JPH0394161A - Latex reagent - Google Patents

Abstract

PURPOSE:To decrease nonspecific agglutination reaction by treating the surface of latex with a large amt. of immunologically inert protein. CONSTITUTION:The latex reagent which is approximately completely suppressed in the nonspecific agglutination reaction is obtd. by treating the latex by using the immunologically inert protein at 3 to 20 weight times the solid content of the latex after the latex is sensitized with an antigen or antibody. The used protein sticks just partly to the latex particle surface. The latex is exemplified by the latex of a polystyrene system. The protein is adequately albumins including bovine serum albumin. Since these albumins adsorb gently on the latex particles, the sensitivity is less degraded. The higher concn. of the protein in the treating liquid contg. such protein is more preferable and the treating liquid of about 2 to 7% concn. is easy to use. For example, the liquid is used by being dissolved in the physiological salt soln. of a phosphoric acid buffer. This reagent is usable for an on-plate measurement method, optical measurement method and microtire method.

Description

DETAILED DESCRIPTION OF THE INVENTION Industrial Application Field The present invention relates to a latex diagnostic reagent for immunoassays that utilizes antigen-antibody reactions used in disease diagnosis.

<Prior Art> Conventionally, in diagnosing diseases, blood or urine from a patient is collected and various tests are performed to assist the clinician in making a diagnosis. Among these tests, immunoassay tests the presence or absence of antigens or antibodies in body fluids such as blood or urine of patients, and uses an antigen-antibody reaction.
This reaction utilizes the property of antibodies to react only with specific antigens, that is, the specificity of antibodies, so it is possible to detect trace amounts of antigens or antibodies.

Immunological tests using antigen-antibody reactions are performed by mixing antibodies against the antigen to be detected or antigens against the antibodies to be detected in body fluids such as blood (normally serum or plasma is used) or urine. Antibody complexes are viable and this is what we are trying to detect. However, in order to directly detect antigen-antibody complexes, both antigen and antibody must be present in large amounts to form an insoluble precipitate (antigen-antibody complexes). )
I don't make it. Therefore, it is often undetectable.

Therefore, various methods have been considered to increase the detection sensitivity.

Currently, the most popular method is to sensitize (adsorb or bind) antigens or antibodies to carriers such as red blood cells or polystyrene-based latex, attach the antigens or antibodies around a large carrier, and perform an antigen-antibody reaction. This method makes it easier to see composites, has high detection sensitivity, and is widely used for general inspection.

The hemagglutination method, also known as the (reverse) passive hemagglutination method using red blood cells, measures antibodies or antigens in body fluids by treating the surface of red blood cells with tannic acid, etc., and then using red blood cells that have been sensitized to antigens or antibodies. This is determined by the agglomeration image at the bottom of the tube. Generally, this is carried out by the microquanta method using a microplate, which has a high detection sensitivity of 1 to 1 Ong/mR for protein concentration in body fluids, and is used for various tests. However, since red blood cells are derived from living organisms, lot differences are likely to occur when used as diagnostic reagents, making reproducibility difficult. Therefore, efforts are being made to replace it with artificial carriers such as Gokai latex. With reagents using latex, antigen-antibody reactions are measured not only by the microtiter method but also by various techniques such as optical measurement methods. The optical measurement method uses a dedicated measuring machine, can perform fully automatic measurement quickly, and has high sensitivity, so it has suddenly become popular in recent years.

Problems to be Solved by the Invention Diagnostic reagents using latex are applied to various test items by various methods. However, it has the disadvantage that agglutination reactions other than antigen-antibody reactions called non-specific agglutination reactions, such as agglutination reactions due to easily adsorbed proteins, are likely to occur.

In order to eliminate non-specific agglutination reactions, we carried out surface treatment of latex particles, control of surface charge of latex particles, devised sensitization methods, addition of bovine serum albumin (hereinafter abbreviated as BS8) to the buffer solution that suspends the latex, Heat inactivation of the test serum, repeated freezing, thawing and shaking of the serum, addition of β and γ globulin fractions of human serum, or 2 above.
Combinations of more than one species have been used together. On the other hand, it is well known that BSA is added when sensitizing antigens or antibodies, but the amount usually added is o. Although it is said that the non-specific agglutination reaction can be suppressed to a considerable extent by adding ooi~1 times the weight, it cannot be completely suppressed.

These non-specific agglutination reactions do not measure the antigen or antibody that is the target of the test, but are agglutination reactions other than antigen-antibody reactions, so accurate measurement values cannot be obtained and there is a large possibility of erroneous diagnosis. It may be the cause. Therefore, when producing test reagents, a major point is how to suppress this non-specific agglutination reaction.

<Means for solving the problem> In order to solve these problems, we conducted various studies on suppressing non-specific agglutination reactions by surface treatment of latex.
The inventors have now completed the present invention by discovering that non-specific agglutination reactions can be reduced by treating the latex surface with a large amount of an immunologically inactive protein such as BSA.

That is, in the present invention, latex is sensitized with an antigen or antibody, and then treated with an immunologically inactive protein in an amount of 3 to 20 times the solid content of the latex, preferably 3 to 10 times the solid content. The invention relates to latex reagents.

In general, polystyrene-based latex used as a latex reagent is said to strongly adsorb antigens or antibodies simply by mixing them with latex when sensitizing antigens or antibodies. In particular, when attaching an antibody, it is said that the Fc portion of the antibody is well adsorbed, and as a result, the Fab portion faces outward, which is favorable for antigen-antibody reactions. This is because the polystyrene surface is hydrophobic, so the highly hydrophobic Fc of the antibody
This is because the parts are easily adsorbed. However, if the hydrophobic surface remains, non-specific agglutination reactions are likely to occur.

Therefore, attempts have been made to make the surface of latex itself hydrophilic by carboxylating it, but this tends to weaken the adsorption of antigens or antibodies, making the sensitization method difficult.

Therefore, the present inventors investigated a method of suppressing the non-specific agglutination reaction while maintaining the strong hydrophobicity of the latex surface. Non-specific agglutination reactions are thought to occur when a highly active hydrophobic surface remains on the surface of a latex reagent, and when mixed with serum, various proteins in the serum adsorb, resulting in the latex reagent aggregating. They discovered that non-specific agglutination reactions can be suppressed by sensitizing antigens or antibodies and treating the remaining surface with a large amount of immunologically inactive protein, and have perfected the present invention. .

As a method of suppressing non-specific agglutination reactions, it is known to add an immunologically inactive protein at the same time when sensitizing an antigen or antibody. The sensitization time is about 48 hours, but it is difficult to almost completely suppress the non-specific agglutination reaction under such conditions. Furthermore, even if treated under the same conditions after sensitization, complete suppression cannot be achieved. However, by treating with the method of the present invention, non-specific agglutination reactions can be almost completely suppressed.

The reason for this is thought to be that the latex latex, which is said to have a strong non-specific aggregation reaction, has a strong latex surface activity, which causes it to more strongly adsorb to easily adsorbable substances such as many proteins. Even in latex where such a non-specific agglutination reaction is likely to occur, the non-specific agglutination reaction can be suppressed by employing the method of the present invention.

In the present invention, after sensitizing antigens or antibodies, non-specific agglutination reactions are almost completely suppressed by treating the solid latex with an immunologically inactive protein 3 to 20 times the weight of the solid latex. be done. This is due to changes in the physical and chemical properties of the surface of the latex particles, which effectively cover the bare parts of the surface of the latex particles that are not sensitized with antigens or antibodies with immunologically inactive proteins. Conceivable.

In the present invention, the immunologically inactive protein used in an amount of 3 to 20 times by weight does not entirely adhere to the surface of the latex particles, but only a portion thereof. However, by using a large amount of immunologically inactive protein during the above treatment, a latex reagent in which non-specific agglutination reactions are significantly suppressed can be obtained.

In the present invention, various known latexes used in latex reagents can be used as the latex, and examples thereof include polystyrene latex, polyacrylate latex, etc., and are not particularly limited.

Furthermore, sensitization with antigens or antibodies can be carried out by conventional methods, and various antigens and antibodies can be used and are not particularly limited.

The immunologically inactive protein used in the present invention is BSA
Fetal calf serum (FCS) or fetal calf serum (FCS), including
) are suitable. These are preferable because they gently adsorb to latex particles and cause less deterioration in sensitivity. Strongly adsorbing materials such as casein and gelatin are not very preferred. When processing with an immunologically inactive protein, the concentration of the protein in the treatment solution containing the protein is preferably high, and a concentration of about 2 to 7% is easy to use. It is used by dissolving it in a buffered saline solution such as (abbreviated as ). The processing conditions are not particularly limited and may be generally used such as about 37°C or 4°C. The processing time is approximately 1 to 8 hours at 37°C and 8 to 48 hours at 4°C. According to the present invention, non-specific aggregation reactions caused by the protein adsorption properties of the latex carrier can be substantially prevented from occurring. Therefore, the reagent of the present invention can be used not only for on-plate measurement methods that do not require high sensitivity, but also for optical measurement methods or microtiter methods that have high sensitivity. In addition, in some cases, BSA or FCS is added to the finished diagnostic reagent to supplement the protein in the buffer solution in which the sensitized latex particles are suspended, and in this case, it is advantageous to use the same kind of protein as the protein used in the treatment. .

Therefore, according to the present invention, conventional heat inactivation of serum;
Measurements can be made without requiring complicated operations such as freezing, thawing, and shaking to remove insoluble fractions.

The concentration of latex particle solids in the latex reagent of the present invention typically ranges from 0.01 to 2% by weight.

The latex reagent obtained by the method of the present invention is extracted with a buffer solution with high pH and high salt concentration, and the eluate is quantified by high performance liquid chromatography.
I can recognize it.

<Example> Examples, comparative examples, and test examples will be given below to specifically explain.

Example 1 9 = 10 - 2 polystyrene latex (average particle size 0.25 μm)
% concentration in 5 ml of PB3, and anti-human fibrinogen antibody (rabbit IgG fraction 4 mg/d
5 (manufactured by Sevac) and shaken at room temperature for 2 hours.

Thereafter, the precipitate obtained by centrifugation at 8000 rpm x 30 min was resuspended in 10 ml of a 4% BSA PBS solution and shaken at room temperature for 8 hours. The precipitate obtained by centrifuging this was centrifuged and washed twice with PBS containing 0.3% BSA, and resuspended in PBS10rd containing 0.3% BSA to obtain an anti-human fibrinogen sensitized latex reagent. Ta.

Example 2 Boriacrolein latex (average particle size 1.5 μm) was suspended in PB310d at a concentration of 1%, and anti-human alphafetoprotein antibody (goat IgG fraction protein concentration 2.5 mg/rd) was added at 0.25 m 3 at 37°C.
Shake slowly for 0 minutes. Then 1500rpm
Centrifuge for 5 ml, add 10 d of 5% BSA in PBS to the sediment, disperse well, and shake slowly at 37°C for 4 hours. The precipitate obtained by centrifuging this was 0.3% B
Centrifugal washing twice with PBS containing SA, 0.3% BSA
An anti-human alpha-fetoprotein sensitized latex reagent was obtained by resuspending it in PBS40d containing the following.

Example 3 Treponema pal grown in Usagi Suimaru
Lidum (hereinafter abbreviated as TP) nichols strain was extracted with citrate buffer, centrifuged and collected, and 1-2X
The cells were resuspended in PBS at 109 cells/d.

This TP bacterial suspension was diluted 10 times with PBS and treated with an ultrasonicator at 9 KHz and 1800 W for 10 minutes to obtain an antigen solution containing TP bacterial fraction.

Polyacrolein latex (average particle size 1.8 μm)
was suspended in PB35mM at a concentration of 2%, and the T
Four P antigen solutions were added and the mixture was slowly shaken at 37°C for 30 minutes. Thereafter, 10 m of 5% BSA dissolved in PBS was added, and the mixture was further slowly shaken at 37°C for 5 hours. This was centrifugally washed twice with PBS containing 0.3% BSA, and
A TP antigen sensitization latex reagent was obtained by suspending it in PBS40d containing 3% BSA.

Comparative Example 1 Anti-human fibrinogen antibody 4. under the same conditions as Example 1.
Example 1 using 8 d and using 10 volumes of 1% BSA in PBS instead of 1 volume of 4% BSA in PBS.
An anti-human fibrinogen antibody sensitized latex reagent was obtained in the same manner as above.

Comparative Example 2 Using anti-human alpha-fetoprotein antibody 0.24 mA under the same conditions as Example 2, 5% BSA in PBS solution 10
A latex reagent sensitized with an anti-human alpha-fetoprotein antibody was obtained by using 10 m2 of a 1% BSA solution in PBS instead of the 1% BSA solution and treating in the same manner as in Example 2.

Comparative Example 3 Sensitized with TP antigen under the same conditions as in Example 3, using 1% BSA PBS solution If) m1 instead of 5% BSA PBS solution 10 ml, and treating in the same manner as Example 3. T.P.
An antigen-sensitized latex reagent was obtained.

Test Example 1 Serum is separated from the subject's blood by a conventional method and diluted 20 times, 40 times, and 80 times with PBS containing 0.3% BSA. Place each diluted serum onto a reaction slide plate at 50 μC.
25μ of the anti-human fibrinogen antibody sensitized latex reagent prepared in Example 1 and Comparative Example 1 was added dropwise to this.
1 was added dropwise, immediately mixed, and gently rotated with a slide ξ mixer. After 3 minutes, the presence or absence of aggregated images was observed with the naked eye. Since this reagent is adjusted to have a sensitivity of 0.25 μg/ml, if agglutination occurs at a dilution of 40 times or more, that is, if the sensitivity is 10 μgird or more, the sensitivity will deviate from the normal value.

The results of a comparison between Example 1 and Comparative Example 1 using the above method using a commercially available latex reagent for FDP testing and measurement using 50 serum samples with normal values (10 μg/1 or less) are shown below.

Note that this value indicates the number of cases in which agglutination occurred up to the dilution multiple out of 50 cases, and the example clearly judged the normal value more correctly.

14 Test Example 2 P containing 0.3% BSA in a 96-well U-shaped microplate
Drop 25 μl of BS from the 1st 6th point onward and add test serum 2.
5 μl was added to the 1st 6th tube to make a 2-fold dilution, and the mixture was serially diluted 2-fold using a GILUTTER. From the 2nd 6th point onwards, Example 2
Then, 25 μl of the anti-human alpha-fetoprotein antibody sensitized latex reagent prepared in Comparative Example 2 was added dropwise, thoroughly shaken with a plate shaker, and left at room temperature for 2 hours. The obtained agglutination image is judged visually, and the quantitative value is the value obtained by multiplying the dilution ratio of serum with agglutination by the sensitivity.

In Example 2 and Comparative Example 2, a 100 ng/ml standard solution of human alphafeI protein was positive for agglutination up to a 64-fold dilution, so the sensitivity was 1.5 ng/ml.

A test was conducted using the microtiter method described above using 50 cases of serum from normal subjects whose serum concentration was 1 Ong/ml or less as measured by RIA, and the results of comparing the two are shown below.

Note that this value indicates the number of cases in which agglutination occurred at the dilution rate among 50 cases of normal subjects, and the example clearly determined the normal value more accurately.

Test Example 3 Tested using the microtiter method in the same manner as Test Example 2,
100 μl of PBS containing 0.3% BSA was added to the first hole, and 25 μl of the test serum was added to make a 5-fold dilution in the first hole.Other operations were performed in the same manner as in Test Example 2. In addition, the dilution rate is the final dilution rate including the reagent, so the second size is 2.
This will be a 0x dilution.

A test was conducted using the above-mentioned microtiter method using 50 cases of serum from normal subjects whose concentration was 40 times or less when measured using a commercially available TPHA reagent, and the results of a comparison between the two are shown below.

Note that this value indicates the number of cases in which agglutination occurred at that dilution rate among 50 normal cases, and 80 times is generally used as the cutoff, so the example clearly shows the normal value more accurately. Judging.

<Effects of the Invention> Diagnostic reagents using latex are used for various test items because of their simple techniques. However, when producing diagnostic reagents, if a bare latex surface remains;
Non-specific agglutination reactions are likely to occur, and conventional non-specific agglutination reaction suppression methods have not been able to completely suppress them. According to the present invention, non-specific agglutination reactions can be almost completely suppressed through relatively simple processing, and only agglutination caused by antigen-antibody reactions can be measured.

Claims (1)

[Claims] A latex reagent obtained by sensitizing latex with an antigen or antibody and then treating the latex with an immunologically inactive protein in an amount of 3 to 20 times the solid content of the latex.