JPH04309864A - Quantitative measurement for antigen or antibody - Google Patents

Abstract

PURPOSE:To quantitatively measure the antigen or antibody with the stable and superior precision without requiring a special device for exclusive use. CONSTITUTION:A sample which is to be measured and contains the antigen or antibody and the insoluble carrier particles which are treated with the substance which does not generate the immunological reaction with the antigen or antibody are mixed, and the optical intensity A1 is measured. Then, the insoluble carrier particles which are treated with the antigen or antibody which generates the immunological reaction with the antigen or antibody are added, and after the coagulation by the immunological reaction is generated, the optical intensity A2 is measured. The antigen or antibody in the sample is quantitatively measured from the value which is obtained by subtracting the optical intensity A1 from the optical intensity A2.

Description

[Detailed description of the invention]

0001

TECHNICAL FIELD The present invention relates to a method for quantifying antigens or antibodies. More specifically, the present invention relates to a method for quantifying antigen or antibody by irradiating an antigen-antibody reaction mixture with light and measuring the optical intensity.

0002

BACKGROUND OF THE INVENTION In recent years, in the medical field, it has become very important to quickly, easily, and accurately quantify trace amounts of substances, especially antibodies and/or antigens, in a specimen for immunodiagnosis. For this reason, immunoserological tests are widely used, which test for the presence of antigens or antibodies in body fluid components by supporting (sensitizing) antibodies or antigens on insoluble carrier particles and reacting them with the antigens or antibodies. There is. Conventionally, latex particles (sensitized latex) on which antibodies or antigens are supported (sensitized) are mixed with a specimen on a glass plate to cause an antigen-antibody reaction with the antigen or antibody in the specimen, and this agglutination state is eliminated. A method has been used to semi-quantitatively measure antigens or antibodies in a specimen by observing with the naked eye. Therefore, a method has been proposed in which latex particles sensitized with antibodies or antigens are used to optically measure the reaction aggregates between the latex and the antigens or antibodies in the specimen (Japanese Patent Publication No. 11575/1983). Publication No. 62-43138, Japanese Patent Publication No. 62-55103). Recently, using this method, antigens or antibodies have also been quantitatively measured using a dedicated analyzer.

0003

[Problem to be solved by the invention] However, the above method
Since it requires a dedicated analyzer, it is expensive and unsuitable for use in immunoserology laboratories where the number of specimens is relatively small. For this reason, reagents that can be applied to general biochemical analyzers have recently been studied. However, there are various problems in adapting it to automatic analyzers developed for biochemical tests. For example, because it is measured at the same time as regular biochemical items, the measured value may fluctuate due to reagent contamination (carry-over) from the cell or dispensing nozzle, etc., and the measurement is susceptible to optical and electrical noise and stirring efficiency. There were problems such as poor accuracy. In addition, although the latex agglutination method is a rapid, simple, and highly accurate method, it suffers from nonspecific reactions caused by interfering substances in the specimen. Thus, it is an object of the present invention to provide a method for quantifying antigens or antibodies that utilizes the immunolatex agglutination method, but that provides stability and good accuracy without requiring special dedicated equipment.

0004

[Means for Solving the Problems] That is, the present invention mixes a sample containing an antigen or antibody to be measured with insoluble carrier particles sensitized with a substance that does not cause an immunological reaction with the antigen or antibody. to measure the optical intensity A1, and then
After adding insoluble carrier particles sensitized with an antibody or antigen that causes an immunological reaction with the antigen or antibody and causing aggregation due to the immunological reaction, optical intensity A2 is measured, and from this optical intensity A2 The present invention relates to a method for quantifying an antigen or antibody, which comprises quantifying the antigen or antibody in the sample from a value obtained by subtracting the optical intensity A1.

In the present invention, as the insoluble carrier particles, organic polymer latexes such as polystyrene and styrene-butadiene copolymers, and inorganic oxides such as silica and alumina are used. Its average particle size is 0.05
A range of ~0.5 μm is preferred. If the particle size of the carrier is too large, the optical intensity of the reagent itself before the immunological reaction is too high, making measurement difficult; if the particle size is too small, sensitivity tends to be low. In addition, media for these insoluble carrier particles include phosphate buffer, glycine buffer, Tris buffer,
It is preferable to use Good's buffer or the like.

In the present invention, first, a sample containing an antigen or antibody to be measured (hereinafter referred to as a specimen sample) is mixed with insoluble carrier particles sensitized with a substance that does not cause an immunological reaction with the antigen or antibody. However, the substances used at this time that sensitize the insoluble carrier particles and do not cause an immunological reaction with the antibodies and antigens to be measured include albumin,
Globulin, casein and gelatin are preferably used. These substances can be used regardless of their animal or other origin. In addition, substances processed so as not to cause an immunological reaction, such as Fc fragments of immunoglobulin, can also be used. As a method of sensitizing these on an insoluble carrier,
As is commonly done, physical adsorption may be performed, chemical bonding may be performed, or both may be used in combination.

[0007] In the present invention, sensitized insoluble carrier particles are then added to generate an immunological reaction.
Antigens that sensitize the insoluble carrier particles used at this time and cause an immunological reaction when the measurement target is an antibody include proteins, polypeptides, polysaccharides, lipids, etc., and there are no particular restrictions. Proteins such as immunoglobulins are usually used as antibodies that cause immunological reactions in cases of
)2 fragment, Fab fragment, etc. can also be used. As a method for sensitizing these on an insoluble carrier, as is commonly practiced, physical adsorption, chemical bonding, or a combination of both may be used.

[0008] The sensitized insoluble carrier particles are maintained as a dispersion in a medium until the immunological reaction, at which time they are dispersed in the medium to a concentration of 0.1 to 1.0% by weight. It is preferable to keep it for storage purposes, and it is generally easier to use. In addition, bovine serum albumin, NaCl, etc. may be added to this medium as appropriate.
etc. may be dissolved.

[0009] In addition, the sensitized insoluble carrier particles are used by being dispersed in a medium at an appropriate concentration during an immunological reaction, but the concentration is 0.5% by weight or less for ease of optical intensity measurement. It is preferable to use it in such a manner that the amount becomes 0.01% by weight or more from the viewpoint of sensitizing amount. In this case, in the medium, bovine serum albumin, NaC, if necessary.
A solution (diluted solution) in which L.

In the present invention, in order to adjust the reactivity of the aggregation reaction, a substance that suppresses the reaction or a substance that promotes the reaction can be used. As the substance used to suppress the aggregation reaction, trialkylamines, salts thereof, quaternary ammonium salts, saccharides, etc. can be used. Trialkylamines include triethylamine, trialkylamine salts include triethylamine hydrochloride, quaternary ammonium salts include choline chloride, choline bromide, acetylcholine chloride, acetylcholine bromide, betaine hydrochloride, and sugars. There are sugars, etc. One or more of these compounds may be used.

[0011] The substance that suppresses the agglutination reaction may be dissolved in a buffer solution and mixed with the specimen sample separately from the insoluble carrier particle dispersion, or may be dissolved in the above-mentioned insoluble carrier particle dispersion. It may be dissolved in a diluent for adjusting the volume of the dispersion liquid and mixed with the dispersion liquid at the time of use. Furthermore, if the reactivity between the sensitized antibody or antigen and the antigen or antibody to be measured in the specimen sample is low, the measurement can be performed without adding a substance that suppresses such an agglutination reaction. As the buffer, phosphate buffer, glycine buffer, Tris buffer, Good's buffer, etc. are preferably used. Further, bovine serum albumin, NaCl, etc. may be dissolved in this medium as appropriate.

[0012] As a substance that promotes the aggregation reaction, polyethylene glycol and the like are used.
The average molecular weight of the polymer is preferably 1,000 or more. As the molecular weight increases, the effect of promoting the aggregation reaction increases; however, when the molecular weight is too small, the effect is small. Polyethylene glycol has a concentration of 0.1 to 10.0% by weight in the final reaction solution.
It is preferable to make it exist in the range of . If the concentration of polyethylene glycol is too high, nonspecific aggregation of sensitized insoluble carrier particles tends to occur, and if it is too low, the effect of promoting the reaction is small. Preferably, the substance that promotes the agglutination reaction is dissolved in the buffer.

Next, the actual quantitative method will be explained in detail. First, a sample containing the antigen or antibody to be measured and a medium dispersion of insoluble carrier particles sensitized with a substance that does not cause an immunological reaction with the antigen or antibody are mixed and stirred. After incubation for a minute, the optical intensity A1 is measured. Next, a medium dispersion of insoluble carrier particles sensitized with an antibody or antigen that causes an immunological reaction with the antigen or antibody to be measured is mixed and stirred to cause an immunoreaction, and after mixing, incubation is performed for 5 seconds to 15 minutes. Measure the optical intensity A2. These reactions are preferably carried out at 20 to 50°C, and preferably at a constant temperature. If the reaction temperature is outside this range, the antigen-antibody reaction tends to become unstable. Furthermore, each of these reactions is preferably carried out for 5 seconds to 15 minutes after mixing, and particularly preferably for 10 seconds to 5 minutes. If it is less than 5 seconds, the above reaction tends to be insufficient, and if it is more than 15 minutes, it is not suitable for rapid measurement.

[0014] Also, after first mixing a sample containing the antigen or antibody to be measured with a buffer containing a substance that suppresses and/or promotes the above-mentioned reaction, an immunological reaction with the antigen or antibody is performed. It is also possible to measure the optical intensity A1 by mixing insoluble carrier particles sensitized with a substance that does not form.

[0015] Here, a medium dispersion of insoluble carrier particles sensitized with a substance that does not cause an immunological reaction with the antigen or antibody to be measured (the former) and an immunological reaction with the antigen or antibody to be measured are used. If the optical intensities of the medium dispersion (the latter) of insoluble carrier particles sensitized with antibodies or antigens that produce the same value are the same, they will be less affected by dispensing errors and the like, resulting in improved accuracy. The optical intensity ratio of this dispersion is 0 for the former/latter.
．． 1 to 10 is preferred, 0.2 to 5 is particularly preferred, and about 1 is most preferred.

In the present invention, optical intensity means absorbance or scattered light intensity. The measurement wavelength is usually 400 to 1
It is appropriately selected from a range of 200 nm. Measurement wavelength is 12
If the wavelength exceeds 00 nm, the sensitivity tends to decrease, and if the measurement wavelength is less than 400 nm, the optical intensity of the medium dispersion liquid itself becomes large and the measurement range becomes narrow.

[0017] Next, purified water,
Using a buffer solution or physiological saline, the measurement values A1' and A2' corresponding to the optical intensities A1 and A2 (meaning the optical intensities due to the medium dispersion) are obtained by performing the same operation. The optical intensity A calculated from the optical intensities A1 and A2 measured using the specimen sample and the optical intensities A1' and A2' caused by the medium dispersion is calculated using the formula (1) A=A2-A1-(A2'-A1) ')......Calculated according to (1).

On the other hand, a sample with a known concentration (
Using a sample containing a known amount of the antigen or antibody to be measured), calculate the calculated optical intensity in the same manner as above, and apply this to the formula (2) below to determine the unknown amount of antigen or antibody in the specimen sample. The quantity (CX) can be determined. CX=AX×CS/AS……(2) (where, AX is the calculated optical intensity of the sample containing an unknown amount of antigen or antibody, and CS is the calculated optical intensity of the sample containing a known amount of antigen or antibody. The amount of antibody and AS are the calculated optical intensities of the sample.) Here, CX is the factor of error in dispensing the sensitized carrier particles from the optical intensity A2 when performing the antigen-antibody reaction. The measurement accuracy is good because it is calculated by subtracting the optical intensity A1 caused by dispensing error, cell dirt, optical system noise, turbidity caused by the sample, non-specific reaction, etc., especially in the low concentration region. The reliability of the measured values is excellent.

[0019] It is also possible to prepare a sample with a known concentration at various concentrations, measure it in the same manner as described above, create a calibration curve, and use this calibration curve to quantify the sample. Furthermore, in order to further improve the measurement accuracy, the optical intensity can be determined by simultaneously measuring two wavelengths of light as described above, and the quantity can be determined from the difference in optical intensity between the two wavelengths.

[0020]

EXAMPLES Next, the present invention will be explained in detail with reference to examples. Hereinafter, % means weight %. Example 1 (1) Preparation of reagent (a) Latex solution Bovine serum albumin was sensitized to 0.05M phosphate buffer (pH 6.50) containing 0.15M NaCl and 1.0% bovine serum albumin. Polystyrene latex particles with an average particle diameter of approximately 0.1 μm are mixed at a latex concentration of 0.1.
% to form a latex liquid. (b) Sensitize anti-human C-reactive protein (hereinafter abbreviated as CRP) antibody to 0.05M phosphate buffer (pH 6.50) containing 0.15M NaCl and 1.0% bovine serum albumin latex test solution. A latex test solution was prepared by dispersing the polystyrene latex particles for diagnostic reagents having an average particle diameter of about 0.1 μm to a latex concentration of 0.1%. (C) Buffer A 0.05M phosphate buffer (pH 6.50) containing 0.15M NaCl and 1.0% bovine serum albumin is prepared and used as a buffer.

(2) Measuring method 250 μl of latex liquid and 3 μl of a specimen sample are dispensed into a reaction cuvette, stirred, and then heated at 37° C. for 5 minutes to determine the absorbance (A1) at a wavelength of 570 nm. Next, 250 μl of latex test solution was added, stirred, and kept at 37°C for 5 minutes. The absorbance (A2) at a wavelength of 570 nm was determined, and the difference in absorbance (A2 - A1 x 250/503) (2
50/503 is a coefficient for correcting the difference in volume before and after addition of the buffer solution (coefficient=latex reagent volume/total volume). As a conventional method for comparison, a method was performed in which the above buffer solution was used instead of the latex solution and the same operation as above was performed.

(3) Actual measurement results Measured values (A1', A2') using physiological saline and a dilution series (1/10 to 10/10) of CRP-containing serum as specimen samples. was used as a reagent blank to examine dilution linearity. The results of this measurement method are shown in FIG. 1, and as shown in FIG. 1, good dilution linearity was obtained. Note that it was almost the same as the conventional method.

Example 2 Using the same reagents as in Example 1, the same operations were carried out to examine simultaneous reproducibility. The absorbance difference when using physiological saline as the specimen sample was determined as AB, and the absorbance difference when using a sample with a CRP concentration of 5.3 mg/dl was determined as AS. Absorbance difference A obtained by repeatedly measuring a sample with unknown CRP concentration 10 times
Concentration CX was calculated by applying X to the formula below to check simultaneous reproducibility. In addition, the reproducibility of a conventional method, that is, a measurement method using a buffer solution instead of a latex solution, was measured and compared with the present invention. CX=(AX-AB)×5.3/(AS-AB) As shown in Table 1, the simultaneous reproducibility of the measurement method of the present invention is higher than that of the conventional measurement method. V. (coefficient of variation) was about 3 times better. Note that the measurement in this example was performed using Hitachi Model 7150, which is an automatic analyzer manufactured by Hitachi, Ltd. This device can automatically perform the above calculations using an analysis program to calculate measurement results.

[0024]

[Table 1]

As described above, according to the quantification method of the present invention, antigens or antibodies can be quantified stably and with good accuracy without the need for special dedicated equipment.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the relationship (dilution linearity) between the measurement results (concentration conversion values) and the dilution series of CRP-containing serum in Example 1 of the present invention.

Claims (5)

[Claims] Claim 1: A sample containing an antigen or antibody to be measured is mixed with insoluble carrier particles sensitized with a substance that does not cause an immunological reaction with the antigen or antibody.
1 is measured, then insoluble carrier particles sensitized with an antibody or antigen that causes an immunological reaction with the antigen or antibody are added, and after aggregation due to the immunological reaction, the optical intensity A2
is measured, and from this optical intensity A2, the optical intensity A1
A method for quantifying an antigen or antibody, comprising quantifying the antigen or antibody in the sample from the value obtained by subtracting . 2. Before measuring the optical intensity A1 by mixing insoluble carrier particles sensitized with a substance that does not cause an immunological reaction with the antigen or antibody to be measured, the antigen or antibody to be measured is first mixed. 2. The method for quantifying an antigen or antibody according to claim 1, wherein the sample containing the antigen is mixed with a buffer containing a substance that suppresses or promotes an immunological reaction. Claim 3: The substance that does not cause an immunological reaction with the antigen or antibody to be measured is albumin, globulin,
The method for quantifying an antigen or antibody according to claim 1 or 2, which is casein or gelatin. Claim 4: Claim 1, wherein the optical intensity is absorbance;
3. The method for quantifying the antigen or antibody according to 2 or 3. 5. The method for quantifying an antigen or antibody according to claim 4, wherein absorbance of light at two wavelengths is measured.