JPS61192299A - Method of dyeing peroxidase - Google Patents

Abstract

PURPOSE:To perform simply he titled dyeing useful for diagnosing acute leukemia, etc., capable of carrying out surely dyeing independently of impure substances in a buffer solution, not fading, having good dyeability, by using pyrophosphoric acid or its salt. CONSTITUTION:In a method using 3,3',5,5'-tetramethylbenzidine of its salt as a substrate, peroxidase is dyed by using pyrophosphoric acid or its salt.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a peroxidase staining method, and is mainly intended for use in medical diagnosis.

[Conventional technology]

Peroxidase staining is used as a routine test for blood diseases, primarily to differentiate between myeloid and lymphocytic leukemia when diagnosing acute leukemia, and is also used for diagnosing miniloba-oxidase deficiency. Peroxidase is an enzyme that catalyzes reactions that oxidize various substances in the presence of hydrogen peroxide. Detection of peroxidase through cell chemical reactions indicates that the substrate hydrogen donor is oxidized by the enzyme.
It is based on the reaction of H2 + H2022 2 A + 2 I-T20 occurring and the oxide developing color.

Traditionally, peroxidase staining methods have often used methods that use benzidine as a substrate, but since benzidine is a carcinogenic substance and its use is problematic, there are several alternative methods that use other substances as a substrate. It has been reported. However, for example, it is difficult to determine weakly positive granules with the method using fluorine derivatives, the color fades if not encapsulated with the method using naphthol derivatives, and the staining procedure is complicated with the method using carbazole derivatives. and technology, Gaku.

327 (1978)). The other one is also 3.3', 5,5'-
There is also a report using tetramethylbenzidine (hereinafter referred to as TMB) as a substrate [Beidai Medical Journal Bulletin, 7.29 (
1981)). However, this method often encounters no staining when the staining reaction is performed with a highly purified phosphate buffer.

[Problem that the invention seeks to solve]

In particular, the present invention is a peroxidase staining method using TMB, which is an easy-to-operate method that allows staining to be performed reliably and without fading due to the dependence on impurities in the buffer used as a staining reagent, and which is stable and has good staining properties. Our goal is to provide the following.

Based on the above report regarding TMB, the present inventor has discovered that TMB
Based on a clinical trial in which no reaction occurred when peroxidase staining was performed with a phosphate-based buffer using did. On the other hand, the content of pyrophosphoric acid in phosphates is not specified in J.E.S., and the availability of phosphates containing pyrophosphoric acid is a matter of chance.
And the probability of that occurring is extremely rare at present. Therefore, if a phosphate containing pyrophosphoric acid or its salts is used as a buffer, no staining reaction will occur. The above report regarding TMB states that staining was achieved only by using a phosphate buffer as a result of consideration regarding the selection of the type of buffer. This means that the phosphate buffer was selected and used without being aware of the presence of pyrophosphoric acid, which is sometimes contained as an impurity in phosphoric acid salts.

The present invention has been completed based on the findings that when TMB is used, peroxidase staining can be performed reliably by adding pyrophosphoric acid or its salts to the staining reagent.

[Failure to solve the problem]

The present invention is a peroxidase staining method using TMB or a salt thereof as a substrate, which is characterized by using pyrophosphoric acid or a salt thereof.

In the peroxidase staining method, a staining solution containing TMB is dropped onto a sample prepared by smearing whole blood onto a slide glass in a conventional manner, and after allowing the sample to stand for several seconds, a buffer solution is added and staining is carried out. At this time, the pyrophosphoric acid used may be added to the sample in advance, or may be added and mixed into the buffer solution used. After completing the staining reaction in this manner, post-staining is performed if necessary, washed with running water, dried, and examined under a microscope.

The staining reaction is carried out under acidic conditions around H5 to 6.5, but any buffer may be used as long as it satisfies these conditions. Pyrophosphoric acid exhibits staining properties if 1-body o II 71 or higher is used, but considering the ease of viewing under a microscope and the beauty of the finished specimen, 2. Ortanol/
There is an optimum concentration around II.

The staining solution consists of TMB as a substrate, alcohol for dissolving TMB, hydrogen peroxide, and metal ions, and is prepared immediately before use.

[Effect]

Conventionally, when performing peroxidase staining using TMB, which is not carcinogenic, staining could only be carried out using phosphoric acid or its salt as a buffer. This uses pyrophosphoric acid, which is contained as an impurity in phosphoric acid salts, in the dyeing reaction. However, the use of phosphates that do not contain pyrophosphate will result in no staining.

Therefore, it was not possible to use anything other than phosphoric acid in the buffer system of the staining solution, which was a limitation.

According to the present invention, by using pyrophosphoric acid, there is no need to use phosphoric acid or its salt in the buffer system of the staining solution, and a wide range of buffer solutions can be selected.

Furthermore, even if a phosphate buffer is used, staining can be performed reliably by using pyrophosphoric acid or its salt.

〔Example〕

The method of the present invention will be specifically explained below using Test Examples and Examples.0 Test Example 1 Differences in staining properties due to lot differences in distrium phosphate Reagent (1) Staining solution 0-2% TMB s o % methanol aqueous solution 10m
Before use, mix 0.04 rnl of 1.3% hydrogen peroxide solution and 7 ml of 10% zinc sulfate aqueous solution to prepare a staining solution.

(2) Buffer solution - I/I'5M potassium dihydrogen phosphate solution and 1/15M
Mix the IJ acid-sodium hydrogen solution to prepare H6.4.

Staining method: Drop 1 ml of staining solution onto the sample (blood smear) and let stand at room temperature for 30 seconds. Next, add 2 ml of buffer solution, mix, and allow to react at room temperature for 5 minutes. Safranin 0 solution is used for post-staining.

(3) Results Table 1 As shown in the table above, there were differences in staining properties depending on the lot of dinylium phosphate used in the phosphate buffer.

Test Example 2 Effect of pyrophosphoric acid on dyeability 5wmall/
One drop of pyrophosphoric acid in an aqueous solution is added to 16 ml of the buffer solution of the lot that was not stained in Comparative Example 1, and staining is carried out using the reagent and staining method used in Comparative Example 1. As a result, it was found that staining can be achieved by adding pyrophosphoric acid. At this time, the results of stainability depending on the concentration of pyrophosphoric acid added are shown in Table 2.

Table 2 Example 1 Reagent (1) Staining solution 0.2 TMBso% methanol aqueous solution 10ml, 3%
Hydrogen peroxide solution 0.04 ml, 10% zinc sulfate aqueous solution o, tml and 0.07 mol/l phosphate buffer (
, H6゜4) 2 oml are mixed before use to make a staining solution.

(2) Sodium pyrophosphate aqueous solution 5rranol
Prepare a l/l aqueous solution.

(3) Post-staining solution 1% safranin 0 aqueous solution and 1/15M phosphate buffer (,
Prepare by mixing H6°4) at a ratio of 1:3.

Staining method: Drop 1 ml of PI-01J aqueous solution onto the sample (blood smear), let stand at room temperature for 30 seconds, and after 1 minute add 2 ml of staining solution.
Mix and leave at room temperature for 5 minutes. After this, post-staining solution 2
ml for post-staining, washing with water, drying, and microscopic examination.

Example 2 A staining solution is a reagent prepared by adding thorium pyrophosphate to the staining solution in Example 1 to a final concentration of 1 to 10 mmol/l.

The post-staining solution was the same as in Example 1.

Staining method: Drop 3 ml of staining solution onto the sample (blood smear) and let stand at room temperature for 5 minutes. After this, post-staining is performed using 2 ml of post-staining solution, washed with water, dried, and examined microscopically.

Example 3 Reagent (1) Staining solution 0.15% TMB 80% methanol aqueous solution 10 ml,
3% hydrogen peroxide solution 1ml, 10% zinc sulfate solution 0,
02 ml is mixed before use to make a staining solution.

(2) Buffer solution 0゜1rrLOl/l sodium pyrophosphate 0.5ml
and 0.1mo 11/It Gutsudovatufu 7 (pHpH
6-4) Mix 11 before use to prepare a buffer solution.

(3) Post-staining solution 1 tisafranin O aqueous solution and 1/15M Guttudo buffer y
(pH 6,4) at a ratio of 1:3.

Staining method: Drop 0°5 ml of staining solution onto the sample (blood smear), leave for 30 seconds, and add 1mA of buffer solution! After leaving it for a while, wash it with water, dry it, and examine it under a microscope.

Example 4 Reagent 1 oynl of 80 timethanol aqueous solution of 0.2% TMB,
3t$ hydrogen peroxide solution 0.04ml1. 10 Zinc sulfate aqueous solution 0.7 m, f! and srazoll/l sodium birophosphate in o,xmog/zgud buffer (p
H6,4) Mix 20 rnl before use to make a staining solution. The post-staining solution was the same as in Example 3.

Dyeing method Same as the dyeing method in Example 2.

Example 5 reagent Same as Example 2.

Staining method: Paste liver tissue sliced with cryostanoto onto a slide glass, fix with formaldehyde as usual, and wash with water. 2 ml of PBS (phosphate buffered saline) containing a peroxidase-labeled anti-HBs antigen antibody is placed on this, left in a humid cylinder at room temperature for 2 hours, and then washed overnight with chilled PBS. Add 3 ml of staining solution to this slide glass.
dropwise and let stand at room temperature for 5 minutes. After this, post-staining solution 2
Post-stain with rnl, wash with water, dry, and examine under a microscope.

Example 6 reagent Same as Example 2.

A paraffin-embedded thin section of liver tissue was deparaffinized using a conventional method, washed with PBS containing anti-HBs antigen antibody at room temperature for 24 hours, reacted with a second antibody labeled with peroxidase for 2 hours, and cooled. Cleanse overnight.

Add 3 ml of this sample staining solution dropwise and let stand at room temperature for 5 minutes. Thereafter, post-staining is performed using 2 ml of post-staining solution, followed by washing with water, drying, and microscopic examination.

Patent applicant Gino Test Institute Co., Ltd. Procedural amendment (method) June 1985 Noah date

Claims (1)

[Claims] 1. A peroxidase staining method using 3,3',5,5'-tetramethylbenzidine or a salt thereof as a substrate, the method comprising using pyrophosphoric acid or a salt thereof.