JPS6324163A - Reagent latex for immunoassay - Google Patents

Abstract

PURPOSE:To detect the antigen or antibody in a bodily fluid by a microtiter method by using a reagent latex for immunoassay obtd. by sensitizing an antibody or antigen with a granular carrier consisting of polyacrolein. CONSTITUTION:The antigen or antibody or the like to be inspected induces an antigen-antibody reaction with the antibody or antigen existing on the surface of the polyacrolein particles to agglutinate the polyacrolein particles to each other if the antigen or antibody or the like to be inspected exists in the bodily fluid such as serum or urine to be inspected. As a result, the agglutinated lumps settle downward as they are to yield the positive agglutination having the spread in the tube bottom. The microtiter method using a microplate is executed by using the microplate having a U- or V-shaped tube bottom. Dilution series of consecutive two-times dilution, etc., of the bodily fluid to be inspected are prepd. and the same amt. of the reagent latex for inspection is dispensed therein. The fluids are rested for 1-3hr after mixing, then the agglutinated images appear on the tube bottoms; therefore, the concn. of the antigen or antibody in the bodily fluid is quantitatively determined by visual decision.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to an immunoassay reagent latex that utilizes an antigen-antibody reaction used in disease diagnosis.

BACKGROUND ART Traditionally, disease diagnosis has involved collecting serum or urine from a patient and conducting various tests to assist clinicians in their diagnosis. Among these tests, immunological tests test the presence or absence of antigens or antibodies in body fluids such as blood (and urine) of patients, and utilize antigen-antibody reactions.
This reaction makes use of the specificity of antibodies, which is the property of antibodies that react only with certain antigens, so it is possible to detect minute amounts of antigens or antibodies.

Tests using antigen-antibody reactions generate antigen-antibody reactions by mixing antibodies against the antigen to be detected, or antigens against the antibodies to be detected, present in body fluids such as serum or urine, and an antigen-antibody complex is generated. However, since it does not directly detect the antigen-antibody complex, it cannot be detected. Therefore, various methods have been considered to increase the detection sensitivity.

Currently, the most popular method is to sensitize (adsorb) antigens or antibodies to carriers such as red blood cells or synthetic latexes mainly made of polystyrene, attach antigens or antibodies to one large carrier, and use antigen-antibody reactions. This method makes it easy to see composites, has high detection sensitivity, and is widely used.

The hemagglutination method, also known as the (reverse) passive red blood cell agglutination method, uses red blood cells that have been treated with tannic acid etc. on their surface and then adsorbed with white matter antigens, or sensitized red blood cells that have been adsorbed with antibodies. This method attempts to measure antibodies or antigens in body fluids, and is generally performed using a microtiter method using a microplate.The detection sensitivity is high, with protein concentrations in body fluids ranging from 1 to 30 n, g/ml. and is used for various tests.

However, since red blood cells are living white rice, there are differences not only between species, but also between individuals and seasons even within the same species, and differences between lots tend to occur, making reproducibility difficult. Furthermore, since it is a biological component, it also has shortcomings in storage stability.

In addition, the latex agglomeration method, called the glass plate method or flat plate method, uses O,
This is a method of visually determining the iJ' collection by sensitizing the surface of the particles with antigens or antibodies, which have a particle size of about 1 to 1 μm, and mixing them with serum or urine diluted on a glass plate. It is widely used because it can determine between 100 ng/ml and 100 ng/ml.

<Problem to be solved by Invention C> Synthetic latex, mainly polystyrene, is widely used in the latex agglutination method, but when red blood cells are used in the microtiter method, red blood cells can be used as a carrier. It also has the disadvantage that it takes a long time for the agglomerated image to appear compared to the case where the agglomerated image appears. In order to alleviate these problems, the small particle size and the low specific gravity of the carrier itself are considered to be the reasons why it takes a long time for sedimentation. -Nox is highly desired.

In order to solve these problems, attempts have been made to make synthetic latex, mainly made of polystyrene, with a high specific gravity so that it can be applied to the microcotiter method. Attempts have been made to copolymerize styrene monomer with a high specific gravity latex to increase the specific gravity and shorten the determination time. However, many of these monomers are difficult to copolymerize with styrene, are expensive, and have the drawback that it is difficult to obtain latex particles with uniform particle sizes.Furthermore, the surface properties of the obtained particles change, making it difficult to copolymerize with antigens. Alternatively, it is said that it is difficult to sensitize antibodies.

Rather than increasing the specific gravity of latex particles by copolymerizing the high specific gravity monomers mentioned above, the present inventors have developed synthetic latex particles that can be applied to the microtiter method, that is, synthetic latex particles that can be detected with short determination time and high sensitivity. explore latex,
We have discovered that polyacrolein can produce a sensitized latex with sensitivity as high as that using red blood cells, a short time of 3 hours, and even better stability, leading to the completion of the present invention. Ta.

The present invention relates to an immunoassay reagent latex for detecting antigens or antibodies in body fluids by a microtiter method, which is obtained by sensitizing a particulate carrier made of polyacrolein with an antibody or antigen. When the immunolateral distortion reagent latex of the present invention is applied to the microtiter method, antibodies or antigens in body fluids can be detected with high sensitivity.

Tests are performed by mixing the latex immunological test reagent of the present invention with body fluids such as serum or urine to be tested. When the antigen or antibody to be tested is present in the body fluid, an antigen-antibody reaction occurs with the antibody or antigen present on the surface of the polyacrolein particles, causing the polyacrolein particles to aggregate with each other, and as a result, the aggregates drop down as they are. A positive agglutination image is obtained in which the particles settle and remain spread at the bottom of the tube. If the target antigen or antibody is not present in the body fluid, the antigen-antibody reaction will not occur and the particles will settle out.Since the particles are spherical, they will settle to the bottom of the tube and the shape of the tube bottom, such as the bottom of a test tube. It further settles and aggregates along a U-shape like this.

The result is a negative agglutination image with no spread, which is different from a positive agglutination image in which there is an antigen-antibody reaction, and allows confirmation of the presence or absence of antigen or antibody.

In the microtiter method using a microplate, a dilution series such as serial 2-fold dilution of the body fluid to be tested is prepared using a microplate with a U- or V-shaped tube bottom, and the same amount of the calibration reagent of the present invention is added. By dispensing the latex and allowing it to stand for 1 to 3 hours after mixing, an agglutination image will appear at the bottom of the tube. This can be visually judged from above or below, and the antigen or antibody in the body fluid can be determined from the maximum dilution concentration of the positive agglutination image. Concentration can be quantified.

Examples of applications of polyacrolein latex in the immune system include cell recognition using fluorescent dye-containing latex and cell separation using magnetic iron-containing latex. (Ind-Eng, Chem, Prod-
(Re5-Dev-21343 (1982)) However, these methods attempt to recognize antigens on the cell surface and are completely different from in vitro immunoassay methods using body fluids.

General methods can be used to sensitize antigens or antibodies to polyacrolein, including RD in physiological saline or phosphate buffered saline (buffers can be glycine, Tris-HCl, etc.) at pH= In the range of about 6.0 to 9.5,
The mixture is slowly heated at room temperature to 60°C for about 1 to 50 hours, with a polyacrolein concentration of about 0.1 to 5%, using an antigen or antibody as a protein amount of polyacrolein of about 0.1 to 10%. Sensitization is achieved by stirring. If necessary, bovine serum albumin (BSA) or immobilized animal serum may be added to compensate for the lack of protein. Polyacrolein has an aldehyde group on its surface, and the aldehyde group is known to bond with an amino group by forming a Schiff base, and it forms a strong bond with the amine group present in the side chain of the antigen or antibody protein. For sensitization, it is preferable to raise the temperature and take a long time to achieve binding, for example, 3 to 40 hours at 37°C or 1 to 30 hours at 45°C. In order to further stabilize the sensitized latex, it is also possible to use a Schiff base reducing agent such as sodium cyanoporohydrate to create a more stable covalent bond.

The degree of sensitizing polyacrolein in the immunoassay reagent latex of the present invention is usually in the range of 0.05 to 2% by weight, particularly preferably in the range of 0.2 to 0.6% by weight.

As described above, water is used as the dispersion medium in the latex immunoassay reagent of the present invention, and for example, sensitized polyacrolein is suspended in a buffered saline such as phosphate buffered saline.

For the reasons mentioned above, the antigen or antibody that sensitizes to polyacrolein is preferably a protein, and in the case of an antibody, there is no problem since it is a protein. However, the antigen may be a lipid antigen, but the surface of polyacrolein is primarily hydrophobic. Therefore, sensitization by adsorption is possible. There is no problem in sensitization whether the antibody is a so-called polyclonal antibody obtained by immunizing various animals with antiserum, or a monoclonal antibody obtained from hybridoma using the cell fusion method.

The antigens and antibodies used in the present invention are not particularly limited, and a variety of antigens and antibodies can be used.

Polyacrolein latex itself is well known and various manufacturing methods have been proposed, but any polymerization method can be used, and generally emulsion polymerization is used, radical polymerization, polymerization with alkali, polymerization with light or radiation The particle size obtained by polymerization is preferably about 0.5 to 5.0 μm, and a monodisperse latex with a narrow particle size distribution is preferable.

<Example> This will be specifically explained below with reference to Examples.

Actual example 1. Measurement of rheumatoid factor 0, polyacrolein latex (alkaline polymer with particle size 0.9 μm) dispersed to a solid content concentration of 05%.
05M') A PBS solution in which 1 part of acid-buffered saline (PBS) and heat-denatured human gamma globulin (Sigma human gamma globulin 63°C x 10 minutes heat treated solubles) were dissolved at 5 q/ml. Mix 1 part with 37℃×
Shake slowly for 1 hour, and add bovine serum albumin (BS).
A) Add 1 part of ]% PBS solution and shake at 37°C for 1 hour. Centrifuge this (1500 rpm
The precipitate from in) was washed three times with PBS and dispersed in a PBS solution containing O, ]% BSA so that the final sensitized latex concentration was 0.5% to obtain a sensitized latex.

Separately, in a 96-well V-type microplate, add 25 μι of PBS containing 0.1% BSA to each well, and add normal human serum diluted 10 times with the same PBS and R to the first well.
25μ of A (-1-1 serum (mixed from 3 people)) was added and serially diluted 2 times using a diluter.

A 25 μm thick layer of sensitizing latex was added to each well, shaken with a mixer, and allowed to stand at room temperature for 2 hours. The aggregation image was as follows.

RAHA (Hemagglutination method) RA: Highland RA test RAHA: Fuji Organ Pharmaceutical ■ was used.

C: Control, reaction with diluent only Example 2.
Measurement of Human Alpha Fetoprotein Anti-human alpha fetoprotein serum (rabbit) was sensitized in the same manner as in Example 1, and sodium cyanoborohydrate 0.1
1IIg was added and the Schiff base bond between the latex and the antibody was reduced by a reaction at 37° C. for 2 hours, followed by washing by centrifugation to obtain a sensitized latex.

The measurement results using this sensitized latex were as follows.

Example 3゜Polyacrolein latex (particle size: 1.2 μm, alkaline polymerization followed by radical polymerization) at a solid content concentration of 1.0%
Monoclonal antibody NCC-8T-439 (manufactured by Nippon Kayaku ■) (-1.0
mg/mi) 2 rnl plus 1% BSA
25 mt of TBS solution was added and shaken at 45° C. for 3 hours. After washing with TBS solution in the same manner as in Example I, a sensitized latex was obtained. Using the same method as in Example 1, 100 cancer patient sera and 100 normal human sera were determined to have a cutoff value of 1.
6 times higher, and the positive rate among cancer patients was 29%.

<Effects of the Invention> As described above, according to the present invention, synthetic latex can be applied to the microtiter method instead of red blood cells in immunoassays, and the measurement time is short, the measurement sensitivity is sufficiently high, and the quality of the immunological test is stable. Test reagents can be obtained.

Claims (1)

[Claims] An immunoassay reagent latex obtained by sensitizing an antibody or antigen to a particulate carrier made of polyacrolein for detecting antigens or antibodies in body fluids by the microtiter method.