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CN-116718451-B - DAB buffer solution for DAB color development and DAB staining solution kit

CN116718451BCN 116718451 BCN116718451 BCN 116718451BCN-116718451-B

Abstract

The application relates to the field of immunohistochemical staining, in particular to DAB buffer solution for DAB color development and a DAB staining solution kit. The application discloses a DAB buffer solution for DAB color development, which comprises the main components of hydrogen peroxide, polyoxyethylene nonylphenol ether, magnesium sulfate and trisodium citrate. The metal ions in the DAB buffer solution can enhance the electron transfer speed between DAB and hydrogen peroxide and improve the color development sensitivity. The polyoxyethylene nonylphenol ether can stabilize hydrogen peroxide, so that the polyoxyethylene nonylphenol ether can be stored for a longer time, and the cost is saved. The application also discloses a DAB staining solution kit, which comprises a blocking agent, a reaction enhancement solution, enzyme-labeled polymer IgG, DAB concentrated solution, DAB buffer solution and hematoxylin, and the DAB buffer solution for DAB color development. The DAB staining solution kit can improve the storage stability of DAB staining solution and reduce the cost while improving the detection sensitivity of immunohistochemical staining.

Inventors

  • HU JIEFENG
  • LIU HENGSHENG
  • WANG PENG
  • LI DEQIANG

Assignees

  • 杭州百殷生物科技有限公司

Dates

Publication Date
20260512
Application Date
20230531

Claims (7)

  1. 1. A DAB buffer solution for DAB color development is characterized in that the DAB buffer solution is prepared by mixing hydrogen peroxide, imidazole, polyoxyethylene nonylphenol ether, chloroalkyl dimethylbenzylamine, magnesium sulfate and trisodium citrate; the volume concentration of the hydrogen peroxide is 0.05-0.3%, The molar concentration of the imidazole is 30-80mM, The mass percentage concentration of the nonylphenol polyoxyethylene ether is 0.04-0.18%, The volume concentration of the chloroalkyl dimethylbenzene methylamine is 0.006-0.015%, The molar concentration of the magnesium sulfate is 0.5-2.2mM, The molar concentration of the trisodium citrate is 0.6-1.8mM.
  2. 2. A DAB staining solution kit comprises a blocking agent, a reaction enhancement solution, enzyme-labeled polymer IgG, DAB concentrated solution, DAB buffer solution and hematoxylin, and is characterized in that the DAB buffer solution is the DAB buffer solution for DAB staining according to claim 1.
  3. 3. The DAB staining solution kit of claim 2, wherein the blocking agent is a hydrogen peroxide solution with a concentration of 1-6%.
  4. 4. A DAB staining solution kit according to claim 2, wherein the reaction enhancing solution comprises rabbit anti-mouse immunoglobulin as a main component.
  5. 5. The DAB staining solution kit of claim 4 wherein the concentration of the reaction enhancing solution is 130-550ug/mL.
  6. 6. The DAB staining solution kit of claim 2, wherein the DAB concentrated solution is prepared from 1, 2-propanediol, water and DAB.
  7. 7. The DAB staining solution kit of claim 2, wherein the enzyme-labeled polymer IgG is prepared by the following steps: S1, uniformly mixing 3-15mg/mL of goat anti-rabbit antibody and 2-10mg/mL of goat anti-mouse antibody, then adding 12-26mg/mL of ethylene glycol chitosan polymer, uniformly mixing, then adding 20-50mg/mL of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and 3-8mg/mL of N-hydroxysulfosuccinimide, uniformly mixing, regulating the pH value to 2.2-5.4 by using 0.03-0.2M HCl, and carrying out light-proof reaction at room temperature for 2-8 hours to obtain a solution A for later use; S2, adding 6-16mg/mL of peroxidase solution into 18-30mg/mL of polyethylene glycol 2000 solution, then adding 2-8mg/mL of polyethylene glycol SMCC cross-linking agent, and carrying out light-shielding reaction for 2-8 hours to obtain solution B for later use; s3, mixing the A, B solutions, adding 1-4mg/mL of polyethylene glycol SMCC cross-linking agent, adjusting the pH value to 6.5-7.5, carrying out light-shielding reaction for 2-6 hours, and carrying out adsorption elution.

Description

DAB buffer solution for DAB color development and DAB staining solution kit Technical Field The application relates to the field of immunohistochemical staining, in particular to DAB buffer solution for DAB color development and a DAB staining solution kit. Background The DAB staining solution kit in the prior art generally comprises components such as a blocking agent, a reaction enhancement solution, enzyme-labeled polymer IgG, DAB concentrated solution, DAB buffer solution, hematoxylin and the like, and is mainly used for immunohistochemical staining of pathology department. Immunohistochemical staining is one of the common techniques in pathology department, and utilizes the immunological principle of antigen-antibody specific binding to make antigen-antibody-enzyme-labeled polymer IgG complex develop in situ by means of enzymatic reaction to determine whether the target antigen exists in tissue cells, and to locate and qualitatively detect the target antigen. In the DAB staining solution kit, enzyme-labeled polymer IgG (commonly called secondary antibody, polymer secondary antibody) is the most important component. The traditional secondary antibody is generally formed by combining goat anti-mouse or goat anti-rabbit antibody with peroxidase (HRP) under the action of a compound, has low combining efficiency and poor detection sensitivity, and can not meet the requirements of in-vitro diagnostic reagents. Most of the recent polymer secondary antibodies are based on polymers to which goat anti-mouse, goat anti-rabbit antibodies and HRP enzymes are coupled via a broad binding site on the polymer. The polymer is taken as a substrate, and a large amount of antibody and HRP enzyme can be combined, so that the detection sensitivity and the dyeing effect of the antibody are greatly improved compared with those of the traditional secondary antibody. However, because of different technology and process of the polymer secondary antigen material, the dyeing quality of different polymer secondary antigen raw materials has larger difference, and the raw material price is generally higher. In the use process of the DAB staining solution kit, how to improve the color rendering is also important. The prior art generally uses 3,3 Diaminobenzidine (DAB) as a chromogenic substrate and uses HRP labeled on polymer IgG to catalyze DAB chromogenic reaction. The DAB color development solution is generally prepared from DAB concentrated solution and DAB buffer solution according to the proportion of 1:20. The DAB buffer solution contains hydrogen peroxide (H 2O2) which is critical to color development, and because H 2O2 has unstable and easily-decomposed characteristics, the DAB buffer solution is often failed due to the decomposition of H 2O2, so that the immunohistochemical staining is shallow or negative. Therefore, how to maintain the stability of H 2O2 in DAB buffer is of great importance. The color development principle of DAB is that HRP catalyzes H 2O2 to oxidize DAB to form reduced DAB, and the reduced DAB is insoluble in water and forms brown yellow precipitate in situ of antigen-antibody-enzyme labeled polymer IgG complex. In the reaction process, because of the higher standard potential difference between H 2O2 and DAB and the instability of the physicochemical property of H 2O2, the prepared DAB color developing solution can be gradually oxidized and lose efficacy, and can not be placed for a long time. Therefore, the existing reagent kit uses the chromogenic reagent which is prepared by preparing DAB into DAB concentrated solution, preparing H 2O2 into DAB buffer solution, preparing and storing separately, and preparing the two solutions according to the ratio of 1:20 when in use. In summary, the DAB staining solution kit has the problems of low sensitivity of the kit caused by instability of H 2O2 of ①, poor stability of the DAB staining solution prepared by ② and short shelf life. At present, the intensity and sensitivity of immunohistochemical detection are improved by adding DAB reinforcing agent, which comprises copper sulfate, sodium oxide, potassium oxide, sucrose and benzalkonium chloride, in the Chinese patent publication No. CN113484313A, hydrogen peroxide is protected and stabilized by using sodium pyrophosphate and EDTA, and the stability is improved by delaying the oxidation rate of DAB by methyl glycol. In chinese patent publication No. CN1595150a, stability is improved by adding carbohydrates and polymers to inhibit the autoxidation activity of the color former. The Chinese patent with publication number of CN110887831A solves the problems of poor stability and short shelf life of DAB color development liquid by providing aminodextran. The chinese patent publication No. CN110361245A increased stability by adding disodium dihydrogen pyrophosphate to the buffer to protect the hydrogen peroxide. The Chinese patent publication No. CN110567785A discloses that the stability of DAB working solution is impr