JPH03118471A - Insulin assay and reagent therefor - Google Patents

Abstract

PURPOSE:To make measurement quick and simple by detecting the degree of inhibition of the agglutination caused by the antigen-antibody reaction of insulin carried by an insoluble carrier and antibodies by the reaction between the insulin in the sample and the antibodies. CONSTITUTION:Insulin is adsorbed in an insoluble carrier, and an insulin- sensitive insoluble carrier reagent is obtained. As the insoluble carrier, e.g. organic macromolecular latex, coccus-type bacteria and inorganic oxide can be used. Furthermore, the reagent containing anti-insulin antibody such as, e.g. immunoglobulin is prepared, and insulin assay reagent is formed of insoluble carrier reagent and antibody reagent. The insoluble carrier reagent and the sample are made to react in the antibody reagent and a liquid medium, and the degree of the inhibition of agglutination caused by antigen-antibody reaction is detected. Thus the concentration of insulin in the sample is measured.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a method and reagent for quantifying insulin that utilizes an immune reaction, and more specifically, to a method and reagent for quantifying insulin that utilizes an immune reaction. The present invention relates to a method for quantifying insulin that detects insulin, and a reagent used therein.

[Conventional technology]

Insulin exists in very small amounts in human blood, so it is extremely difficult to accurately measure it.

Conventionally, radioimmunoassay method (hereinafter referred to as RIA method) and enzyme immunoassay method (hereinafter referred to as EIA method) have been known as methods for measuring minute amounts of insulin (for example, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai: Enzyme Immunoassay Measurement method 2nd edition: Igaku Shoin, pages 211 to 226 (19B2)).

In the RIA method, Ig labeled with a radioactive isotope
This method measures insulin concentration by reacting 'G with insulin in a sample.It has excellent sensitivity and can detect trace amounts of insulin, and is widely used for measuring blood insulin.

On the other hand, in the EIA method, IgG bound to an enzyme with excellent detection sensitivity is reacted with insulin in a sample, and the reacted I
Insulin concentration is measured by measuring the activity of an enzyme bound to gG.

[Problem to be solved by the invention]

Although the RIA method has excellent sensitivity, it requires special equipment and equipment to ensure safety, and it also has many restrictions in handling, such as the need to be careful about waste disposal and contamination containing isotopes.

On the other hand, in the measurement using the EIA method, the enzyme-bound rg
In order to measure not only the immunoreaction between G and insulin in the sample, but also the enzyme activity, it is necessary to react the enzyme with the substrate. Therefore, the measurement takes a long time (at least 3 hours),
Moreover, the procedure was complicated.

Therefore, an object of the present invention is to provide a novel insulin quantification method and quantification reagent that enable rapid and simple measurement of insulin in a sample.

[Means and effects for solving the problem] The method for quantifying insulin of the present invention involves reacting insulin supported on an insoluble carrier and insulin in a sample with an anti-insulin antibody in a liquid medium. It is characterized by quantifying the insulin concentration in a sample by detecting the extent to which agglutination due to the antigen-antibody reaction between insulin and anti-insulin antibodies is blocked by the antigen-antibody reaction between insulin and anti-insulin antibodies in the sample. That is.

That is, in the present invention, when aggregation occurs due to the reaction between insulin supported on an insoluble carrier and an anti-insulin antibody, the insulin in the sample also competes with the insulin supported on the insoluble carrier and immunoreacts with the anti-insulin antibody. By focusing on the fact that the above-mentioned aggregation is inhibited by the insulin concentration in the sample in order to produce the above-mentioned aggregation, the insulin concentration in the sample is quantified by detecting the degree of inhibition of aggregation.

The insulin quantitative reagent of the present invention comprises an insulin sensitizing insoluble carrier reagent containing an insulin-supported insoluble carrier;
and an anti-insulin antibody reagent containing an anti-insulin antibody. In use, the insulin concentration in the sample is measured by reacting a sample containing an insulin sensitizing insoluble carrier reagent and insulin with an anti-insulin antibody reagent in a liquid medium and detecting the degree of inhibition of aggregation.

The insoluble carrier used in the present invention is substantially insoluble in the liquid medium used in the measurement of the present invention,
and the particle size is 0.05 μm x 1. Various organic and inorganic substances on the order of 0 μm can be used.

Examples of organic substances used as insoluble carriers include organic polymer latices obtained by polymerization, such as polystyrene or styrene-butadiene copolymers, individually dispersed cocci-type pores or cell membranes such as staphylococci or streptococci. Examples include pieces. Further, examples of inorganic substances include silica, inorganic oxides such as silica alumina or alumina, other mineral powders, and metals.

In the present invention, insulin is supported (sensitized) on the above-mentioned insoluble carrier. This loading method may be carried out by physically adsorbing insulin to the insoluble carrier, or may be carried out chemically. It may also be carried out by adsorption.

As the anti-insulin antibody used in the present invention, for example, immunoglobulin can be used, and not only the entire immunoglobulin can be used, but also "Revised New Edition Immunochemistry" edited by Yu Yamamura and 3 others: pp. 457-544 (1977) Year)
Fabs derived from immunoglobulins, Fab' and F(ab'), etc., may also be used, as described in .

In the present invention, the liquid medium used to react the insulin supported on the insoluble carrier and the insulin in the sample with the anti-insulin antibody is a phosphate buffer, a Tris buffer, a glycine buffer, or an ammonia buffer. A variety of buffers may be used, such as buffer solutions. The pH during the aggregation reaction is usually maintained in the range of 5 to 10, especially pH 6 to 10.
It is preferable to set it to 8. In addition, the reaction temperature is 0 to 50°
C1 is preferably 20 to 40°C.

Additionally, exacerbating agents may be added to the liquid medium.

Examples of the sensitizer include polyethylene glycol having a molecular weight of 30,000 or more.

In addition, in the quantitative reagent of the present invention, as the insulin sensitizing insoluble carrier reagent, a suspension having a concentration of 0.01% by weight or more of an insulin-supported insoluble carrier is used, preferably 0.05 to 1% by weight. A suspension with a concentration of % by weight is used. On the other hand, anti-insulin antibodies are provided as anti-insulin antibody reagents contained in a liquid medium as described above.

To explain the present invention more specifically, for example, when using polystyrene latex as an insoluble carrier and physically adsorbing insulin to the polystyrene latex, pH 5
,0 to 10.0, preferably pH 6.0 to 9.
．． In a buffer solution of 0.0, the concentration is 0.05% by weight to 2.0% by weight.
The polystyrene latex is compressed to a concentration of 0.1 to 1.0% by weight, more preferably 0.1 to 1.0% by weight.

Further, at a temperature of 1 to 50°C, preferably 20°C
0.00% insulin with stirring at a temperature of ~35°C.
1 deep view 1.0 deep view%, preferably 0°01~0.5
Add so that it becomes % by weight. After that, IO minutes ~ 48
Continue stirring for a period of time, preferably 30 minutes to 2 hours, to sensitize the polystyrene latex with insulin.

Next, centrifugation is performed under cooling, the precipitate is decanted, and the separated insulin-sensitized polystyrene latex particles are suspended in a bovine serum albumin solution.
% insulin sensitization latex reagent is prepared.

An anti-insulin antibody reagent (described later) and an insulin-containing sample are added to the insulin-sensitized latex reagent prepared as described above.

Optical detection methods are usually used to detect the degree of aggregation of insoluble carriers. When using an optical detection method, 0.1 μg of the above-mentioned anti-insulin antibody is usually added to a solution containing an appropriate buffer with a pH of 5.0 to 10.0.
/mj! ~50mg/mI! Prepare an anti-insulin antibody reagent containing: In addition, since the anti-insulin antibody reagent also functions as a reaction diluent, it will be hereinafter referred to as an anti-insulin antibody-containing reaction dilution liquid or simply as a reaction dilution liquid. That is, after diluting the sample with a reaction diluent, the above-mentioned insulin sensitizing latex reagent is mixed.

The degree of inhibition of aggregation is usually detected by an optical detection method, which is a method of measuring scattered light intensity or absorbance. The measurement wavelength is 300
~2400 nm can be used.

As for the photometric method, the decrease in scattered light intensity or absorbance is measured according to known methods by selecting the size or concentration of the insoluble carrier particles used or the reaction time. It is also possible to quantify by measuring the increase or rate of increase in absorbance using known methods. Furthermore, the degree of aggregation may be detected using a photoacoustic method. In addition, in the above-mentioned detection, the reaction time can be appropriately selected in the quantitative method of the present invention.

Hereinafter, the present invention will be explained more specifically by showing examples.

[Example 1] Insulin latex A polystyrene latex (manufactured by Sekisui Chemical Co., Ltd.) with a diameter of 0.3 μm was prepared, and a 50 mM glycine buffer solution (pH =
9.0) so that the concentration is 1% by weight. Take 1 m2 of the diluted solution and stir at a temperature of 30"C. Add 100 μg of human insulin to the stirred solution,
Thereafter, the polystyrene latex is sensitized to insulin by stirring for 2 hours at a temperature of 30°C.

After sensitization, centrifuge at 4°C, 18.00 rpm for 1 hour, and collect the precipitate. On the other hand, bovine serum albumin (hereinafter referred to as
BSA) was added to prepare a 1% by weight BSA solution, and added to the above precipitate for 1 m! The mixture was added and further stirred for 1 hour at a temperature of 30°C.

After 1 hour, centrifugation was performed under the same conditions as above to collect the precipitate, which was then added to 50mM Tris-HCl buffer (pH=8
．． 0) 2mj! to prepare an insulin sensitization latex reagent.

Amount of 1 While stirring 5 ml of guinea pig-produced anti-porcine insulin antiserum, slowly drip 0.1M phosphate buffer (pH = 7°4) with saturated ammonium sulfate until it reaches 30% saturation. After leaving at room temperature for 3 hours, 18.00 Orp at 4℃
Centrifuge for 1 hour, remove the supernatant, and collect the precipitate.

The obtained precipitate was added to 5 ml of O, 1M phosphate buffer (pH
= 7.4) Next, the above-mentioned ammonium sulfate precipitation was performed again, and after centrifugation under the same conditions as above, the precipitate was mixed with 1 mf of 0.1M phosphate buffer (pl (= 7.4).The resulting solution was diluted with 0.1M phosphate buffer (pH=
7.1) to desalt.

0.1M of the anti-insulin antibody purified as above
Phosphate buffer (pH=7.0) at a concentration of 2 mg/
The density is adjusted so that m j2.

The above anti-insulin antibody solution was mixed with 50mM glycine buffer (pH=9.0) at 100mj! Dissolve the solution in a volume of 1 ml to obtain a diluted solution for reaction.

0.1M phosphate buffer containing 5% BSA (pH-7,
0) to dilute standard human insulin to a concentration of 0.
10.20.40.80.160, and 320μU/
Prepare ml of each specimen.

The above sample is 20μ! , the aforementioned insulin sensitizing latex 50μ! 350 μl of the diluted reaction solution was weighed into a cell, and the reactivity was measured for 10 minutes by absorbance at a wavelength of 550 nm. The results are shown in Table 1 and Figure 1 below. In these results, the absorbance value multiplied by 104 is expressed as reactivity.

(The following is a margin.) Table [Example 2] Insulin-tex polystyrene latex with a diameter of 0.3 μm (manufactured by Sekisui Chemical Co., Ltd.) was prepared, and 50 mM glycine buffer ( pH=9.0).

Take 1 ml of the diluted solution, stir it at a temperature of 30 °C, add 100 μg of human insulin to the stirred solution, and then
Sensitize by stirring for 2 hours at a temperature of 0°C.

After sensitization, incubate at 4°C for 18. Centrifuge at OOOrpm for 1 hour and collect the precipitate. A 1 wt % BSA solution is prepared by adding BSA to 50 mM glycine buffer (pH = 9.0), 1 m 2 of which is added to the above precipitate, and the mixture is further stirred at a temperature of 30° C. for 1 hour. After stirring, the precipitate is collected by centrifugation under the same conditions as above and suspended in 2 mf of 50 mM Tris-HCl buffer (pH=8.0) to provide an insulin sensitizing latex reagent.

Preparation of dilute solution for reaction 1 An anti-insulin antibody solution was prepared in the same manner as in Example 1,
Molecular weight 5 in 50mM glycine buffer (pH=9°0)
Example 1 was added to 100 ml of a solution in which 0.0000% polyethylene glycol was dissolved to a concentration of 0.1% by weight.
Dissolve 1 ml of the anti-insulin antibody solution obtained in step 1 and use it as a diluted solution for reaction.

Concentration 0110.20.40.8 in the same manner as in Example 1
Prepare specimens of 0.160 and 320 tt U/m 1.

20μ2 of these specimens, 50μ of insulin sensitization latex reagent! 350 μl of the diluted reaction solution was measured into a cell, and the reactivity was measured for 10 minutes by absorbance at 550 nm. The results are shown in Table 2 and Figure 2 below.

In addition, in these results, the reactivity is expressed by multiplying the absorbance value by 10'.

Table 2 [Effects of the Invention] As described above, in the method for quantifying insulin of the present invention, insulin supported on an insoluble carrier and insulin in a sample are reacted with an anti-insulin carrier in a liquid medium, and the insulin is supported on an insoluble carrier. Insulin in a sample can be quantified by detecting the extent to which insulin in the sample inhibits aggregation caused by the reaction between insulin and anti-insulin antibodies. Can be quantified. In other words, there is no need for special equipment or equipment as in the case of the RIA method, and the RIA method and E
Unlike the IA method, it does not take a long time to measure.

In addition, the insulin quantitative reagent of the present invention consists only of an insulin sensitizing insoluble carrier reagent and an anti-insulin antibody reagent, and does not require a substrate solution or a reaction stop solution as in the case of a reagent based on the EIA method. The insulin concentration in a sample can be measured extremely easily, and the procedure is also extremely simple.

[Brief explanation of drawings]

Figure 1 shows the results of Example 1 and shows the relationship between the insulin concentration in the sample and the absorbance (XIO'). Figure 2 shows the results of Example 2 and shows the relationship between the insulin concentration in the sample and the absorbance (XIO').
It is a figure showing the relationship of XIO'). Figure 1 A> Surin $il (pU/m1) Figure Z

Claims (3)

[Claims] (1) The insulin supported on the insoluble carrier and the insulin in the sample are reacted with an anti-insulin antibody in a liquid medium, and aggregation based on the antigen-antibody reaction between the insulin supported on the insoluble carrier and the anti-insulin antibody is caused. . A method for quantifying insulin, which comprises quantifying insulin in a sample by detecting the degree of inhibition due to an antigen-antibody reaction between insulin in the sample and an anti-insulin antibody. (2) The method for quantifying insulin according to claim 1, wherein polyethylene glycol having a molecular weight of 30,000 or more is used as the sensitizer. (3) An insulin quantitative reagent comprising an insulin sensitizing insoluble carrier reagent containing an insoluble carrier carrying insulin and an anti-insulin antibody reagent containing an anti-insulin antibody.