JPH0658935A - Immunoassay and reagent therefor - Google Patents

Abstract

PURPOSE:To obtain a measuring method and a reagent therefor which increase remarkably the production of an antigen-antibody coagulation block in an immunological analysis, complete an antigen-antibody reaction in a short time and make the reaction proceed uniformly on the occasion of measurement of a target constituent in a specimen sample and a trace constituent therein, in particular, thus making it possible to furnish the result of the analysis stably. CONSTITUTION:An immunoassay wherein an antigen-antibody reaction is effected in the existence of dextran or a dextran sulfate and a surfactant, and an immunoassay assisting reagent which contains the dextran or the dextran sulfate and the surfactant, can be obtained.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an immunological assay method and an auxiliary reagent used therein.

[0002]

2. Description of the Related Art Conventionally, for the diagnosis of diseases, blood, urine, etc. are collected and various tests are carried out and used as an index for clinical diagnosis. Among these tests, the immunological measurement is a test for the presence or amount of an antigen or antibody in a body fluid component such as blood or urine, and an antigen-antibody reaction is used. This reaction utilizes the property that an antibody reacts only with a specific antigen, that is, the specificity of an antibody, and it is possible to specify only one component out of various components and measure it in a minute amount.

Immunological assay methods utilizing the antigen-antibody reaction include immunodiffusion method, immunoturbidimetric method, immunoturbidimetric method, hemagglutination method, latex method, enzyme immunoassay method (EIA method), and radioimmunoassay. The law (RIA law) is now widely practiced. Of these, the immunodiffusion method is time-consuming and inaccurate.
The red blood cell agglutination method and the latex agglutination method using a carrier require difficulty in producing a reagent. The EIA method and RIA method require complicated measuring operations and special measuring equipment and facilities. In addition to these, the immunoturbidimetric method and the nephelometric method are widely used in the field of clinical examination at present, and an antigen-antibody reaction is caused in a solution to generate an aggregate resulting from the transmitted light intensity or scattered light intensity. It is a method to catch it as a change of. This method has few drawbacks as described above and is applied to a general automatic analyzer, which enables measurement of multiple samples and multiple items, and has been widely used in the field of clinical examination in recent years.

However, the immunological analysis used today requires a long time for reaction, and it is difficult to observe the reaction until the reaction is completed when using an automatic analyzer which is widely used at present. Even if it is possible to observe the sample to the end, the disadvantage that a huge amount of time is required when a large number of sample specimens are subjected to the measurement occurs. In addition, when measuring trace components in a specimen sample, the reaction time becomes longer, and the antigen-antibody aggregates grow nonuniformly in the reaction solution, resulting in inconsistent analysis results. ing. Therefore, when manufacturing a test reagent, how to quickly complete the reaction and how to perform a uniform reaction are important points.

[0005]

The object of the present invention is to significantly increase the production of antigen-antibody aggregates in immunological analysis, to allow the reaction to be completed in a short time, and to analyze the target component in the specimen sample. In particular, it is an object of the present invention to provide a measuring method and a reagent therefor which can stably provide an analysis result by allowing an antigen-antibody reaction to uniformly proceed in measuring a trace amount component.

[0006]

[Means for Solving the Problems] In solving the above-mentioned problems, various studies were conducted on the conditions for enhancing the antigen-antibody reaction, and by adding dextran or its sulfate to the reaction system, the agglutination reaction remarkably increased. I found that it would be enhanced. However, in the case of a small amount of reaction, the effect of the additive on the reaction becomes non-uniform and the reliability of the analysis result is lost. Therefore, the inventors of the present application have further studied, and found that stable results can be obtained even in trace measurement by mixing a surfactant with an immunological measurement reagent containing the additive, and completed the present invention.

That is, the present invention provides an immunological assay method characterized by carrying out an antigen-antibody reaction in the presence of dextran or dextran sulfate and a surfactant. The present invention also provides an immunological measurement auxiliary reagent containing dextran or dextran sulfate and a surfactant.

The present invention will be described in detail below.

The dextran in the method of the present invention preferably has a molecular weight of about 10,000 to 2,000,000, more preferably about 100,000 to 2,000,000, but is not particularly limited. Further, the dextran sulfate also has a similar effect. In addition, the concentration of dextran or dextran sulfate in the liquid in which the antigen-antibody reaction is performed is about 0.1
It is preferably 10% by weight / volume (w / v%), more preferably 2 to 7 w / v%, but is not particularly limited.

The surfactant used in the present invention may be any of anionic surfactants, cationic surfactants, nonionic surfactants and amphoteric surfactants. Ethylene primary alkyl ether (for example, Brij-based (trade name)), polyoxyethylene octylphenyl ether (for example, Triton-based (trade name)), alkylphenol polyethylene glycol,
Polyoxyethylene lauric acid ester, polyoxyethylene sorbitan alkyl ester (for example, Tween
System (trade name)), sodium dodecyl sulfate, and the like, but are not limited thereto.

The concentration of the surfactant in the liquid for carrying out the antigen-antibody reaction is preferably about 0.1-10 w / v%, more preferably 0.1-5 w / v%, but is not particularly limited.

As described above, the present invention also provides an immunoassay auxiliary reagent containing dextran or dextran sulfate and a surfactant. The auxiliary reagent of the present invention is preferably in the form of a solution in order to ensure that the respective components are uniformly and rapidly mixed in the antigen-antibody reaction system. In this case, a single solution may contain dextran or dextran sulfate and a surfactant, or a plurality of separate solutions may be included such that the dextran solution is the first liquid and the surfactant is the second liquid. It may be one. Furthermore, an antibody or an antigen for carrying out an antigen-antibody reaction may be added in advance to the solution to serve as an immunological measurement reagent. In the following examples, a solution containing dextran and a surfactant is used as a first liquid, and a reagent containing an antibody and a surfactant is used as a second liquid. Various buffers can be preferably used as the solvent of the solution. The amount of the solution used is preferably such that the concentration of each component in the antigen-antibody reaction system falls within the above-mentioned preferred range.

According to the method of the present invention, the antigen-antibody reaction is promoted and the variation in the measurement results is reduced,
It can be applied to all immunological measurement methods. That is, for example, immunodiffusion method, immunoturbidimetric method, immunoturbidimetric method, hemagglutination method, latex method, enzyme immunoassay method (EIA
Method), radioimmunoassay (RIA method), etc. Among these, the immunoturbidimetric method, the immunoturbulent method, and the agglutination methods such as the red blood cell agglutination method and the latex agglutination method exert a great effect.

Next, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

EXAMPLES Example 1 Of dextran having a molecular weight of about 2,000,000, 0, 1,
Prepared with 0.17M glycine buffer pH 8.0 to give 2, 3, 4, 5 w / v% and 1% for each buffer.
Polyoxyethylene (20) sorbitan monooleate was added. Next, 1% polyoxyethylene (20) sorbitan monooleate was added to an aqueous solution containing an anti-human albumin antibody as a main component to prepare a reagent. The former of the reagents described here is the first liquid, and the latter is the second liquid. First of all, 5 kinds of human urine samples with known albumin values were used in 3 ml of each 1% solution, 0.1 ml of each was added and mixed, incubated at 37 ° C, and then 1 ml of the second solution was added and mixed. The change in absorbance at 340 nm was measured at 37 ° C. The result was as shown in FIG.

From FIG. 1, it can be seen that the absorbance increases as the concentration of dextran increases. When this dextran concentration is converted to the final concentration, it is 0, 0.73, 1.
46, 2.20, 2.93 and 3.66%. Also,
When the volume ratio of the first liquid, the second liquid and the sample is changed, the final concentration also changes. That is, in order to obtain the same degree of absorbance, the dextran concentration can be lowered by increasing the volume ratio of the first liquid, and the opposite can be achieved by decreasing the ratio of the first liquid.

Example 2 A reagent was obtained under the same conditions as in Example 1 except that dextran of about 10,000 was used instead of the molecular weight of 2,000,000. The results are shown in Figure 2.

Example 3 A reagent was obtained under the same conditions as in Example 1, except that dextran of about 40,000 was used instead of the molecular weight of 2,000,000. The results are shown in Figure 3.

Example 4 A reagent was obtained under the same conditions as in Example 1 except that about 70,000 dextran was used instead of the molecular weight of 2,000,000. As shown in FIG.

Example 5 Under the same conditions as in Example 1, instead of having a molecular weight of 2,000,000, about 50
A reagent was obtained using ten thousand dextran. The results are shown in Figure 5.

Example 6 Under the same conditions as in Example 1, instead of a molecular weight of 2,000,000, about 20
A reagent was obtained using ten thousand dextran sulfates. As shown in FIG.

It can be seen from FIGS. 1 to 6 that the greater the molecular weight of dextran, the greater the amount of change in absorbance. For reactions with weak aggregation, it is preferable to use one with a large molecular weight, one for a strong one with a small molecular weight, or a large one with a low concentration, but considering the viscosity of the reagent, the labor of preparation, etc. It is preferable to use a large one. Further, since the amount of change in absorbance, that is, the sensitivity is remarkably increased as compared with the conventional method, that is, 0%, it is possible to measure a sample having a lower concentration than before.

Example 7 0.17M glycine buffer containing 3% (W / V) of molecular weight of about 2,000,000 in dextran, and further adding 1% Tw to pH 8.0.
een20, Tween60, Tween80, Bri
j-35, TritonX-100, and SDS were added to prepare 6 types of reagents (first solution). Next, 1% of each of the above surfactants was added to a solution containing an anti-human albumin antibody as a main component to prepare a reagent (second solution). First of all, 0.1 ml of two kinds of human urine samples having different albumin concentrations were added to each of the first liquids 3 of the respective surfactants, mixed and mixed at 37 ° C
Incubate at room temperature and add 1 ml of the second solution and mix.
The change in absorbance at 0 nm was measured and the concentration was calculated from a standard curve prepared in advance. The results of the multiplex measurement are shown in Table 1. A control without addition of a surfactant was used as a control.

[Table 1]

From Table 1, it is possible to obtain more stable results with the addition of the surfactant as compared with the control, and to exert a great effect in the measurement of a sample having a lower concentration. Depending on the type of surfactant and the degree of its effect, it can be appropriately selected depending on measurement conditions, items, types of measurement samples, and the like.

Example 8 0 to 5% for Tween 80 under the same conditions as in Example 7.
Was added to obtain reagents (first liquid, second liquid). Multiple measurements were performed under the same conditions as in Example 7 for each concentration. The results are shown in Table 2.
Shown in.

[Table 2]

Example 9 0 to 5% of Triton X-100 was added under the same conditions as in Example 8 to obtain reagents (first solution, second solution). Multiple measurements were performed under the same conditions as in Example 7 for each concentration. The results are shown in Table 3.

[Table 3]

From Tables 2 and 3, more stable measurement values can be provided as the concentration of the surfactant is increased. However, if the concentration is excessively increased too much, the viscosity of the reagent increases remarkably, which adversely affects the measured value. Therefore, while considering the measurement conditions and items, dextran,
It is preferred to determine the type and concentration of surfactant.

[0027]

As is clear from the above, the immunological measuring method and the reagent thereof according to the present invention can effectively promote the antigen-antibody reaction and allow the reaction to proceed uniformly. In particular, trace components contained in body fluid components such as serum and urine can be simply, accurately and rapidly measured.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between albumin concentration and absorbance change in each% of dextran having a molecular weight of about 2 million in Example 1.

FIG. 2 is a diagram showing a relationship between albumin concentration and absorbance change in each% of dextran having a molecular weight of about 10,000 in Example 2.

FIG. 3 is a diagram showing the relationship between albumin concentration and absorbance change amount for each% of dextran having a molecular weight of about 40,000 in Example 3.

FIG. 4 is a diagram showing a relationship between albumin concentration and absorbance change amount in each% of dextran having a molecular weight of about 70,000 in Example 4.

FIG. 5 is a diagram showing the relationship between albumin concentration and absorbance change in each% of dextran having a molecular weight of about 500,000 in Example 5.

FIG. 6 is a diagram showing the relationship between albumin concentration and absorbance change in each% of dextran sulfate having a molecular weight of about 200,000 in Example 6.

Claims (8)

[Claims] 1. An immunological assay method, which comprises performing an antigen-antibody reaction in the presence of dextran or dextran sulfate and a surfactant. 2. The immunological measuring method according to claim 1, wherein the concentrations of the dextran or dextran sulfate and the surfactant in the liquid in which the antigen-antibody reaction is carried out are 0.1 to 10% by weight / volume, respectively. 3. The immunoassay method according to claim 1, wherein the dextran or dextran sulfate has a molecular weight of 10,000 to 2,000,000. 4. The immunological measurement method according to any one of claims 1 to 3, wherein the immunological measurement is performed by a turbidimetric method, a nephelometric method or an agglutination method. 5. An immunological measurement auxiliary reagent containing dextran or dextran sulfate and a surfactant. 6. The reagent according to claim 5, which is in the form of a solution. 7. The reagent of claim 6, which comprises a plurality of separate solutions. 8. An immunological measurement reagent containing the antigen or antibody involved in an antigen-antibody reaction in the immunological measurement auxiliary reagent according to any one of claims 5 to 7.