antibody [eg Annals of the Ne
w York Academy of Science, vol.254, 203 (1975)],
Biotin having a hydrazino group introduced therein is reacted with an aldehyde group of an aldehyde-modified antigen or antibody [for example, J. Biol. Chem., Vol. 172, 71 (1948); Biotech. Appl. Bi
ochem., vol.9, 488-496 (1987)] and the like are preferable.

The degree of modification of the antigen or antibody with biotin is 0.2 to 1 in molar ratio with respect to the antigen or antibody.
It is about 0 times, preferably about 1 to 5 times. If the amount of modification with biotin is large, problems such as high insolubility of the antigen or antibody or interference with the antigen-antibody reaction occur. Conversely, if the amount of modification is small, the sensitivity may not reach the desired target. As it occurs, caution is required.

In the method of the present invention, examples of the antigen modified with biotin include streptolysin O (SLO), rheumatoid factor (RF), hepatitis B virus surface antigen (HBs) and the like.

The antibody modified with biotin may be either a monoclonal antibody or a polyclonal antibody and is not particularly limited. Specific examples thereof include anti-C-reactive protein (anti-CRP) antibody, anti-immunoglobulin G (anti-IgG) antibody, anti-immunoglobulin A (anti-IgA) antibody, anti-immunoglobulin M (anti-IgA) antibody.
M) antibody, anti-albumin antibody, anti-C3 antibody, anti-C4 antibody, anti-α-fetoprotein (anti-AFP) antibody and the like.

All other reagents and measurement conditions (reaction temperature, reaction time, measurement wavelength, measuring device, etc.) used in carrying out the measurement method of the present invention are all known per se by the immunoturbidimetric method and the immunoratio method. It suffices to select according to those in the above. That is, the measurement method of the present invention may be carried out in accordance with the measurement operation method of the immunoturbidimetric method or immunohistochemistry method known per se except that the antigen or antibody modified with a hapten as described above is used, The automatic analyzers, spectrophotometers, etc. used in this field can be used without exception.

Specific examples of the buffer solution used in the assay method of the present invention include normal antigen-antibody reactions such as Tris buffer solution, phosphate buffer solution, veronal buffer solution, borate buffer solution and Good buffer solution. All the buffers used in the assay method used are listed, and the pH is not particularly limited as long as it does not suppress the antigen-antibody reaction, but usually 5
The range of -9 is preferably used.

The reagent composition for immunological measurement of the present invention can be used in both the immunoturbidimetric method and the immunoparasitic method.

The immunological assay reagent composition of the present invention can be used in a single reagent system, but since the reaction between avidin (or streptavidin) and biotin gradually progresses, avidin or streptavidin is added. It is preferable from the standpoint of stability of the reagents that the reagent group prepared above and the reagent group to which the biotin-modified antigen or the biotin-modified antibody is added are separated into reagent forms.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

[0021]

【Example】

Example 1. Measurement of anti-streptolidine O (ASO) value (1) Modification of streptolysin O (SLO) 1.3 mg protein / ml of commercially available SLO (manufactured by Wako Pure Chemical Industries, Ltd.)
30 mM N-Hydroxysuccinimido Biotin (NHS) was dissolved in 10 ml of 30 mM phosphate buffer (pH 7.5)
-1 ml of N, N-dimethylformamide solution containing -biotin, manufactured by PIERCE) was added and reacted at 37 ° C for 1 hour. After the reaction was completed, the reaction solution was dialyzed against a 0.9% NaCl solution to remove unreacted NHS-biotin to obtain a biotin-modified SLO solution. (2) Measurement of anti-streptolidine O (ASO) value (Preparation of measurement reagent) Measurement buffer 3.5% polyethylene glycol 6,000, 0.9% NaCl and 0.1
A 50 mM 3- (N-morpholino) propanesulfonic acid (MOPS) -NaOH buffer solution (pH 7.4) containing% NaN3 was used as a measurement buffer solution. First reagent solution Avidin (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in the above measurement buffer solution to a protein concentration of 20 μg / ml, and the first reagent solution was prepared.
Used as a test solution. Second Reagent Solution The biotin-modified SLO obtained in (1) above was dissolved in the above measurement buffer solution to a protein concentration of 150 μg / ml, and was used as the second reagent solution. (Measurement equipment) Hitachi automatic analyzer 7070 was used. (Measurement operation) 20 μl of a sample containing a predetermined concentration of ASO and 350 μl of the first reagent solution were mixed and incubated at 37 ° C. for 5 minutes, and then the absorbance at the measurement main wavelength of 340 nm and the sub-wavelength of 700 nm was measured (sample blind value). . Next, add 50 μl of the 2nd test solution to this, incubate at 37 ° C for 5 minutes, measure the absorbance at the measurement main wavelength of 340 nm, and the sub wavelength of 700 nm. The subtracted absorbance was determined (reaction absorbance). In addition, as a control, the same sample and reagent solution were used except that the second reagent solution prepared in the same manner using unmodified SLO was used, and the same operation was performed to determine the reaction absorbance. (Results) FIG. 1 shows a calibration curve showing the relationship between the obtained reaction absorbance and ASO concentration. In addition, in FIG. 1, + is a modified S
The results obtained using the second reagent solution containing LO are
Indicate the results obtained using the second reagent solution containing unmodified SLO, respectively. As is clear from FIG. 1, unmodified SL
It can be seen that when O is used (conventional method), the reaction sensitivity decreases in the region of 200 U / ml or less, and measurement becomes impossible. On the other hand, according to the method of the present invention using modified SLO, there is sufficient reaction sensitivity even in the region of 100 U / ml or less,
It turns out that measurement is possible.

Reference Example 1. Anti-human C-reactive protein (anti-CR
P) Preparation of monoclonal antibody (1) Immunization A commercially available human CRP (manufactured by Wako Pure Chemical Industries, Ltd.) was used to inoculate mice three times at 2-week intervals (inoculation amount per administration: 50 μg / mouse). (2) Preparation of hybridoma Cell fusion of spleen cells and myeloma cells (P3 / NS1-1-Ag4-1) extracted from the mouse on the third day after the final immunization was performed by the method of Keller and Milstein [Nature, vol.256, 495 (1975)]
The screening was performed by the following method. That is, 50 μl of a 0.2% human CRP solution prepared by dissolving it in 50 mM carbonate buffer (pH 9.6) was dispensed into a 96-well microplate and incubated at 37 ° C. for 1 hour to immobilize it. After washing with 10 mM phosphate buffered saline (pH 7.4), 50 μl of culture supernatant of various hybridomas obtained by cell fusion was dispensed into the microplate and reacted at 37 ° C. for 1 hour. After washing with 10 mM phosphate buffered saline (pH 7.4), 50 μl of peroxidase-labeled anti-mouse immunoglobulin antibody (manufactured by DAKO) appropriately diluted with 10 mM phosphate buffered saline (pH 7.4) was added to the micro Dispense into plates and 3
The reaction was carried out at 7 ° C for 1 hour. Then, after washing with 10 mM phosphate-buffered saline (pH 7.4), a citrate buffer solution containing 2 mg / ml o-phenylenediamine and 0.017% hydrogen peroxide (pH 4.
9) 50 μl was dispensed to the microplate, left to stand at room temperature for 10 minutes to cause a color reaction, and then 6N sulfuric acid was dispensed to the microplate to stop the color reaction. Based on the results obtained above, a hybridoma producing a culture supernatant that reacts with human CRP was selected and used as a parent hybridoma. This parent hybridoma was cloned by the limiting dilution method to produce anti-human CRP monoclonal antibody-producing hybridoma 1-
3 and 2-6 were established. (3) Preparation of Monoclonal Antibody 0.5 ml of pristane (2,6,10,14-tetramethylpentadecane, manufactured by Wako Pure Chemical Industries, Ltd.) was intraperitoneally injected into the abdominal cavity of a mouse 3 days after the injection. Hybridoma 1 obtained in 2)
-3 or 2-6 1 x 106 cells were inoculated. Twelve days after hybridoma inoculation, the ascites fluid stored in the abdominal cavity was collected,
10 ml of the obtained ascites was subjected to a 40% ammonium sulfate fractionation treatment, and then dialyzed against 10 mM phosphate buffered saline (pH 7.4) (1 liter x
3 times) to obtain an anti-human CRP monoclonal antibody solution.

Example 2. C-reactive protein (CRP) measurement (1) Preparation of biotin-modified anti-human CRP monoclonal antibody The anti-human CRP monoclonal obtained in Reference Example 1 was used instead of SLO.
A biotin-modified anti-human CRP monoclonal antibody was obtained by the same procedure as in Example 1 except that the null antibody (clone 1-3) was used. (2) Measurement of C-reactive protein (CRP) (Preparation of reagent solution for measurement) Measurement buffer solution Same as in Example 1. First reagent solution Avidin (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in the above measurement buffer solution to a protein concentration of 50 μg / ml
Used as a test solution. Second reagent solution The biotin-modified anti-human CRP monoclonal antibody obtained in (1) above and the anti-human CRP monoclonal antibody obtained in Reference Example 1 (clone 2-6) were each added to a protein concentration of 1 μg / ml. The above-mentioned measurement buffer solution added so that
Used as a test solution. (Measurement equipment) Hitachi automatic analyzer 7070 was used. (Measurement operation) 15 μl of a sample containing CRP at a predetermined concentration and 350 μl of the first reagent solution were mixed and incubated at 37 ° C. for 5 minutes, and then the absorbance at a measurement main wavelength of 340 nm and a sub-wavelength of 700 nm was measured (sample blind value). . Next, add 50 μl of the 2nd test solution to this, incubate at 37 ° C for 5 minutes, measure the absorbance at the measurement main wavelength of 340 nm, and the sub wavelength of 700 nm. The subtracted absorbance was determined (reaction absorbance). Also, as a control, the same was used except that the second reagent solution prepared in the same manner using an unmodified anti-human CRP monoclonal antibody (clone 1-3) instead of the biotin-modified anti-human CRP monoclonal antibody was used. Using the sample and the test solution, the same operation was performed to determine the reaction absorbance. (Result) A calibration curve showing the relationship between the obtained reaction absorbance and CRP concentration is shown in FIG. In FIG. 2, + indicates the result obtained by using the second reagent solution containing the biotin-modified anti-human CRP monoclonal antibody, and □ indicates the unmodified anti-human CR.
The results obtained using the second reagent containing only the P monoclonal antibody are shown. As is clear from FIG. 2, it is difficult to measure CRP of 5 mg / dl or less when the second reagent containing only unmodified anti-human CRP monoclonal antibody is used (conventional method). On the other hand, according to the method of the present invention using the second reagent solution containing the biotin-modified anti-human CRP monoclonal antibody, it is possible to measure CRP of 5 mg / dl or less.

Example 3. Measurement of anti-human IgG antibody (1) Preparation of biotin-modified human IgG Biotinamidocaproate-N-Hydroxysucci was prepared by dissolving 100 mg of commercially available human IgG in 9 ml of 50 mM carbonate buffer (pH 9.0).
A solution prepared by dissolving 9 mg of nimidoester (BHS, manufactured by PIECE) in 1 ml of N, N-dimethylformamide solution was added, and the mixture was reacted at 5 ° C for one day. After the reaction was completed, the reaction solution was dialyzed against a 0.9% NaCl solution to remove unreacted BHS to obtain biotin-modified human IgG. (2) Measurement of anti-human IgG antibody (Preparation of measurement reagent) Measurement buffer The same as in Example 1. First reagent solution Avidin (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in the above measurement buffer solution to a protein concentration of 10 μg / ml, and the first reagent solution was prepared.
Used as a test solution. Second Reagent Solution The biotin-modified anti-human IgG obtained in (1) above was added to the above measurement buffer solution at a protein concentration of 75 μg / ml to give a second reagent solution. (Measurement equipment) Hitachi automatic analyzer 7070 was used. (Measurement procedure) Sample containing anti-human IgG antibody at a predetermined concentration 20μ
1 and 350 μl of the first reagent solution were mixed and incubated at 37 ° C. for 5 minutes, and then the absorbance at the main measurement wavelength of 340 nm and the sub-wavelength of 700 nm was measured (sample blind value). Next, add 50 μl of the 2nd test solution to this, incubate at 37 ° C for 5 minutes, measure the absorbance at the measurement main wavelength of 340 nm, and the sub wavelength of 700 nm. The subtracted absorbance was determined (reaction absorbance). Reaction absorbance was determined by performing the same procedure using the same sample and reagent solution except that the second reagent solution prepared in the same manner using unmodified human IgG was used as a control. (Result) A calibration curve showing the relationship between the obtained reaction absorbance and the anti-human IgG antibody concentration is shown in FIG. In addition, in FIG.
+ Indicates the results obtained by using the second reagent solution containing biotin-modified human IgG, and □ indicates the second specimen containing unmodified human IgG.
The results obtained using the test solutions are shown below. As is clear from FIG. 3, the calibration curve obtained by the method of the present invention using the second reagent solution containing biotin-modified human IgG is the unmodified human I
It can be seen that the curvature in the low concentration range was eliminated as compared with the calibration curve obtained when the second reagent solution containing gG was used (conventional method). Moreover, from the result of FIG. 3, 0.2 m is obtained by the method of the present invention.
It is also understood that it is possible to measure anti-human IgG antibody at g / ml or less.

[0025]

INDUSTRIAL APPLICABILITY As described above, the present invention is a method capable of accurately and reproducibly measuring a component to be measured in a low concentration range, which cannot be measured by the conventional method, quickly and simply. And a reagent composition used therefor, which is a great invention that contributes to the industry.

[Brief description of drawings]

FIG. 1 shows the calibration curve obtained in Example 1. FIG. 2 shows the calibration curve obtained in Example 2. FIG. 3 shows the calibration curve obtained in Example 3.

[Explanation of symbols]

In FIG. 1, + represents modified streptolidine O (SLO)
Shows the result obtained by using the second reagent solution containing ## STR4 ## and □ shows the result obtained by using the second reagent solution containing unmodified SLO. In FIG. 2, + is the result obtained by using the second reagent solution containing biotin-modified anti-human C-reactive protein (anti-human CRP) monoclonal antibody, and □ is only the unmodified decorated anti-human CRP monoclonal antibody. The results obtained using the second reagent solution containing In FIG. 3, + indicates the results obtained using the second reagent containing biotin-modified human IgG, and □ indicates the results obtained using the second reagent containing unmodified human IgG.

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[Procedure amendment]

[Submission date] September 1, 1994

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0022

[Correction method] Change

[Correction content]

Reference Example 1. Anti-human C-reactive protein (anti-CR
P) Preparation of monoclonal antibody (1) Immunization A commercially available human CRP (manufactured by Wako Pure Chemical Industries, Ltd.) was used to inoculate mice three times at 2-week intervals (inoculation amount per administration: 50 μg / mouse). (2) Preparation of hybridoma Cell fusion of spleen cells and myeloma cells (P3 / NS1-1-Ag4-1) extracted from the mouse on day 3 from the final immunization was performed by the method of Keller and Milstein [Nature,
vol. 256, 495 (1975)],
Screening was performed by the following method. That is, 50
50 μl of a 0.2% human CRP solution prepared by dissolving in a mM carbonate buffer (pH 9.6) was dispensed into a 96-well microplate and incubated at 37 ° C. for 1 hour to immobilize it. 10 mM phosphate buffered saline (pH
After washing with 7.4), 50 μl of culture supernatant of various hybridomas obtained by cell fusion was dispensed into the microplate and reacted at 37 ° C. for 1 hour. After washing with 10 mM phosphate-buffered saline (pH 7.4), 50 μl of peroxidase-labeled anti-mouse immunoglobulin antibody (manufactured by DAKO) appropriately diluted with 10 mM phosphate-buffered saline (pH 7.4) was used for the micro. The mixture was dispensed on a plate and reacted at 37 ° C. for 1 hour. Then, after washing with 10 mM phosphate buffered saline (pH 7.4), 50 μl of a citrate buffer (pH 4.9) containing 2 mg / ml o-phenylenediamine and 0.017% hydrogen peroxide was added to the micro. After being dispensed to the plate and allowed to stand for 10 minutes at room temperature to cause a color reaction, 6N sulfuric acid was dispensed to the microplate to stop the color reaction. Based on the results obtained above, human CRP
A hybridoma producing a culture supernatant that reacts with was selected and used as a parent hybridoma. This parent hybridoma was cloned by the limiting dilution method to establish anti-human CRP monoclonal antibody-producing hybridomas 1-3 and 2-6. (3) Preparation of Monoclonal Antibody 0.5 ml of pristane (2,6,10,14-tetramethylpentadecane, manufactured by Wako Pure Chemical Industries, Ltd.) was intraperitoneally injected into the abdominal cavity of a mouse 3 days after the injection. The hybridoma 1-3 or 2-6 1 × 10 ⁵ obtained in (2) was inoculated. Twelve days after the inoculation of the hybridoma, the ascites fluid accumulated in the abdominal cavity was collected, and 10 ml of the obtained ascites fluid was 40%.
After the ammonium sulfate fractionation treatment, it was dialyzed against 10 mM phosphate buffered saline (pH 7.4) (1 liter x 3 times) to obtain an anti-human CRP monoclonal antibody solution.

Claims (2)

[Claims] 1. An immunological assay method for trace components, which is performed based on a change in turbidity or a change in scattered light intensity caused by an antigen-antibody reaction, and an antigen against an antibody (or antigen) to be measured. As (or antibody), an antigen (or antibody) previously modified with biotin is used, and the measurement is performed in the presence of avidin or streptavidin.
The measurement method. 2. A combination of a reagent preliminarily modified with biotin, comprising an antigen (or antibody) against an antibody (or antigen) to be measured, and a reagent comprising avidin or streptavidin. Reagent composition for immunological measurement.