JP2684425B2 - Latex reagent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial field of application> The present invention relates to a latex diagnostic reagent for immunoassay using an antigen-antibody reaction used for diagnosis of epidemics.

<Prior Art> Conventionally, in order to diagnose a plague, a test for collecting blood or urine of a patient and performing various tests to assist a clinician in diagnosis has been performed. Of these tests, the immunological test is for examining the presence or absence of antigens or antibodies in body fluids such as blood or urine of patients, and the antigen-antibody reaction is used. It is possible to detect a very small amount of antigen or antibody because it utilizes the property of not reacting, that is, the specificity of the antibody.

An immunoassay utilizing the antigen-antibody reaction is carried out by mixing an antibody against an antigen to be detected or an antigen against the antibody to be detected present in body fluid such as blood (usually serum or plasma) or urine. Although an antibody complex is generated and it is intended to detect it, in order to directly detect the antigen-antibody complex, an insoluble precipitate (antigen-antibody complex) unless both the antigen and the antibody are present in abundance Do not make Therefore, it is often undetectable. Therefore, various techniques have been considered to increase the detection sensitivity.

At present, the most popular method is to sensitize (adsorb or bind) an antigen or antibody to a carrier such as erythrocyte or a synthetic latex mainly composed of polyethylene, attach the antigen or antibody around a large carrier, and perform an antigen-antibody reaction. Is a method that makes it easier to see the compound, and has a high detection sensitivity, and is widely used in general inspection.

The hemagglutination method called (reverse) passive hemagglutination method using erythrocytes measures the antibody or antigen in body fluid using erythrocytes that have been sensitized with antigen or antibody after treating the surface of erythrocyte with tannic acid etc. It is judged by the agglutination image of the bottom of the tube. Generally, it is carried out by a microtiter method using a microplate, and its detection sensitivity is as high as 1 to 10 ng / ml as a protein concentration in body fluid, and it is used for various tests. However,
Since erythrocytes are of biological origin, lot differences easily occur when used as a diagnostic reagent, and reproducibility is difficult. Therefore, the replacement of synthetic latex with an artificial carrier has been devised. With a reagent using latex, the antigen-antibody reaction is measured not only by the microtiter method but also by various methods such as an optical measurement method. The optical measuring method, which uses a dedicated measuring machine, can be measured fully automatically and quickly, and has high sensitivity.

<Problems to be Solved by the Invention> A diagnostic reagent using latex is applied to various inspection items by various methods. However, it has a drawback that an agglutination reaction other than an antigen-antibody reaction, which is called a non-specific agglutination reaction, such as an agglutination reaction due to a protein that is easily adsorbed, is likely to occur.

In order to eliminate non-specific agglutination reaction, surface treatment of latex particles, control of surface charge of latex particles, devising of sensitization method, addition of bovine serum albumin (hereinafter abbreviated as BSA) in a buffer in which latex is suspended, Heat inactivation of test serum, repeated freezing, thawing and shaking of serum, addition of β and γ globulin fraction of human serum, or the above 2
Combinations of more than one species are used together. On the other hand, it is well known that BSA is added when sensitizing an antigen or an antibody, but as a normal addition amount, 0.001 to 1 times by weight of latex solids gives a considerable amount of nonspecificity. It is said that the agglutination reaction can be suppressed, but it cannot be completely suppressed.

These non-specific agglutination reactions do not measure the antigen or antibody to be tested, but are agglutination reactions other than the antigen-antibody reaction, so accurate measurement values cannot be obtained, resulting in a large diagnostic error. It may be a cause. Therefore, how to suppress this non-specific agglutination reaction is a major point when manufacturing test reagents.

<Means for Solving the Problems> In order to solve such problems, various studies have been conducted on the suppression of non-specific agglutination reaction by surface treatment of latex, and a large amount of immunologically inactive proteins such as BSA have been investigated. It was found that the non-specific agglutination reaction is reduced by treating the surface of the polyacrolein latex with, and the present invention has been completed.

That is, in the present invention, polyacrolein latex is sensitized with an antigen or an antibody and then treated with 3 to 20 times by weight, preferably 3 to 10 times by weight, of immunologically inactive protein based on the latex solid content. It relates to the obtained latex reagent.

As a method of suppressing non-specific agglutination reaction, it is known to add an immunologically inactive protein at the same time when sensitizing an antigen or an antibody, and the addition amount is 0.001 relative to latex solid content. Although the sensitization time is about 1 times the weight and the sensitization time is about 48 hours, it is difficult to almost completely suppress the non-specific agglutination reaction under such conditions. Furthermore, after sensitization, even if treated under the same conditions, it cannot be completely suppressed. However, the treatment by the method of the present invention can almost completely suppress the non-specific agglutination reaction.

It is considered that polyacrolein latex, which is said to have a strong non-specific agglutination reaction, has a strong activity on the surface of the latex, and is strongly adsorbed to substances such as many proteins that are easily adsorbed. Polyacrolein latex is
In this way, although non-specific agglutination reaction is likely to occur,
By performing the treatment of the present invention, non-specific agglutination reaction can be suppressed.

In the present invention, after sensitizing with an antigen or an antibody, a protein that is immunologically inactive with respect to the latex solid content is used in an amount of 3 to 20 times by weight to treat the nonspecific agglutination reaction almost completely. To be done. This is because the physical and chemical properties of the surface of latex particles were changed by effectively covering the unsensitized naked part of the antigen or antibody with immunologically inactive protein. Conceivable.

In the present invention, 3 to 20 times by weight of the immunologically inactive protein used is not entirely attached to the surface of the latex particles, but only a part thereof. However, when a large amount of immunologically inactive protein is used in the treatment, a latex reagent in which the non-specific agglutination reaction is significantly suppressed can be obtained.

Sensitization with an antigen or an antibody can be carried out by a conventional method, and various kinds of antigens and antibodies can be used without any particular limitation.

The immunologically inactive protein used in the present invention is BSA.
And other albumins or fetal calf serum (FCS)
Fetal sera such as are suitable. Since these gently adsorb to the latex particles, the sensitivity is less likely to decrease, which is preferable. Those that strongly adsorb such as casein and gelatin are not preferable. When treated with an immunologically inactive protein, the concentration of the protein in the treatment solution containing the protein is preferably higher, and about 2 to 7% is easy to use, and a phosphate buffered saline (hereinafter referred to as PBS) is used. Abbreviated) and used in a buffered saline solution. The conditions for treatment can be about 37 ° C. or 4 ° C. which is generally used, and is not particularly limited. Processing time is 1 to 8 hours at 37 ℃, 4 ℃
It takes about 8 to 48 hours. According to the present invention, the non-specific agglutination reaction caused by the protein adsorption property of the latex carrier can be almost eliminated. Therefore, the reagent of the present invention can be used not only for on-plate measurement, which requires less sensitivity, but also for highly sensitive optical measurement or microtiter method. In addition, the resulting diagnostic reagent supplements the protein in the buffer that suspends the sensitized latex particles.
BSA or FCS may be added in some cases, and in this case, it is advantageous to use the same type of protein as that used for the treatment.

Therefore, according to the present invention, it is possible to carry out the measurement without requiring a complicated operation such as heat inactivation of serum and removal of an insoluble component by freezing, thawing and shaking.

The latex particle solid content in the latex reagent of the present invention is usually in the range of 0.01 to 2% by weight.

The latex reagent obtained by the method of the present invention can be confirmed by performing extraction treatment with a buffer solution having a high pH and a high salt concentration, and quantifying the eluate by high performance liquid chromatography.

<Examples> Examples, comparative examples, and test examples will be specifically described below.

Example 1 Polyacrolein latex (average particle size: 1.5 μm) was suspended in 10 ml of PBS at a concentration of 1%, and an anti-human alpha fetoprotein antibody (goat IgG fractionated protein concentration 2.5 mg /
0.25 ml) and shake gently at 37 ° C for 30 minutes. After that, centrifuge at 1500 rpm for 5 min and deposit 5% BSA in PBS on the sediment.
Add 10 ml of the solution, disperse well, and gently shake at 37 ° C for 4 hours. The precipitate obtained by centrifugation was centrifuged and washed twice with PBS containing 0.3% BSA, and resuspended in 40 ml of PBS containing 0.3% BSA to obtain an anti-human alphafetoprotein-sensitized latex reagent.

Example 2 Treponema pallidum grown in rabbit testis
Abbreviated as TP) nichols strain is extracted with citrate buffer solution, centrifuged and collected to collect the cells, and PBS is adjusted to 1 to 2 × 10 ⁹ cells / ml.
Re-floated in. This TP bacterial suspension is diluted 10 times with PBS,
Using an ultrasonic crusher, process at 9KHz, 1800W, 10 minutes, TP
An antigen solution containing bacterial components was obtained.

Polyacrolein latex (average particle size 1.8 μm) was suspended in 5 ml of PBS at a concentration of 2%, and the TP antigen solution 4 m prepared above was suspended.
l was added and the mixture was gently shaken at 37 ° C. for 30 minutes. Then, 10 ml of 5% BSA dissolved in PBS was added, and the mixture was further shaken slowly at 37 ° C for 5 hours. This was centrifugally washed twice with PBS containing 0.3% BSA and suspended in 40 ml of PBS containing 0.3% BSA to obtain a TP antigen-sensitized latex reagent.

Comparative Example 1 0.24 ml of the anti-human alphafetoprotein antibody was used under the same conditions as in Example 1, and 10 ml of 1% BSA in PBS was used instead of 10 ml of 5% BSA in PBS. , Anti-human alpha fetoprotein antibody-sensitized latex reagent was obtained.

Comparative Example 2 TP antigen was sensitized under the same conditions as in Example 2, and PBS containing 5% BSA was used.
Instead of 10 ml of the solution, 10 ml of 1% BSA in PBS was used and treated in the same manner as in Example 2 to obtain a TP antigen-sensitized latex reagent.

Test Example 1 To the 96-well U-type microplate, PBS containing 0.3% BSA was dropped by 25μ after the first hole, and 25μ of test serum was added first.
It was added to the holes and diluted 2-fold, and serially 2-fold diluted with a diluter. The anti-human alpha-fetoprotein antibody-sensitized latex reagent (25 μm) produced in Example 1 and Comparative Example 1 was dropped into the second and subsequent holes, shaken well with a plate mixer, and allowed to stand at room temperature for 2 hours. The obtained agglutination image is visually determined, and the value obtained by multiplying the dilution ratio of serum with aggregation by the sensitivity is the quantitative value.

In Example 1 and Comparative Example 1, 100 ng / ml standard solution of human alphafetoprotein was positive for aggregation up to a 64-fold dilution, so the sensitivity was 1.5 ng / ml. 10n as measured by RIA
The test was carried out by the above-mentioned microtiter method using 50 cases of serum of a normal person who had g / ml or less, and the results of comparing both are shown below.

Incidentally, this numerical value shows the number of cases in which aggregation occurred up to the dilution ratio in 50 normal subjects, and the example clearly clearly determines the normal value correctly.

Test Example 2 Tests were carried out using the microtiter method in the same manner as in Test Example 1, 100 μ of PBS containing 0.3% BSA was added to the first well, and 25 μ of test serum was added to make the first well a 5-fold dilution. Others were the same as in Test Example 1. Since the dilution ratio is the final dilution rate including the reagents, the second hole is 20 times diluted.

The test by the above-mentioned microtiter method was carried out by using 50 cases of serum of a normal person who was 40 times or less as measured by a commercially available TPHA reagent, and the results of comparing both are shown below.

In addition, since this value shows the number of cases in which agglutination occurred up to the dilution ratio in 50 normal subjects, since the cutoff is generally 80 times, it is clear that the example is correct from the normal value. Making a decision.

<Effects of the Invention> A diagnostic reagent using latex is used for various inspection items because of its simple method. However, when a bare latex surface remains during the production of a diagnostic reagent, nonspecific agglutination reaction is likely to occur, and it has not been possible to completely suppress nonspecific agglutination reactions by conventional methods. According to the present invention, the non-specific agglutination reaction can be almost completely suppressed by a relatively simple treatment, and only the agglutination due to the antigen-antibody reaction can be measured.

Claims (1)

(57) [Claims] 1. A latex reagent obtained by sensitizing a polyacrolein latex with an antigen or an antibody and treating the polyacrolein latex with 3 to 20 times by weight of an immunologically inactive protein based on the solid content of the latex.