JP3423085B2 - Immunoassay - Google Patents

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunoassay method capable of avoiding the influence of a rheumatoid factor which is a substance that interferes with an antigen-antibody reaction. [0002] In the immunoassay method, biological components collected from humans, such as urine, serum, and tissue fragments, are analyzed and identified as samples, and trace components in the components are detected to detect biological components such as diseases. This is a clinical test method for diagnosing abnormalities of the liposome, and is widely used as a method utilizing the reaction specificity of antigen-antibody reaction, binding strength, and the like. However, the specimens to be measured by the immunoassay include rheumatoid factor (hereinafter referred to as “RF”), which is a biological component, depending on individual characteristics, collection conditions, and the like.
Since these interfere with the antigen-antibody reaction and cause a misdiagnosis, various techniques for eliminating or reducing the effects of these interfering substances have been studied. In an immunoassay using a biological component as a sample, the pH of a reagent system is adjusted to around 6.0 to 8.0 in order to bring the conditions for causing an antigen-antibody reaction closer to those in a living body. Was customary. However, under these conditions,
In rare cases, the RF in the specimen may react with an antigen or antibody contained in the reagent system and cause self-aggregation. RF is
Although it is an autoantibody, it is known to react with the Fc portion of a denatured human antibody molecule. However, in some cases, it reacts with a heterologous antibody molecule under specific conditions such as RF concentration and purity. Blocks specific reaction of antigen-antibody in clinical test using antibody. In order to avoid such RF interference with the measurement, pretreatment such as inactivating or removing endogenous RF in serum is usually required before the assay,
Without this pre-processing, the results of the measurements may have significant errors. Japanese Patent Application Laid-Open No. 54-119292 discloses an immunoassay using an F (ab ') ₂ molecule obtained by removing the Fc portion of a heterologous antibody molecule by an enzyme reaction as an antibody in order to avoid this interference reaction. Is disclosed. However, such an immunoassay involves complicated steps for preparing the raw materials such as enzyme reaction and purification, so that the cost is increased,
In some cases, this led to differences between production lots. [0005] As described above, the conventional immunoassay method has problems in that the test is complicated and delayed, the reagent manufacturing process is complicated, and a difference between manufacturing lots is caused. Therefore, in order to avoid these problems, it has been desired to establish a method for measuring a collected biological component using a reagent which does not treat an antibody molecule with an enzyme. DISCLOSURE OF THE INVENTION [0006] In view of the above, the present invention provides an immunoassay method capable of reducing the interaction between a rheumatoid factor and a heterologous antibody molecule and avoiding the influence on the measurement of a rheumatoid factor. The purpose is to provide. The gist of the present invention is to provide an immunoassay method in which a latex reagent in which goat-derived antibodies are supported on latex particles and a sample are mixed, and the antigen is measured by an immunoagglutination reaction. Once the pH of the sample has been reduced to 1-4
PH of the reaction solution to 4.0 to 4.0 without any pretreatment.
6.0. [0008] The antibody carried on the latex particles is
It is a goat-derived antibody. The latex particles are not particularly limited, and include, for example, organic polymer powder and the like. Examples of the material of the organic polymer powder include polystyrene, styrene-sulfonate copolymer, methacrylic acid polymer, acrylic acid polymer, acrylonitrile-butadiene-styrene copolymer, and vinyl chloride-acrylate copolymer. And a polymer such as a polyvinyl acetate-acrylate copolymer. In particular, a latex in which a particle powder of these polymers is uniformly suspended is preferably used. The average particle size of the latex particles is appropriately selected depending on the method for detecting the antigen in the specimen, the concentration of the antigen detected, the detection equipment, and the like, but is usually 0.05 to 1.0 μm, and particularly 0.05 to 1.0 μm. 0.5 μm is preferred. In order to carry the antibody on the latex particles, ordinary chemical bonding or physical adsorption can be applied, and the latex reagent can be obtained. PH of the reaction solution for performing an immunoagglutination reaction between the latex reagent and the antigen in the sample
Is 4.0 to 6.0. When the pH is less than 4.0, self-aggregation of latex, suppression of antigen-antibody reaction and the like occur, the calibration curve is high, the reproducibility is poor, and the desired reactivity cannot be obtained. Above zero, RF is limited to the above range because RF affects CRP measurements and the object of the present invention cannot be achieved. The buffer constituting the reaction solution is appropriately selected depending on the measurement conditions, and examples thereof include a phosphate buffer, a phosphate-citrate buffer, and an acetate-sodium acetate buffer. For the purpose of improving storage stability, quaternary ammonium salts such as bovine serum albumin (BSA) and choline chloride; and stabilizers such as saccharides can be added to the latex reagent. In order to promote the agglutination reaction of the latex reagent, polyethylene glycol,
It is also effective to add a water-soluble polymer such as dextran. The reaction temperature for carrying out the above reaction is 4-5.
0 ° C is preferred, and 20 to 40 ° C is more preferred. In the immunoassay of the present invention, a usual optical assay can be applied. For example, any one of the scattered light intensity, the absorbance and the number of particles may be measured. The measurement wavelength is generally 300 to 1000
nm can be used. The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Examples 1-2 and Comparative Examples 1-3 The pH of the reaction solution of the latex reagent for measuring human CRP was adjusted, and the effect of RF on the measured CRP was examined. Preparation of latex reagent 0.1 g of goat-derived anti-human CRP antibody per 1 g of polystyrene latex particles having a particle size of about 0.15 μm
After the B / F separation, human CR suspended in 36 mM phosphate buffer (pH 6.5) containing 1% bovine serum albumin so as to have a latex concentration of 5.0 g / l.
A latex reagent for P measurement was used. Preparation of Latex Diluent When measuring CRP in serum using the above-mentioned latex reagent for measuring human CRP, a latex diluent may be used for the purpose of adjusting reactivity, pH and the like. For the purpose of examining the effect, the measurement was carried out using buffer solutions having different pHs as diluents and changing the pH of the reaction solution. As the diluent, a 20 mM citrate-phosphate buffer containing 1% bovine serum albumin was used, and the pH at the final measurement was 3.5 by changing the mixing ratio of citrate-phosphate (Comparative Example 1). ), 4.
The pH was adjusted to 5 (Example 1), 5.5 (Example 2), 6.5 (Comparative Example 2), and 7.5 (Comparative Example 3). Measurement Method The measurement was performed using a Hitachi 7150 type automatic analyzer (manufactured by Hitachi, Ltd.) at a measurement wavelength of 700 nm. The procedure is shown below. First, the absorbance of a water blank was measured, and then the sample (S) and 250 μl of the first reagent (R1) were added. After the addition of R1, the absorbance of the reaction solution for 10 minutes until the end of the measurement was measured at intervals of 20 seconds. The value obtained by subtracting the water blank value was used as the absorbance. 250 μl of the second reagent (R2)
Was performed 5 minutes after the addition of R1. That is, the absorbance measurement within 5 minutes from the addition of R1 was performed on the reaction solution of S and R1, and the absorbance measurement after 5 minutes from the addition of R1 was performed on the reaction solution of S and R2. Here, a zero concentration standard product (position)
n 1) is physiological saline, CRP standard product (positio)
The output was corrected using 5.0 mg / dl CRP standard serum (manufactured by Far East Pharmaceutical Co., Ltd.) in n2). That is, 5.0 mg
/ Dl The increase in absorbance due to the standard serum was determined by arithmetic processing, this was read as 5.0 [mg / dl], and the data output after the correction was converted from the ratio of the increase in absorbance between the sample and the standard serum. Value. For example, if the increase in absorbance due to the sample is の of the standard serum, the output data is 5.0 × １ ／ = 2.5 [mg / dl]. [0020] The confirmation test of the presence or absence of the effects of RF of influence confirmation test RF was carried out by the addition test of RF. That is, a known concentration of CRP-positive serum (about 0.5 m
g / dl), and the presence or absence of a change in the measured CRP value was confirmed. It is very rare that RF-positive samples affect the measured values in daily clinical tests, but the use of highly purified RF solutions can reproduce similar phenomena. In this example, an interference check RF (manufactured by Kokusai Reagent Co., Ltd.) was used as a purified RF. The interference check RF reconstituted with purified water was added to the CRP-positive serum at a ratio of 1 volume of an RF solution (5 concentrations) to 4 volumes, and finally the RF was 0, 100, 200, 300, 400 IU / m.
The test sample contained was prepared and measured after calibration with a standard product. The results are shown in Table 1. Comparative Examples 2 and 3 (pH 6.5 to 7.
Under the measurement conditions of 5), an increase in the measured value was observed with an increase in the RF addition concentration. That is, it was found that RF affected the CRP measurement value. On the other hand, in Comparative Example 1, Example 1 and Example 2 (pH 3.5 to 5.5), the measured values hardly changed. [Table 1] Examples 3 and 4 and Comparative Examples 4 and 6 The reagent performance when the pH of the reaction solution was changed was confirmed by a dilution linearity test. A latex reagent and a latex diluent for human CRP measurement were prepared in the same manner as in Example 1, and the pH was adjusted.
The latex diluent of 3.5 was used in Comparative Example 4, the latex diluent of pH 4.5 was Example 3, the latex diluent of pH 5.5 was Example 4, the latex diluent of pH 6.5 was Comparative Example 5, and the pH was 7.5. The measurement was performed using the latex diluent No. 5 as Comparative Example 6 to compare the reagent performances. About 20 mg / dl CRP-positive serum (AT
AB) was diluted with physiological saline, and 1/10 to 1
Ten serial CRP dilution trains of 0/10 CRP concentration were prepared. After calibration with standard product, 0 concentration (saline) and 1
The zero-step concentration CRP dilution series was measured at n = 3, and the average was calculated and plotted on a graph. The results are shown in FIG. In the reaction solution of Comparative Example 4 (pH 3.5), CR
A decrease in the measured value at a high P value was observed, and normal reactivity could not be obtained. Such deterioration is considered to be due to latex self-aggregation, suppression of antigen-antibody reaction, and the like. On the other hand, Examples 3 to 4 and Comparative Examples 5 to 6 (pH
Under the measurement conditions of 4.5 to 7.5), substantially the same dilution linear performance was exhibited. Since the immunoassay of the present invention has the above-described configuration, it is possible to specifically and accurately measure a target antigen without performing pretreatment of a sample or processing of a reagent material. Can be.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a calibration curve of Examples 3 to 4 and Comparative Examples 4 to 6. The vertical axis represents measured values (mg / dl), and the horizontal axis represents concentration (mg / d).
l) respectively.

Claims (1)

(57) [Claims 1] In an immunoassay method in which a latex reagent in which goat-derived antibodies are supported on latex particles and a sample are mixed, and the antigen is measured by immunoagglutination, the pH of the sample is determined.
An immunoassay wherein the pH of the reaction solution is maintained at 4.0 to 6.0 without performing a pretreatment for once lowering the pH to 1 to 4 .