JPS61213673A - Production of reagent for immunological measurement - Google Patents

Abstract

PURPOSE:To improve the sensitivity of specific agglutination in a reagent for measuring an antigen and antibody by an agglutination reaction of agglutination inhibition reaction by treating an antibody or hapten-deltaglobulin composite sensitized with a carrier with a specific compd. CONSTITUTION:The reagent is produced by sensitizing the high-polymer antigen (protein, polypetide, etc.) and low-polymer hapten (steroid)-gammaglobulin composite with the carrier. A high polymer of pulverous particles, red blood cells, etc. are used for the carrier. The antibody, etc. are treated by bringing the same into contact with the specific compd., 2-mercaptoethanol, 2-mercaptoethylamine, thioglycolic acid, cysteine, dithiothreitol, dithioerythritol, etc. for a prescribed period in fore, middle and post processes for production, by which the antibody and composite are sensitized with the carrier. Since the reagent is obtd. in the above-mentioned manner, the non-specific agglutination inhibition reaction is suppressed even if extraneous materials are contained in the specimen. The agglutination or agglutination inhibition reaction is thus induced with good sensitivity and the reliability of the immunological measurement is improved.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application-1 The present invention relates to a method for producing a novel immunoassay reagent.
In detail, it is an immunoassay reagent that suppresses nonspecific agglutination reactions (aggregation other than specific agglutination based on antigen-antibody reactions) caused by contaminants present in the specimen and has increased specific agglutination reactivity. Concerning the manufacturing method.

"Technical Background and Prior Art" In recent years, it has become an extremely important issue in the medical field to quickly, easily, and accurately measure trace components in body fluids such as blood and urine for diagnosis. For this purpose, an immunologically active substance such as an antibody or an antigen is supported on a microparticle carrier by adsorption or binding (hereinafter referred to as "sensitization"), and the resulting sensitized carrier and the subject are Immunological measurement methods are widely used to measure the presence of a corresponding antigen or antibody by observing agglutination or agglutination inhibition reactions due to antigen-antibody reactions between the antigen and the corresponding immunologically active substance. Measurements using these sensitized microparticle carriers involve mixing and reacting with the specimen in a test tube or on a reaction plate to cause agglutination or agglutination inhibition reactions due to antigen-antibody reactions, and then capturing the agglutination (li!1 stop) image using an optical 7 instrument. The antigen or antibody in the specimen is measured either quantitatively or qualitatively based on the mechanical or macroscopic observation using the method.

In this method of detecting and measuring trace components in body fluids using immunological agglutination or agglutination inhibition reactions using sensitized particulate carriers, the most influential factor is nonspecific agglutination reactions caused by contaminants present in the specimen. It is. This becomes a particularly serious problem when the carrier is sensitized with an antibody or a hapten-gamma globulin complex (hereinafter abbreviated as "complex").

In other words, if non-specific agglutination occurs, a component in the sample may be incorrectly determined to be present when it is not originally present, or a component with a high abundance may be determined to be low, leading to problems with accuracy, reproducibility, etc. This results in a lack of reliability.

Therefore, it has a significant impact on diagnosis, determining prognosis, determining treatment methods, etc.

Many methods have been proposed for the purpose of preventing these non-specific agglutination reactions. For example, the method of adding various proteins (
JP-A-55-12419 and JP-A-57-972
Publication No. 3), method of selecting salt concentration or its type (
JP-A-57-35754, JP-A-57-1821
No. 68 and JP-A-58-75063), methods for modifying antibody molecules (JP-A-54-139595 and JP-A-59-224565), and methods for modifying antibody molecules such as choline chloride, penicillamine, and mercollidine. Method of adding specific substances (JP-A-54-26327, JP-A-5
9-92354 and JP-A-59-192962). As is clear from such conventional methods, the problem regarding the appearance of non-specific aggregation is a serious concern in the field of clinical analysis.

On the other hand, 2-mercaptoethanol, 2-mercaptoethylamine, thioglycolic acid, cysteine, dithiothreitol, and dithioerythritol have properties that are particularly susceptible to air oxidation in a solution state, and they also have the property of being susceptible to air oxidation when mixed with proteins, etc. Disulfide (s-s) in the molecule
) Reduces bonds, destroys higher-order structure, and eliminates biological activity.

Therefore, conventionally, the carrier was not sensitized by contact treatment of antibodies or macropurine with these compounds.

As a result of repeated research on the prevention of non-specific agglutination reactions in immunological agglutination (inhibition) reactions, the present inventors found that contact treatment of antibodies or complexes with the above compound for a relatively short period of time (within 24 hours) The present invention was achieved based on the discovery that non-specific agglutination reactions, which are disadvantageous to immunological agglutination (inhibition) reactions, can be eliminated and the sensitivity of specific agglutination can be increased.

``Object of the present invention and summary of the invention'' The object of the present invention is to provide a reagent that enables immunoassays to be performed more easily than before and without being affected by contaminants in a sample. be.

Another object of the present invention is to provide a method for producing an immunoassay reagent that suppresses nonspecific a-aggregate reactions and has high sample sensitivity.

The present invention is for immunologically measuring trace components in a specimen by an agglutination reaction or an agglutination inhibition reaction using a sensitized carrier sensitized with an antibody carrier or a sensitized carrier sensitized with a complex as a carrier. 2-mercaptoethanol, 2-mercaptoethylamine, thioglycolic acid, cysteine, dithiothreitol, dithioglycolic acid, etc. This is a method for producing an immunoassay reagent, which comprises contacting with at least one compound selected from the group consisting of erythritol and mixtures thereof, and then removing the compound.

[Detailed Description of the Invention] In the method of the present invention, the antibody used is a polymeric antigen (
(e.g. proteins, polypeptides, polysaccharides, lipids, etc.) or low molecular haptens (e.g. steroids, peptides, drugs, etc.)
It may be an antiserum obtained by immunizing an animal by a conventional method with a complex made by binding a complex to a molecular substance such as serum albumin, or a culture solution of antibody-producing cells, and the type of antibody does not matter. Although it is possible to use the solution as it is, it is preferable to use it after fractionation and purification using a known method.The haptens in the complex include steroid hormones, low-molecular peptide hormones, non-peptide hormones, and catecholamines. lemma, enzymes, vitamins, pharmaceutical substances, antibiotics, and their metabolites, etc. Gamma globulin is commercially available normal gamma globulin or its components from various mammals, or gamma globulin or its components obtained by fractionation and purification from the blood of normal animals that have not received hyperimmunity. These haptens and gamma globulins are chemically bonded by the known carbodiimide method, mixed acid anhydride method, incyanate method, etc., and used as a composite. The macropurine moiety in these antibodies or complexes can also be chemically modified with an acylating agent such as acetic anhydride, succinic anhydride, maleic anhydride, etc.
24565).

The carrier used in the method of the present invention is fine particles of organic polymer compounds, red blood cells, inorganic compounds, etc., and preferably latex fine particles such as polystyrene, polyvinyltoluentadiene, styrene-butadiene copolymer, styrene-acrylonitrile copolymer, etc. Or these polymers partially have functional groups such as carboxyl groups and amide groups on their surfaces.

The buffer used in the method of the present invention is a pH 6-10 buffer commonly used in immunology, and is usually glycine buffered saline (hereinafter abbreviated as [GBsJ) or the like.

Next, the carrier is sensitized by physically adsorbing the antibody or the complex or chemically covalently bonding it to the carrier by a conventional method. For example, when using an adsorption method, a microparticle carrier is added to a buffer solution in which the antibody or complex is dissolved, stirred or shaken, and heated if necessary to sensitize the carrier with the antibody or complex. In the case of a covalent bonding method, for example, a water-soluble carboimide or the like as a coupling agent is mixed with a suspension of latex particles having carboxyl groups on the surface while stirring under water cooling or at room temperature. Thereafter, a solution of the antibody or complex is added to this solution and stirred for sensitization.

Next, the sensitized carrier solution obtained in this way is centrifuged, and the precipitate is diluted with a buffer solution containing proteins, etc., usually at a concentration of 0.01-
3% (w/v), preferably 0.05-1.5% (w/v)
Resuspend at a concentration of V) to obtain a reagent.

All of these production methods are well known, but the present invention provides a process for producing reagents using these known techniques, in which the antibody or complex that sensitizes the carrier is made of the six compounds mentioned above and a mixture thereof. It is characterized by contact treatment with at least one compound selected from the group, and then removing the compound.

In the method of the present invention, the contact treatment means that the reagent is produced while the above-mentioned compound is present.

Specific embodiments of the contact treatment in the method of the present invention are as follows.

1) Method of contact treatment before sensitizing the carrier The antibody or complex before sensitizing the carrier is added to a buffered salt solution containing the compound and maintained under the conditions described below.

2) Method of contact treatment during sensitization The compound is added to the carrier in the step of sensitizing the antibody or complex.

3) Method of contact treatment after sensitization After sensitizing the carrier with the antibody or complex, adding the compound,
It is held under the conditions described below.

4) Method of contact treatment until sensitization The antibody or complex is allowed to coexist with the carrier until the step of sensitizing the carrier.

5) Method of continuous contact treatment after sensitization After the step of sensitizing the antibody or complex to the carrier, the compound is allowed to coexist continuously after the step of sensitizing the carrier.

6) Method of continuous contact treatment throughout the entire process All steps are carried out in the presence of the compound.

Among the above six contact treatment methods, method 4) is the most desirable.

Since all the compounds used in the treatment of the present invention are susceptible to air oxidation in a solution state, it is desirable to prepare and use them freshly. In addition, poorly soluble compounds are dissolved in the form of metal salts or hydrochlorides, and used in an aqueous solution or buffer having a pH of 6-10, preferably p++ 7-9.

The contact treatment concentration with the compound was 5-5- as the final concentration.
1000, preferably 225-500a. The treatment concentration is 2-56°C, preferably 15-37°C. The treatment time is 1 minute to 24 hours, preferably 5 minutes to 5 hours. These contact treatment conditions can be appropriately selected depending on the type of compound used, the properties of the antibody or complex, and the sensitivity of the intended reagent.

After the contact treatment, the compound is removed, and it is necessary to remove the used compound by dialysis, ultrafiltration, centrifugation, etc. If the contact treatment is continued for a long time, non-specific agglutination reactivity will reoccur and specific reactivity will disappear.

The reagent thus produced by the method of the present invention is used in immunoassays using the known agglutination (inhibition) reaction. For example, a certain amount of a test liquid such as a body fluid or urine or a diluted test liquid is taken on a reaction plate, and a certain amount of a buffer solution is added as necessary. When the component to be measured is a molecular antigen, when a certain amount of the antibody-sensitized microparticle carrier reagent according to the method of the present invention is added and reacted, if the component is present at or above the detection sensitivity of the reagent, agglutination will occur. If the component to be measured is a low-molecular-weight hapten, the hapten in the specimen is neutralized by adding a certain amount of an appropriately diluted hapten antibody or a hapten antibody-sensitized microparticle carrier according to the method of the present invention, and then the method according to the present invention If the hapten is present at or above the detection sensitivity of the reagent, it is used for agglutination inhibition measurement. The reagent produced by the method of the present invention suppresses non-specific agglutination reactions caused by contaminants in the specimen, and causes agglutination (inhibition) reactions with good sensitivity, allowing fine particle components to be detected more accurately and reliably. It is highly measurable and has great benefits in the field of clinical analysis.

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 A goat was immunized with purified human chorionic gonadotropin (hereinafter abbreviated as jHcG-1) by a conventional method to obtain anti-11c serum. To 50 ml of the obtained anti-11cc serum was added 1125 ml of a saturated solution of ammonium sulfate. After stirring for 20 minutes under water cooling, centrifugation was performed for 20 minutes at 1G and 0OOOr4, and the resulting precipitate was collected.The same operation was repeated two more times, and the final precipitate was diluted with 10mM +7N. Acid buffer a (
The immunoglobulin was dissolved in 100% pH 8) and fractionated by Delfiltration using a Sephadex G-25 column equilibrated with the same buffer, and the resulting fraction was lyophilized.
Approximately 0.5 g of 1cG antibody was obtained. This anti-HCG antibody
CBS containing 0 mM 2-mercaptoethanol (pH
8,2) at a concentration of 0.1% (,/V) and heated at 37°C.
It was kept warm for 30 minutes. One volume of this solution was mixed with one volume of polystyrene latex particles dispersed in CBS at a concentration of 2% (w/v), kept at 37°C for 1 hour, and then 10t
Centrifugation was carried out at OOOrpm for 30 minutes, and the resulting precipitate was mixed with 0.2% (-/V) bovine serum albumin (rBS^).
1% (tw/V) in Cll5 containing
Resuspend the latex reagent for HCG detection at a concentration of approx.
1 was manufactured.

Example 2 CBS containing 200ml 2-mercaptoethanol
About 19 ml of a latex reagent for HCG detection was produced in the same manner as in Example 1, except that CBS containing 2-mercaptoethylamine hydrochloride at the same concentration was used instead.

Example 3 HCG detection latex reagent approximately 19-1 was prepared in the same manner as in Example 1, except that CBS containing the same concentration of sodium thioglycolate was used instead of Gas containing 200 mM 2-flucaptoethanol. Manufactured.

Example 4 About 19 ml of a latex reagent for HCG detection was produced in the same manner as in Example 1, except that instead of GBs containing 200 mM 2-mercaptoethanol, CBS containing cysteine hydrochloride at the same concentration was used.

Example 5 Instead of CBS containing 200-2-mercaptoethanol, CB containing 100a+H dithiothreitol
A latex reagent for HCG detection, about 19a1, was produced in the same manner as in Example 1 except that S was used.

Example 6 CBS containing 200n+H 2-mercaptoethanol
IIc in the same manner as in Example 1 except that CBS containing 100+aM notothioerythritol was used instead of
Approximately 19 ml of latex reagent for GIc was produced.

Example 7 Antiserum was obtained by immunizing a rabbit with highly purified human fibrinodenum (hereinafter abbreviated as rFgJ). 50 ml of the obtained antiserum was added to Fg-Cef70 prepared by a conventional method.
-The solution was passed through a column packed with 4B. Then 10m
Wash the column by passing M phosphate buffer (pH 8) through it,
After removing unadsorbed substances other than anti-Fg immunoglobulin, anti-F and immunoglobulin were eluted with glycine-hydrochloric acid buffer a (pH 2, 3), and the pH of the eluate was adjusted to 7.5. This liquid was subjected to ultrafiltration to remove unnecessary substances, and then lyophilized to obtain about 75 mg of anti-F antibody. 3mg of this antibody
was dissolved in GBs (pH 8) at a concentration of 0.03% (w/V) and 2% (w/V) polystyrene latex particles.
/V) mixed with 1 volume of B suspended in GBS at a concentration of 4
Hold at 5°C for 30 min, then incubate at 10,0 OOr4 for 30 min.
After centrifugation for 1 min, the resulting precipitate was resuspended in CBS containing 100 mM 2-mercaptoethanol at a concentration of 1% (w/v), kept at room temperature for 1 h, and then resuspended at 10,000 r
The precipitate obtained by centrifugation for 30 minutes at 0.
1% (w/v) CBS containing 2% (w/v) BS^
Resuspend at the concentration of v) and make about 19m of latex for Fg detection.
l was produced.

Example 8 In Example 7, instead of using the purified anti-Fg antibody to sensitize the carrier, succinyl anti-F, which was prepared as described below, was used in the same manner as in Example 7. , 19ml of latex reagent for detection was manufactured.

The method for preparing the succinyl anti-Fg antibody is as follows.

1 g of purified anti-F antibody 30II obtained in Example 7 was added to 1% (w
/v) in water and adjusted to pH using N-NaOH.
was adjusted to 8. While stirring this solution in ice water, 1 g of succinic anhydride powder 2II was gradually added, and N-Na0
11 to dissolve the succinic anhydride while maintaining the pH above 7, then continue stirring for an additional 30 minutes, dialyze against water overnight, and lyophilize to obtain succinyl anti-F, antibody approx.
8 mg was obtained.

Example 9 Estriol-16α-glucuronide (hereinafter referred to as [E.

-16 (abbreviated as X-GJ) 58 g was dissolved in 1 ml of trimethylformamide, 30 μm of tri-n-butylamine was added thereto, and then 17 μm of inbutyl chloroformate was added and mixed with stirring to prepare Solution A. did. On the other hand, beef tuna groin purine (hereinafter abbreviated as BGGJ) 1. O
Dissolve g in 50 ml of water, adjust I)H to 9.5 with N-NaOH, and after dissolving, dimethylformamide for 4 hours.
1 was added to prepare Solution B. The previously prepared solution A was added dropwise to this solution B, stirred for 3.5 hours, and E3-16cr-
A solution containing the G-[ICG complex was prepared. Add 1 volume of 7 setone to 2 volumes of the obtained solution, mix uniformly, and add N
-IC+ was added to precipitate the complex and centrifuged. The separated precipitate was dissolved in water, dialyzed against water, and unnecessary substances were removed to give an E, -16a -G-BCG complex of about 0.9. I got it. All of these operations were performed at 5°C. This E
, 0.1% G complex Z5+mH to 1-16α-G-B
Cm/V) in GBs (pH 8) containing 300 mM 2-mercaptoethanol and kept at room temperature for 1 hour. One volume of this solution was mixed with one volume of polystyrene latex particles dispersed in G[lS at a concentration of 2% (w/v), kept at 37°C for 1 hour, and then
Centrifuge for 30 minutes at r4, and the resulting precipitate is 0.2%
Resuspend at a concentration of 1% (w/v) in CBS containing (w/v) BS^, Tex reagent for detection of estroden, approximately 4
7ml was produced.

Example 10 Anti-HCG immunoglobulin about 85 I
I got IFI. This antibody was succinylated in the same manner as in Example 8, and the succinyl anti-HCG antibody was approximately 80Il1
g was prepared. Antibody 15 was added to the obtained succinyl anti-+1C.
+ag 0°159! Solution 1 dissolved at a concentration of (w/v)
1 volume of a 4% (w/v) suspension of latex particles containing carboxyl groups centrifugally washed twice with water and 500-
of 2-mercaptoethanol was added and mixed.

Next, 1 volume of a freshly prepared 1% (W/V) 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimidometh-p-toluenesulfonic acid solution was added, and the mixture was allowed to react overnight with stirring at room temperature for sensitization. . After centrifuging the reaction solution and washing twice with Ga5 (pH 8,2) to remove 2-furcaptoethanol, the precipitate was diluted to 0.1% (tu/V
) was suspended in CBS containing BS^ at a concentration of 1% (w/v) to produce about 40 ml of a latex reagent for 1fcG detection.

Comparative Example 1 IIC (: detection A latex reagent was manufactured for

This is substantially equivalent to conventional reagents. The detection sensitivity and non-specific agglutination reactivity of this conventional reagent and the latex reagents for ICG detection produced in Examples 1 to 6 were measured and compared as follows, and the results are shown in Table 1.

Standard HCG in 0.1% (u+/v) BS at the concentration shown in the table.
Three types of standard solutions, non-pregnant women A and B, dissolved in ＾containing CBS (pH 8).

C urine or pregnant woman's urine, E urine 50 μm each as buffer solution, CBS 25 μm and conventional method reagent or Example 1
25 μm of each of the reagents produced in Example 6 was dropped onto a reaction plate and mixed. The reaction plate was then gently rocked for 3 minutes, and the presence or absence of aggregation was visually observed.

Table 1 As is clear from Table 1, the reagents produced by the method of the present invention all showed increased measurement sensitivity, whereas the reagents of the conventional method showed non-specific @ collection in non-pregnant women's urine. The results show that pregnancy or non-pregnancy can be accurately determined, with no non-specific agglutination caused by contaminants in the specimen. Furthermore, the results of determination using a standard solution that does not contain contaminants indicate that the detection sensitivity has been improved.

Comparative Example 2 E, -16α-G-BCG complex in Example 9,
An estrogen detection reagent was produced in the same manner as in Example 9, except that instead of the contact treatment with CBS containing 2-furcaptoethanol, a fake treatment was performed with only CBS. This is substantially equivalent to conventional reagents. The non-specific reactivity of this conventional reagent and the estrogen detection reagent produced by the method of Example 9 was measured and compared as follows, and the results are shown in Table 2.

First, 3!mM standard solutions of E, -16α (-16α) were dissolved in CBS (pt18) containing 0.1% (w/v) BS^ at different concentrations (as shown in the table) and Ea prepared by a conventional method.
-16ff<-BS^ complex was used as an antigen to immunize a family member, and the obtained anti-E, -16a (antiserum was absorbed with BS8, then anti-E, -16α (antibody) was diluted appropriately with a buffer solution). Using these, the measurement sensitivity of the estrogen detection reagents produced by the conventional method and the method of the present invention was measured as follows. E3-16α-G (with a detection sensitivity of 0.2 μl/ml) (as estriol) was prepared.

50 μm each of multiple types of E and -16α-G standard solutions with different concentrations and 20 μm each of appropriately diluted anti-E3-16α (antibody) were dropped onto the reaction plate and mixed. Next,
Then, 20 μm of the reagent produced by the conventional method or the method of the present invention was dropped into the mixed solution, and the reaction plate was gently rocked for 2 minutes to visually observe whether or not aggregation was inhibited. The lowest concentration that inhibits aggregation is the detection sensitivity of the reagent. By the above method, using the reagent of the conventional method and the reagent according to Example 9, which have the same detection sensitivity for a standard solution free of contaminants, o
r 5teroid BiocbemisLry+10
Vol., p. 443, 1979) and estrogen is 5 μge.
The presence or absence of non-specific agglutination was compared by measuring 50 μm of each of the urine of pregnant women A, B, and C, which was confirmed to contain ml or more, and the results are shown in Table 2.

! 22 As is clear from the table, the conventional reagent does not prevent agglutination even though estrogen is present in urine of pregnant women at a level exceeding the reagent's detection sensitivity, resulting in nonspecific agglutination and erroneous determination. It turns out.

However, the reagent produced by the method of the present invention showed inhibition of aggregation in pregnant women's urine, indicating that estrogen was present at a level exceeding the detection sensitivity of the reagent, and also showed aggregation in men's urine, where the estrogen concentration was extremely low, indicating that the estrogen concentration was higher than that of the reagent. It is correctly determined that the detection sensitivity is below. This also shows the usefulness of the present invention.

"Effect of the invention" The effects achieved by the present invention are as follows.

(1) A reagent can be obtained that allows accurate determination without being affected by contaminants such as non-specific agglutination factors present in the specimen.

(2) A reagent with improved measurement sensitivity and excellent specificity can be obtained.

(3) Reagent production is extremely simple.

Claims (8)

[Claims] (1) Using a sensitized carrier sensitized with an antibody or a hapten-gamma globulin complex as a reagent, the antigen or antibody in the sample is immunized by an agglutination reaction or an agglutination inhibition reaction. In the method for producing the above-mentioned reagent for chemical measurement, the antibody or hapten-gamma globulin complex that sensitizes the carrier is mixed with 2-mercaptoethanol, 2-mercaptoethylamine, thioglycolic acid, 1. A method for producing an immunoassay reagent, which comprises contacting with at least one compound selected from the group consisting of cysteine, dithiothreitol, dithioerythritol, and mixtures thereof, and then removing the compound. (2) The method for producing an immunoassay reagent according to claim 1, wherein the contact treatment is performed before sensitization. (3) The method for producing an immunoassay reagent according to claim 1, wherein the contact treatment is performed at the time of sensitization. (4) The method for producing an immunoassay reagent according to claim 1, wherein the contact treatment is performed after sensitization. (5) The method for producing an immunoassay reagent according to claim 1, wherein the contact treatment is continued until sensitization. (6) The method for producing an immunoassay reagent according to claim 1, wherein the contact treatment is carried out continuously from the time of sensitization. (7) The method for producing an immunoassay reagent according to claim 1, wherein the contact treatment is carried out throughout the entire process. (8) Contact treatment at a concentration of 5-1000mM, 2-56℃
8. The method for producing an immunoassay reagent according to claim 1, wherein the method is carried out at a temperature of 1 minute to 24 hours.