JPS59102161A - Antigen detection reagent by anti-passive agglutination - Google Patents

Abstract

PURPOSE:To enable the detection of an antigen at high accuracy by adding serum or serum globulin fraction treated with a high density salt solution, a low pH solution or a high pH solution to an antigen detection reagent by an antipassive agglutination to eliminate obstruction of a factor causing a non- specific coagulation. CONSTITUTION:1 or more mol of a high density solution of a chloride of alkali (earth) metal, thiocyanate, acetate or nitrate, a more than 3 mol of urea solution, a solution or a buffer with pH 4 or less of glycine-hydrochloride, a solution or a buffer liquid with pH 9 or more of boric acid-borax, glycine-NaOH or the like is used as a treating liquid. A 1-3W/V% solution of serum of serum globurin fraction of a human being or an animal subjected to a dialysis with any treating liquid as mentioned above and then, with a saline solution or a phosphoric acid buffer salt to obtain a treated serum (globurin fraction) solution. This solution is added to an anti-passive agglutination reagent (or a diluted solution) having an antibody sensitized on a carrier (blood cell or the like) at the rate of 1mug/1mg/ ml in the case of the treated serum, and 0.2mug-0.2mg/ml. Thus, non-specific coagulation is eliminated.

Description

Detailed Description of the Invention The present invention relates to an antigen detection reagent and an antigen detection method using reverse passive agglutination reaction, and more specifically, a special treatment for the reaction solution (and/or dilution solution) used for reverse passive agglutination reaction. The present invention relates to an antigen detection reagent that eliminates non-specific agglutination reactions, which is obtained by adding human or animal serum that has been subjected to a treatment, and an antigen detection method using the treated serum.

One method of detecting antigens using an immunological reaction is generally to use a reaction called reverse passive agglutination reaction, in which a carrier is sensitized (adsorbed) with an antibody, and the corresponding antigen is applied to the carrier. This method detects the aggregated image of the antibody-sensitized carrier produced by the method, and is widely used for detecting and quantifying trace amounts of antigens contained in biological body fluids because of its ease of operation and good sensitivity and accuracy.

In this method, for example, when using blood cells as a carrier,
Blood cells, which serve as carriers, are first fixed with formalin or glugulaldehyde, and the fixed blood cells are sensitized (adsorbed) with antibodies corresponding to the antigen to be tested. A reaction solution containing an antibody sensitized carrier is prepared by suspending it in a buffered saline solution (abbreviated as CPBS), which is also referred to as a diluent because it is also used to dilute the specimen. Diluted test serum 5 is added and reacted, and after a certain period of time, the sensitized hemagglutination image produced by reverse passive agglutination reaction is determined. However, in addition to the specific antigen-antibody reaction between a specific antibody and antigen, this reverse passive agglutination reaction is often accompanied by a non-specific agglutination reaction, which poses a problem in testing.

This non-specific aggregation reaction includes natural agglutination that is not mediated by any antibodies, agglutination reaction that is mediated by true antibodies called heterogeneous agglutination,
Furthermore, there are aggregations caused by rheumatoid factors (antibodies against immunoglobulins). Among these, it is known that natural aggregation can be removed by various methods6, but a sufficient method for removing antibody-mediated non-specific reactions has not yet been found. In general, as a method for removing non-specific aggregation, for example, one animal serum or its components, various animal blood cell membrane components, a surfactant such as Triton However, this method does not completely remove antibody-like substances that cause non-specific reactions, and the solution containing such unremovable antibody-like substances remains. The reality is that an extremely large number of serum samples are tested positive for the antigen.

Under these circumstances, the present inventors have conducted various studies in order to completely eliminate the non-specific agglutination caused by antibodies as described above and to obtain an antigen detection reagent that can always perform accurate detection. We have discovered that by adding human or animal serum that has been subjected to a specific treatment to a reaction solution containing an antibody-sensitized carrier, a reagent for detecting an antigen can be obtained by a reverse passive agglutination reaction that is not accompanied by non-specific agglutination, and has developed the present invention. It was completed.

That is, in the present invention, a reaction solution (and/or dilution solution) used in an antigen detection method using a reverse passive agglutination reaction is treated with a treatment solution selected from a high concentration salt solution, a low p)I solution, and a high pH solution. The present invention provides an antigen detection reagent to which human or animal serum is added.

The present invention also provides reverse passive agglutination by adding human or animal serum subjected to the above specific treatment to a reaction solution containing an antibody sensitized carrier and/or a diluted solution containing a test antigen used in an antigen detection method by reverse passive agglutination reaction. The present invention also provides an antigen detection method that eliminates non-specific agglutination reactions by performing a reaction.

In this specification, the reagent for antigen detection refers to a reaction solution containing an antibody-sensitized carrier, which is generally used for antigen detection by this type of reverse passive agglutination reaction, and a control solution containing an unsensitized carrier that is not sensitized with an antibody. It means a reaction solution for diluting a sample serum, a diluent for diluting a sample serum, a reaction solution before suspending the carrier, and a freeze-dried product of the reaction solution and dilution solution. Therefore, in the present invention, human or animal serum or serum globulin fraction that has been subjected to a specific treatment is added to either or both of the reaction solution containing or not containing the carrier and the dilution solution. Note that the reaction solution and dilution solution used in this reverse passive agglutination-based antigen detection method generally have the same composition, and therefore both the reaction solution and dilution solution are sometimes simply referred to as the reaction solution. . In addition, serum that has been subjected to specific treatments contains treated serum globulin fractions in addition to treated serum, and these are sometimes simply referred to as treated serum.

The present invention is characterized by using human or animal serum treated with a specific treatment solution, preferably the immunoglobulin fraction of the serum, and adding this to a reaction solution and/or dilution solution for antigen detection by reverse passive agglutination reaction. This method eliminates nonspecific agglutination reactions that often accompany reverse passive agglutination reactions, making it possible to detect antigens accurately and with high sensitivity.

In addition to human serum, the above-mentioned serum used in the invention includes guinea pig, rat, rabbit, sheep, goat, monkey, pig,
Contains serum from animals such as cow, horse, chicken) IJ, duck, turkey, and pigeon. As mentioned above, this human or animal serum also includes a serum globulin fraction, and may be hereinafter simply referred to as human or animal serum.

The processing liquid for treating human or animal serum used in the present invention includes alkali metal or alkaline earth metal chloride, thiocyanate-acetate or nitrate;
or highly concentrated salt solutions having a concentration range from 1 molar to close to the saturation concentration of urea, such as 2-6 molar magnesium chloride solution, 2-6 molar calcium chloride solution, 2-6 molar sodium chloride solution, 2-6 molar chloride solution. potassium solution,
2-18 molar lithium chloride solution, 2-15 molar sodium thiocyanate solution, 2-15 molar potassium thiocyanate solution, 2-15 molar magnesium thiocyanate solution -
2-15 molar lithium thiocyanate solution, 2-15 molar calcium thiocyanate solution, 2-15 molar sodium acetate solution, 2-15 molar magnesium acetate solution, 1-9
Mol? in2 sodium solution - 1-3.5 molar potassium nitrate solution, 1-4.5 molar magnesium nitrate solution, 3-
No molar urea solution, etc.; Low pH solution below pI-14, such as glycine-hydrochloric acid, citric acid-sodium phosphate-sodium citrate-hydrochloric acid, etc., preferably pH below 4, preferably PI-(2.0 to 4.0). Aqueous acid solution or buffer;
High pH solutions with a pl-19 or higher, such as boric acid-borax, boric acid-sodium hydroxide, borax-sodium hydroxide, glycine-sodium hydroxide, etc., with a pH of 9 or higher, preferably pH 9,0-12,0. Examples include aqueous alkaline solutions or buffer solutions. Treatment with these treatment solutions usually involves treatment with a concentration of 0.1 to 0.1 to
10 w/v%, preferably 1-3 w/v% solution,
Dialysis is performed in the above saline solution, preferably a 2 to 5 molar solution, a low pH solution or a high pH solution, preferably a 0.05 to 0.3 molar solution, for about 10 minutes to 5 hours, followed by physiological saline or phosphate buffer. This is done by dialysis against a saline solution.

By combining the human or animal serum (or serum globulin fraction) obtained by the above treatment with the reaction solution and/or dilution solution for reverse passive agglutination reaction, certain non-contaminants that could not be removed by conventional methods can be removed. Antibody-like substances that cause specific agglutination reactions can be removed very effectively. In other words, this non-specific antibody-like substance is a type of anti-globulin antibody present in the sample, which reacts with antibody-sensitized blood cells whose properties (antigenicity) have been partially changed during the purification or sensitization process. Although this anti-globulin antibody cannot be absorbed by untreated animal serum, it is absorbed by human or animal serum that has undergone the above specific treatment, especially in the serum clopurin fraction. High affinity allows for effective absorption and removal. This treated oil serum or serum globulin fraction competes with the sensitized antibody and reacts with the anti-globulin antibody, but the treated serum or serum globulin fraction, which has smaller molecules and greater motility, binds more easily to the anti-globulin antibody.

The treated serum or serum globulin fraction in the present invention is
It can be added to either or both of the reaction solution for the carrier and the dilution solution for the analyte.

The effect of suppressing non-specific agglutination reactions is best when added to both, but a sufficient suppressive effect can be seen even when added to only one. The carrier-containing solution to which this treated serum or globulin fraction has been added can also be lyophilized, without reducing the non-specific aggregation inhibiting effect. The part weight of this treated serum is 1 μg to 1 μg/ml, preferably 5 μg to 1 μg/ml.
00μg/resistant. In the case of treated serum globulin fraction, 0.2μy-~0.2μ, preferably lμg~20μQ
/ys/.

As the carrier, commonly used carriers can be used as they are, such as red blood cells, synthetic latex (usually particle size 0.1 to 1.3μ, preferably 05 to 1μ), etc., which are usually used in the reaction solution. It is used in the form of a suspension. An antibody-sensitized carrier can be obtained by adsorbing and sensitizing an antibody corresponding to the antigen to be detected onto the carrier using a conventional method.

Although a significant anti-globulin antibody absorption effect can be achieved even when the antibodies used for sensitization and the treated serum or serum globulin fraction added are obtained from animals of different species, it is preferable to use serum from animals of the same species.

Also, a reaction solution (diluent) that is commonly used may be used, such as physiological saline, phosphate buffered saline (CPBS), and the like.

The uninvented antigen detection reagent can be used for the detection and quantification of various antigens, such as hepatitis B, irrigin antigen, α-fetoprotein, C-reactive flotin (CRP), and other trace antigens. Applicable.

EXAMPLES Next, the present invention will be specifically explained with reference to Examples, but the present invention is not limited thereto.

Example 1 (a) Preparation of reaction solution (diluent) PBS (disodium monohydrogen phosphate 17.08f, monopotassium dihydrogen phosphate 2.59 IQ and salt 4.9
Buffer solution prepared by dissolving 1 g in 1 liter of water, pH 7
, 2) to prepare a reaction solution (diluent) by adding healthy rabbit serum lV/V% - sheep blood cell membrane component 0.01 W/V% and nitriding) 11um (preservative) 0.1w/v%. do.

(b) Preparation of reaction solution with addition of untreated guinea pig serum and the guinea pig serum globulin fraction 8 Add 0.5 q/w of healthy guinea pig serum to the above reaction solution,
1 (in terms of protein content) to obtain a "control reaction solution". Similarly, the healthy guinea pig serum globulin fraction was measured at 0.
A "control reaction solution (■)" was obtained by adding the reaction mixture at a ratio of (1■/1alc protein amount).

(c) Preparation of guinea pig serum addition reaction solution treated with magnesium chloride - Dialyze guinea pig serum in 100 times volume of 5M aqueous magnesium chloride solution at room temperature for 2 hours, then use 1000 times volume of physiological saline solution Magnesium chloride is removed by dialysis at ℃ for 16 hours to obtain magnesium chloride-treated guinea pig serum. This serum was added to the reaction solution obtained in (a) above at a ratio of 0.05rng/+x/(in terms of protein amount).
Obtain the serum-added reaction solution ■.

Example 2 Preparation of reaction solution to which globulin fractions of human and various animal sera treated with various treatment solutions were added ~ Example 1 (c) above
Similarly, human, guinea pig-rabbit, goat, and chicken serum globulin fractions (1 w/v % physiological saline) were added to 100 times the volume of a 5 molar aqueous magnesium chloride solution, a 5 molar calcium chloride aqueous solution, and a 3 molar sodium thiocyanate solution, respectively. Aqueous solution - Dialyzed in 5 molar urea aqueous solution, 0.1 molar glycine-hydrochloric acid buffer (pH 2,5) and 0.1 molar boric acid-borax buffer CPHIOI at room temperature for 2 hours, and further dialyzed in each physiological saline solution. Each drug is removed and the pH is returned to neutral to obtain various treated serum globulin fractions. These treated serum globulin fractions were 0.01
Example 1 (above) at the ratio of 'f/l (as protein amount)
Add to the reaction solution prepared in step (b) to obtain a reaction solution containing various serum globulin fractions.

Example Suppression experiment of non-specific reaction in detection of HBs antigen-α-fetoprotein by reverse passive hemagglutination reaction Reaction solution obtained in Example 1 above, control reaction solution ■ and ■
, serum addition reaction solution ■, and serum globulin fraction addition reaction solution obtained in Example 2, guinea pig magnesium chloride solution-treated serum globulin fraction addition reaction solution C (hereinafter referred to as serum addition reaction solution ■). Example 1 (a)
Using the reaction solution (conventional product), three specimens (human serum) A, B, and C showing nonspecific agglutination reactions were tested.

Pour each reaction solution into 8 wells of a microplate (CV type) with a 25μ
Place 25 μl of the sample into the first hole, serially dilute it with diluent, and sensitize it with guinea pig HBs antibody or guinea pig α-fetoprotein antibody highly purified by affinity chromatography. 0,
25 μm of 6% sheep-fixed red blood cell solution (carrier) was added dropwise, mixed, and allowed to stand at room temperature for 2 hours, after which the agglutination image was observed.

The results are shown in Table 1.

As is clear from Table 1, agglutination due to non-specific reaction was observed in the conventional product reaction solution and control reaction solutions ■ and ■ containing untreated guinea pig serum or its globulin fraction, whereas agglutination due to non-specific reaction was observed. Alternatively, when using serum-added reaction solutions (■) and (■) to which a serum globulin fraction was added, the agglutination reaction was negative, indicating that non-specific reactants were removed.

Example Non-specific reaction suppression experiment in detecting HBs antigen by reverse passive hemagglutination reaction The reaction solution obtained in the above example] (a) (conventional product) and the reaction solution obtained in Example 2 with addition of various serum globulin fractions sample B (which shows a non-specific reaction with the conventional reaction solution)
Human serum) was tested in the same manner as in Example 1 above. The results are shown in Table 2.

As is clear from Table 2, all of the reaction solutions to which the purified globulin fraction treated according to the present invention was added exhibited the effect of suppressing non-specific reactions, and in particular, the effect of suppressing non-specific reactions was particularly high when the reaction solution was added to the treated globulin fraction of the same species as the antibody used for blood cell sensitization. The strongest inhibitory effect was obtained by adding a serum globulin fraction obtained by treating guinea pig serum with a magnesium chloride solution, a sodium thiocyanate solution, or a glycine-hydrochloric acid buffer.

Table 2 (continued) Example 3 Sheep blood cell fluid C sensitized with guinea pig HBs antibody:
The magnesium chloride-treated guinea pig serum globulin fraction obtained in the same manner as in Example 2 was added to the control blood cell fluid at a ratio of 0.001 mm/ml protein amount), and the treated serum globulin fraction was added to prepare antibodies. A sensitized carrier-containing reaction solution c (hereinafter simply referred to as a serum-added phase reaction solution) is obtained.

Furthermore, after adding bovine serum albumin to the serum-added carrier reaction solution described in Section 2 at a ratio of IW/V%, lyophilize it by a conventional method to obtain a lyophilized product c of the serum-added carrier reaction solution, which is simply lyophilized. (referred to as a carrier).

Example I-! by reverse passive agglutination reaction in the same manner as in Experimental Example 1 above.
The effect of suppressing non-specific reactions before and after freeze-drying the above serum-added carrier solution in Bs antigen detection was tested.

That is, the reaction solution (conventional product) of Example 1 (A) was serially diluted on a microplate for three samples (human serum) A = B and C that showed a non-specific reaction, and then the reaction solution of Example 3 was diluted serially on a microplate. Add dropwise "A phosphorocyte solution,""serum-added carrier reaction solution," and "freeze-dried carrier" (redissolved in purified water to the original 1.), mix, and let stand at room temperature for 2 hours. After the infection, the aggregation image was observed.The results are shown in Table 3.

As is clear from Table 3, both the serum-added carrier reaction solution and the lyophilized carrier had a clear suppressive effect on non-specific reactions compared to the control blood cell solution to which no treated serum globulin fraction was added. No difference was observed before and after freeze-drying.

Example 4 (a) Preparation of reaction solution (diluent) 0.1 w/v % glycine and sodium nitride (preservative) in the same PBS as used in Example 1 (a).
A reaction solution (conventional product) is obtained by adding O, 1 w/v%.

(b) Preparation of reaction solution with guinea pig serum and serum globulin fraction treated with magnesium chloride The reaction solution obtained in (a) above was treated with the magnesium chloride solution obtained in the same manner as in Example 1 (c) above. 0.05 guinea pig serum
'W,/ltl (in terms of protein amount) to obtain treated serum addition reaction solution C (hereinafter referred to as serum addition reaction solution ■).

Similarly, a guinea pig serum globulin fraction obtained in the same manner as in Example 2 and treated with a magnesium chloride solution was added to the above reaction solution at a ratio of 0.0], Q/rxl (as protein amount) to obtain the treated serum. Globulin fraction addition reaction solution C
Hereinafter, a serum-added reaction solution (referred to as ■) is obtained.

(c) Preparation of reaction solution with addition of untreated guinea pig serum and serum globulin fraction Add healthy guinea pig serum to the reaction solution of (a) above at 0.5μ/cm
(in terms of protein amount) to obtain "control reaction solution ■". Similarly, the healthy guinea pig serum globulin fraction was measured at 0.
1"I/me (as protein amount) to obtain "control reaction solution ■".

Example Using the reaction solution obtained in Example 4 above, serum-added reaction solutions ■ and ■, and control reaction solutions ■ and ■, the reaction solution of Example 4 (a) (conventional product) shows a non-specific agglutination reaction. The effect of suppressing non-specific reactions was tested on three specimens (human serum) A and B-C.

In the same manner as in Experimental Example 1, each reaction solution was dropped into a microplate and serially diluted, and then a latex suspension sensitized with guinea pig HBs antibody or guinea pig C-reactive protein CCRP"+ was added to this as a sensitization carrier. Add dropwise the latex suspension sensitized with antibodies, mix, and incubate at room temperature for 1 hour.
After standing still for 6 hours, the aggregation image was observed. The results are shown in Table 4.

As is clear from Table 4, a strong non-specific reaction suppression effect was observed in both the treated serum of the present invention and the reaction solution added with the treated serum globulin fraction, and even when antibody-sensitized latex was used as the sensitization carrier. It showed the same effect as the red blood cell carrier in Experimental Example 1 above.

Example A suspension of latex sensitized with the same guinea pig HBs antibody as used in Experimental Example 4 and a suspension of latex sensitized with the same guinea pig CRP antibody were obtained in the same manner as in Examples 1 and 2, respectively. Magnesium chloride-treated guinea pig serum and guinea pig serum globulin fraction were incubated at 0.05'V/me and 0.0'9/m, respectively.
t (in terms of protein amount) to obtain treated serum-added latex suspension C (hereinafter referred to as serum-added reaction solution ■) and treated serum globulin fraction-added latex suspension C (hereinafter referred to as serum-added reaction solution ■). .

Using the above serum-added reaction solutions (1) and (2), the effect of suppressing non-specific reactions was tested in the same manner as in Experimental Example 4.

The results are shown in Table 5. As is clear from Table 5,
A similar inhibitory effect was also shown when treated serum or treated globulin fraction was added to the sensitized latex tuber suspension. It should be noted that when it was added to both the sensitized Lattetsunu suspension and the diluent for diluting the specimen (reaction liquid), a more remarkable effect was exhibited.

Example 1 Specific antigen detection experiment in detecting HBs antigen by reverse passive hemagglutination reaction The reaction solution obtained in Example 1 (conventional product) and serum Two hepatitis B patient sera showing positive for HBs antigen (E) were tested in the same manner as in Experimental Example J using the added reaction solution (■). The results are shown in Table 6.

As is clear from Table 6, even when using the reaction solution added with the treated guinea pig serum according to the present invention, the agglutination value was the same as when using the conventional reaction solution, and the specific reaction between the sensitized blood cells and the antigen was observed. did not interfere with the reaction.

Similarly, when the freeze-dried carrier of Example 3 was used instead of the sensitized blood cells of Example 1, the specific reaction was not interfered with.

Table 6 Example: Specific antigen detection experiment in detecting HBs antigen by latexnu agglutination reaction Using the reaction solution (conventional product) obtained in Example 4 above and the solution and serum addition reactions ■ and ■, HBs antigen positivity was determined. Two cases of hepatitis B patient sera showing 1 and 2 cases of hepatitis H were tested in the same manner as in Experimental Example 4 above. The results are shown in Table 7.

In the Latetsuknu agglutination reaction, as in the case of the hemagglutination reaction,
The guinea pig serum treated according to the invention did not interfere with the specific reaction between the sensitized latexnu and the antigen.

Table 7

Claims (1)

[Scope of Claims] (1) A reagent for antigen detection by reverse passive agglutination reaction, characterized in that it contains serum or serum globulin fraction treated with a high concentration salt solution, a low pH solution, or a high pH solution. (2) The reagent according to item (1) above, wherein the highly concentrated salt solution is a 1 mol or more highly concentrated solution of an alkali metal or alkaline earth metal salt, thiocyanate, acetate, or nitrate. (3) Item (1) above, wherein the highly concentrated salt solution is a urea solution.
Section reagents. (4) The reagent according to item (1) above, wherein the low pH solution is an aqueous acid solution or a buffer solution with a pH of 4 or less. (5) The reagent of item (1) above, wherein the high pH solution is an alkaline aqueous solution or buffer with pI-(9 or more). The reagent of paragraph (1). (7) The amount of the treated serum globulin fraction added is 0.2 μg.
The reagent of item (1) above, which is ~0.2~/g. (8) The reagent of item (1) above, which is a lyophilized product. (9) High concentration salt solution, low p
Antigen characterized by adding serum or serum globulin fraction treated with H solution or high pH solution and subjecting it to a reverse passive agglutination reaction using the treated serum or serum globulin fraction addition reaction solution and/or dilution solution. Detection method. (10) The detection method according to item (9) above, wherein the high concentration salt solution is a 1 mol or more highly concentrated solution of an alkali metal or alkaline earth metal chloride, thiocyanate-acetate or nitrate. (11) The above-mentioned item (9), wherein the highly concentrated salt solution is a urea solution.
) term detection method. (12) The detection method according to item (9) above, wherein the low pH solution is an acid aqueous solution or a buffer solution with a pH of 4 or less. (13) The detection method according to item (9) above, wherein the high pH solution is an alkaline aqueous solution or a buffer solution with a pH of 9 or more. (14) The detection method according to paragraph (9) above, wherein the amount of the treated serum added is 1μf"lq/me. [15) The amount of the treated serum globulin fraction added is 0.2μ
The detection method according to item (9) above, wherein g~0.2/g.