JPH06300761A - Reagent and method for immunonephelometry - Google Patents

Abstract

PURPOSE:To accurately measure an insoluble immune complex generated through an antigen-antibody reaction by suppressing the nonspecific reaction with the use of a reagent containing a quaternary ammonium salt. CONSTITUTION:One or more kinds of quaternary ammonium salt among choline chloride, choline bromide, choline iodine, choline bitartrate, acetylcholine chloride, betaine hydrochloride, etc., are selected. A using reagent contains the selected quaternary ammonium salt. An insoluble immune complex generated in an antigen-antibody reaction is optically measured with the use of this reagent without using an insoluble carrier such as latex or the like. The using amount of the quaternary ammonium salt is adjusted to keep 0.1-10.0%, preferably, 0.3-3.0%. If the quaternary ammonium salt is used smaller than 0.1%, the nonspecific reaction cannot be sufficiently restricted. If the quaternary ammonium salt is used not smaller than 10.0%, it hinders the generation of the insoluble immune complex required for the measurement.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunoturbidimetric measuring reagent and a measuring method used for measuring an immunologically active substance using an antigen-antibody reaction. More specifically, it relates to a reagent composition and method for suppressing nonspecific reactions other than the intended antigen-antibody reaction in the immunoturbidimetric assay method that does not use an insoluble carrier.

[0002]

2. Description of the Related Art An immunological assay method for assaying an immunologically active substance in a biological sample using an antigen-antibody reaction is widely used in the field of clinical examination. For example, single immunodiffusion method (S
RID), immunonephelometry (TIA), immunonephelometry, haemagglutination (HA), latex agglutination (LA), enzyme immunoassay (EIA), radioimmunoassay (RIA), and the like, They are used according to the purpose of use due to their operability, sensitivity, accuracy, etc.

Among them, an immunoturbidimetric method (hereinafter referred to as TI, which optically measures an insoluble immune complex produced by an antigen-antibody reaction)
A is abbreviated as A) is a washing operation (B
/ F separation step) is not necessary and is easy to automate, so it is one of the analysis methods that is attracting attention. Since TIA does not need to label the immune component, it is more advantageous than other immunoassays in the preparation of reagents and is widely used.

For example, immunoglobulin (I
g) G, A, M, transferrin, complement, apolipoprotein, β-lipoprotein, Lp (a), C-reactive protein (CR
P), anti-streptolysin O (ASO), rheumatoid factor (RF), plasminogen, albumin and the like.

The principle is that the antigen / antibody in the sample reacts with the antibody / antigen in the reagent, and as a result of the antigen-antibody reaction, an insoluble reaction product (suspended particles) consisting of the antigen-antibody complex is quantitatively produced. This suspended particle is an analysis method that optically quantifies by utilizing the fact that it blocks (scatters) the transmission of light.

Therefore, the sensitivity, accuracy, and time required for the analysis depend largely on the degree of formation of suspended particles. Generally, when an antigen solution and an antibody specific to the antigen are mixed, an antigen-antibody reaction occurs in the order of 10 ⁻³ seconds. However, thereafter, it may take several minutes to several hours, or several days until optically suspended particles of a measurable size are formed.

Therefore, for the purpose of accelerating the formation of suspended particles, speeding up the measurement, increasing the sensitivity, and increasing the accuracy, methods of using various additives have been studied in order to assist the reaction. For example, Clinical CH
EMISTRY) Volume 20, page 1071 (1974), use of polyethylene glycol (PEG)
No. 938 describes a method of performing an immune reaction in the coexistence of PEG and a nonionic surfactant.

JP-A-2-103466 describes the use of compounds represented by the general formula R ₁ O-[(CH ₂ CH ₂ O) _m (AO) _n ] -R ₂ .

Further, JP-A-61-25062 describes the use of polyanions such as chondroitin sulfate, heparin and dextran.

However, since the use of these known additives does not completely suppress the non-specific reaction, satisfactory results have not always been obtained. For example, when an antigen and an antibody are reacted in a solution and the resulting immune complex is measured by an optical method such as a turbidimetric method or an absorptiometry method, other than an aggregation product caused by the reaction between the target antigen and the antibody In addition, turbidity occurs due to a non-specific reaction between a reagent and another substance (so-called interfering substance) that is not a measurement target in the sample. It is also known that additives such as PEG itself react with contaminant proteins and cause turbidity, and such a reaction is also called a non-specific reaction.
Further, the turbidity of the biological sample itself (particularly lipid) is also a cause of error.

As a countermeasure against this, Japanese Patent Laid-Open No. 64-15656
In No. 6, a non-specific reaction is suppressed by adding a surfactant to the reagent. However, a surfactant alone is easily affected by an additive substance such as PEG used as a reaction accelerator, and measures against the influence of complement and other turbidity whose cause is unknown are insufficient.

As described above, the conventional technique has not been able to obtain satisfactory results in suppressing and avoiding the non-specific reaction that causes turbidity that causes an error.

[0013]

DISCLOSURE OF THE INVENTION The present invention solves the problems of the prior art as described above, suppresses non-specific reactions, and accurately and accurately measures an object to be measured. The purpose is to provide a method.

[0014]

The present invention provides an immunological activity in a biological sample by optically measuring an insoluble immune complex produced by an antigen-antibody reaction without using an insoluble carrier such as latex. A reagent used in a method for measuring a substance, which comprises a quaternary ammonium salt in the reagent, and an immunoturbidimetric measuring reagent, and a measuring method.

Examples of the quaternary ammonium salt used in the present invention include choline chloride, choline bromide, choline iodide, choline bitartrate, acetylcholine chloride, betaine hydrochloride and the like. These quaternary ammonium salts may be used alone or as a mixture of two or more kinds.

The amount of the quaternary ammonium salt used in the present invention is adjusted so that the final concentration during the reaction is 0.1 to 10.0%, preferably 0.3 to 3.0%. To do. (% Means weight / volume% unless otherwise specified).

If the amount of the quaternary ammonium salt used in the present invention is less than 0.1%, the non-specific reaction cannot be sufficiently suppressed, and if it exceeds 10.0%, the insoluble immunocomplex necessary for the measurement can be obtained. Not suitable as it inhibits body formation.

In the present invention, the quaternary ammonium salt may be present in a required amount in the reaction field. Therefore, the quaternary ammonium salt may be added to a sample processing solution such as an antiserum-containing reagent or a diluent. You can also leave it.

Further, the reaction system is optionally selected from the group consisting of polyethylene glycol (PEG), polyoxyethylene-polyoxypropylene-block copolymer (hereinafter abbreviated as block copolymer), and polyanion. In addition, one or more conventional additives may be added. Further, these may be used in combination with a nonionic surfactant.

Commercially available PEG can be used as the PEG used in the present invention, and those having an average molecular weight of about 400 to 500,000, preferably 1500 to 100,000 are suitable, and the amount used is a concentration of 1 to 15 in the reaction field. The addition amount is adjusted so that it becomes%.

As the block copolymer used in the present invention, commercially available products are Pluronic L34 and Pluronic L4.
4, Pluronic F68, Pluronic F88, Pluronic F108 (all manufactured by Wyandot Chemicals, trade name), Pronon 105 and Pronone 208 (both manufactured by NOF Corporation, trade name), Emulgen PP250, Emulgen PP290 (both manufactured by Kao) , Trade name), etc., and the amount used is 1 to 15 at the reaction site.
The addition amount is adjusted so that it becomes%.

Examples of the polyanion used in the present invention include dextran sulfate, phosphotungstic acid, phosphomolybdic acid, and the like.
The addition amount is adjusted so as to be 001 to 1.000%.

These conventional additives may be used alone or in combination of two or more. In addition,
It may be used in combination with a nonionic surfactant as described below.

As the nonionic surfactant used in the present invention, polyoxyethylene alkylphenyl ether,
There are polyoxyethylene sorbitan fatty acid ester, polyoxyethylene alkyl ether and the like, which are commercially available under the names Triton, Tween, Bridge and the like.
Among these, a nonionic surfactant having good water solubility is suitable for the present invention. The amount used is 0 at the reaction site.
Adjust the addition amount so that it becomes 01-5.00%, and add PEG.
Used in combination with additives such as.

In the immunoturbidimetric assay reagent of the present invention, conventionally known substances may be used as the other components. For example, as a buffering agent, a phosphate buffer solution having a pH around neutrality or HE
Good's buffer such as PES can be used, and if necessary, preservatives and stabilizers such as sodium azide and paraben may be added to the reagent.

[0026]

Although the detailed mechanism of action of the present invention is unknown, it is presumed as follows. It is known that the quaternary ammonium salt has an action of inhibiting the formation of a molecular aggregate by hydrogen bond or ionic bond. The antigen-antibody reaction is also a kind of molecular association formation reaction due to hydrogen bonding and the like, and is inhibited by the quaternary ammonium salt. However, it is considered that by selecting an appropriate concentration range, the quaternary ammonium salt of the present invention does not affect the intended immune reaction and specifically suppresses only the non-specific reaction.

[0027]

EXAMPLES The present invention will be described in detail based on the following examples, but the present invention is not limited to these examples. The reagents used in this example were commercial products purchased from Wako Pure Chemical Industries, Ltd., Tokyo Kasei Co., Ltd., etc., except for those specified.

Example 1 The effect of suppressing the non-specific reaction by adding the quaternary ammonium salt was examined. The change in absorbance due to a non-specific reaction was measured under the analysis conditions shown below.

[0029]

[Chemical 1]

320 μl of the reagent 1 was added to 20 μl of the sample, and the mixture was incubated at 37 ° C. for 5 minutes.
The absorbance at m was measured. Similarly, the measurement was performed in the case where the quaternary ammonium salt was not added, and the two were compared. Also, choline chloride was replaced with choline iodide and the same procedure was performed. The results are shown in Table 1 and FIG.

[0031]

[Table 1]

As shown in Table 1 and FIG. 1, an increase in absorbance due to a non-specific reaction was observed without addition, but an increase in absorbance was not observed due to the addition of the quaternary ammonium salt. Was suppressed.

Example 2 Apolipoprotein B was measured using the SRID method (manufactured by Daiichi Pure Chemicals Co., Ltd.), which is said to be unaffected by non-specific reactions, as a control, and the apolipoprotein B was measured using a quaternary ammonium salt. The suppression effect of specific reaction was investigated. Apolipoprotein B was measured by the immunoturbidimetric method under the analysis conditions shown below. Human serum having a known concentration of apolipoprotein B was used as a standard.

[0034]

[Chemical 2]

320 μl of Reagent 1 was added to 5 μl (sample) of human serum containing apolipoprotein B and mixed at 37 ° C.
Incubated for minutes. Then add 80 μl of Reagent 2 and add 5
The amount of change in absorbance at two wavelengths of a main wavelength of 340 nm and a sub wavelength of 700 nm per minute was measured. Measurement is Hitachi 7150
An automatic analyzer was used. Anti-human apolipoprotein B serum
What was obtained by immunizing a goat with apolipoprotein B by a conventional method was used. At the same time, measurement was carried out for the case where no quaternary ammonium salt was added, and the two were compared. The results are shown in Table 2.

[0036]

[Table 2]

As shown in Table 2, in the case of no addition, a positive error is caused by the non-specific reaction, but in the case of adding the quaternary ammonium salt, the non-specific reaction is suppressed, and therefore SRID
Little difference from the law was observed.

Example 3 Apolipoprotein C-III was measured using the SRID method (manufactured by Daiichi Pure Chemicals Co., Ltd.), which is said to be unaffected by nonspecific reaction, as a control, and a quaternary ammonium salt was obtained. The effect of suppressing non-specific reaction was investigated. Apolipoprotein C-III was measured by the immunoturbidimetric method under the analysis conditions shown below. Human serum of known concentration was used as a standard.

[0039]

[Chemical 2]

Human serum containing apolipoprotein C-III 10 μm
300 μl of reagent 1 was added to and mixed with 1, and the mixture was incubated at 37 ° C. for 5 minutes. Next, 100 μl of Reagent 2 was added, and 5 minutes after the addition, a main wavelength of 340 nm and a sub wavelength of 700 nm
The amount of change in absorbance at the wavelength was measured. Measurement is Hitachi 71
Anti-human apolipoprotein C-III using a 50 type automatic analyzer
The serum used was obtained by immunizing a goat with apolipoprotein C-III by a conventional method. At the same time, measurement was carried out for the case where no quaternary ammonium salt was added, and the two were compared.
The results are shown in Table 3.

[0041]

[Table 3]

As shown in Table 3, in the case of no addition, a positive error is caused by the non-specific reaction, but in the case of adding the quaternary ammonium salt, the non-specific reaction is suppressed, and therefore SRID
Little difference from the law was observed.

Example 4 The CRP was measured using the SRID method (manufactured by Nissui Pharmaceutical Co., Ltd.), which is said to be unaffected by the nonspecific reaction, as a control, and the nonspecific reaction by the quaternary ammonium salt was confirmed. The inhibitory effect was investigated. C by immunoturbidimetric assay under the following analysis conditions
The RP was measured. Human serum of known concentration was used as a standard.

[0044]

[Chemical 4]

320 for 20 μl of CRP-containing human serum
μl of reagent 1 was added and mixed, and the mixture was incubated at 37 ° C. for 5 minutes. Next, 80 μl of the reagent 2 was added, and the amount of change in absorbance at two wavelengths of a main wavelength of 340 nm and a sub wavelength of 700 nm was measured for 5 minutes after the addition. Hitachi 7150 automatic analyzer was used for measurement, and anti-human CRP serum obtained by immunizing a goat with CRP by a conventional method was used. At the same time, measurement was carried out for the case where no quaternary ammonium salt was added, and the two were compared. The results are shown in Table 4.

[0046]

[Table 4]

As shown in Table 4, a positive or negative error is caused by the non-specific reaction without addition, but the addition of the quaternary ammonium salt suppresses the non-specific reaction.
Almost no difference from the RID method was observed.

Next, a specimen (No.
24), the change in absorbance with time was examined. The results are shown in Table 5.
And shown in FIG.

[0049]

[Table 5]

As shown in Table 5 and FIG. 2, during the incubation time of 5 minutes, a considerably large increase in absorbance was observed without addition of the reagent 1 which contained no antibody and originally did not react with serum. With Reagent 1 containing choline chloride, no increase in absorbance due to non-specific reaction was observed. Similarly, the reagent 1 containing choline iodide was also measured, but as in the case of choline chloride, no increase in absorbance due to nonspecific reaction was observed.

Example 5 LATEX agglutination method (manufactured by Eiken Chemical Co., Ltd.) with high measurement accuracy
Was used as a control to measure the anti-streptolidine O (ASO) value, and the value was compared with the value measured by the quaternary ammonium salt addition reagent. ASO was measured by the immunoturbidimetric method under the analysis conditions shown below. Human serum of known concentration was used as a standard.

[0052]

[Chemical 5]

320 for 20 μl of human serum containing ASO
μl of reagent 1 was added and mixed, and the mixture was incubated at 37 ° C. for 5 minutes. Next, 80 μl of reagent 2 was added, and the amount of change in absorbance at two wavelengths of a main wavelength of 340 nm and a sub wavelength of 700 nm was measured for 5 minutes after the addition. A Hitachi 7150 type automatic analyzer was used for the measurement. At the same time, measurement was carried out for the case where no quaternary ammonium salt was added, and the two were compared. The results are shown in Table 6.

[0054]

[Table 6]

As shown in Table 6, in the case of no addition, a positive or negative error is caused by the non-specific reaction, but in the case of adding the quaternary ammonium salt, the non-specific reaction is suppressed, so that L
Almost no difference from the ATEX method was observed.

Next, the sample (N
o. 24), the change in absorbance with time was examined. The results are shown in Table 7 and FIG.

[0057]

[Table 7]

As shown in Table 7 and FIG. 3, an increase in absorbance was observed in Reagent 1 containing no SLO and originally not reacting with serum, but in Reagent 1 containing choline chloride, the absorbance due to a nonspecific reaction was observed. No rise was observed. Similarly, the reagent 1 containing choline iodide was also measured, but as in the case of choline chloride, no increase in absorbance due to nonspecific reaction was observed.

Example 6 ELISA method with high measurement accuracy (Biopool, USA)
Lp (a) was measured using a product manufactured by Mitsui Chemical Co., Ltd. as a control, and a comparison was made between the quaternary ammonium salt and the value measured by a reagent to which a nonionic surfactant was added. Lp (a) was measured by the immunoturbidimetric method under the analysis conditions shown below. Human serum of known concentration was used as a standard.

[0060]

[Chemical 6]

3 for 15 μl of human serum containing Lp (a)
20 μl of reagent 1 was added and mixed, and the mixture was incubated at 37 ° C. for 5 minutes. Next, 80 μl of reagent 2 was added, and the amount of change in absorbance at two wavelengths of a main wavelength of 340 nm and a sub wavelength of 700 nm was measured for 5 minutes after the addition. The measurement was carried out using a Hitachi 7150 type automatic analyzer, and anti-human Lp (a) serum was tested for Lp by a conventional method.
What was obtained by immunizing a rabbit with (a) was used. The results are shown in Table 8.

[0062]

[Table 8]

As shown in Table 8, in the case of no addition, a negative error is caused by the non-specific reaction, but in the case of adding the quaternary ammonium salt and the nonionic surfactant, the non-specific reaction is suppressed and thus the ELISA is performed. Little difference from the law was observed.

Next, the specimen (No.
49), the change in absorbance with time was examined. The results are shown in Table 9
And shown in FIG.

[0065]

[Table 9]

As shown in Table 9 and FIG. 4, in the case of no addition, a large increase in absorbance was observed only with the reagent 1 containing no antibody and originally not reacting with serum, and without addition of a surfactant alone. Reagent 1 (Fig. 4) also showed a considerable increase in absorbance. However, with Reagent 1 containing both the quaternary ammonium salt and the nonionic surfactant, no increase in absorbance due to the nonspecific reaction was observed. The non-ionic surfactant alone can suppress the non-specific reaction to some extent, but the reagent in combination with the quaternary ammonium salt was superior in the suppression effect, and good results were obtained.

[0067]

EFFECTS OF THE INVENTION Turbidity occurs due to non-specific reaction between an antibody / antigen in a reagent and an interfering substance in a sample. In addition, Example 1,
From 4, 5, and 6, even if there is no antibody / antigen in the reagent, a non-specific reaction occurs due to an additive such as polyethylene glycol and an interfering substance in the sample, and the absorbance (turbidity) increases. These non-specific reactions are suppressed by the addition of the quaternary ammonium salt.

According to the present invention, the non-specific reaction with the interfering substance in the sample which is not the measurement object is suppressed, so that the measurement object can be accurately measured, and the immunoturbidimetric assay reagent whose accuracy, reliability and precision are improved. Can be supplied.

[0069]

[Brief description of drawings]

FIG. 1 shows the effect of suppressing a non-specific reaction by the quaternary ammonium salt of Example 1.

FIG. 2 shows the effect of suppressing non-specific reaction by the quaternary ammonium salt of Example 4.

FIG. 3 shows the effect of suppressing a non-specific reaction by the quaternary ammonium salt of Example 5.

FIG. 4 shows the effect of suppressing the non-specific reaction by the quaternary ammonium salt of Example 6.

[Chemical 3]

Claims (14)

[Reference number] P276 [Claims] 1. A reagent used in a method for measuring an immunologically active substance in a biological sample by optically measuring an insoluble immune complex produced by an antigen-antibody reaction without using an insoluble carrier. An immunoturbidimetric assay reagent, which contains a quaternary ammonium salt therein. 2. The quaternary ammonium salt is choline chloride,
The immunological ratio according to claim 1, wherein the reagent is contained in the reagent as one or a mixture of two or more selected from the group consisting of choline bromide, choline iodide, choline bitartrate, acetylcholine chloride, and betaine hydrochloride. Turbidity measurement reagent. 3. A quaternary ammonium salt is added to the reaction site in an amount of 0.
The immunoturbidimetric assay reagent according to claim 1 or 2, which is contained in the reagent so as to be present in a concentration of 1 to 10.0% by weight / volume. 4. (1) together with a quaternary ammonium salt,
(2) One or two or more substances selected from the group consisting of polyethylene glycol, polyethylene-polypropylene-block copolymer, and polyanion are contained in the reagent. 3. The immunoturbidimetric assay reagent described in 3. 5. Polyethylene glycol is used as a reaction site in the reaction.
The immunoturbidimetric reagent according to claim 4, wherein the reagent is contained in the reagent so as to be present at a concentration of -15% by weight / volume. 6. A polyethylene-polypropylene-block copolymer is included in the reagent such that it is present in the reaction at a concentration of 1 to 15% w / v.
The immunoturbidimetric assay reagent as described. 7. A polyanion is added to the reaction site in an amount of 0.001-.
The immunoturbidimetric assay reagent according to claim 4, which is contained in the reagent such that it is present at a concentration of 1.000% by weight / volume. 8. (1) a quaternary ammonium salt, and (2)
One or more substances selected from the group consisting of polyethylene glycol, polyethylene-polypropylene-block copolymer and polyanion, and (3) a nonionic surfactant are contained in the reagent. The immunoturbidimetric assay reagent according to any one of claims 1 to 3. 9. A nonionic surfactant is added to the reaction site at a rate of 0.
The immunoturbidimetric assay reagent according to claim 8, which is contained in the reagent so that it is present at a concentration of 01 to 5.00% by weight / volume. 10. A reagent containing a nonionic surfactant together with a quaternary ammonium salt and polyethylene glycol so that it is present at a concentration of 0.01 to 5.00% by weight / volume in the reaction field. The immunoturbidimetric assay reagent according to claim 9. 11. A nonionic surfactant together with a quaternary ammonium salt and a polyethylene-polypropylene-block copolymer is used in a reaction field in an amount of 0.01 to 5.00% by weight / weight.
The immunoturbidimetric assay reagent according to claim 9, which is contained in the reagent so as to be present in a concentration of volume%. 12. A nonionic surfactant is used together with a quaternary ammonium salt and a polyanion in an amount of 0.01 in the reaction field.
The immunoturbidimetric assay reagent according to claim 9, wherein the reagent is contained in the reagent so as to be present at a concentration of ˜5.00% by weight / volume. 13. A method for measuring an immunologically active substance in a biological sample by optically measuring an insoluble immunocomplex produced by an antigen-antibody reaction without using an insoluble carrier, in a reaction field. An immunoturbidimetric measuring method, which comprises the presence of a quaternary ammonium salt. 14. A method for measuring an immunologically active substance in a biological sample by optically measuring an insoluble immunocomplex produced in an antigen-antibody reaction without using an insoluble carrier, which is used as a reaction field. A method for suppressing nonspecific reaction other than the intended antigen-antibody reaction by allowing the presence of a quaternary ammonium salt.