JPS62218865A - Quantitative analysis of rheumatic factor - Google Patents

Abstract

PURPOSE:To obtain a quantity detecting line of good linearity, by allowing the specific compound to co-exist in a mixture liquid of specimen and carrier sensitized with human gamma-globulin and generating a latex agglutinating reaction caused by the antigen-antibody reaction. CONSTITUTION:A mixture liquid is prepared by mixing a specimen and an unsoluble carrier sensitized with human gamma-globulin and a allowing co-existence, in this mixture liquid, of at least one kind of water-soluble compounds selected from a group consisting of polyethylene glycol, trialkylamine, its salt and No.4 grade ammonium and the latex agglutinating reaction is generated by the antigen-antibody reaction for measurement of an optical intensity and a rheumatic factor is analyzed quantitatively in the specimen from afore said value. As the specimens, blood serum, etc. can be used. As the unsoluble carriers polystyrene-series latex grain, etc. can be used. As the sensitized unsoluble carrier, content of 0.05wt% and less is preferable and as the water-soluble compound, 0.1-10wt% is preferable.

Description

[Detailed description of the invention] (Industrial application field) The present invention relates to a method for quantifying rheumatoid factor, and in particular to a method for quantifying rheumatoid factor.

This invention relates to a method for quantifying rheumatoid factor using immunolatex agglutination method.

(Prior art) Rheumatoid factor
r: hereinafter abbreviated as F) is a substance that appears in the blood of patients with chronic rheumatoid arthritis and is said to be a large protein with a molecular weight of about 1 million.

Measurement of RF has been designated as a standard item for the diagnosis of rheumatoid arthritis by the American Rheumatology Association, and is considered clinically essential for testing for the diagnosis of rheumatoid arthritis.

Currently, for RF measurement, the 几A test method that uses latex agglutination reaction and the 几A-HA test method that uses passive hemagglutination reaction using sheep red blood cells are widely used. Scandinavia 7 (A
cta Path, Microbiol.

5cand, ) Vol. 17, p. 172, 1940). However, since these measurement methods are qualitative reactions, it is impossible to understand changes in RF values over time, and there are also flaws in terms of operability.

In recent years, advances in detection methods have made it possible to quantitatively measure various trace proteins in plasma. One of these detection methods is immunoturbidimetry. In immunoturbidimetry, IgG has a relatively high blood concentration among plasma proteins.

IgA, IgM, C3, C4, and CRP are measured.

In terms of sensitivity, there was a limit to the measurement of low concentrations and plasma proteins.

Due to these trends, the immunolatex agglutination method has been developed to measure various low-concentration plasma proteins (Special Publication No. 58).
-11575, Japanese Patent Application Laid-open No. 53-62826)
.

(Problems to be Solved by the Invention) Although the immunolatex agglutination method uses laser light and is highly sensitive, when quantifying RF using this method,
In order to rapidly carry out the immune reaction (antigen-antibody reaction), it is preferable to carry out the reaction in the presence of polyethylene glycol. However, in this case, a test fk#J having good linearity cannot be obtained, and therefore, there are problems in that the preparation of a calibration curve is complicated during quantitative determination, and the accuracy of quantitative determination is reduced.

(Means for Solving the Problems) The present invention involves mixing a sample and an insoluble carrier sensitized with human r-globulin to form a mixed solution, and adding polyethylene glycol, trialkylamine, a salt thereof, and At least one type of water-based compound selected from the group consisting of quaternary ammonium salts is allowed to coexist to cause a latex agglutination reaction by an antigen-antibody reaction, and the optical intensity is measured. From this measurement value, the rheumatoid factor in the sample is determined. This invention relates to a constant lid method for quantifying rheumatoid factors.

The above-mentioned sample includes serum and the like.

As the above-mentioned insoluble carrier, known carriers such as polystyrene latex particles for diagnosis can be used.
The insoluble carrier is sensitized with human globulin by adsorbing globulin physically or chemically. The particle size of the insoluble carrier is 0.05 to 0.2μ to improve measurement sensitivity.
Preferably, it is m.

This sensitized insoluble carrier is used in the above mixed solution at an appropriate concentration, but from the viewpoint of ease of absorbance measurement, 0.05
It is preferably used in an amount of 5% by weight or less, and from the viewpoint of sufficient reaction, 0.01% by weight or more is preferable.

The polyethylene glycol is usually 2.

Those having an average molecular weight of 3.000 or more are preferred. As the molecular weight increases, the optical intensity due to latex aggregation increases. Add polyethylene glycol to the 8-pi mixture,
Preferably, it is present in an amount less than or equal to % by weight. If the concentration of polyethylene glycol becomes too high, nonspecific aggregation of the sensitized insoluble carrier tends to occur. Preferably, polyethylene glycol is present in the mixed liquid at a concentration of 0.5% by weight or more. If the amount is too small, the effect of promoting the reaction will be small.

Examples of the water-soluble compounds include triethylamine as a trialkylamine, triethylamine hydrochloride as a salt of trialkylamine, and choline chloride, choline bromide, acetylcholine chloride, and acetylcholine bromide as quaternary ammonium salts.

Examples include betaine hydrochloride. These compounds may be used alone or in combination. This water-soluble compound is.

It is used in the mixed solution at an appropriate concentration, preferably 0.1 to 10i1i%. If the amount of the water-soluble compound is too small, the effect of adding it will be small, and if it is too large, the measurement sensitivity will tend to decrease.

The sensitized insoluble carrier, polyethylene glycol, and water-soluble compound described above are preferably used as a reagent after being dispersed and dissolved in a suitable medium.

In this case, the reagent has a primary form.

(1) A reagent (one-liquid latex reagent) in which a sensitized insoluble carrier, polyethylene glycol, and a water-soluble compound are both dispersed or dissolved in the same medium.

(2) A reagent in which a sensitized insoluble carrier is dispersed in a medium (
A two-component reagent consisting of a latex reagent), polyethylene glycol, and a water-soluble compound dissolved in the same medium.

(3) A reagent in which a sensitized insoluble carrier and a water-soluble compound are both dispersed or dissolved in the same medium (latex reagent) A two-component reagent consisting of a reagent in which polyethylene glycol is dissolved.

(4) A reagent (latex reagent) in which a sensitized insoluble carrier is dispersed in a medium.

A reagent in which polyethylene glycol is dissolved in a medium and A reagent containing a water-soluble compound dissolved in a medium A three-component reagent consisting of:

Among these, forms (2) and (3) are most preferred.

In the above, the medium is a phosphate buffer.

Glycine buffer, Tris-HCl buffer, Gud buffer, etc. are preferred, and those whose pH is adjusted to 6 to 10 are preferred.

In addition, the reagents include bovine serum albumin and salt concentration n1, as appropriate.
You may dissolve NaCl etc. for ``1 adjustment''.

In the one-liquid latex reagent and multi-liquid latex reagent, the sensitized insoluble carrier is used at an appropriate concentration so that the concentration in the mixed liquid can be easily adjusted.
A concentration of 0.1 to 0.5% by weight is generally easy to use. The polyethylene glycol and water-soluble compound mentioned above.

In one-part latex reagents and multi-part reagents.

It is dissolved at an appropriate concentration so that the concentration in the liquid mixture can be easily adjusted.

Add bovine serum albumin to the above medium as appropriate.

Dissolve NaCl etc. together with the above reagent.

It may also be used for liquid fl-regulation.

In the present invention, a sample, the sensitized insoluble carrier,
Polyethylene glycol and the water-soluble compound are mixed in an appropriate order to form a mixed solution.

The above antigen-antibody reaction is preferably carried out at a temperature of 25 to 37°C, preferably at a constant temperature during one reaction.

Outside this range, the antigen-antibody reaction tends to become unstable. Furthermore, this reaction is preferably carried out for 30 seconds to 15 minutes. In less than 30 seconds.

The above reaction is insufficient, making it difficult to quantify F from absorbance, and if the time exceeds 15 minutes, the advantage of short-time measurement is diminished.

After the latex aggregation reaction starts, the optical intensity of the mixed liquid is measured by selecting an appropriate wavelength.

Here, optical intensity means absorbance or scattered light intensity. The wavelength is usually selected from the range of 500 to 1,000 nm.

The optical intensity is measured once after the start of one reaction [end point assay].

A method of measuring the increase in optical intensity after the start of the reaction] and a method of measuring at least two times after the start of the reaction and determining the increase in optical intensity or the increase per unit time during that time, and - reaching the optical intensity Any method f for measuring the time up to this point can be adopted.

For quantitative determination, perform the above measurements on a sample with a known amount of F (for example, BP standard serum and its dilution series), create a calibration curve from the measured values and the RF amount, and then perform the measurement on a sample with an unknown amount of F. This can be done by determining the corresponding Itle amount from the measured values measured under the same conditions using the calibration curve.

When the above-mentioned two-component reagent is used in a method in which measurement is performed once after the start of the reaction, quantitative determination can be further carried out in the following manner.

That is, measuring the optical intensity (ET) of the sample,
From this value, subtract the absorbance attributable to the sample (EBB) and the absorbance attributable to the latex reagent to obtain the calculated intensity (ER
Find F).

Here, the optical intensity caused by the sample is the optical intensity of a solution obtained by using physiological saline instead of the latex reagent in the preparation of the above-mentioned mixed solution, for example.

What is the optical intensity caused by the latex reagent?1 For example, in preparing the above-mentioned mixed solution, from the optical intensity (E an) of the solution obtained by using physiological saline instead of the sample, the optical intensity of the sample in the above-mentioned mixed solution is calculated. and minus the optical intensity (EBB) obtained using saline instead of the latex reagent.

The optical intensity Ear regarding RF is determined from the above ET, Esa*Eia, and Eaa using the following formula.

Enp= ET −Ess (ERB E
The method of quantifying RF from BBB and Ear is the same as the method of quantitative determination described above.

(Example) Next, the method of the present invention will be explained in detail in relation to reagents, measurement methods, actual measurement results, etc. The following 1% means the amount of M.

Example 1 1) Reagent diluent 0.9% polyethylene glycol (average molecular weight 7500
), 0.05 M phosphate buffer containing 0.15 M NaCl, and 0.1% bovine serum albumin 11)
Latex reagent Remove polyethylene glycol from the diluted solution in 1) above,
A test solution in which 0.1M choline chloride is dissolved and polystyrene latex sensitized with human γ-globulin and having a particle size of 0.2 μm or less is dispersed (latex concentration 0.4% by weight). This latex reagent is stable for at least 12 months when stored at 0-4°C.

2) Measurement method: Full white table below Composition and absorbance Prepare the four solutions shown in the table above, and after reacting each solution at 37°C for 10 minutes, measure the absorbance at a wavelength of 570 nm, and determine whether the sample serum (sample) Absorbance (ARF) is calculated from the formula %. Rheumatoid factor positive serum (
The above ⇠F secondary standard blood-ff) O115 (24IU/di
) to 515 (120 IU/dI) to create a calibration curve of absorbance and F value (IU/ml). ), and the above measurement principle was applied.

In the Hitachi Model 705, when the 2-point end method of the analysis method program is used, the device automatically performs calculations based on the above measurement method and calculates the measurement results. Reaction temperature is 25℃~37℃
℃, the measurement wavelength was 57 Onm, and -wavelength photometry was adopted.

3) Actual measurement results The calibration curve obtained above is shown in FIG. 1 as Graph 1.

Example 2 In Example 1, 0.1M choline chloride in the latex reagent was replaced with 0.12M acetylcholine chloride.

The same procedure as in Example 1 was carried out except that 0.08M betaine hydrochloride and 0.13M 1-ethylamine were used. In each case, a calibration curve with excellent linearity similar to Graph 1 in FIG. 1 was obtained.

Comparative Example 1 The same procedure as in Example 1 was conducted except that the diluent in Example 1 did not contain choline chloride.

The obtained calibration curve is shown in FIG. 1 as Graph 2.

As is clear from Figure 1, even if the calibration curve does not show linearity when polyethylene glycol is used (Graph 2), a good result can be obtained by using a water-soluble compound (such as choline chloride). A calibration curve with linearity can be obtained.

(Effects of the Invention) According to the present invention, a calibration curve having a good straight line can be obtained in the measurement of F, making it easy to create a calibration curve.
Quantitative accuracy also improves.

[Brief explanation of drawings]

FIG. 1 shows the calibration curves obtained in Example 1 and Comparative Example 1. Explanation of symbols

Claims (1)

[Claims] 1. A sample and a human insoluble carrier sensitized with γ-globulin are mixed to form a mixed solution, and in the mixed solution at least one selected from the group consisting of polyethylene glycol, trialkylamine, its salt, and quaternary ammonium salt is added. Quantification of rheumatoid factor, which involves causing a latex agglutination reaction by an antigen-antibody reaction in the coexistence of a type of water-soluble compound, measuring optical intensity, and quantifying rheumatoid factor in a sample from this measured value. Law.