JPH03162669A - Method for determining antigen or antibody - Google Patents

Abstract

PURPOSE:To make stable and accurate determination by inducing an immunological reaction in the antibody or antigen sensitized on insoluble carrier particle and measuring the difference in optical intensity before and after agglutination. CONSTITUTION:The antibody or antigen sensitized on the insoluble carrier particle is dispensed into a reaction vessel and the optical intensity A1 is measured. A sample contg. the antigen or antibody which generates the immunological reaction with this antibody or antigen is then added to thereto and after the agglutination by the immunological reaction is effected, the optical intensity A2 is measured. The antigen or antibody in the sample is determined from the difference between the optical intensities A1 and A2. The latex of an org. high polymer, such as polystyene having 0.05 to 0.5 mum average grain size and an inorg. oxide, such as silica are used as the insoluble carrier particle and the reaction is effected at a constant temp. of 20 to 40 deg.C. Desirable results are obtainable if a buffer soln., refined water or ion exchange water is simultaneously mixed with the specimen sample at the time of adding the sample.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application 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.

(Prior Art) In recent years, in the medical field, it has become extremely important to quickly, easily, and accurately quantify trace substances, fP, antibodies, and/or antigens in specimens for immune diagnosis.

For this reason, immunoserological tests are widely used that 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. . Traditionally, antibodies or antigens support (sensitization)
The sensitized latex particles (sensitized latex) and the specimen are mixed on a glass plate to cause an antigen-antibody reaction with the antigen or antibody in the specimen, and the state of agglutination is observed with the naked eye. Alternatively, methods have been used to measure antibodies semi-quantitatively. As an improvement to this method. A method has been proposed in which latex particles sensitized with antibodies or antigens are used to optically measure reaction aggregates between latex and antigens or antibodies in specimens (Japanese Patent Publication No. 58-11575, Japanese Patent Publication No. 11575/1983). -43138 Publication, % Publication 62-55
103, etc.). Let's try this method. recently. Quantitative measurement of antigens or antibodies using dedicated analyzers has also become common practice.

(Problem to be Solved by the Invention) However, the above method is expensive because it uses a dedicated analyzer, and is not suitable for use in an immunoserological laboratory 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, reagent contamination from cells and dispensing nozzles, etc.
The problem was that the measured value fluctuated depending on the amount of carry opa. However, the conventional method is. Previously, the measurement sample was first dispensed into the reaction vessel, and then the latex solution was added. When carried out in this manner, there were problems such as poor measurement accuracy due to being susceptible to the effects of optical and electrical noise and stirring efficiency.

Thus, an object of the present invention is to provide a method for quantifying antigens or antibodies that utilizes the immunolatex agglutination method, but that is stable and has good accuracy without the need for sophisticated dedicated equipment.

(Means for Solving the Problem) That is, the present invention dispenses an antibody or antigen sensitized to insoluble carrier particles into a reaction container, measures the optical intensity A1, and then immunologically interacts with the antibody or antigen. Add a sample containing the antigen or antibody that produces a reaction. After agglutination by immunological reaction. The present invention relates to a method for quantifying an antigen or antibody, which comprises measuring an optical intensity A2 and quantifying the antigen or antibody in the sample from the value of the difference between the optical intensity A2 and the optical intensity A1.

In the present invention, polystyrene is used as the insoluble carrier particles. Organic polymer latex such as styrene-ptadiene copolymer, and inorganic oxides such as silica and alumina are used. Its average particle size is 0.05~0.5μm
A range of 1 is preferable. 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 decrease.

Additionally, the medium for these insoluble carrier particles is phosphate buffer. It is preferable to use glycine buffer, Tris buffer, Good's buffer, etc.

In the present invention, it is supported (sensitized) on insoluble carrier particles.
Antigens that cause an immunological reaction with the antibody to be measured include proteins, polypeptides, polysaccharides, lipids, etc. There are no particular restrictions, and antibodies that cause an immunological reaction with the antigen to be measured usually include proteins, polypeptides, polysaccharides, lipids, etc. Immunoglobulin is used. In some cases, the Fab fragment. Fab' fragment. F(ab')'' fragments, Pc fragments, etc. can also be used.As a method for sensitizing these on an insoluble carrier, physical adsorption may be used as is commonly done, or chemical adsorption may be used. It may be combined with , or both may be used together.

The sensitized insoluble carrier particles. During the immunological reaction, it is retained as a medium dispersion. In that case. 0.1 in medium
In terms of preservation, it is preferable to disperse the carcasses to a concentration of ~1.0 wt/f%. Generally easy to use. 1. Bovine serum albumin, NaC, etc. may be dissolved in the medium as appropriate.

1. Also, sensitized insoluble carrier particles can be used during immunological reactions. It is used after being dispersed in a medium at an appropriate concentration, but from the viewpoint of ease of optical intensity measurement, it is preferable to use it at a concentration of 0.5 weight or less. from 0
．． 01 Weight or more is preferable! Yes. At this time, a solution (diluent) in which bovine serum albumin, NaCl, etc. are dissolved may be used to adjust the volume, if necessary.

In order to adjust the reactivity of the immunological reaction (agglutination reaction) in the method of the present invention, a substance that suppresses the reaction or a substance that promotes the reaction can be used. Substances that inhibit aggregation reactions include:
Trialkylamines, their salts, quaternary ammonium salts, sugars, etc. can be used. Trialkylamines include triethylamine, trialkylamine salts include triethylamine hydrochloride, quaternary ammonium salts include choline chloride, furin bromide, acetylcholine chloride, acetylcholine bromide, and petaine hydrochloride, and sugars include sulfur. There are sugars, etc. One or more of these compounds may be used.

A substance that suppresses the aggregation reaction may be dissolved in the above-mentioned dispersion of insoluble supported particles. 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.

However, if the reactivity between the sensitized antigen or antibody and the antibody or antigen in the sample is low. Measurements can be performed without adding substances that inhibit such aggregation reactions.

As a substance that promotes aggregation reactions. Polyethylene glycol etc. are used. The average molecular weight of polyethylene glycol is preferably 1,000 or more. The larger the molecular weight, the greater the effect of promoting the aggregation reaction, but if the molecular weight is too small, the effect is small.

The concentration of the substance that promotes the aggregation reaction in the final reaction solution is 0.1
Preferably, the amount is in the range of 5.0 to 5.0% by weight. If the concentration of the substance that promotes the agglutination reaction is too high, nonspecific aggregation of the sensitized insoluble carrier is likely to occur; if it is too low, the effect of promoting the reaction will be small. Substances that promote aggregation reactions are similar to insoluble carrier particles. Phosphate buffer. It is preferable to use it by dispersing it in a medium such as glycine buffer, Tris buffer, or Good's buffer. I cut it. Bovine serum albumin, NaCj', etc. may be dissolved in this medium as appropriate.

Next, the actual quantitative method of the present invention will be explained in detail. i
figure. Dispense 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. After mixing and incubation for 5 seconds to 15 minutes, the optical intensity A is measured. Next, the specimen sample is mixed and stirred to cause agglutination due to immunological reaction, and after mixing,
Optical intensity A2 after incubation for seconds to 15 minutes
Measure. By mixing and measuring in this way, the accuracy of quantitative determination is greatly improved. This reaction is 20~
It is preferable to carry out the reaction at 40°C, and the reaction is preferably carried out at a constant temperature. If the reaction temperature is outside this range, the immunological reaction tends to become unstable. Furthermore. This reaction 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 reaction is likely to be insufficient, and if it is more than 15 minutes, it is unsuitable for rapid measurement. 1. When dispensing the medium dispersion of insoluble carrier particles, a buffer solution, purified water, or ion-exchanged water may be further mixed at the same time as adding the casing or the specimen sample.

Among these, buffer solution is used when adding the specimen sample. If purified water or ion-exchanged water is mixed at the same time, the accuracy of sample dispensing will be high (preferably). In particular, simultaneous dispensing and mixing of the specimen sample by pushing it out with a buffer solution or the like is advantageous because it prevents the specimen sample from adhering to the dispensing nozzle.

here. Optical intensity as used in the present invention means absorbance or scattered light intensity. The measurement wavelength is. 400-1200nm
It is preferable to appropriately select from the range of . Measurement wavelength is 1
If the wavelength exceeds 200 nm, the sensitivity tends to decrease, and if the measurement wavelength is 400 nm or less, the optical intensity of the medium dispersion itself becomes large. The measurement range becomes narrower. From the standpoint of sensitivity, it is preferable to measure the intensity of scattered light or absorbance.

Next, in order to make a correction by subtracting the optical intensity due to the medium dispersion of insoluble carrier particles from the measured optical intensity. Optical intensity AI determined in the same manner using purified water, buffer solution, or physiological saline instead of the specimen sample described above.
Measured time values AH' and A,/t corresponding to and were determined.

Then, the optical intensity Ax is calculated from the optical intensities A and A2 measured using the specimen sample and the optical intensities A, j, and A2' caused by the medium dispersion of the insoluble carrier particles using the formula (1) Ax = At - A1 (Ax' - As')
--"{1) to calculate the value of the difference in optical intensity.

On the other hand, as a specimen sample. Using a sample with a known concentration (a sample containing a known amount of antigen or antibody C8), calculate the calculated optical intensity A8 in the same manner as above, and apply this to the following equation (2), for example, to determine the amount of the antigen or antibody in the specimen sample. The amount of unknown antigen or antibody (Cx) can be determined.

Cx = Ax X (Cs/Aa)
--...(2) (wherein +Ax is the calculated optical intensity of the sample containing an unknown amount of antigen or antibody, Cs is the amount of antigen or antibody of the sample containing a known amount of antigen or antibody, and As is (This is the calculated optical intensity of the sample.) The Cx determined in the above manner is due to cell contamination, optical system noise, etc. that cause errors when dispensing sensitized carrier particles. Because the derived optical intensity Al is calculated using the value subtracted from the optical intensity A2 resulting from the antigen-antibody reaction, compared to the conventional method in which the specimen sample is dispensed first. High measurement accuracy. In particular, the method of the present invention
Excellent reliability of measured values in low concentration areas.

Note that it is also possible to prepare a sample with a known concentration at various concentrations, measure it in the same manner as described above, release a calibration curve, press the button, and use this calibration curve to quantify the specimen sample.

Furthermore, in the present invention, in order to further improve accuracy, the optical intensity is determined by simultaneously measuring two wavelengths of light as described above, and from the difference in optical intensity between the two wavelengths, unknown unknowns in the specimen sample are determined. The amount of antigen or antibody can also be determined.

(Example) Next, let's look at an example. The present invention will be explained in detail. Hereinafter, 多 means heavy fly.

Example 1 1) Preparation of Reagent a) Latex Reagent 0.15M NaCl, 1.0% bovine serum albumin and 1.5% choline chloride. 0 5 M
Prepare a phosphate buffer (pH 6, 50) and use it as a latex dispersion solvent. In this solution. Polystyrene latex particles for diagnostic reagents with an average particle diameter of about 0.26 μm sensitized with anti-human α-fetoprotein (hereinafter abbreviated as AP'P) antibody were prepared at a latex concentration of o. Disperse the latex reagent so that it is o s s. Made by Iil.

b) Buffer 0. 15 M NaC/and l. (0.05M phosphate buffer containing bovine serum albumin (pH 6.5)
0) polyethylene glycol (average molecular weight 7500)
was dissolved at 3.0% and used as a buffer solution.

2) Measurement method Dispense 150 μl of the latex test solution into a reaction cupette, heat it at 37° C. for 5 minutes, and then determine the absorbance (Al) at a wavelength of 570 nm. Next, 8 μl of the specimen sample and 80 μl of buffer were added, stirred, and kept at 37°C for 5 minutes.
The absorbance (A2) at a wavelength of 570 nm is determined. From the obtained absorbance, the formula (A2 AIXI50/23
8) (150/238 is a coefficient for correcting the volume difference before and after addition of the specimen sample and buffer solution; coefficient = (latex test solution volume)/total volume]) is the absorbance of the sample.

3) Actual Measurement Results Measurements were carried out by the above measurement method using physiological saline and AFP-containing serum of known concentrations (10, 40, 100, 200 ng, /m/m) as specimen samples. A calibration curve was created by subtracting the measured value when using physiological saline as a reagent blank from the measured value of each sample.

A good calibration curve was obtained as shown in FIG.

(Example 2) Using the same reagents as in Example 1, the same operations were performed to examine simultaneous reproducibility. The above absorbance difference when using physiological saline as a sample was defined as Am, and the absorbance difference when using a sample with an AFP concentration of 100 ng/ml was defined as As. The absorbance difference Ax obtained by repeating the same operation 10 times to measure the absorbance of a sample with unknown AFP concentration was applied to the formula below to calculate the concentration Cx to check simultaneous reproducibility. On the other hand, the conventional dispensing order (i.e., first dispense the sample and buffer solution and measure the absorbance, then add the latex reagent and measure the absorbance after reaction, and measure the concentration based on the difference in absorbance). The reproducibility was compared with the method of the present invention when the same method was used except that the measurement method was changed. The results are shown in Table 1.

Cx = (Ax-As)
As shown in Table 1, the simultaneous reproducibility of the measurement method of the present invention is higher than the coefficient of variation (C.V,
) was about 3 times better.

In this example, COBASFARA, an automatic analyzer manufactured by Keizokusha and Roche, was used. This device can automatically perform the above calculations using an analysis program and calculate the measurement results.

Table 1 (Unit: ng/d) (Effects of the invention) As described above, according to the quantitative method of the present invention. Stable and highly accurate antigen or antibody quantification can be performed without the need for special dedicated equipment.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the relationship between absorbance and AFP concentration, which is the measurement result of Example 1 of the present invention.ゝ・１

Claims (1)

[Claims] 1. Dispense the antibody or antigen sensitized to the insoluble carrier particles into a reaction container, measure the optical intensity A_1, and then measure the optical intensity A_1. After adding a sample containing antibodies and causing agglutination by immunological reaction, optical intensity A_2 is measured, and the antigen or antibody in the sample is quantified from the difference between optical intensity A_2 and optical intensity A_1. A method for quantifying an antigen or antibody, characterized by: 2. When adding a sample containing an antigen or antibody, a buffer solution,
2. The method for quantifying antigens or antibodies according to claim 1, wherein purified water or ion-exchanged water is mixed at the same time. 3. The method for quantifying an antigen or antibody according to claim 1 or 2, wherein the optical intensity is absorbance.