JPH05123190A - Reagent for detecting labeled peroxidase for immunoblotting analysis - Google Patents

Abstract

PURPOSE:To obtain the subject reagent having excellent color-developing stability and extremely high sensitivity and free from carcinogenecity by dissolving methyl-benzothiazolinone hydrazone salt, a specific phenolic compound and H2O2 in a buffering solution and adjusting the pH of the mixture. CONSTITUTION:The objective reagent is produced by dissolving (A) 3-methyl-2- benzothiazolinone hydrazone salt, (B) a phenolic compound of formula (B is CH3, H, etc.; A is NH2, NHCH3, etc.; X is HCl, HCOOH, etc.; (n) is 0, 1/2 or 1) (preferably p-dimethylaminophenol, etc.) and (C) hydrogen peroxide in a buffering solution (preferably a phosphate buffering solution composed of a monobasic phosphate) and adjusting the pH of the solution to 7.1-9. The amounts of the components A and B are preferably 1-2mM and 5-20mM based on 1L of the solution, respectively. The concentration of the component C is preferably 0.2-0.4mM.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an immunoblot method used as a useful analytical method for proteins,
The present invention relates to a reagent for detecting peroxidase (hereinafter abbreviated as POD) that is widely used as a labeling enzyme.

[0002]

BACKGROUND ART Proteins are separated by various gel electrophoresis methods, transferred to a membrane such as nitrocellulose, and an antigen-antibody reaction is carried out on the membrane to detect and identify a specific protein (antigen). The method is called immunoblot analysis. To detect proteins on the membrane, there are a method of performing autoradiography using an antibody labeled with a radioisotope and a method of chemically detecting using an antibody to which an enzyme is bound, but the latter is easy to handle. This method is often adopted. Conventionally, diaminobenzidine (hereinafter abbreviated as DAB) has been used as a POD detection reagent that has been used as a labeling enzyme for antibodies in immunoblotting.
It has been used for a long time because of its high detection sensitivity. However, since its carcinogenicity was pointed out, various reagents other than DAB have been investigated and 4-chloro-1-naphthol, nitrotetrazolium salt, etc., which have no carcinogenicity have been developed,
The detection sensitivity of these reagents was not always sufficient.
As a POD detection reagent composition developed from this viewpoint,
In JP-B-61-75, 3-methyl-2-benzothiazolinone hydrazone hydrochloride and a phenol compound are added at a pH of 5 to 5.
The use under the conditions of 7 is disclosed. This detection reagent was not carcinogenic and was excellent in terms of sensitivity.

[0003]

However, since this detection reagent was intended to detect POD of a tissue specimen, it could not be directly applied to the immunoblotting method. That is, in the case of the immunoblotting method, when the compound is used under the condition of pH 5 to 7, the solubility of the compound is not always sufficient, so that the efficiency of the color reaction is lowered and the protein detectability is lowered. Became. Further, it became clear that the color development stability was also poor.

The present invention has been made in view of the above circumstances, and enables highly sensitive POD detection in immunoblotting. That is, the present invention has no carcinogenicity, excellent coloration stability, and much higher sensitivity than known reagents in labeled blotting in immunoblotting.
It is intended to provide a D detection reagent.

[0005]

MEANS FOR SOLVING PROBLEMS AND EFFECTS OF THE INVENTION The present invention provides a 3-methyl-2-benzothiazolinone hydrazone salt (hereinafter abbreviated as MBTH), a phenol compound represented by the following general formula, and a peroxide. It is a reagent for detecting labeled peroxidase for immunoblot analysis, which is prepared by dissolving hydrogen and a buffer solution and adjusting the pH to 7.1 to 8.0.

[0006]

[Chemical 2]

MBTH is a compound having the following structure.

[0008]

[Chemical 3]

Although the above structural formula shows the hydrochloride salt of MBTH, other water-soluble salts may be used for MBTH in the detection reagent of the present invention.

The phenol compound used in the detection reagent of the present invention is represented by the above general formula.
-Dimethylaminophenol, o-aminophenol,
m-aminophenol, p-methylaminophenol sulfate, 2-chloro-6-methylphenol, 4-chloro-3-methylphenol, 2,3-dichlorophenol, 2,6-dichlorophenol, o-chloro Phenol, o-methoxyphenol, p-methoxyphenol and the like are particularly effective.

The ratio of the two is within the range of 0.5 to 5 millimoles of MBTH and 3 to 25 millimoles of the phenol compound (both are amounts per 1 L of the solution). If there is less than this range, there is a problem in the color development of the protein, and if there is more than one, the solution is in a suspended state, which is not preferable. Within this range, MBTH is 1-2 mM and phenolic compounds are 5
The range of up to 20 mM is particularly preferred.

The concentration of hydrogen peroxide is an important factor affecting the degree of color development. Usually, the concentration of hydrogen peroxide is 0.
If the amount is in the range of 1 to 0.6 mM, it is not preferable if the amount is out of this range, the color development of the protein decreases. Especially hydrogen peroxide concentration is 0.2
~ 0.4 mM is preferred.

As a buffer solution for dissolving MBTH and a phenol compound, a phosphate buffer solution, a maleate buffer solution, a sodium cacodylate buffer solution, etc. are used. Of these, the phosphate consisting of the primary phosphate is used. Salt buffer is preferred. It is important to adjust the pH of the solution to 7.1 to 9.0. When the pH is 7.1 or less, the solubility of the phenol compound is lowered, the efficiency of the color reaction is low, the color of the protein to be detected is low, and the sensitivity is lowered. Further, the coloration stability is also lowered, which is not preferable. pH is 9.
When it is 0 or more, the coloring reaction is hindered, which is not preferable. A particularly preferable pH is in the range of 7.2 to 7.6. The salt concentration in the buffer is preferably as low as possible, and
A range of 05 to 0.1M is generally preferred.

When the detection reagent of the present invention is used, each compound and buffer in the detection reagent may be mixed and stored so as to have a predetermined concentration, but MBTH, a phenol compound and hydrogen peroxide are respectively stored. You may make it melt | dissolve in a buffer solution separately so that it may become a predetermined | prescribed density | concentration, preserve | save, and mix each solution at the time of use so that it may become a predetermined concentration and predetermined pH, and it may be made to use for a detection (color development) reaction. The detection reagent of the present invention is, for example, a nitrocellulose membrane, PVDF (Po
It can be used for various blot transfer films such as lyvinylidene difluoride) film and nylon film.

The present invention not only provides a reagent for substituting the carcinogen DAB used as a detection reagent for POD which is a labeling enzyme in immunoblotting, but also a conventionally known detection reagent such as DAB. It provides a reagent with a much higher sensitivity than the conventional one, and enables detection of an extremely small amount of protein which cannot be detected by conventional protein detection by immunoblotting, and the effect is extremely large. The detection reagent according to the present invention is particularly useful for detecting / identifying tumor markers such as α-fetoprotein by immunoblotting.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples. It should be noted that the following Examples and Comparative Examples all assume only the staining step after transfer (blotting) to the membrane after electrophoresis, and the steps before the immunoblotting, that is, the operations of electrophoresis and blotting are omitted. did.

[0017]

Example Purified human α-fetoprotein (αFP, IB
L company) 12.0, 6.0, 3.0, 1.5, 0.7, 0.35, 0.17 and 0.
1 μL each of 08 ng / ml phosphate buffer (pH 7.1)
It was spotted on a PVDF (Polyvinylidene difluoride) film. Next, the PVDF membrane with αFP added was mixed with 4% BSA.
The membrane was immersed in a (bovine serum albumin) -PBS (phosphate buffered saline) solution and shaken at 37 ° C. for 60 minutes to block all unadsorbed protein on the membrane with BSA. 4% BSA-P
After removing the BS solution, the PBS solution was added, and the mixture was similarly agitated for 5 minutes. Further, this operation was repeated 3 times in place of a new PBS solution. After washing, transfer the PVDF membrane to the detection reaction tray, dissolve the primary antibody solution (5 mg of anti-human αFP monoclonal antibody in 500 μL of distilled water) on the membrane, and then 20 times with the antibody diluted solution (phosphate buffer solution). Diluted) 5 mL was poured. After reacting at 37 ° C for 60 minutes, the PVDF membrane was washed 3 times for 5 minutes each with a PBS solution containing 0.05% Tween 20, and then P
POD-labeled secondary antibody on VDF membrane (anti-human IgG (rabbit)
Dilute 5 μL of POD-labeled IgG (manufactured by DAKO) solution with 5 mL of antibody dilution solution. ) 5 mL was poured. After reacting at 37 ℃ for 45 minutes, add 0.05% Tween 20 in PBS to PVDF
The membrane was washed 5 times for 5 minutes each.

6.3 mM MBTH (0.1 M phosphate buffer: pH
7.1) 5 mL, 25.2 mM 2,6-dichlorophenol (0.
A detection reagent was prepared by mixing 25 mL of 1 M phosphate buffer: pH 7.1 and 1.5 mL of 6.3 mM hydrogen peroxide (0.1 M phosphate buffer: pH 7.1). The concentrations after mixing were 1 mM for MBTH, 20 mM for 2,6-dichlorophenol, and 0,0 for hydrogen peroxide.
It is 3 mM. To detect labeled POD activity, PVD
1.5 mL of detection reagent per spot on F membrane
The mixture was poured to about 3 minutes and reacted at room temperature for 2 to 5 minutes. The membrane was washed with tap water for 1-2 minutes to stop the reaction, and after drying, the degree of color development of each spot was measured with a densitometer (OD _555nm ). The measurement result is shown in FIG.

[0019]

[Comparative Example] 24.0, 12.0, 6.0, 3.
1 μL of each of 0, 1.5 and 0.7 ng / mL phosphate buffer (pH 7.1) was spotted on the PVDF membrane, and αFP was labeled with the primary antibody and the POD-labeled secondary antibody in the same manner as in Example. Thereafter, detection reagents of 0.05% DAB, 0.04% hydrogen peroxide and Tris-HCl buffer (pH 7.5) were poured on the PVDF membrane at about 1.5 mL per spot and reacted at room temperature for 2 to 5 minutes. The membrane was washed with tap water for 1-2 minutes to stop the reaction, and after drying, the degree of color development of each spot was measured with a densitometer at 480 nm, which is the maximum absorption wavelength. The measurement result is shown in FIG.

As is clear from the results of (A) and (B) in FIG. 1, the reagent of the present invention can detect αFP up to 0.08 ng / mL, whereas the DAB method does not exceed 3.0 ng / mL. With the detection limit (cutoff value: 0.01) or less, only up to about 6.0 ng / mL could be detected, and the detection sensitivity of the reagent of the present invention was remarkably improved as compared with DAB. Further, the degree of fading of spots developed by the reagent of the present invention was smaller than that of DAB, and the coloration stability was excellent.

[Brief description of drawings]

FIG. 1 is a diagram showing a color density of each spot of an example and a comparative example. (A) shows an example, (B) shows a comparative example.

Claims (3)

[Claims] 1. A 3-methyl-2-benzothiazolinone hydrazone salt, a phenol compound represented by the following general formula, and hydrogen peroxide are dissolved in a buffer solution to give a pH of 7.
A reagent for detecting labeled peroxidase for immunoblot analysis, which is adjusted to 1 to 9.0. General formula: 2. The concentration of the 3-methyl-2-benzothiazolinone hydrazone salt is 0.5 to 5 mM, the concentration of the phenol compound is 3 to 25 mM, and the concentration of the hydrogen peroxide is 0. The detection reagent according to claim 1, which is 1 to 0.6 mM. 3. The detection reagent according to claim 1, wherein the buffer solution is a phosphate buffer solution.