JPH0672888B2 - Rheumatoid factor quantification reagent - Google Patents

Description

TECHNICAL FIELD The present invention relates to a rheumatoid factor assay reagent.

More specifically, the present invention relates to a rheumatoid factor quantification reagent suitable for quantifying rheumatoid factors in a sample such as serum by utilizing a latex agglutination reaction caused by an antigen-antibody reaction.

(Prior Art) Rhematoid Factor (hereinafter abbreviated as RF) is
It is a substance that appears in the blood of patients with chronic rheumatoid arthritis and is said to be a giant protein with a molecular weight of approximately 1 million.

The measurement of RF is designated as a standard item for diagnosis of rheumatoid arthritis by the American College of Rheumatology, and is clinically indispensable for the test for diagnosis of rheumatoid arthritis.

At present, RA using the latex agglutination reaction is used for RF measurement.
The test method and the RA / HA test method using the passive hemagglutination reaction using sheep red blood cells are widely used [Acta Path Microviol Scandinavian (Acta Path.Mi
crobiol.Scand.) Volume 17, p. 172, 1940). However, since these measurement methods are qualitative reactions, it is impossible to grasp the variation of the RF value over time.
There were also defects 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 the detection methods is the immunoturbidimetric method. In turbidimetric immunoassay, relatively high blood levels IgG of plasma proteins, IgA, IgM, although C _3, C _4, CRP is measured, a low concentration from the sensitivity surfaces, limit the measurement of plasma protein Atsuta

Due to these trends, an immunolattice agglutination method using an insoluble carrier sensitized with an antibody has been developed for the measurement of various low-concentration plasma proteins (JP-B-58-11575, JP-A-53-62826). Issue).

(Problems to be Solved by the Invention) The immunolatex agglutination method has high sensitivity because it uses laser light, but when quantifying RF using this method, an insoluble carrier sensitized with human γ-globulin is detected. In order to promptly carry out the immune reaction (antigen-antibody reaction), the reaction is preferably carried out in the presence of polyethylene glycol. However, in this case, a calibration curve having good linearity cannot be obtained. Therefore, in the case of quantification, there is a problem that the preparation of the calibration curve becomes complicated, and the accuracy of quantification decreases, which is conventionally known. Reagents cannot solve this problem.

(Means for Solving Problems) The present invention provides at least one water-soluble compound selected from the group consisting of an insoluble carrier sensitized with human γ-globulin, a trialkylamine, a salt thereof and a quaternary ammonium salt. And a combination of polyethylene glycol
Relating to RF quantitative reagents.

As the above-mentioned insoluble carrier, known ones such as diagnostic polystyrene-based latex particles can be used, and human γ-
The globulin is physically or chemically adsorbed to form an insoluble carrier sensitized with human-γ globulin. The particle size of the insoluble carrier is preferably 0.05 to 0.2 μm or less from the viewpoint of improving the measurement sensitivity.

As the polyethylene glycol, one having an average molecular weight of 3,000 or more is usually preferable. As the molecular weight increases, the optical intensity due to the latex agglomeration increases.

Examples of the water-soluble compounds include triethylamine as a trialkylamine, triethylamine hydrochloride as a trialkylamine salt, and choline chloride, choline bromide, acetylcholine chloride, acetylcholine bromide, betaine hydrochloride as a quaternary ammonium salt. is there. These compounds are used alone or in combination of two or more.

The following method is preferable for quantifying RF using the reagent for quantifying RF according to the present invention.

That is, a sample and an insoluble carrier sensitized with human γ-globulin are mixed to prepare a mixed solution, and polyethylene glycol and the water-soluble compound are allowed to coexist in the mixed solution to cause a latex-aggregation reaction by an antigen-antibody reaction. Then, the optical intensity is measured, and the RF in the sample is quantified from the measured value.

Serum or the like is used as the sample.

The above sample, sensitized insoluble carrier, polyethylene glycol and water-soluble compound are mixed in any order to form a mixed solution.

The sensitized insoluble carrier is used in the mixed solution at an appropriate temperature, but is preferably used in an amount of 0.05% by weight or less in order to easily measure the absorbance, and the reaction is performed sufficiently. From the viewpoint, 0.01% by weight or more is preferable. Polyethylene glycol is preferably present in the mixed solution in an amount of 3% by weight or less. When the concentration of polyethylene glycol is too high, nonspecific aggregation of the sensitized insoluble carrier is likely to occur. Polyethylene glycol is preferably present in the mixed solution at a concentration of 0.5% by weight or more. If it is too small, the effect of promoting the reaction is small. The water-soluble compound is used in the mixed solution so as to have an appropriate concentration, but preferably 0.1 to 10% by weight. If the amount of the water-soluble compound is too small, the effect due to the addition is small, and if it is too large, the measurement sensitivity tends to decrease.

The above-mentioned antigen-antibody reaction is preferably carried out at 25 to 37 ° C, and it is preferable to keep the temperature constant during the reaction. If it deviates from this range, the antigen-antibody reaction tends to be unstable. further,
This reaction is preferably carried out for 30 seconds to 15 minutes. 30
If it is less than 2 seconds, the above reaction is insufficient and it becomes difficult to quantify RF from the absorbance, and if it exceeds 15 minutes, the merit of short-time measurement decreases.

After the initiation of the latex agglutination reaction, the optical intensity of the mixed solution is measured by selecting an appropriate wavelength.

Here, the 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 reaction is started [end point measurement (end point assay), optical intensity increase after the reaction is started] and twice or more after the reaction is started. Either a method of obtaining an increase in the optical intensity or an increase per unit time during that time, and a method of measuring the time until the constant optical intensity is reached can be adopted.

For quantification, the above-mentioned measurement is performed on a sample with a known RF amount (for example, RF standard serum and its dilution series), and the measured value and RF
This can be performed by preparing a calibration curve from the quantity and the RF quantity corresponding to the calibration curve from the measured values of the sample of unknown RF quantity measured under the same conditions.

In the present invention, the sensitized insoluble carrier, polyethylene glycol and water-soluble compound are preferably dispersed and dissolved in a medium and used as a reagent.

In this case, the reagent has the following forms.

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

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

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

(4) A three-pack type reagent consisting of 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 in which a water-soluble compound is dissolved in a medium.

Of these, the forms (2) and (3) are preferable. The form (1) is preferably adjusted before use.

In the above, a buffer is mainly used as the medium,
A phosphate buffer solution, a glycine buffer solution, a Tris-hydrochloric acid buffer solution, a good buffer solution and the like are preferable, and those having a pH adjusted to 6 to 10 are preferable.

Further, bovine serum albumin, NaCl or the like for adjusting the salt concentration may be dissolved in the reagent as appropriate.

In the 1-liquid latex reagent and the multi-liquid type latex reagent, the sensitized insoluble carrier is used at an appropriate concentration so that the concentration in the mixed liquid can be easily adjusted. It is necessary to make the concentration of weight%
Generally easy to use. The polyethylene glycol and the water-soluble compound are dissolved in an appropriate concentration so that the concentration in the mixed solution can be easily adjusted in the one-part latex reagent and the multi-part reagent.

Further, a solution prepared by dissolving bovine serum albumin, NaCl or the like in the medium may be used together with the reagent for adjusting the liquid volume.

Further preferable forms of the reagent according to the present invention are shown below.

(1) (a) Buffer solution 50 to 200 mM, pH 6.5 to 7.5 (b) Polyethylene glycol 0.5 to 3% by weight (c) NaCl Physiological ionic strength, and (d) Bovine serum albumin 0.05 to 1% by weight (E) Buffer solution 50-200 mM, pH 6.5-7.5 (f) The sensitized insoluble carrier 0.2-0.5% by weight (g) The water-soluble compound 0.1-10% by weight ( A reagent obtained by combining h) a physiological ionic strength of NaCl and (i) a second reagent (latex reagent) containing 0.05 to 4% by weight of bovine serum albumin.

(2) (a) Buffer 50-200 mM, pH 6.5-7.5 (b) Polyethylene glycol 0.5-3 wt% (c) Water-soluble compound 0.01-2 wt% (d) NaCl Physiological ionic strength And (e) a first reagent containing 0.05 to 4% by weight of bovine serum albumin, and (f) a buffer solution of 50 to 200 mM, pH 6.5 to 7.5 (g) 0.2 to 0.5% by weight of the sensitized insoluble carrier. A reagent obtained by combining (h) an amount having a physiological ionic strength of NaCl and (i) a second reagent (latex reagent) containing 0.05 to 4% by weight of bovine serum albumin.

These are examples that are easy to use, and the concentrations of the respective substances described together also exemplify the ranges that are easy to use, and these do not limit the present invention.

When RF is quantified using the reagent according to the present invention, in the method of measuring once after starting the reaction, the quantification can be performed as follows using the above-mentioned two-component type reagent.

That is, by measuring the optical intensity (F _T) for the sample by subtracting the absorbance attributed absorbance attributed from this value in the sample and (E _SB) to latexes reagent, determining the calculated strength (E _RF).

Here, the optical intensity due to the sample is, for example, the optical intensity of a liquid obtained by using physiological saline instead of the latex reagent in the preparation of the mixed liquid.

The optical intensity due to the latex reagent is, for example, from the optical intensity (E _RB ) of the solution obtained by using physiological saline instead of the sample in the preparation of the above-mentioned mixed solution, from the sample in the above-mentioned mixed solution. And the optical intensity (E _BB ) obtained by using physiological saline instead of the latex reagent.

From the above E _T , E _SB , E _RB , and E _BB , the optical intensity E _RF related to _RF is obtained by the following formula.

E _RF = E _T −E _SB − (E _RB −E _BB ) The method of quantifying _RF from E _RF is the same as the method of quantification described above.

(Examples) Next, the method of the present invention will be described in detail with reference to reagents, measurement methods, and measurement results. Hereinafter,% means% by weight.

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

2) Measuring method Prepare 4 kinds of liquids in the above table, incubate each liquid for 10 minutes at 37 ℃, measure the absorbance at the wavelength of 570nm, and calculate the absorbance (A _RF ) of the sample serum (sample) by the formula A _RF = Calculated from A _T −A _SB − (A _RB −A _BB )
A calibration curve of absorbance and RF value (IU / ml) was prepared by using 5 dilution series from 1/5 (24 IU / dl) to 5/5 (120 IU / dl). For the measurement, Hitachi automatic analyzer 705 type (hereinafter referred to as Hitachi 705 type) was used, and the upper pre-measurement principle was applied.

In the Hitachi 705 model, when the 2-point end method of the analysis method program is used, the device automatically calculates based on the upper pre-measurement method and calculates the measurement result. The reaction temperature was 25 ° C to 37 ° C, the measurement wavelength was 570 nm, and single wavelength photometry was adopted.

3) Results of measurement The calibration curve obtained above is shown as graph 1 in FIG.

Example 2 In Example 1, 0.1M choline chloride in the latex sample was replaced with 0.12M acetylcholine chloride, 0.08M betaine hydrochloride and 0.
The same procedure as in Example 1 was repeated except that 13M triethylamine was used instead. Graph 1 in Fig. 1 in each case
A calibration curve with excellent linearity similar to that obtained was obtained.

Comparative Example 1 The procedure of Example 1 was repeated, except that the latex reagent in Example 1 did not contain choline chloride. The calibration curve obtained is shown as graph 2 in FIG.

As is clear from FIG. 1, even when the calibration curve does not show linearity when polyethylene glycol is used (Graph 2), by using a water-soluble compound (choline chloride, etc.), a good linearity is obtained. It can be used as a calibration curve having properties.

(Effects of the Invention) By using the RF quantification reagent according to the present invention, a calibration curve having a good straight line can be obtained in RF measurement, so that the calibration curve can be easily prepared and the quantification accuracy can be improved.

[Brief description of drawings]

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

Claims (7)

[Claims] 1. A rheumatoid factor comprising a combination of at least one water-soluble compound selected from the group consisting of an insoluble carrier sensitized with human γ-globulin, a trialkylamine, a salt thereof and a quaternary ammonium salt, and polyethylene glycol. Quantitative reagent. 2. The reagent for quantifying rheumatoid factor according to claim 1, which comprises a human γ-globulin-sensitized insoluble carrier, polyethylene glycol and a water-soluble compound dispersed and dissolved in a medium. 3. A latex reagent in which a human γ-globulin-sensitized insoluble carrier and a water-soluble compound are dispersed and dissolved in a medium, and a reagent in which polyethylene glycol is dissolved are combined with each other. Reagent for quantification of rheumatoid factor. 4. The method according to claim 2, wherein a latex reagent in which an insoluble carrier sensitized with human γ-globulin is dispersed in a medium and a reagent in which polyethylene glycol and a water-soluble compound are dissolved in the medium are combined. A reagent for quantifying rheumatoid factors of 5. A patent comprising a combination of a latex reagent in which an insoluble carrier sensitized with human γ-globulin is dispersed in a medium, a reagent in which polyethylene glycol is dissolved in the medium, and a reagent in which a water-soluble compound is dissolved in the medium. The rheumatoid factor assay reagent according to claim 2. 6. A method for quantifying rheumatoid factor according to claim 2, which comprises a human γ-globulin-sensitized insoluble carrier, a polyethylene glycol and a latex reagent in which a water-soluble compound is dispersed and dissolved in the same medium. reagent. 7. The second aspect of the invention wherein the medium is a buffer solution.
The reagent for quantifying rheumatoid factor according to the item 3, item 3, item 4, item 5, or item 6.