JPH0666798A - Method for measuring antigen or antibody in specimen - Google Patents

Abstract

PURPOSE:To reduce the occurrence of a nonspecific reaction caused by an interference substance and contamination of a container used for immunological reactions by adding a specific surface-active agent selected from among nonionic surface-active agents to an immunological measuring system by a specific amount. CONSTITUTION:The antibody or antigen which is formed in a solid state by bonding the antibody or antigen to a water insoluble substrate, for example, beads, magnetic particles, etc., used for the method is selected in accordance with the substance to be measured in a specimen. The method is performed by using the enzyme immunoassay under a condition of 4-45 deg.C in temperature and 4-9 in pH, but, at the time of making an immunological reaction to take place, a nonionic surface-active agent having an HLB value of 12-19 is made to exist in an antigen-antibody reaction system at a concentration of 1.5-6W/W%. The nonionic surface-active agent is selected from the group of polyoxyethylene or polyhydric alcohol nonionic surface-active agents. The preferable adding amount of the surface-active agent to the immunological reaction system is 2-5W/W%.

Description

Detailed Description of the Invention

[0001]

The present invention is based on the principle of immune reaction.
The present invention relates to a method for highly sensitively measuring a component of an immune reaction which is present in a living body in a trace amount.

[0002]

2. Description of the Related Art For example, a radioimmunoassay method is used for measuring trace substances contained in biological samples such as various body fluids such as blood and spinal fluid, excrements such as feces and urine, and extracts of various tissues. Immunoassays such as (RIA) and enzyme immunoassay (EIA) have been adopted.

[0003]

Since the immunoassay method has high specificity and extremely high sensitivity, it is becoming more important as a method for measuring trace components. However, it is known that the immunoassay in the heterogeneous phase has various interferences that hinder the detection of the specific reaction between the antigen and the antibody. These are called "matrix effect", "background" or "non-specific adsorption". This non-specific interference is caused by a component contained in the measurement sample, and this component is usually called "interfering substance" in the immunoassay.

Hitherto, in order to reduce nonspecific interference by interfering substances, a method of adding a hydrolyzate of gelatin or keratin (JP-A-56-42142) and a method of adding a surfactant (JP-A-SHO-42142). 58-187862, JP-A-6-6
0-53846 and JP-A-61-260163) are already known, and a partial improvement in measurement accuracy can be expected by utilizing these methods, but they are still unsatisfactory.

Another problem is the contamination of the container in which the immune reaction takes place. When using a fully automated immunoassay device, perform all reactions from immunoreactions to detection reactions such as color development, fluorescence, luminescence, etc. in the same reaction vessel, or use the immunoreaction vessel repeatedly after a simple washing process. Is often done. In this case, the contamination of the container during the immune reaction causes a significant increase in the blank value and a decrease in measurement accuracy.

[0006] In order to promote the immune reaction, attempts have been made to improve the accuracy of the immunoassay by adding a surfactant or the like. For example, a method using polyethylene glycol (P. TIJSSEN, translated by Eiji Ishikawa, Biochemistry Experimental Method 1
1 (“Enzyme Immunoassay”, Tokyo Kagaku Dojin), and a method using a surfactant (JP-A-62-71861).
However, it was not at the same time satisfactory to reduce the nonspecific reaction (nonspecific interference) by the above-mentioned interfering substances and to reduce the contamination of the container for carrying out the immune reaction.

[0007]

As a result of various studies to solve the above problems, the present inventors have found that one of the components of an immune reaction is a nonionic surface active agent in an immunoassay system which exists in a solid phase. The present invention has been completed by finding that the above problems can be solved at the same time by adding a specific amount of a specific surfactant selected from the agents.

[0008] That is, the present invention is for measuring HLB (Hydrophile Lipophile) of 12 to 19 when measuring an antigen or an antibody in a sample using the antibody or the antigen bound to a water-insoluble support.
Balance in the presence of a nonionic surfactant having a value of 1.5 to 6 w / w% in the antigen-antibody reaction system to carry out the antigen-antibody reaction, and the antigen in the specimen. Or, it relates to an antibody measuring method.

According to the present invention, in the immunoassay in the heterogeneous phase, the background of the reaction container can be prevented from being contaminated during the immune reaction and the nonspecific interference by the interfering substance can be reduced. In particular, the reproducibility of the measurement results can be improved, especially at low values, and the immune reaction is promoted, which improves the measurement accuracy at low values and shortens the analysis time.

The present invention will be described in detail below.

In the present invention, an antibody or antigen bound to a water-insoluble support, that is, a solid-phased antibody or antigen is used. Examples of the water-insoluble support, that is, a solid-phased carrier include beads, magnetic particles, Various materials such as microtiter plates, tubes, and membranes can be used and are not particularly limited.

As the antibody or antigen to be bound to the solid-phased carrier, various ones can be used and are not particularly limited, but it goes without saying that they are selected according to the substance to be measured in the sample. The binding of the antibody or antigen to the solid-phased carrier is
It may be a chemical bond or a physical bond (adsorption).

Various samples can be used as the sample and are not particularly limited. For example, various body fluids such as serum, plasma, blood, and spinal fluid, excrements such as urine, those obtained by removing solids from diluted substances such as feces, and extracts of various tissues are not particularly limited.

In the present invention, the antigen-antibody reaction, that is, the measurement of the antigen or antibody in the sample by the immune reaction can be carried out by a known method. For example, it can be performed by an immunoassay method such as an enzyme immunoassay method (EIA), a radioimmunoassay method (RIA), and a fluorescence immunoassay method (FIA). Further, it can be carried out by various methods such as a sandwich method, a competition method and a two antibody method, and any method may be used as long as it uses a solid-phased antibody or antigen. These are, for example, edited by Eiji Ishikawa et al., “Enzyme Immunoassay”, 2nd Edition, Medical Institute, 1982; Yuichi Yamamura, Laboratory of Medical Chemistry, Volume 4, “Immunochemistry” Nakayama Shoten, 1972. ;
Minoru Irie, "Continued Radioimmunoassay", Kodansha, 19
It is described in 1979 etc.

The immune reaction is usually at 4 to 45 ° C. and pH.
= 4-9, and after immunoreaction, B / F separation and detection are performed by a conventional method.

In the present invention, various containers can be used as the container for carrying out the immune reaction, and are not particularly limited.

When a microtiter plate or a tube is used as the solid-phased carrier, the solid-phased carrier itself can be used as the reaction container, but various reaction vessels can be used separately from the solid-phased carrier. . These various reactors used separately include a glass or plastic tube, a dedicated tray in which many tubes are integrally molded,
Microtiter plate, plastic container (for example, reagent container for AIA series manufactured by Tosoh Corporation) that seals freeze-dried magnetic beads once for each measurement and also serves as a storage container, designed exclusively, and repeatedly used while washing A reaction tube for a fully-automatic EIA measuring device (a reaction tube for PK seeds manufactured by Olympus Corp.) and the like can be mentioned.

In order to save money, these reaction vessels need to be replaced without replacement in the whole process (for example, in the case of EIA, the primary (2
Next, the same reaction vessel can be used throughout the following) (immune reaction, BF separation, and detection reaction), or the once used reaction vessel can be repeatedly used after simple washing.

A feature of the present invention is that, when carrying out the above-mentioned immunoreaction, an antigen-antibody reaction is carried out so that the concentration of the nonionic surfactant having an HLB value of 12 to 19 is 1.5 to 6 w / w%. The point is to make it exist in the system.

The nonionic surfactant is not particularly limited as long as it has an HLB value of 12 to 19, but preferably,
Examples thereof include compounds selected from the group consisting of polyoxyethylene-based nonionic surfactants and polyhydric alcohol-based nonionic surfactants.

For example, polyoxyethylene nonylphenyl ether (specifically, nonion NS-208.
5, Nonion NS-220, and Nonion NS-270
(Manufactured by NOF CORPORATION), polyoxyethylene oleyl ether (specifically, nonion E-215 (manufactured by NOF CORPORATION), etc.), polyoxyethylene monostearate (specifically, nonion S-15.4), And Nonion S-4
0 (manufactured by NOF CORPORATION) and polyoxyethylene sorbitan monolaurate (specifically, Tween 20)
Etc.), etc.

The amount of the nonionic surfactant used in the present invention added to the immune reaction system is 1.5 to 6 w / w%, preferably 2 to 5 w / w%.

If the amount added is less than 1.5 w / w%, the effect of preventing contamination of the immune reaction container and promoting the immune reaction cannot be obtained. On the other hand, if it exceeds 6 w / w%, the blank value is rather increased due to the nonspecific reaction.

In the present invention, when a nonionic surfactant is added to an immune reaction system, when the immune reaction system is divided into several steps, it is added to only a specific step or to all immune reaction systems. Although it may be added to the step, any of these may be added, and there is no particular limitation.

The timing of adding the nonionic surfactant is not particularly limited as long as the nonionic surfactant is present in the system at least during the immune reaction. For example, the immune reaction medium,
It may be added to the labeling component, the specific immune component (antibody or antigen), or the sample, etc., and at the time of the immune reaction, the sample,
You may add separately in the immune reaction medium (namely, immune reaction system) which added the labeling component and the specific immune component as needed.

The nonionic surfactant may be added as it is or as an aqueous solution or dispersion.

[0027]

EXAMPLES Next, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these.

Example 1 The effect of various surfactants was evaluated by the sandwich method described below.
It was examined by enzyme immunoassay for CC-ST-439 antigen.

Immune reaction buffer (phosphate saline containing 1% bovine serum albumin) and peroxidase-labeled N
Various surfactants were added to an enzyme-labeled antibody solution prepared by diluting the CC-ST-439 antibody with an immunoreaction buffer at 3 w / w%.

The measurement operation was performed as follows.

NCC-ST-43 by sandwich method
Enzyme immunoassay of 9 antigens. A tray for reaction with NCC-ST-439 was charged with polystyrene beads having a diameter of 6.25 mm and added with 0 u.
50 μl of standard antigen solutions of 1 / ml, 2.5 u / ml and 100 u / ml were sampled, 200 μl of buffer solution for immune reaction was added, and an immune reaction was carried out at 37 ° C. for 2 hours. Wash the beads 3 times with 1 ml of physiological saline, and then 250 μl of enzyme-labeled antibody solution containing the same surfactant as the immune reaction buffer
Was added and reacted at room temperature for 1 hour. Furthermore, 1 ml of beads
After washing 3 times with physiological saline solution, transfer to another test tube,
300 μl of a coloring solution containing 2.87 mM 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) and 0.014% hydrogen peroxide was added and reacted at room temperature for 1 hour. Then, 2 ml of a reaction stop solution containing 0.14 mM sodium azide was added, and the absorbance at 420 nm was measured using purified water as a control.

The measurement is carried out by multiple measurements of 5 times, and the average value of the absorbance, the coefficient of variation (CV) and the ratio of the absorbance at 2.5 u / ml to the absorbance at 0 u / ml (SN ratio) are obtained and compared. did.

As a control, the same measurement was carried out in a system in which no surfactant was added and polyoxyethylene glycol (PEG), which is known to have an immune reaction promoting effect, was added.

The results are shown in Table 1.

[0035]

[Table 1]

As is clear from Table 1, by adding a nonionic surfactant having an HLB value of 12 to 19, the color development at the NCC-ST-439 antigen concentration of 0 u / ml was lowered, and 2. In the cases of 5 and 100 u / ml, the color development was high and the coefficient of variation was small. The SN ratio was also larger than that of the control.

This indicates that the background was lowered while promoting the immune reaction, and the measurement sensitivity and measurement accuracy of the immune reaction at low values were improved.

Example 2 Effect of surfactant concentration on NCC-by sandwich method
It was examined by enzyme immunoassay for ST-439 antigen.

As surfactants, polyoxyethylene nonylphenyl ether (HLB value 18.7) and polyoxyethylene sorbitan monolaurate (HLB value 1)
6.7) was added to the same immunoreaction buffer and enzyme-labeled antibody solution as in Example 1 at a concentration of 0.1 to 8.0 w / w%, and the same measurement operation as in Example 1 was performed. It was measured at.

The measurement was carried out by multiple measurements five times, and the average value of the absorbance and the coefficient of variation (CV) were obtained and compared.

As a control, the same measurement was carried out in a system in which no surfactant was added and polyoxyethylene glycol 20000 was added.

The results are shown in Table 2.

Addition of a low concentration of the surfactant is not sufficient for the effect of lowering the background and the effect of promoting the immune reaction,
On the contrary, it was found that the background was higher at higher concentrations. A good effect was obtained with the addition of 1.5 to 6.0 w / w%.

[0044]

[Table 2]

Example 3 The effect of preventing contamination of the reaction vessel by NCC by the sandwich method
-ST-439 antigen was examined by enzyme immunoassay.

2 w / w% of polyoxyethylene sorbitan monolaurate (HLB value 16.7) was added to the same immunoreaction buffer and enzyme-labeled antibody solution as in Example 1 and examined.

Olympus automatic enzyme immunoassay device PK
The reaction U-tube for 310 was charged with polystyrene beads having a diameter of 6.25 mm to which the NCC-ST-439 antibody was bound, 50 μl of a standard antigen solution of 0 u / ml was sampled, and 200 μl of the buffer solution for immune reaction was added, 37 An immunoreaction was carried out at 0 ° C for 2 hours. The beads were washed three times with 1 ml of physiological saline, 250 μl of the enzyme-labeled antibody solution was added, and the mixture was reacted at room temperature for 1 hour.
Further, the beads were washed 3 times with 1 ml of physiological saline and then transferred to another test tube. 2.87 mM 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) was added to the beads and the reaction U-tube used for the immunoreaction, respectively.
And 300 μl of coloring solution containing 0.014% hydrogen peroxide
In addition, the reaction was carried out at room temperature for 1 hour. Then, 2 ml of a reaction stop solution containing 0.14 mM sodium azide was added, and the absorbance at 420 nm was measured using purified water as a control.

As a control, the same measurement was carried out in a system containing no surfactant.

The results are shown in Table 3.

[0050]

[Table 3]

Contamination of the reaction U-tube was prevented by adding polyoxyethylene sorbitan monolaurate (HLB value 16.7) to the immune reaction system.

Example 4 Reproducibility by continuous use of the same reaction vessel was examined by an enzyme immunoassay method for NCC-ST-439 antigen by the sandwich method.

As surfactants, polyoxyethylene nonylphenyl ether (HLB value 18.7) and polyoxyethylene sorbitan monolaurate (HLB value 1)
6.7) was added to the same immunoreaction buffer and enzyme-labeled antibody solution as in Example 1 at a concentration of 2.0 w / w% for study.

For the measurement operation, an automatic enzyme immunoassay analyzer PK310 manufactured by Olympus Corporation was used, and 50 μl of a standard antigen solution or serum sample of 0 u / ml and 210 μl of a buffer solution for immune reaction were used.
The NCC-ST-439 antibody-bound polystyrene beads having a diameter of 6.25 mm were put into a reaction U-shaped tube, and the first reaction was carried out at 37 ° C. for 15.75 minutes. After washing with B / F separation buffer, 300 μl of enzyme-labeled antibody solution was dispensed,
The second reaction was carried out at 37 ° C. for 16.75 minutes. B / F again
After washing with a separation buffer, o-phenylenediamine-
Dispense 300 μl of hydrogen peroxide-based coloring agent and
It was allowed to react for 6 minutes. Dispense 700 μl of diluted solution into 492
The difference in absorbance between nm and 800 nm was measured. The same reaction tube was used to measure three consecutive times to determine the coefficient of variation.

As a control, the same measurement was carried out in a system containing no surfactant.

The results are shown in Table 4.

[0057]

[Table 4]

The system to which the surfactant was added had a low background, and there was no tendency for the absorbance to increase due to continuous use. Also, the coefficient of variation was small, and the effect of addition was clearly seen.

Example 5 Polyoxyethylene sorbitan monolaurate (HLB value: 16.
The effect of 7) was investigated.

2.0 w / w% of polyoxyethylene sorbitan monolaurate (HLB value 16.7) was added to the enzyme-labeled antibody solution of the HBs antigen measurement kit manufactured by Dynabot Co., Ltd. Positive samples were measured by the sandwich method.

As a control, the same measurement was carried out in a system containing no surfactant.

The results are shown in Table 5.

[0063]

[Table 5]

The absorbance of the negative sample decreased and the absorbance of the positive sample increased, and the difference between the negative and positive samples was obviously large.

Example 6 The effect of adding polyoxyethylene nonylphenyl ether (HLB value 18.7) to the enzyme-labeled antibody solution of the CEA measurement kit was examined.

3.0 w / w% of polyoxyethylene nonylphenyl ether (HLB value 18.7) was added to the enzyme-labeled antibody solution of the CEA measuring kit manufactured by Roche, and 0 and 20 ng / ml were added according to a predetermined operation method. The standard antigen of was measured by the sandwich method.

As a control, the same measurement was carried out in a system containing no surfactant.

The results are shown in Table 6.

[0069]

[Table 6]

The absorbance of 0 ng / ml standard antigen decreased,
The absorbance of the ng / ml standard antigen was high. The decrease in background indicates an improvement in measurement sensitivity and measurement accuracy at a low concentration, and it is also apparent that the immune reaction is promoted.

[0071]

According to the present invention, contamination of the reaction vessel is prevented, the background is lowered, and the immune reaction is promoted.
In particular, it is possible to improve measurement accuracy, measurement accuracy, and reproducibility at low values.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Toshio Tanabe 16-3 Iwahana Town, Takasaki City Gunma Prefecture (72) Inventor Hisako Takagi 13-5 Tatsumi Town Takasaki City Gunma Prefecture (72) Masayuki Iwamoto Takasaki Gunma Prefecture 239 Iwahanacho, Fukui City

Claims (2)

[Claims] 1. When measuring an antigen or antibody in a specimen using the antibody or antigen bound to a water-insoluble support, 12
A nonionic surfactant having an HLB value of 19
A method for assaying an antigen or an antibody in a specimen, which comprises allowing an antigen-antibody reaction to occur in an antibody reaction system at a concentration of 1.5 to 6 w / w%. 2. The assay method according to claim 1, wherein the nonionic surfactant is present in the antigen-antibody reaction system at a concentration of 2 to 5 w / w%.