CN116008530A - Preparation method and application of HRP (HRP) conjugated antibody dilution stabilizer - Google Patents

Abstract

The invention discloses a preparation method and application of a HRP-coupled antibody dilution stabilizer: firstly prepare PBS buffer solution, then add protective protein, PEG, Tween and preservative into it, mix well and then filter and sterilize to obtain The HRP-conjugated antibody is diluted with stabilizer. This dilution stabilizer can effectively slow down the reduction of the enzyme-labeled antibody activity, and is used for the diluted storage of the enzyme-labeled antibody in the BSA sandwich ELISA antibody detection kit. The diluted enzyme-labeled antibody can be stored at 2–8°C for at least 12 months. Basically no change. This dilution stabilizer can also be used for the dilution of some common enzyme-labeled antibodies, so as to maintain their biological activity for a long time.

Description

A kind of preparation method and application of HRP-coupled antibody dilution stabilizer

technical field

The invention belongs to the technical field of diagnostic biological products, and in particular relates to a preparation method of an HRP-coupled antibody dilution stabilizer and its application in a BSA detection kit.

Background technique

Enzyme-conjugated antibodies, especially horseradish peroxidase (HRP)-conjugated antibodies, generally can only be stored at -20°C or even lower temperatures and cannot be repeatedly frozen and thawed, so that their immune activity can be maintained at a certain level. Time (generally one year) will not be greatly reduced, thereby effectively ensuring the reliability of the detection method results. In recent years, a large number of studies have shown that some protective proteins, stabilizers, etc. have a little protective effect on the activity of enzyme-conjugated antibodies. However, a large number of applications have proved that each enzyme-conjugated antibody stabilizer is not broad-spectrum, and the relevant formula needs to be adjusted to verify its effect. Especially for the detection of BSA, the storage of its enzyme-conjugated antibody cannot use the conventional protective protein BSA.

Therefore, it was explored to develop a dilution stabilizer that can effectively protect the immune activity of the BSA monoclonal antibody enzyme-conjugated antibody Mars when it is stored at 2-8°C or even room temperature for a long time, so as to ensure the stability of the kit. Furthermore, ensuring the accuracy of the results and the convenience of operation is crucial to the commercialization of the kit.

Contents of the invention

Compared with the prior art, the present invention provides a dilution stabilizer of HRP-coupled antibody, which can preserve the diluted HRP-coupled antibody stock solution at 2-8°C for at least 12 months. The specific implementation techniques are as follows:

A preparation method of an HRP-coupled antibody dilution stabilizer, the preparation method comprising the following steps:

Step S1: Add 80g of NaCl, 2g of KCl, 14.2g of Na ₂ HPO ₄ , and 2.7g of KH ₂ PO ₄ into 800mL of deionized water. After completely dissolving, adjust the pH to 7.2, and then add deionized water to make it up to 1L to obtain 10× PBS solution;

Step S2: Take 300mL of ultrapure water, add 100mL of 10×PBS solution, 1-10g of protective protein, and 1-2g of high molecular polymer. After fully dissolving, add 0.5-1mL of surfactant, 500mL of antifreeze, and 0.3- 0.5mL, mix thoroughly, dilute to 1L with ultrapure water, and then filter and sterilize with a 0.2μm filter membrane to obtain the HRP-conjugated antibody dilution stabilizer.

Preferably, the protective protein described in step S2 is gelatin, the polymer is polyethylene glycol (PEG), the surfactant is Tween-20, the antifreeze is glycerin, and the preservative For Proclin-300.

Preferably, the step S2 is: each 1L of HRP-conjugated antibody dilution stabilizer contains 100mL of 10×PBS solution, 2g of protective protein, 1g of high molecular weight polymer, 0.5mL of Tween-20, 80mL of glycerin, and 0.5mL of Proclin-300 .

In a preferred embodiment of the present invention, the step S2 is: add 100mL of 10×PBS solution, 2g of gelatin, and 1g of polymer to 300mL of ultrapure water, and after fully dissolving, add 0.5mL of Tween-20 and 500mL of glycerin , 0.5mL Proclin-300, after mixing thoroughly, dilute to 1L with ultra-pure water, filter and sterilize through a 0.2μm filter membrane to obtain a diluted stabilizer.

The present invention is based on the above-mentioned dilution stabilizer, which is used in bovine serum albumin (BSA) detection kits, which include anti-BSA monoclonal antibody coated microwell plates, HRP labeling reagents, HRP labeling reagent dilutions, 15 times concentrated Wash solution, sample diluent, BSA standard solution, TMB substrate solution and stop solution. Wherein, the HRP labeling reagent is obtained by diluting the HRP-conjugated antibody stock solution by 1:20 with the HRP-conjugated antibody dilution stabilizer.

Preferably, the specific method of use of the bovine serum albumin (BSA) detection kit is:

P1: Take out the kit, and equilibrate all reagents at room temperature for 30 minutes.

P2: Take out the coated microwell plate, add 350 μL of washing solution to each well to wash the plate, and repeat 2 times.

P3: Add 0, 5, 10, 20, 30, 40, 50, 60ng/mL standard solutions and samples to be tested one by one, 50 μL/well. .

P4: Seal the plate with a sealing film, and incubate for 1 hour at 25°C in the dark.

P5: At the end of the incubation, discard the liquid in the wells of the enzyme labeling plate, add 350 μL of washing solution to each well to wash the plate, and repeat 2 times.

P6: Add 100 μL of HRP-labeled antibody working solution diluted with HRP-labeled reagent diluent to each well.

P7: Seal the plate with the sealing film, and incubate at 25°C in the dark for 30 minutes.

P8: After incubation, discard the liquid in the microplate wells, add 350 μL of washing solution to each well to wash the plate, and repeat 4 times.

P9: Add 50 μL TMB substrate solution to each well, and let it stand at 25°C in the dark for 15 minutes.

P10: Add 50 μL of reaction termination solution to each well.

P11: Use a microplate reader to read the absorbance at dual wavelengths (450nm and 630nm).

The present invention solves the defects existing in the background technology, and the present invention has the following beneficial effects: the dilution stabilizer of the HRP-coupled antibody provided by the present invention contains protective protein gelatin, high molecular polymer PEG, and surfactant Tween-20 , antifreeze glycerin, and preservative Proclin-300, the five complement each other, and the HRP labeling reagent obtained by diluting this dilution stabilizer can be stored at 2-8°C for 12 months. In the field of reagent kits, the combination of these five reagents has not been reported in literature and patents.

Detailed ways

In order to more clearly understand the above objects, features and advantages of the present invention, the following specific embodiments will further describe the present invention in detail. It should be noted that, in the case of no conflict, the embodiments of the present application and the features in the embodiments can be combined with each other.

Apparently, the described embodiments are only some of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative work, all belong to the protection scope of the present invention, and many specific details are set forth in the following description so as to fully Understand the present invention, however, the present invention can also be implemented in other ways than those described here, therefore, the protection scope of the present invention is not limited by the specific embodiments disclosed below.

In the following examples and comparative examples, the gelatin, PEG, and Proclin-300 used were purchased from Sigma, and glycerin and Tween-20 were purchased from Sinopharm.

In the following application examples, the method of using the kit is as follows:

P1: Take out the kit, and equilibrate all reagents at room temperature for 30 minutes.

P2: Take out the coated microwell plate, add 350 μL of washing solution to each well to wash the plate, and repeat 2 times.

P3: Add 0, 5, 10, 20, 30, 40, 50, 60ng/mL standard solutions and samples to be tested one by one, 50 μL/well. .

P4: Seal the plate with a sealing film, and incubate for 1 hour at 25°C in the dark.

P5: At the end of the incubation, discard the liquid in the wells of the enzyme labeling plate, add 350 μL of washing solution to each well to wash the plate, and repeat 2 times.

P6: Add 100 μL of HRP-labeled antibody working solution diluted with HRP-labeled reagent diluent to each well.

P7: Seal the plate with the sealing film, and incubate at 25°C in the dark for 30 minutes.

P8: After incubation, discard the liquid in the microplate wells, add 350 μL of washing solution to each well to wash the plate, and repeat 4 times.

P9: Add 50 μL TMB substrate solution to each well, and let it stand at 25°C in the dark for 15 minutes.

P10: Add 50 μL of reaction termination solution to each well.

P11: Use a microplate reader to read the absorbance at dual wavelengths (450nm and 630nm).

Example 1

The stable diluent of the HRP-conjugated antibody provided in this example, its preparation method is:

Step S1: Add 80g of NaCl, 2g of KCl, 14.2g of Na ₂ HPO ₄ , and 2.7g of KH ₂ PO ₄ into 800mL of deionized water. After completely dissolving, adjust the pH to 7.2, and then add deionized water to make it up to 1L to obtain 10× PBS solution;

Step S2: Take 300mL of ultrapure water, add 100mL of 10×PBS solution, 2g of gelatin, and 1g of PEG. After fully dissolving, add 0.5mL of Tween-20, 500mL of glycerin, and 0.5mL of Proclin-300, mix well, and set After the volume was reduced to 1 L, filter and sterilize with a 0.2 μm filter membrane to obtain the HRP-conjugated antibody dilution stabilizer.

Example 2

The stable diluent of the HRP-conjugated antibody provided in this example, its preparation method is:

Step S1: Add 80g of NaCl, 2g of KCl, 14.2g of Na ₂ HPO ₄ , and 2.7g of KH ₂ PO ₄ into 800mL of deionized water. After completely dissolving, adjust the pH to 7.2, and then add deionized water to make it up to 1L to obtain 10× PBS solution;

Step S2: Take 300mL of ultrapure water, add 100mL of 10×PBS solution, 2g of gelatin, and 1g of PEG. After fully dissolving, add 0.5mL of Tween-20, 500mL of glycerin, and 0.3mL of Proclin-300, mix well, and set After the volume was reduced to 1 L, filter and sterilize with a 0.2 μm filter membrane to obtain the HRP-conjugated antibody dilution stabilizer.

Example 3

The stable diluent of the HRP-conjugated antibody provided in this example, its preparation method is:

Step S1: Add 80g of NaCl, 2g of KCl, 14.2g of Na ₂ HPO ₄ , and 2.7g of KH ₂ PO ₄ into 800mL of deionized water. After completely dissolving, adjust the pH to 7.2, and then add deionized water to make it up to 1L to obtain 10× PBS solution;

Step S2: Take 300mL of ultrapure water, add 100mL of 10×PBS solution, 2g of gelatin, and 1g of PEG. After fully dissolving, add 1mL of Tween-20, 500mL of glycerin, and 0.5mL of Proclin-300, mix well, and dilute to volume with ultrapure water After reaching 1 L, filter and sterilize with a 0.2 μm filter membrane to obtain the HRP-conjugated antibody dilution stabilizer.

Example 4

The stable diluent of the HRP-conjugated antibody provided in this example, its preparation method is:

Step S1: Add 80g of NaCl, 2g of KCl, 14.2g of Na ₂ HPO ₄ , and 2.7g of KH ₂ PO ₄ into 800mL of deionized water. After completely dissolving, adjust the pH to 7.2, and then add deionized water to make it up to 1L to obtain 10× PBS solution;

Step S2: Take 300mL of ultrapure water, add 100mL of 10×PBS solution, 2g of gelatin, and 2g of PEG. After fully dissolving, add 0.5mL of Tween-20, 500mL of glycerin, and 0.5mL of Proclin-300. After the volume was reduced to 1 L, filter and sterilize with a 0.2 μm filter membrane to obtain the HRP-conjugated antibody dilution stabilizer.

Example 5

The stable diluent of the HRP-conjugated antibody provided in this example, its preparation method is:

Step S1: Add 80g of NaCl, 2g of KCl, 14.2g of Na ₂ HPO ₄ , and 2.7g of KH ₂ PO ₄ into 800mL of deionized water. After completely dissolving, adjust the pH to 7.2, and then add deionized water to make it up to 1L to obtain 10× PBS solution;

Step S2: Take 300 mL of ultrapure water, add 100 mL of 10×PBS solution, 5 g of gelatin, and 1 g of PEG. After fully dissolving, add 0.5 mL of Tween-20, 500 mL of glycerin, and 0.5 mL of Proclin-300. After the volume was reduced to 1 L, filter and sterilize with a 0.2 μm filter membrane to obtain the HRP-conjugated antibody dilution stabilizer.

Example 6

The stable diluent of the HRP-conjugated antibody provided in this example, its preparation method is:

Step S1: Add 80g of NaCl, 2g of KCl, 14.2g of Na ₂ HPO ₄ , and 2.7g of KH ₂ PO ₄ into 800mL of deionized water. After completely dissolving, adjust the pH to 7.2, and then add deionized water to make it up to 1L to obtain 10× PBS solution;

Step S2: Take 300mL of ultrapure water, add 100mL of 10×PBS solution, 10g of gelatin, and 1g of PEG. After fully dissolving, add 0.5mL of Tween-20, 500mL of glycerin, and 0.5mL of Proclin-300, mix well, and set After the volume was reduced to 1 L, filter and sterilize with a 0.2 μm filter membrane to obtain the HRP-conjugated antibody dilution stabilizer.

Comparative example 1

The stable diluent of HRP conjugated antibody provided in this comparative example, its preparation method is:

Step S1: Add 80g of NaCl, 2g of KCl, 14.2g of Na ₂ HPO ₄ , and 2.7g of KH ₂ PO ₄ into 800mL of deionized water. After completely dissolving, adjust the pH to 7.2, and then add deionized water to make it up to 1L to obtain 10× PBS solution;

Step S2: Take 300mL of ultrapure water, add 100mL of 10×PBS solution, 2g of gelatin, and 1g of PEG. After fully dissolving, add 0.5mL of Tween-20 and 0.5mL of Proclin-300, mix well, and dilute to 1L with ultrapure water Afterwards, filter and sterilize with a 0.2 μm filter membrane to obtain the HRP-conjugated antibody dilution stabilizer.

Comparative example 2

The stable diluent of HRP conjugated antibody provided in this comparative example, its preparation method is:

Step S1: Add 80g of NaCl, 2g of KCl, 14.2g of Na ₂ HPO ₄ , and 2.7g of KH ₂ PO ₄ into 800mL of deionized water. After completely dissolving, adjust the pH to 7.2, and then add deionized water to make it up to 1L to obtain 10× PBS solution;

Step S2: Take 300mL of ultrapure water, add 100mL of 10×PBS solution, 1g of PEG, after fully dissolved, add 0.5mL of Tween-20, 500mL of glycerin, 0.5mL of Proclin-300, mix well, and dilute to 1L with ultrapure water Afterwards, filter and sterilize with a 0.2 μm filter membrane to obtain the HRP-conjugated antibody dilution stabilizer.

Comparative example 3

The stable diluent of HRP conjugated antibody provided in this comparative example, its preparation method is:

Step S1: Add 80g of NaCl, 2g of KCl, 14.2g of Na ₂ HPO ₄ , and 2.7g of KH ₂ PO ₄ into 800mL of deionized water. After completely dissolving, adjust the pH to 7.2, and then add deionized water to make it up to 1L to obtain 10× PBS solution;

Step S2: Take 300mL of ultrapure water, add 100mL of 10×PBS solution, 2g of gelatin, after fully dissolved, add 0.5mL of Tween-20, 500mL of glycerin, 0.5mL of Proclin-300, mix well, and dilute to After 1 L, filter and sterilize with a 0.2 μm filter membrane to obtain the HRP-conjugated antibody dilution stabilizer.

Comparative example 4

The stable diluent of HRP conjugated antibody provided in this comparative example, its preparation method is:

Step S1: Add 80g of NaCl, 2g of KCl, 14.2g of Na ₂ HPO ₄ , and 2.7g of KH ₂ PO ₄ into 800mL of deionized water. After completely dissolving, adjust the pH to 7.2, and then add deionized water to make it up to 1L to obtain 10× PBS solution;

Step S2: Take 300mL of ultrapure water, add 100mL of 10×PBS solution, 0.5mL of Tween-20, 500mL of glycerin, and 0.5mL of Proclin-300, mix thoroughly, dilute to 1L with ultrapure water, and filter with 0.2μm Sterilize by membrane filtration to obtain the HRP-conjugated antibody dilution stabilizer.

Comparative example 5

The stable diluent of HRP conjugated antibody provided in this comparative example, its preparation method is:

Step S1: Add 80g of NaCl, 2g of KCl, 14.2g of Na ₂ HPO ₄ , and 2.7g of KH ₂ PO ₄ into 800mL of deionized water. After completely dissolving, adjust the pH to 7.2, and then add deionized water to make it up to 1L to obtain 10× PBS solution;

Step S2: Take 300 mL of ultrapure water, add 100 mL of 10×PBS solution and 0.5 mL of Tween-20, mix thoroughly, and dilute to 1 L with ultrapure water, then filter and sterilize with a 0.2 μm filter membrane to obtain HRP pair Antibody dilution stabilizer.

Comparative example 6

The stabilizing diluent for the HRP-conjugated antibody provided in this comparative example is a commercially available HRP-conjugated antibody stabilizer, and the specific formula is unknown.

Application example 1:

Use the diluted stabilizer prepared in Examples 1-6 and Comparative Examples 1-6 to dilute the stock solution of the HRP-conjugated antibody at 1:20 to obtain the HRP-labeled reagent, which is subjected to an accelerated destruction test at 37°C and tested once every 2 days. A total of 5 tests were performed. In this way, the protection of the diluted stabilizers prepared in Examples 1-6 and Comparative Examples 1-6 to HRP-conjugated antibodies was tested, and the specific results are shown in Table 1.

Table 1: OD value of BSA standard solution 60, 0ng/mL in accelerated destruction test at 37°C

It can be seen from Table 1 that the diluted stabilizer prepared in Example 1 is significantly better than Examples 2-6 and Comparative Examples 1-6 in protecting HRP-conjugated antibodies in the accelerated destruction test at 37°C. Although Examples 2-6 have a certain protective effect, the signal value at 60ng/mL is relatively low. The signal value of Comparative Example 1 is relatively weak. Comparative examples 2, 4 and 5 have poor protection effects. Although Comparative Examples 3 and 6 have better protective effects, the background value is higher at 0 ng/mL, resulting in a significantly lower signal-to-noise ratio than Example 1.

Application example 2: The storage life experiment of HRP labeling reagent diluted with dilution stabilizer at 2-8°C

The diluted stabilizer prepared in Examples 1-6 and Comparative Examples 1-6 was selected to dilute the stock solution of HRP-conjugated antibody at 1:20 to obtain HRP labeling reagent, which was stored at 2-8°C for later use.

Experiment 1: Standard solution 60, 0ng/mL test experiment

Take out the HRP labeling reagent every 1-2 months, and test it according to the experimental detection method for a total of 12 months. The test results are shown in Table 2.

Table 2: Results of HRP labeling reagent test standard solution 60, 0ng/mL stored at 4°C

Experiment 2: Physical Properties Experiment

(1) Detection of solution clarity: the HRP labeling reagent prepared by using the diluted stabilizer prepared in Example 1 was stored at 2-8°C. Test every 1 month for a total of 12 months to see if it is easy to clear. The results showed that all were clear solutions.

(2) Sterility test: the HRP labeling reagent prepared by using the diluted stabilizer prepared in Example 1 was stored at 2-8°C. They were inoculated on plates every 1 month and cultured at 37°C. A total of 7 days of culture and observation, a total of 12 months of inspection. The test results were all sterile.

Claims (9)

1. A preparation method of HRP-coupled antibody dilution stabilizer, characterized in that, its preparation method may further comprise the steps: Step S1: Add 80g of NaCl, 2g of KCl, 14.2g of Na ₂ HPO ₄ , and 2.7g of KH ₂ PO ₄ into 800mL of deionized water. After completely dissolving, adjust the pH to 7.2, and then add deionized water to make it up to 1L to obtain 10× PBS solution; Step S2: Take 300mL of ultrapure water, add 100mL of 10×PBS solution, 1-10g of protective protein, and 1-2g of high molecular polymer. After fully dissolving, add 0.5-1mL of surfactant, 500mL of antifreeze, and 0.3- 0.5mL, mix thoroughly, dilute to 1L with ultrapure water, and then filter and sterilize with a 0.2μm filter membrane to obtain the HRP-conjugated antibody dilution stabilizer. 2. The preparation method of a kind of HRP-coupled antibody dilution stabilizer according to claim 1, characterized in that: the protective protein in step S2 is gelatin. 3. The preparation method of a kind of HRP-coupled antibody dilution stabilizer according to claim 1, characterized in that: the high molecular polymer described in step S2 is polyethylene glycol (PEG). 4. The preparation method of a HRP-coupled antibody dilution stabilizer according to claim 1, characterized in that: the surfactant in step S2 is Tween-20. 5. The preparation method of a kind of HRP-coupled antibody dilution stabilizer according to claim 1, characterized in that: the antifreeze agent in step S2 is glycerol. 6. The preparation method of a HRP-coupled antibody dilution stabilizer according to claim 1, characterized in that: the preservative in step S2 is Proclin-300. 7. The preparation method of a kind of HRP-coupled antibody dilution stabilizer according to claim 1, characterized in that, the step S2 is: add 100mL10×PBS solution, 2g gelatin, 1g polymer to 300mL ultrapure water After the polymer is fully dissolved, add 0.5mL Tween-20, 500mL glycerin, and 0.5mL Proclin-300, mix thoroughly, and then dilute to 1L with ultrapure water, and filter and sterilize through a 0.2μm membrane filter to obtain a diluted stabilizer. 8. The application of a HRP-coupled antibody dilution stabilizer, characterized in that: the dilution stabilizer is used in bovine serum albumin (BSA) detection kits, and the kits include anti-BSA monoclonal antibody coated microwell plates, HRP Labeling Reagent, HRP Labeling Reagent Diluent, 15-fold Concentrated Wash Solution, Sample Diluent, BSA Standard Solution, TMB Substrate Solution and Stop Solution. Wherein, the HRP labeling reagent is obtained by diluting the HRP-conjugated antibody stock solution by 1:20 with the HRP-conjugated antibody dilution stabilizer. 9. the application of HRP-coupled antibody dilution stabilizer according to claim 8, is characterized in that: the concrete use method of described bovine serum albumin (BSA) detection kit is: P1: Take out the kit, and equilibrate all reagents at room temperature for 30 minutes. P2: Take out the coated microwell plate, add 350 μL of washing solution to each well to wash the plate, and repeat 2 times. P3: Add 0, 5, 10, 20, 30, 40, 50, 60ng/mL standard solutions and samples to be tested one by one, 50 μL/well. . P4: Seal the plate with a sealing film, and incubate for 1 hour at 25°C in the dark. P5: At the end of the incubation, discard the liquid in the wells of the enzyme labeling plate, add 350 μL of washing solution to each well to wash the plate, and repeat 2 times. P6: Add 100 μL of HRP-labeled antibody working solution diluted with HRP-labeled reagent diluent to each well. P7: Seal the plate with the sealing film, and incubate at 25°C in the dark for 30 minutes. P8: After incubation, discard the liquid in the microplate wells, add 350 μL of washing solution to each well to wash the plate, and repeat 4 times. P9: Add 50 μL TMB substrate solution to each well, and let it stand at 25°C in the dark for 15 minutes. P10: Add 50 μL of reaction termination solution to each well. P11: Use a microplate reader to read the absorbance at dual wavelengths (450nm and 630nm).