JPH0815258A - Immunological determination reagent and determination method - Google Patents

Abstract

PURPOSE:To carry out highly precise determination with high reproducibility, within a narrow dispersion, and without bubbling during the determination by determining an antigen-antibody reaction based on optical turbidity by using a reagent component produced by either containing or combining a soluble acyclic monoalcohol. CONSTITUTION:Wonoalcohols of acyclic hydrocarbons with 1-30 carbon number are among soluble acyclic monoalcohols to be contained in or combined with a constituent reagent and above all, n-octylalcohol is most effective and most preferable. It is preferable to adjust the concentration of a soluble acyclic monoalcohol existing in the reaction solution during the reaction to be 0.001-10%v/v, especially to be 0.001-1%V/V. As a reaction medium for a sample to be determined, water or a buffer of a physiological saline solution containing 0.1-0.2M/l NaCl at pH 6-9 is preferable. To determine an antigen-antibody reaction from the optical turbidity, for example, an immunological turbidimetric method, a latex immunological turbidimetric method, an optochemical enzyme immunological method, etc., can be applied.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an immunological measuring reagent and a measuring method for measuring an antigen-antibody reaction as optical turbidity. More specifically, the present invention relates to an immunological measuring reagent and a measuring method with little variation in measured values.

[0002]

2. Description of the Related Art Currently, in the fields of medicine and clinical examination,
Various biological substances in human samples (blood, urine), for example,
Plasma proteins and various coagulation / fibrinolytic system proteins, hormones, drugs,
An immunological assay method based on an antigen-antibody reaction is widely used for the measurement of various items such as various bacteria, syphilis, chlamydia, viruses and the like, and antibodies thereof. Further, in recent years, detection methods and reagents have been developed and improved in response to customer needs such as miniaturization, speedup, simplification, and automation.

The immunological assay utilizes the antigen-antibody reaction, and usually, an antibody against a test antigen is used for detecting an antigen, and an antigen against a test antibody is used for detecting an antibody. Is used as a reagent. As the optical immunoassay, various immunoassays such as an immunoturbidimetric assay, a latex immunoturbidimetric assay, an enzyme immunoassay, and a chemiluminescent enzyme immunoassay are used depending on the detection means. A method of optically measuring a reaction aggregate of a latex and a test antigen or a test antibody in a sample using latex particles sensitized with various antibodies or antigens has been proposed and spread (Japanese Patent Publication No. 58-11).
575, JP-B-62-43138, JP-B-62-55103, etc.), and recently, an antigen or antibody has been quantitatively detected using a dedicated analyzer or automatic analyzer. There is.

Further, an antigen or antibody is supported (sensitized) on an insoluble carrier, and this is reacted with the antibody or antigen to be detected, and after washing, an antibody labeled with an enzyme, a fluorescent substance, biotin or the like is reacted and washed. Afterwards, fluorescent antibody methods and enzyme immunoassays have been widely performed to detect the presence of antigens or antibodies in body fluid components by measuring the intensity of each activity or enzyme activity. A chemiluminescent enzyme immunoassay, which is a combination of these, has also been used.

[0005]

However, in the above-mentioned optical immunoassay, sometimes abnormalities and reproducibility (dispersion) of measured values become a problem. To improve the reproducibility of measured values,
Further, in order to avoid the influence of turbid substances, addition of various surfactants, polyethylene glycol, etc. has been attempted. However, on the contrary, these may increase the viscosity of the reagent and the like, which may cause abnormal measurement values. In addition, JP-A-4-340
Japanese Patent No. 467 discloses a method in which an immunoassay reagent containing a reaction sensitizer such as polyethylene glycol is heated at a temperature of 10 ° C. or higher for 10 minutes and then measured. However, this method requires a thermostat and a thermostat, and has a problem that the operation is troublesome. As a result of various studies to solve the above problems, the present inventors have found that many causes are generation of bubbles at the time of detection, and have completed the present invention.

[0006]

That is, the present invention relates to the following and: An immunological measuring reagent for measuring an antigen-antibody reaction as an optical turbidity, which contains or combines a soluble chain monoalcohol as a reagent component. An immunological measuring method for measuring an antigen-antibody reaction as optical turbidity, which comprises allowing a soluble chain monoalcohol to exist in a reaction solution at the time of measurement.

Hereinafter, the present invention will be described in detail. The immunological measurement reagent of the present invention contains a soluble chain monoalcohol, but may be mixed as one component of the constituent reagents or may be stored as a combination with another reagent. In addition, when there are multiple constituent reagents (for example, the reagent consists of a diluent and an antiserum solution), all may contain soluble chain alcohols, or only some reagents may contain them. You can also After all, it may be present in the reaction solution at the time of measurement. As the soluble chain monoalcohol used in the present invention, methanol, ethanol, n-propyl alcohol, isopropyl alcohol, tert-butyl alcohol, isobutyl alcohol, sec-butyl alcohol, n-amyl alcohol, isoamyl alcohol, hexyl alcohol, A chain hydrocarbon monoalcohol having 1 to 30 carbon atoms such as heptyl alcohol, capryl alcohol, n-octyl alcohol, nonyl alcohol, and decyl alcohol is preferable, and a chain monoalcohol having 6 or more carbon atoms is particularly preferable. Of these, n-octyl alcohol is the most effective and preferred.

When a chain monoalcohol soluble in advance is contained as one component of the reagent and is stored for a long period of time, it has 6 carbon atoms.
The above chain higher monoalcohol or a mixture of the chain higher monoalcohol having 6 or more carbon atoms and the chain lower monoalcohol having 1 to 5 carbon atoms is preferable. The amount of the soluble chain monoalcohol used is such that the amount present in the reaction during the reaction is 0.
001-10% (v / v), further 0.001-5%
(V / v), particularly preferably 0.001 to 1% (v / v). If a lower monoalcohol having about 1 to 5 carbon atoms is used at the time of measurement, 1% (v /
v) Even if it is a very small amount below, the effect will be exhibited, but if it is mixed as one component of the reagent and stored for a long time, it will volatilize and its effect will weaken with time, so it can be stored as another reagent and used for measurement. preferable. Further, a higher monoalcohol having 6 or more carbon atoms has a high viscosity, and the effect of the present invention cannot be sufficiently exerted, so that 10% (v / v)
v) It is preferably used below.

As a reaction medium of the measuring reagent of the present invention, water or a buffer solution having a physiological NaCl concentration (0.1 to 0.2 M / liter) and a pH (6 to 9) is preferably used. Specific buffers include phosphate buffers, borate buffers, glycine buffers, tris [tris (hydroxymethyl) aminomethane] hydrochloric acid buffers, ammonia buffers, etc. in the fields of ordinary biochemistry and immunology. The buffer used can be used and is not particularly limited. In addition, the measurement reagent of the present invention, to the extent that it does not interfere with the antigen-antibody reaction and its detection,
Additives such as a stabilizer, a preservative, a surfactant, a chelating agent, a reaction accelerator such as polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), and polylysine can be appropriately selected and contained.

The present invention can be applied to various immunoassay methods as long as the antigen-antibody reaction is measured as optical turbidity, and the measurement method is not limited. For example, an immunoturbidimetric method, a latex immunoturbidimetric method, an enzyme immunoassay method, a chemiluminescent enzyme immunoassay method and the like can be mentioned. The antigen-antibody reaction to be measured is also not particularly limited, and various biological substances in human samples (blood, urine), such as plasma proteins and various coagulation / fibrinolytic system proteins, hormones, drugs, and various bacteria. It can be applied to a large number of measurement items such as antigens of syphilis, chlamydia, virus, etc. and their antibodies.

As the optical turbidity measuring device, a usual spectrophotometer or biochemical automatic analyzer can be used. The light to be actually measured may be so-called transmitted light, scattered light, or a combination of both. The amount of change in turbidity is
One-point end measurement method that measures at one point after a certain time after the reaction starts, two-point end measurement method that measures at two points at the start of the reaction and after a certain time, rate measurement method that measures at two points after the reaction starts, It can be measured and quantified by a method such as a multipoint measuring method which measures at two or more points.
Further, in the measuring method of the present invention, the wavelength for measuring the optical turbidity is appropriately selected depending on the measuring device, the measuring method, the use or non-use of carrier particles, the particle diameter of the carrier particles in that case, the concentration of the reaction component, etc. To be done. Generally, monochromatic light or polychromatic light of 400 to 2400 nm is used in the immunoturbidimetric method that does not use carrier particles, and 300 to 400 nm in the latex immunoturbidimetric method that uses carrier particles.

[0012]

The present invention will be described in detail below with reference to examples. Example 1 [Detection of Apoprotein CII in Serum] The following apoprotein CII immunoturbidimetric reagent was prepared to confirm the effects of the present invention. 1) Preparation of measurement reagents, etc. a. Diluting liquid n-octyl alcohol 0%, 0.002%, 0.0
1%, 0.1%, 1%, 10%, 50% (all v / v)
Containing 0.15M sodium chloride and polyethylene glycol 6000 4 (v / v)
% 20 mM potassium phosphate buffer (pH 7.
3) was used as a diluent. b. Antiserum Solution 0.02M phosphoric acid containing 0.1% (w / v) of anti-human apoprotein CII IgG antibody, 0.15M sodium chloride buffer solution (pH 7.3) was used as an antiserum solution. c. Apoprotein CII standard serum Two types of apoprotein CII concentrations of 0 mg / dl and 4.5 mg / dl were used. d. Human serum containing apoprotein CII was used.

2) Apoprotein CII measurement in serum (automatic analyzer) It was measured by a Hitachi 7150 type automatic analyzer (manufactured by Hitachi, Ltd.). The analysis conditions are as follows. Sample 3
μl and n-octyl alcohol each in an amount of 0 to 50% (v /
v) 200 μl of the contained diluent was dispensed and stirred in the reaction cell and kept at a constant temperature (37 ° C.) for 5 minutes, then 200 μl of the antiserum solution was dispensed and stirred in each reaction cell, and further incubated for 5 minutes at a constant temperature (37 ° C.). (° C). The two-point end method was used in which the difference in absorbance (wavelength 370 nm) 10 minutes and 5 minutes after the addition of the sample was automatically measured. The calibration method was a two-check method in which the apoprotein CII standard serum (two kinds) was measured in advance. The test sample was continuously measured 10 times by the same measurement method, and the above-mentioned concentration was automatically obtained as a measurement value from the results of the two inspection amounts, and the average value, standard deviation and coefficient of variation were calculated.

[0014]

[Table 1]

[0015]

EFFECTS OF THE INVENTION According to the measuring method using the immunological measuring reagent of the present invention, bubbles are not generated during the measurement, and the measurement is highly accurate.
It is possible to quantify antigen-antibody reaction with high reproducibility with little variation in measured values.

Claims (8)

[Claims] 1. An immunological measuring reagent for measuring an antigen-antibody reaction as optical turbidity, which contains or combines a soluble chain monoalcohol as a reagent component. 2. The chain monoalcohol has 1 to 30 carbon atoms.
The immunoassay reagent according to claim 1, which is 3. The immunoassay reagent according to claim 2, wherein the chain monoalcohol has 6 or more carbon atoms. 4. The immunoassay reagent according to claim 1, wherein the chain monoalcohol is n-octyl alcohol. 5. An immunological measuring method for measuring an antigen-antibody reaction as optical turbidity, wherein a soluble chain monoalcohol is present in a reaction solution at the time of measurement. Measuring method. 6. The immunological assay method according to claim 5, wherein the amount of the chain monoalcohol present is 0.001 to 10% (v / v) in the reaction solution. 7. The chain monoalcohol has 1 to 30 carbon atoms.
The immunological assay method according to claim 5 or 6, wherein 8. The immunological assay method according to claim 5, 6 or 7, wherein the chain monoalcohol is n-octyl alcohol.