JP2001074739A - Immunological measurement method, method for removing immunological interference substances, and measurement reagent - Google Patents

Abstract

(57) [Summary] (Modifications) [PROBLEMS] The present invention provides an immunological method capable of sufficiently suppressing the interference effect even when a sample to be measured contains a high concentration of an interfering substance. Provided are a measuring method, a method for removing an immune reaction interference substance, and a measuring reagent. An immunoassay method comprising performing an antigen-antibody reaction in the presence of a lower oxyacid salt such as sulfurous acid, hyposulfuric acid, phosphorous acid, hypophosphorous acid, and a measuring reagent used therefor.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunological method for measuring a substance to be measured, which avoids the influence of interference such as heterophilic antibodies and rheumatoid factors and measures the exact antigen or antibody concentration. The present invention relates to an immunological measurement method, a method for removing an immune reaction interference substance, and a measurement reagent that can be used.

[0002]

2. Description of the Related Art In recent years, it has been widely used to detect immunologically active substances such as proteins appearing in a living body in connection with various diseases by utilizing an antigen-antibody reaction and to use them for diagnosis.
As a measurement method using such an antigen-antibody reaction,
Radioimmunoassay (RIA), enzyme immunoassay (EIA),
Fluorescence immunoassay (FIA), latex agglutination (L
A), various methods such as immunoturbidimetry (TIA) have been put to practical use.

[0003] However, a specimen to be measured by the immunoassay may contain a mixture of biogenic heterophilic antibodies and rheumatoid factors depending on individual characteristics and collection conditions. The presence of many substances that cause these interferences causes errors in the measurement results, especially in the field of clinical testing.
Methods to eliminate or mitigate the effects of these interfering substances are being studied, as they can lead to misdiagnosis.

[0004] For example, an immunoassay method using an F (ab ') 2 molecule obtained by removing the Fc portion of a heterologous antibody molecule by an enzymatic reaction as an antibody has been proposed (JP-A-54-119292). However, this immunoassay method can be used only when measuring an antigen, and requires a complicated process for preparing a raw material such as an enzyme reaction or purification, which leads to an increase in cost and a difference between production lots. there were.

Also, Be, Mg, Ca, Sr, Ba, F
An aqueous solvent for an agglutination test has been proposed which removes the influence of interfering substances such as lipoproteins and fibrinogen in the presence of divalent metal ions such as e, Zn, and Cd (Japanese Patent Application Laid-Open No. Sho 5).
No. 6-2555), but there is no suggestion at all about avoiding the effects of heterophilic antibodies or rheumatoid factors.

Further, in order to remove the influence of the heterophilic antibody which is an interfering substance, a polymerized or agglutinated antibody molecule is used. Since a reagent is required, the cost is high and the method is not suitable (Japanese Patent Application Laid-Open No. 4-221762).

[0007] In order to solve the problem that rheumatoid factor is mixed, a sample is pretreated in advance with an animal-derived antibody capable of binding to a reaction site of human rheumatoid factor, and non-specific reaction caused by human rheumatoid factor in the sample is performed. An immunoassay method for reducing the concentration has been proposed (Japanese Patent Application Laid-Open No. Hei 7-12818).

Further, an immunoassay method for avoiding the influence on the measurement of rheumatoid factor by maintaining the pH of the reaction solution at 4.0 to 6.0 (JP-A-8-146000, JP-A-9-49839) Japanese Patent Application Laid-Open No. 8-29420), a method of removing an immune reaction interference effect in which coexistence of mucopolysaccharide to remove the effect of an immune reaction interference substance contained in a biological sample (Japanese Patent Application Laid-Open No. 8-29420), and using α-globulin as an interference inhibitor A combined immunoassay method (JP-A-9-49840) and the like have been proposed.

[0009]

However, in the methods described in these publications, although the effect on the interfering substance is certainly improved, when the interfering substance is contained in a high concentration in the sample to be measured, The inhibitory effect is still insufficient, and a further improved method has been strongly desired.

[0010] Accordingly, the present invention has been made in view of such conventional problems, and even when the interfering substance is contained at a high concentration in the sample to be measured, the interference effect is sufficiently reduced. An object of the present invention is to provide an immunological measurement method that can be suppressed and a measurement reagent used for the method.

[0011]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, by adding a lower oxyacid salt or a transition metal to a conventional immunoassay reagent, an interference substance was obtained. It has been found that a high suppression effect can be obtained, and the present invention has been achieved.

The above object of the present invention has been attained by an immunological assay method and an assay reagent characterized in that an antigen-antibody reaction is carried out in the presence of a lower oxygenate.

Another object of the present invention is to provide a method for treating rheumatoid factor and / or rheumatoid factor contained in a biological sample in the presence of transition metal.
Alternatively, the present invention has been attained by a method for removing an immunological reaction interfering substance, which comprises removing the influence of a heterophilic antibody.

Hereinafter, the present invention will be described in more detail.

The lower oxyacid salt used in the present invention can be appropriately selected from known lower oxyacid salts and used. Examples of these lower oxyacid salts include at least one selected from the group consisting of sulfites, hyposulfites, phosphites, and hypophosphites, with sodium sulfite being particularly preferred.

In the present invention, the lower oxyacid salt is 1 to 1
000 mM, preferably 150 to 250 mM, more preferably 200 mM. When the concentration of the lower oxyacid salt is less than 1 mM, the interfering substance which affects the immune reaction cannot be effectively suppressed. On the other hand, when the concentration exceeds 1000 mM, the viscosity of the solution increases, which adversely affects the measured value.

The transition metal used in the present invention can be appropriately selected from known ones. Specific examples include at least one selected from the group consisting of copper, nickel, and cobalt, or salts thereof, with nickel acetate being particularly preferred.

In the present invention, the transition metal is 0.1 to 1
It is contained so as to be 00 mM, preferably 12 to 20 mM, more preferably 16 mM. When the concentration of the transition metal is less than 0.1 mM, the rheumatoid factor and the heterophilic antibody cannot be suppressed effectively. On the contrary, when the concentration exceeds 100 mm, the measured value is adversely affected and accurate measurement cannot be performed.

In the present invention, although not particularly required, the effect of stabilizing the substance to be measured can be further enhanced by adding a known protein protective agent as needed. Examples of such a protein protecting agent include inactive proteins represented by albumin and gelatin.

A sample which has been prevented from being affected by an interfering substance according to the present invention is directly used as a measurement sample based on a known immunological measurement method. Specific examples of the measurement method include a sandwich method, an RIA method and an ELISA method based on the principle of a competition method,
Examples include those selected from the group consisting of immunological particle agglutination such as latex agglutination and immunoturbidimetry.

Among these immunological measurement methods, in particular, an antigen (or an antibody) which is a substance to be measured in a biological sample is reacted with an antibody (or an antigen) in a measurement reagent, and the resulting immunoaggregate is obtained. Preference is given to immunoturbidimetry and latex agglutination, which are based on the principle of detection by optical means.

The test sample is appropriately diluted according to the measurable range of the reagent to be used to prepare a sample for measurement. For dilution, a diluting solution that gives a pH or a salt concentration suitable for an immunological reaction may be used.

In the present invention, the antigen or antibody to be sensitized to the solid phase carrier is not particularly limited, and may be appropriately selected from known ones and used. Specific examples thereof include antibodies against plasma proteins such as C-reactive protein (CRP) and transferrin, thyroid stimulating hormone (TSH), triiodothyronine, thyroxine, thyroxine-binding protein, thyroglobulin, insulin, estriol, Antibodies to hormones such as human placental lactogen, carcinoembryonic antigen (CEA), β2
-Macroglobulin, α-fetoprotein (AFP)
Antibodies against tumor-related substances such as HBs antigen, HBs antibody, HBe antigen, antibodies against viral hepatitis antigens such as HBe antibody and antigens against antibodies, viruses such as mumps, herpes, measles, rubella, antibodies or antigens against various biological components , Phenobarbital, acetaminophenone, salicylic acid, cyclosporine, and other various drugs.

The above-mentioned antibodies that can be used in the present invention are not particularly limited to animal species and clones derived therefrom, and include antibodies derived from rabbits, goats, mice, rats, horses, sheep, and the like. A monoclonal antibody may be used.

According to the present invention, there can be provided an immunoassay reagent containing a lower oxygenate and / or a transition metal which acts as an inhibitor of an interfering substance such as an heterophilic antibody or a rheumatoid factor. In this case, the lower oxyacid salt and / or the transition metal can be appropriately selected from known ones as described above and used.

[0026]

The present invention makes it possible to carry out an immunoassay with high accuracy even when a specimen to be measured contains a high concentration of an interfering substance such as an heterophilic antibody or a rheumatoid factor. . Therefore, according to the present invention, complicated operations such as pretreatment of a measurement sample and processing of a measurement reagent material are not required.

[0027]

Hereinafter, the present invention will be described based on examples.
The present invention is not limited by these.

Embodiment 1 FIG. Confirmation test of avoidance of the interference effect of rheumatoid factor using sodium sulfite To a sample 3 μl of a rheumatoid factor-positive human serum sample containing 4716 IU / ml of rheumatoid factor, which was diluted by a factor of 64 with physiological saline, 0, 50, 100, 150, 20
50 mM H containing 0,250 mM sodium sulfite
150 μl of EPES buffer (pH 7.4) was added into the reaction cell and mixed. Five minutes after mixing, 150 μl of a latex solution containing latex particles sensitized with an anti-human CRP antibody (animal name: goat) was added to the mixture, reacted at 37 ° C., and fully automated after 0.5 to 2 minutes. Hitachi 717 equipment
The absorbance was measured at a wavelength of 570 nm using 0 (manufactured by Hitachi, Ltd.), and the change in absorbance between each measurement point was determined. CRP
In order to quantify the concentration of CRP, the CRP concentration in the sample was calculated based on a calibration curve obtained by measuring a standard substance having a known concentration of CRP. The result is shown in FIG.

Comparative Example 1 Effect of Sodium Sulfite on Immune Response The CRP concentration was calculated in exactly the same manner as in Example 1 except that a rheumatoid factor negative sample was used. The result is shown in FIG. As shown in FIG. 2, it can be seen that sodium sulfite has almost no effect on the immune response. Therefore, in view of FIGS. 1 and 2, when 150, 200, and 250 mM sodium sulfite are added, the measurement error caused by the rheumatoid factor is remarkably improved, and the interference effect in the immunological measurement method is sufficiently avoided. It is understood that it was possible. In contrast, when 50 or 100 mM sodium sulfite was added, although the effect of suppressing the interference was observed,
It is still insufficient, and when sodium sulfite is not contained, it can be seen that an interference action has occurred.

Embodiment 2 FIG. Confirmation test for avoidance of interference effect of rheumatoid factor using nickel (II) acetate Instead of sodium sulfite used in Example 1, 0,
A confirmation test for avoiding the interference effect of rheumatoid factor was performed in exactly the same manner as in Example 1 except that 4, 8, 12, 16, and 20 mM of nickel (II) acetate was used. The result is shown in FIG.

Comparative Example 2 Effect of Nickel (II) Acetate on Immune Response The CRP concentration was calculated in exactly the same manner as in Example 2 except that a rheumatoid factor negative sample was used. FIG. 4 shows the results. As shown in FIG. 4, it is understood that nickel (II) acetate has almost no effect on the immune reaction. Therefore, in view of FIGS. 3 and 4, when 12, 16, and 20 mM of nickel (II) acetate was added, the measurement error caused by the rheumatoid factor was remarkably improved, and the interference effect in the immunological assay was reduced. It is understood that this was sufficiently avoided. In contrast, when 4.8 mM nickel acetate (II) was added, the effect of suppressing the interference was observed, but the effect was still insufficient. It can be seen that the action is occurring.

[Brief description of the drawings]

FIG. 1 is a graph showing the measurement results of the CRP concentration of Example 1 in a dilution series of a rheumatoid factor-positive specimen.

FIG. 2 is a graph showing the measurement results of the CRP concentration of Control Example 1 in a dilution series of a rheumatoid factor-negative specimen.

FIG. 3 is a graph showing the results of measuring the CRP concentration of Example 2 in a dilution series of a rheumatoid factor-positive specimen.

FIG. 4 is a graph showing the results of measuring the CRP concentration of Control Example 2 in a dilution series of a rheumatoid factor-negative specimen.

Claims (10)

[Claims] 1. An immunoassay method comprising performing an antigen-antibody reaction in the presence of a lower oxygenate. 2. The immunoassay according to claim 1, wherein the concentration of the lower oxygenate is in the range of 1 to 1000 mM. 3. The immunoassay according to claim 1, wherein the lower oxyacid salt is at least one selected from the group consisting of sulfurous acid, hyposulfuric acid, phosphorous acid, and hypophosphorous acid. 4. The immunoassay according to claim 3, wherein the lower oxyacid salt is sodium sulfite. 5. A method for removing an immunological reaction interfering substance, which comprises removing the influence of a rheumatoid factor and / or heterophilic antibody contained in a biological sample in the presence of a transition metal. 6. The method according to claim 1, wherein the concentration of the transition metal is in the range of 0.1 to 100 mM. 7. The method according to claim 5, wherein the transition metal is at least one selected from the group consisting of copper, nickel, and cobalt, or salts thereof. 8. The method according to claim 7, wherein the transition metal is nickel (II) acetate. 9. An immunoassay reagent comprising a lower oxyacid salt. 10. The immunoassay reagent according to claim 9, wherein the lower oxyacid salt is at least one selected from the group consisting of sulfurous acid, hyposulfurous acid, phosphorous acid, and hypophosphorous acid.