JPH0354466A - Immunoassay reagent and immunoassay - Google Patents

Abstract

PURPOSE:To enhance storage stability and to avoid cross reaction by containing an insoluble carrier having an antibody supported thereon and the scrum albumin of the animal to be immunized used in the preparation of said antibody in a measuring reagent. CONSTITUTION:An insoluble carrier on which the antibody corresponding to the antigen (e.g., human albumin) in a specimen to be measured is supported and the serum albumin originating from the animal used in the preparation of the antibody are contained in a measuring reagent. The content of this serum albumin is determined according to the use mode of this reagent but about 0,01-8 wt.% in general, pref., about 1-2 wt.%. By this method, the storage stability of the reagent is effectively enhanced as compared with a case using serum albumin of a different kind of an animal. The cross reaction generated between serum albumin to be used and the antigen according to circumstances can be avoided.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention provides a reagent for measuring an antigen in a sample by detecting the degree of agglutination caused by an immunological specific reaction of an insoluble carrier carrying an antibody. The present invention relates to a reagent and a measurement method, and particularly to a reagent and a measurement method that have good storage stability and can avoid the effects of cross-reactivity.

[Conventional technology]

Conventionally, immunoassay methods have been widely used in which an insoluble carrier supports an antibody corresponding to an antigen in a sample to be measured, and the degree of aggregation of the insoluble carrier caused by an immune reaction is detected. In order to increase the measurement accuracy in this type of immunological measurement method, it is necessary to increase the specificity of the immune reaction, and various methods have been proposed. In the method disclosed in JP-A No. 57-9723, a component of immunoglobulin is used as a stabilizing agent in order to increase specificity, and in addition to the component of immunoglobulin, An example has been disclosed in which bovine serum albumin was also present.

On the other hand, JP-A-60-256057 discloses serum albumin as a substance having a stabilizing effect as a protective colloid in order to suppress non-specific reactions in immunoassays. Here, too, it is disclosed that bovine serum albumin is used as the serum albumin.

[Problem to be solved by the invention] The above-mentioned JP-A-57-9723 and JP-A-60-25
In all of the methods described in No. 6057, it has been shown that bovine serum albumin can be used as a stabilizer to suppress non-specific reactions. However, regardless of the certified species of antibody used, when bovine serum albumin is used, the storage stability of the reagent is not sufficient. Furthermore, there was always the possibility that the antibody used would react not only with the corresponding antigen but also with bovine serum albumin, a so-called cross-reactivity. In particular, when the antigen is human albumin, this cross-reactivity cannot be avoided. The object of the present invention is to provide a reagent and a measuring method for measuring antigens, which have excellent storage stability and can avoid the effects of cross-reactivity. [Means for Solving the Problems] The present inventors have developed a reagent and method for supporting an antibody corresponding to an antigen in a sample on an insoluble carrier and detecting the degree of agglutination of the insoluble carrier caused by an immune reaction. The present inventors have discovered that if the reaction system contains serum albumin from the immunized animal species used during the vaccination, storage stability is good and cross-reactivity can be avoided, and the present invention has been achieved. That is, the present invention provides an immunology method comprising an insoluble carrier carrying an antibody corresponding to an antigen in a sample to be measured, and serum albumin of the immunized animal species used to prepare the antibody. The antigen contained in the sample to be measured is detected by supporting a specific measurement reagent and an antibody corresponding to the antigen in the sample to be measured on an insoluble carrier, and detecting the degree of aggregation of the insoluble carrier caused by an immune reaction. This is an immunological determination method characterized by containing serum albumin of the immunized animal species used during antibody production in the reaction system. Objects to be measured in the reagent and measurement method of the present invention include haptens, polypeptides, steroids, polysaccharides, and various antigenic proteins such as human albumin and CRP. The present invention is particularly suitable for use in measuring human albumin.

In addition, the antibodies that can be used in the present invention include antibody proteins specific to the various antigens mentioned above. Examples include immunoglobulins and fragments thereof, such as F(ab')2. Insoluble carriers include inorganic powder, organic polymer powder,
Possible sources include microorganisms, blood cells, and cell membrane fragments. More specifically, examples of the inorganic substance powder include silica, alumina, and metal pieces such as gold, titanium, iron, and Nisokel. Examples of the organic polymer powder include latex, insoluble agarose, cellulose, and insoluble dextran, and preferably latex is used. Examples of the latex include polystyrene, styrene-styrene sulfonate copolymer, methacrylic acid polymer, acrylic acid polymer, acrylonitrile butadiene copolymer, and vinyl chloride-acrylic acid ester copolymer.

Latex is usually used in the form of a latex suspension in a reaction medium. The average particle size of the above-mentioned insoluble carrier is appropriately selected depending on the detected concentration of the substance to be measured or the measuring instrument used, but a particle size of about 0.05 to 1.0 μm is preferably used. Examples of reaction media for dispersing or suspending insoluble carriers include aqueous media such as water and buffer solutions. Further, in some cases, a specimen such as urine can be used as a reaction medium as it is.

In the present invention, to support (sensitize) the antibody on the above-mentioned insoluble carrier, a known method using physical or chemical bonding can be used.

The measurement reagent of the present invention contains an insoluble carrier sensitized with the above antibody and serum albumin derived from a reptile species used to sensitize the above antibody. The content of serum albumin in the reagent is determined depending on the manner in which the reagent is used, so it cannot be determined uniformly, but it is usually 0. O
The amount is about 1 to 8% by weight, preferably about 0.1 to 2% by weight.

In addition, in the measurement method of the present invention, serum albumin of the immunized animal species used during antibody production is contained in the reaction system.
The method of incorporating the serum albumin into the reaction system includes adding the serum albumin into a reaction medium in which an insoluble carrier is dispersed or suspended after the above-mentioned sensitization, and adding the serum albumin into a diluted solution of the sample to be measured. Various methods can be considered, such as a method of adding the serum albumin to the sample, and any method may be used. . Serum albumin may be added by adding a solution containing serum albumin or by adding powdered serum albumin. The content of serum albumin in the reaction system is appropriately selected depending on the detected concentration of the substance to be measured, etc., but is usually 0.01 to 8% by weight, preferably 0.1 to 5.0% by weight. It is said that the concentration is about 100%.

In the immunoassay method of the present invention, specific methods for detecting the degree of agglutination include a method of visually determining the presence or absence of agglutination, and a method of irradiating the reaction solution with light and detecting scattered or transmitted light. Any optical method of measuring can be used.

That is, the presence or absence of an antigen in a sample to be measured may be determined by a visual determination method, or the antigen may be detected semi-quantitatively, or the antigen may be quantified by an optical measurement method. If the invention is applied to the measurement of human albumin, it will not only have good storage stability, but also reduce the cross-reactivity between human albumin and serum albumin that occurs when serum albumin from an animal species different from the immunized animal species is used. Since a reagent and a measuring method can be obtained that avoid this, the effects of the present invention are particularly exhibited. Any specimen containing human albumin can be used, and examples thereof include urine and serum specimens. [Action and Effects of the Invention] The immunoassay reagent of the present invention contains serum albumin of the same animal species as that used in preparing the antibody supported on the insoluble carrier. The storage stability of the reagent is effectively increased compared to when serum albumin of different species is used. The reason for this is assumed to be that non-specific interaction between the antibody and serum albumin is unlikely to occur when serum albumin of the same species as that used in preparing the antibody is used. Furthermore, in the immunoassay reagent of the present invention, possible cross-over between the serum albumin used and the antigen is avoided because serum albumin of the same animal species as used for producing the antibody is used. Similarly, in the immunoassay method of the present invention, since the reaction system contains serum albumin of the immunized animal species, nonspecific interactions and cross-reactions between antibodies and serum albumin can be avoided. can. Therefore, according to the reagent and measurement method of the present invention, immunological specificity can be effectively increased, making it possible to accurately measure antigens in a sample to be measured. In addition, when applying the immunological measurement reagent and measurement method of the present invention to the measurement of human albumin, it is important to not only have good storage stability, but also to improve the results when using serum albumin of an animal species different from the immunized animal species. Since the present invention provides a reagent and a measurement method that avoids the interaction between human albumin and serum albumin, human albumin can be measured accurately at all times. [Explanation of Examples] Anti-human albumin antibody (derived from rabbit) was added to lmg/m
0.1 M glycine buffer containing at an Igcl degree of 12 (
pH=9.0). In 10mf of this anti-human albumin antibody-containing glycine buffer, the average particle size was 0.13μ.
Add 1m2 of polystyrene latex (manufactured by Sekisui Chemical Co., Ltd., solid content 10%), stir at 37°C for 60 minutes, and then centrifuge at 4°C for 60 minutes.
1 (1800 rpm).

After centrifugation, the supernatant was discarded and the precipitate was added to a concentration of l. 2% by weight
of rabbit serum albumin in 0.1M glinone buffer (
Add LOMF (pH = 9.0) to make the latex cloudy and obtain a measurement reagent. Store the above measurement reagent at a temperature of 4''C, mix 900 μl of the above rabbit serum albumin-containing glycine 11 buffer solution as a diluent with 100 μl of the reagent,
Absorbance at 700 nm was measured using a glass cell with a cell length of 5 mm.

Anti-human albumin antibody (derived from rabbit) in the history of sharks at lmg/m
l of Ig (0.1M glycine buffered fi to 4 degrees
In 10 ml of glycine buffer containing anti-human albumin antibody (pH = 9.0), the average particle size was 0.13 μm.
polystyrene latex (manufactured by Sekisui Chemical Co., Ltd.,
10% solids) was added, stirred at 37 °C for 60 min, and then centrifuged at 4'C for 60 min (18000 °C).
rpm). After centrifugation, add 1 to the precipitate after discarding the supernatant.
．． 0.1M glycine buffer containing 2% by weight bovine serum albumin (pH=9.0)lor+/! is added to make the latex cloudy to obtain a reagent. This measurement reagent was stored at a temperature of 4°C, and 100 μe of the reagent was mixed with 900 μl of the above glycine buffer containing live serum albumin as a diluent, and the absorbance at 700 nm was measured using a glass cell with a cell length of 5 μm. Reagent storage period and 70 days in Example 1 and Comparative Example 1 above
The relationship between changes in turbidity at 0 nm is shown in Table 1 below.

Table I From Table 1, it can be seen that in the conventional method, that is, Comparative Example 1, self-aggregation of the measurement reagent itself occurs, and the turbidity increases rapidly as the storage period becomes longer. On the other hand, it can be seen that the measurement wipe of Example 1 does not cause self-aggregation, has almost no upper limit of turbidity, and has excellent storage stability.

Inutoike 1 Anti-human CRP antibody (goat-derived) IgGi degree is 1
0.1M glycine 1M so that mg/mE
fIi1 (pH=9.0) contains 7 certain anti-! =}Lmffi latex (manufactured by Sekisui Chemical Co., Ltd., solid content 10%) with an average particle size of 0.24 μm was added to 10 m of glycine buffer containing CRP antibody, stirred for 60 minutes at a temperature of 37°C, and then Then, centrifuge M (18,000 rpm) for 60 minutes at a temperature of 4°C. After centrifugation, the supernatant was discarded and the precipitate was mixed with 0.1M glycine 7M street solution (pH = 9.0) containing goat serum albumin at a concentration of 0.7% by weight.
) Add 10 mf and suspend the latex to obtain a measurement reagent. This measurement reagent was stored at a salinity of 4'C, and 100 μl of the measurement reagent was mixed with 900 μl of the above glycine buffer containing goat serum albumin as a diluent, and the absorbance at 700 nm was measured using a glass cell with a cell length of 5. was measured. Comparative Example 2 IgG4 degree of anti-human CRP antibody (goat origin) is 1 m
The anti-human CRP antibody was added to O.B/1 ml. Mix with IM glycine buffer (pH 9.0) and add 10rr+j of this glycine buffer solution containing anti-human CRP antibody!
1 ml of latenox (manufactured by Sekisui Chemical Co., Ltd., solid content 10%) with an average particle size of 0.24 μm was added to the mixture, and heated at 37°C.
Stir for 60 minutes and then centrifuge (18,000 rpm) for 60 minutes at a temperature of 4°C. After centrifugation, the supernatant was discarded and the precipitate was added to the precipitate containing 0.7% by weight of bovine serum albumin. tM glycine buffer (pH=9.0) 10rr+
j! is added to suspend the latex to obtain a measurement reagent.

Store this measurement reagent at 4°C, and store 100 μi of this measurement reagent.
900 μl of the above-mentioned glycine buffer containing live serum albumin was mixed as a diluent, and the absorbance at 700 nm was measured using a glass cell with a cell length of 5IIIm.

Reagent storage period and 7 in Example 2 and Comparative Example 2 above
The changes in thirst at 00 nm are shown in Table 2 below.

Table 2 From Table 2, it can be seen that in the measurement reagent of the conventional method (Comparative Example 2), self-aggregation occurs in the measurement reagent itself, which causes a rapid increase in turbidity as the storage period becomes longer. Recognize. On the other hand, in the measurement reagent of the present invention (Example 2), since self-aggregation does not occur, there is almost no increase in turbidity, and it can be seen that it has excellent storage stability.

Claims (4)

[Claims] (1) An immunological assay reagent comprising an insoluble carrier carrying an antibody corresponding to an antigen in a sample to be measured, and serum albumin of the immunized animal species used to prepare the antibody. (2) The immunoassay reagent according to claim 1, wherein the antigen in the sample to be measured is human albumin. (3) A method of detecting an antigen contained in a sample to be measured by supporting an antibody corresponding to the antigen in the sample to be measured on an insoluble carrier and detecting the degree of aggregation of the insoluble carrier caused by an immune reaction. An immunoassay method characterized in that the reaction system contains serum albumin of the immunized animal species used in producing the antibody. (4) The immunoassay method according to claim 3, wherein the antigen in the sample to be measured is human albumin.