JPH04299263A - Stabilizing method for rheumatoid factor measuring reagent - Google Patents

Abstract

PURPOSE:To stably preserve a modified human gamma globulin solution measuring a rheumatoid factor. CONSTITUTION:Modified human gamma globulin in a rheumatoid factor measuring reagent is stably preserved by adding modified human globulin to an aqueous solution with a salt concn. of 0.3-1.0M. The measuring error due to precipitation or turbidity is reduced by lowering the concn. of modified human gamma globulin but becomes easy to receive the effect of a coexisting substance. Even when the concn. of modified human gamma globulin is not lowered, by the use of an aqueous solution with salt concn., stable preservation becomes possible.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] In order to react denatured human gamma globulin in a solution at a high concentration of 0.5 mg/ml or more, denatured human gamma globulin, which has poor stability under the same conditions, is reacted with 0.3 mg/ml or more. This invention relates to a rheumatoid factor measuring reagent that is stabilized at a salt concentration of ~1.0M.

0002

[Prior art] Synge is a rheumatoid factor detection method.
A latex agglutination reaction method using a reagent in which human gamma globulin is adsorbed to latex by applying the r-Plotz method, and a hemagglutination reaction using a reagent in which sheep red blood cells are sensitized with immune gamma globulin by applying the Waaler-Rose method. There are various methods, especially the latex agglutination reaction method, which is widely adopted in general clinical and daily tests as a screening test for patients with rheumatoid arthritis, and various reagents are manufactured and sold by many manufacturers. In addition, as a modification of the latex aggregation reaction, products using ion exchange resins, activated carbon, etc. instead of latex are also commercially available.

However, these measurement methods that utilize agglutination reactions require diluting the specimen to an appropriate multiple, and the agglutination images are visually determined and are usually divided into four stages: negative, false positive, weak positive, and strong positive. This method is only a judgment quantity method for making judgments based on the accuracy of the judgment, and it also involves extremely serious problems in accuracy control due to measurement conditions, measurement techniques, subjectivity of the judge, etc. Furthermore, according to recent literature, Fc obtained by decomposing immunoglobulin with papain
A method for quantifying rheumatoid factor is to place a sample in a fragment-coated plastic tester to bind rheumatoid factor, then react with anti-IgG labeled with a radioisotope and measure the radioactivity of the bound radioisotope. Although it has been reported (Journal of Immunology, Vol. 119, No. 1, p. 295 (1977)), the measurement techniques are still far from being put to practical use.

According to Japanese Patent Publication No. 63-45066, when denatured human gamma globulin denatured under certain conditions is incubated in a solution containing rheumatoid factor and an aggregation promoter, a uniform turbid liquid having a turbidity corresponding to the amount of rheumatoid factor is formed. A rheumatoid factor measurement method has been disclosed that utilizes the ability to obtain (hereinafter referred to as immunoturbidimetry).

[0005]

[Problems to be Solved by the Invention] However, in the method of Japanese Patent Publication No. 63-45066, when a reagent containing modified human gamma globulin at an effective concentration for rheumatoid factor measurement is stored for a long period of time, precipitation and turbidity occur. There are problems such as abnormally high measured values of rheumatoid factors, which are thought to be low concentrations, and measurement equipment that becomes clogged. Furthermore, in order to prevent precipitation and turbidity from occurring, it is possible to freeze-dry the modified human gamma globulin preparation, but the stability of the freeze-dried solution remains a problem. Therefore, improvement can be achieved by lowering denatured human gamma globulin, which is the direct cause of turbidity. However, in the reaction with the analyte, the concentration change per unit time decreases,
As a result, a problem arises in that it becomes susceptible to the influence of coexisting substances such as chyle. See Table 3. That is, the concentration of denatured human gamma globulin must be at least 0.5 mg/ml or more.

[0006]

[Means for Solving the Problems] In view of these drawbacks of conventional methods, the present inventors have focused on the stability of a solution containing modified human gamma globulin of 0.5 mg/ml to 20 mg/ml in immunoturbidimetry. As a result of repeated research, 0.
0.5mg/ml at salt concentrations from 2M to 1.0M
The present invention was completed based on the finding that it is possible to stabilize a solution containing ~20 mg/ml denatured human gamma globulin.

That is, in the present invention, human maglobulin modified by various methods is dissolved in a solvent with a salt concentration between 0.2M and 1.0M, and the solution is 0.5mg/ml to 20mg/ml.
l It provides a stable denatured immunoglobulin solution, which can be used directly or diluted to measure rheumatoid factor. As the modified human gamma globulin, heat-denatured products and products modified by chemical substances such as glutaraldehyde are used. Examples of the solvent used in the present invention include water, phosphate buffer, Tris buffer, HEPE
There are S buffers, citrate buffers, acetate buffers, etc. Salts added to adjust the salt concentration include, for example, sodium chloride, potassium chloride, phosphates, citrates, acetates, etc. .

[0008]

[Example] Example 1 Human gamma globulin FRII (
Miles) 100mg in 0.01M phosphate buffer pH
7.2 Dissolved in 10 ml, heated at 61°C for 20 minutes, and filtered off insoluble matter. Add salt to this, 0.1M and 0.
．． 3M and 0.5M to prepare a denatured human gamma globulin preservation solution (containing 5 mg/ml of denatured human gamma globulin). Table 1 shows the results of storing this storage solution at 4° C. and examining the presence or absence of precipitates and the turbidity at an absorption wavelength of 610 nm over time.

[0009]

[Table 1] At 0.1M, a precipitate was formed and the turbidity significantly increased after 2 months of storage, but at 0.3M and 0.5M, good storage conditions were maintained even after 4 months of storage.

Example 2 A denatured human gamma globulin storage solution prepared in the same manner as in Example 1 was diluted 5 times with 0.01M phosphate buffer pH 7.2, and polyethylene glycol 6000 was added to this to a concentration of 1%. It was dissolved to obtain a reagent for RF measurement. The obtained R
Mix 200 μl of sample serum with 2 ml of F measurement reagent, and measure the change in absorbance using a spectrophotometer at a wavelength of 340 nm at 37° C. for 15 minutes (ΔDs). At the same time, a similar operation is performed using a standard sample to measure the change in absorbance (ΔDstd), and the amount of rheumatoid factor in the specimen is calculated using the following formula. Sample rheumatoid factor amount (U/ml) =ΔDs/ΔDst
Table 2 shows an example of measuring the rheumatoid factor amount (U/ml) of the dx standard sample.

[0011]

[Table 2] For 0.5M and 0.3M, little change in measured values was observed after 2 months of storage. On the other hand, with 0.1M, a significant increase in the measured value was observed after 2 months of storage.

Example 3 Human gamma globulin FRII (Miles) in a test tube
100mg in 0.01M phosphate buffer pH 7.2, 1
The mixture was dissolved in 0 ml and heated at 61°C for 20 minutes, and insoluble matter was removed by filtration. Salt was dissolved in this to a concentration of 0.1 M to obtain a 5 mg/ml modified human gamma globulin solution. 0.01M phosphate buffer containing 0.1M salt pH
Diluted with 7.2 to 1.0mg/ml, 0.5mg/m
A denatured human gamma globulin solution of 0.3 mg/ml was prepared, and polyethylene glycol 6 was added to each solution.
Dissolve 000 to 1% and add 3% of RF measurement reagent.
I got the seeds. 0.9 ml of human serum with a rheumatoid factor concentration of 4.5 U/ml and 0.1 ml of 0.01 M phosphate buffer (abbreviated as PBS) containing 0.9% sodium chloride or formazin turbidity 2
0.1 ml of Chyle 8300 serum was added to prepare two types of sample serum. Using the three types of RF measurement reagents obtained, two types of sample serum were measured in the same manner as in Example 2, and the amount of rheumatoid factor was calculated. The measurement results are shown in Table 3.

[0013]

[Table 3] At denatured human gamma globulin concentrations of 0.5 ml/ml or higher, there was no effect of Chyle, but at 0.3 mg/ml, the rheumatoid factor concentration measurements were significantly reduced by the addition of Chyle serum. This result shows that the concentration of denatured human gamma globulin is 0.5 mg.
This shows that if it is less than /ml, it will be affected by chyle.

[0014]

[Effects of the Invention] A modified human gamma globulin solution for measuring rheumatoid factor that is stable for a long period of time can be obtained.

Claims (2)

[Claims] 1. A method for stabilizing a denatured human gamma globulin solution for a rheumatoid factor measurement reagent, which comprises containing denatured human gamma globulin in an aqueous solution with a salt concentration of 0.3 to 1.0 M. 2. The denatured human gamma globulin solution prepared by the method of claim 1 is directly or diluted to 0.0%.
Rheumatoid factor measuring reagent characterized by reacting at a concentration of 5 mg/ml or more