Enhanced BCIP / NBT chromogenic substrate as well as preparation method and application thereof

Abstract

The invention provides an enhanced BCIP / NBT chromogenic substrate. The chromogenic substrate comprises the following components: substrate components BCIP and NBT, an enhancer, a stabilizer, iodonitrotetrazole chloride, a buffer system with the pH value of 8.5-10.0, and metal ions, wherein the reinforcing agent is polyethylene glycol or dextran; the stabilizer is composed of homosalate and gelatin; the metal ions are magnesium ions and zinc ions. According to the chromogenic substrate provided by the invention, by introducing a specific enhancer and a stabilizer, the detection sensitivity is remarkably improved, the non-specific background is reduced, the chromogenic reaction speed is accelerated, a chromogenic product is more stable, and methods for detecting the activity of alkaline phosphatase, such as ELISA, ELISPOT, Western Blot and IHC, are universal and have very high commercial application value.

Description

Technical Field

[0001] This invention belongs to the field of immunological detection technology, specifically relating to an enhanced BCIP / NBT chromogenic substrate, its preparation method, and its application. Background Technology

[0002] 5-Bromo-4-chloro-3-indole phosphate (BCIP) / nitroblue tetrazolium (NBT) is a widely used enzyme-catalyzed chromogenic substrate system, primarily used for detection coupled with alkaline phosphatase. Its chromogenic principle is as follows: alkaline phosphatase hydrolyzes BCIP to produce indophenol, which is then oxidized by NBT under alkaline conditions to form an insoluble blue / blue-violet dimethyl sulfoxide precipitate, thus indicating the position of the target molecule.

[0003] However, traditional BCIP / NBT substrates have the following significant drawbacks: 1) Limited sensitivity: Insufficient detection capability for low abundance targets, weak colorimetric signal, and difficulty in accurate quantification.

[0004] 2) High background staining: Background staining is easily generated during long-term incubation or at non-specific sites, which reduces the signal-to-noise ratio and the specificity of detection.

[0005] 3) Slow color development speed: It takes a long time to reach the ideal color development intensity, which affects the detection efficiency.

[0006] 4) Poor precipitate stability: The formed dimethyl sulfoxide precipitate may dissolve or fade under certain conditions, which is not conducive to the long-term preservation of the results.

[0007] Therefore, there is an urgent need in the field for an enhanced BCIP / NBT chromogenic substrate that can overcome the above-mentioned defects, has higher sensitivity, lower background, faster reaction speed, and more stable products. Summary of the Invention

[0008] In view of this, the present invention provides an enhanced BCIP / NBT chromogenic substrate, which significantly improves the detection sensitivity, reduces nonspecific background, accelerates the chromogenic reaction rate, and makes the chromogenic product more stable by introducing specific enhancers and stabilizers.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: An enhanced BCIP / NBT chromogenic substrate, the chromogenic substrate comprising the following components: substrate components BCIP and NBT, enhancer, stabilizer, iodonitrobenzyl tetrazolium chloride, a buffer system with a pH of 8.5~10.0, and metal ions; in, The reinforcing agent is polyethylene glycol or dextran; The stabilizers are homosalate and gelatin; The metal ions are magnesium ions and zinc ions.

[0010] Furthermore, the concentration of BCIP is 0.5~2 mg / mL; The concentration of NBT is 0.2~1 mg / mL.

[0011] Furthermore, the polyethylene glycol has a molecular weight of 500-4000 and a concentration of 0.5%-10% (w / v).

[0012] Furthermore, the concentration of homosalate is 0.1~2mM; The concentration of the gelatin is 0.1% to 5% (w / v).

[0013] Furthermore, the concentration of the iodonitroxytetrazolium chloride is 0.05~0.2mM.

[0014] Furthermore, the buffer system includes Tris-HCl buffer and diethanolamine buffer.

[0015] Furthermore, the magnesium ions are derived from magnesium chloride and have a concentration of 2-10 mM; The zinc ions are derived from zinc chloride and have a concentration of 0.1~1mM.

[0016] A method for preparing the above-mentioned chromogenic substrate includes the following steps: adding each component sequentially to ultrapure water, stirring until completely dissolved, filtering with a 0.22 μm filter membrane for sterilization, and storing at 2~8℃ in the dark to obtain the substrate.

[0017] The application of the above-mentioned chromogenic substrate in the preparation of a kit for detecting alkaline phosphatase activity.

[0018] Furthermore, the methods for detecting alkaline phosphatase activity include ELISPOT, ELISA, Western Blot, Dot Blot, and IHC.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Ultra-high sensitivity: By adding enhancers such as PEG, the effective concentration of local reactants is increased, and through the catalytic effect of INT, a BCIP / NBT / INT ternary synergistic catalytic system is formed, which enables the signal to be cascaded and amplified, greatly improving the detection capability of low abundance targets.

[0020] (2) Extremely low background: The selective adsorption of stabilizers such as gelatin can effectively inhibit the non-specific deposition of NBT on the membrane or plate, thereby significantly reducing background coloration and improving the signal-to-noise ratio.

[0021] (3) Rapid color development: INT, as an efficient electron acceptor, accelerates the redox process of conversion from indophenol to dimethyl sulfoxide, enabling the color development reaction to be completed in a shorter time and shortening the detection cycle (clear target spots appear within 5 minutes, and the best color development effect is achieved at 10 minutes).

[0022] (4) Product stability: The optimized formula and buffer system make the generated blue / blue-purple precipitate more stable and less likely to be dissolved by organic solvents or dehydration process. The color development results can be preserved for a long time without fading (the signal strength is still effectively guaranteed after 10 days under the simulated aging test at 37℃).

[0023] (5) High versatility: The enhanced substrate of the present invention can directly replace the traditional BCIP / NBT substrate and is suitable for a variety of alkaline phosphatase-based detection platforms (ELISPOT, ELISA, Western Blot, Dot Blot, IHC, etc.) without changing the original experimental procedure, making it easy to use. Attached Figure Description

[0024] Figure 1 This is a comparison chart of the color development results of the colorimetric substrate prepared in Example 1 of the present invention and a commercially available conventional colorimetric solution; wherein, column 1A represents the commercially available conventional product, and column 1B represents the product of the present invention.

[0025] Figure 2 This illustrates the effect of different molecular weights and concentrations of PEG on the results in Example 2 of the present invention; where A represents the effect of different molecular weights of PEG, and B represents the effect of different concentrations of PEG.

[0026] Figure 3 The effect of different stabilizers on the results in Example 2 of the present invention is shown; where A is the effect of different gelatin concentrations when the standard is 0, B is the effect of different concentrations of homosalate, and C is the effect of different concentrations of homosalate under accelerated aging conditions. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and all reagents and consumables are commercially available products.

[0028] Example 1 This embodiment provides an enhanced BCIP / NBT chromogenic substrate, the preparation and application of which are as follows: 1.1 Preparation of chromogenic substrates Prepare 100mL of colorimetric solution with the following components: 1) Tris-HCl buffer (pH 9.5): 10 mL; 2) Magnesium chloride solution (1M): 0.5mL (final concentration 5mM); 3) Zinc chloride solution (0.1M): 0.5mL (final concentration 0.5mM); 4) BCPI (50 mg / mL in DMF): 2 mL (final concentration 1 mg / mL); 5) NBT (50 mg / mL in DMF): 1 mL (final concentration 0.5 mg / mL); 6) Polyethylene glycol PEG-1000: 3g (final concentration 3% w / v); 7) Homosalate: 0.013g (final concentration 0.5mM); 8) Gelatin: 1g (final concentration 1% w / v); 9) INT (iodonitrosyltetrazole chloride) 1mM: 10mL (final concentration 0.1mM); 10) Ultrapure water: Add to 100mL.

[0029] Add the above components in sequence, stir magnetically until completely dissolved, filter sterilize using a 0.22μm filter membrane, dispense into 10mL / bottle, and store at 2-8℃ protected from light.

[0030] 1.2 Application in ELISPOT detection Furthermore, the following tests were conducted to understand the properties of the chromogenic substrate prepared above: Using the ELISPOT kit (Elabscience, catalog number ESP-H0009), under the same cell culture stimulation conditions (background: complete culture medium only, 100 μL / well; negative: 1 × 10⁶), the following were observed: background: complete culture medium for cell culture only, 100 μL / well. 4 PBMCs / well, 100 μL / well. Positive: 1 × 10⁻⁶ 4 PBMCs / well + 1 mg / mL LPS, 100 μL / well), cultured for 20 h, then removed cells. Biotin-labeled detection antibody was added, followed by incubation with streptavidin-conjugated alkaline phosphatase. After washing, incubated at 37°C in the dark. One copy ( Figure 1 Column A) was developed using a commercially available traditional BCIP / NBT developer, and the other ( Figure 1 (Column B) The enhanced colorimetric solution prepared according to Example 1 of this invention was used for color development.

[0031] The results are as follows Figure 1 As shown: the film using the substrate of this invention exhibits clear target spots within 5 minutes, and reaches optimal color development at 10 minutes, with a clean background. In contrast, the film using commercially available conventional substrates shows relatively faint spots after 25 minutes, and the entire film is stained by the background.

[0032] Example 2 This embodiment further investigates the influence of different materials on the performance of the chromogenic substrate based on the chromogenic substrate prepared in Example 1, as detailed below: 2.1 Effects of different molecular weights and concentrations of PEG on performance Using the chromogenic substrate materials from Example 1, different chromogenic substrates were prepared by sequentially changing the molecular weight and concentration of PEG. The Human IL-6 ELISA experimental protocols for different molecular weight PEGs were designed according to Table 1 below; the Human IL-6 ELISA experimental protocols for different concentrations of PEG were designed according to Table 2 below.

[0033] The ELISA testing steps are as follows: ① Add 100 μL of standard, quality control working solution or sample to the corresponding well and incubate at 37℃ for 90 min; ② After discarding the liquid in the plate, immediately add 100uL of biotinylated antibody working solution and incubate at 37℃ for 60min; ③ Discard the liquid inside the plate and wash the plate 3 times; ④ Add 100 μL of ARP enzyme conjugate working solution to each well, incubate at 37°C for 30 min, discard the liquid in the plate, and wash the plate 5 times; ⑤ Add 90 μL of the prepared chromogenic substrate solution to each well and incubate at 37°C for about 15 min; ⑥ Add 50 μL of stop solution to each well; ⑦ Readings from the ELISA reader.

[0034] The test results obtained by following the above testing steps are shown in Table 1 and Table 2. Figure 2 As shown.

[0035] Table 1. ELISA experimental design and results showing the effect of different molecular weight PEGs on substrates.

[0036] Table 2. ELISA experimental design and results showing the effect of different PEG concentrations on the substrate.

[0037] From Tables 1 and 2 Figure 2 The results show that substrates with different molecular weights of PEG affect ELISA detection. The signal reaches its peak in the PEG1000-PEG2000 range, but the background caused by non-specific adsorption also increases accordingly. Figure 2B indicates that the signal gradually increases with increasing PEG concentration, then begins to weaken after reaching 5% w / v, while the background increases with increasing concentration. Therefore, the molecular weight and concentration of PEG in the substrate affect the ELISA signal and increase background problems caused by nonspecific adsorption. The selection of PEG molecular weight and concentration is particularly important, with the molecular weight preferably between 500 and 4000 and the concentration preferably between 0.5% and 10% (w / v).

[0038] 2.2 Effect of different concentrations of stabilizer on performance Using the chromogenic substrate material from Example 1, and based on the optimal PEG molecular weight and concentration obtained in 2.1 (PEG molecular weight 1000, addition amount 3% w / v), different chromogenic substrates were prepared by sequentially varying the gelatin addition concentration in the stabilizer. The Human IL-6 ELISA experimental protocol was designed according to Table 3 for different gelatin addition concentrations. The detection steps were the same as in 2.1, and the test results are shown in Table 3. Figure 3 .

[0039] Table 3. ELISA experimental design and results showing the effect of different gelatin concentrations on the substrate.

[0040] From Table 3, Figure 3 The results show that the addition of gelatin can effectively reduce the background value caused by non-specific adsorption while ensuring the signal value. The optimal concentration is 0.1%w / v to 5%w / v. Increasing the concentration further may lead to new non-specific adsorption due to the viscosity of gelatin.

[0041] Furthermore, to investigate the stability of the substrate itself and the stability of the color after development, under the conditions of optimal PEG (molecular weight 1000, concentration 3% w / v) and gelatin (concentration 1% w / v), 0 and 0.5 mM concentrations of homosalate were set sequentially, and the Human IL-6 ELISA experimental protocol was designed according to Table 4. The detection steps were the same as in 2.1.

[0042] Table 4. ELISA experimental design and results showing the effect of homosalate on substrates.

[0043] Furthermore, the stability of the substrate itself and the stability of the color after development were investigated by accelerated aging test at 37℃. The Human IL-6 ELISA experimental protocol was designed according to Table 5, and the detection steps were the same as in 2.1.

[0044] Table 5. Experimental design and results under accelerated aging conditions.

[0045] From Tables 4 and 5 Figure 3 It can be seen that the addition of homosalate can effectively maintain the signal intensity for a period of time after color development, thus ensuring the authenticity of the experiment. Accelerated aging tests at 37℃ simulate storage conditions of 2-8℃. Figure 3 C indicates that the addition of homosalate significantly increases its preservation stability.

[0046] Through the above series of investigations, it can be seen that the present invention innovatively introduces an enhancer (polyethylene glycol or dextran), a stabilizer (homosalate and gelatin), and a catalyst (INT) into the BCIP / NBT chromogenic substrate, which can significantly improve the signal, achieve faster color development and extremely low background, and at the same time, the substrate can be effectively preserved for a long time, and the results after color development can be stable. In addition, it is compatible with methods for detecting alkaline phosphatase activity such as ELISA, ELISPOT, Western Blot, and IHC.

[0047] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0048] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An enhanced BCIP / NBT chromogenic substrate, characterized in that, The chromogenic substrate comprises the following components: substrate components BCIP and NBT, enhancer, stabilizer, iodonitroxytetrazolium chloride, a buffer system with a pH of 8.5~10.0, and metal ions; in, The reinforcing agent is polyethylene glycol or dextran; The stabilizers are homosalate and gelatin; The metal ions are magnesium ions and zinc ions.

2. The chromogenic substrate according to claim 1, characterized in that, The concentration of BCIP is 0.5~2 mg / mL; The concentration of NBT is 0.2~1 mg / mL.

3. The colorimetric substrate according to claim 1, characterized in that, The polyethylene glycol has a molecular weight of 500-4000 and a concentration of 0.5%-10% (w / v).

4. The colorimetric substrate according to claim 1, characterized in that, The concentration of homosalate is 0.1~2mM; The concentration of the gelatin is 0.1% to 5% (w / v).

5. The colorimetric substrate according to claim 1, characterized in that, The concentration of the iodonitroxytetrazolium chloride is 0.05~0.2mM.

6. The chromogenic substrate according to claim 1, characterized in that, The buffer system includes Tris-HCl buffer and diethanolamine buffer.

7. The chromogenic substrate according to claim 1, characterized in that, The magnesium ions are derived from magnesium chloride and have a concentration of 2-10 mM. The zinc ions are derived from zinc chloride and have a concentration of 0.1~1mM.

8. A method for preparing the chromogenic substrate according to any one of claims 1-7, characterized in that, The process includes the following steps: adding each component sequentially to ultrapure water, stirring until completely dissolved, filtering with a 0.22μm filter membrane for sterilization, and storing at 2~8℃ in the dark to obtain the final product.

9. The use of the chromogenic substrate according to any one of claims 1-7 in the preparation of a kit for detecting alkaline phosphatase activity.

10. The application according to claim 9, characterized in that, The methods for detecting alkaline phosphatase activity include ELISPOT, ELISA, Western Blot, Dot Blot, and IHC.

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