Preservative solution for alkaline phosphatase-labeled conjugate and preparation method thereof

Abstract

The present invention relates to the field of biochemical detection technology, and particularly relates to a preservation solution for alkaline phosphatase-labeled conjugate and a preparation method thereof. The preservation solution is composed of water and the following components: 10-100 mmol / L buffer salt, 2-20 g / L protein, 0.5-5 g / L surfactant, 0.1-0.5 mol / L salt ion, 0.5-5 g / L preservative, 0.1%-1% protective agent; the protective agent is diglycine. The preservation solution of the present invention can significantly improve the stability of the alkaline phosphatase-labeled conjugate solution during storage and use. At the same time, it can also improve the signal-to-noise ratio of some project tests, thereby reducing the cost of reagent preparation.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and particularly relates to a preservation solution for alkaline phosphatase-labeled conjugate and a preparation method thereof. Background Art

[0002] With the development of medical technology, immunoassay has attracted more and more attention. Among them, chemiluminescence immunoassay is a non-radioactive immunoassay method that has developed rapidly in the past thirty years. As a highly sensitive microassay technology, it has gradually replaced ELISA immunoassay. Chemiluminescence immunoassay has also been applied in many aspects such as thyroid, hormones, metabolism, cardiovascular, inflammation and tumors. Among them, enzyme-labeled antibody conjugate, as one of the key components of enzymatic reactions, has defects such as easy degradation during storage, reduction of enzyme activity and reaction activity of antibody-antibody specific binding. Therefore, improving the stability and sensitivity of enzyme-labeled antibody conjugate is crucial for the application of chemiluminescence immunoassay.

[0003] Currently, the methods for improving the stability of enzyme-labeled antibody conjugate mainly include chemical modification and addition techniques. Among them, chemical modification techniques are relatively complex and difficult to control; addition techniques are simple and easy to implement. Therefore, addition techniques are more widely used.

[0004] Existing addition techniques mainly include the addition of various stabilizers such as alcohols, sugars, and lipids, and some also add IgG protection. Different enzyme-labeled conjugates may require the addition of one or several combinations of stabilizers such as alcohols, sugars, and lipids, and IgG may also be one or more combinations, that is, different enzyme-labeled conjugates require different formulations. On the one hand, the preparation process is relatively cumbersome and complex; on the other hand, IgG is generally relatively expensive, and adding one or more IgG will increase the cost; more importantly, after adding these components, it may also affect the reagent performance, such as sensitivity, minimum detection limit, etc. Summary of the Invention

[0005] In view of this, the present invention provides a preservation solution for alkaline phosphatase-labeled conjugate and a preparation method thereof. This preservation solution can significantly improve the stability of alkaline phosphatase-labeled conjugate solution during storage and use.

[0006] In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0007] A preservation solution for alkaline phosphatase-labeled conjugate, which is composed of water and the following components:

[0008] 10 - 100 mmol / L buffer salt, 2 - 20 g / L protein, 0.5 - 5 g / L surfactant, 0.1 - 0.5 mol / L salt ion, 0.5 - 5 g / L preservative, and 0.1% - 1% protective agent;

[0009] The protective agent is diglycine.

[0010] In the present invention, the mass fraction of the protective agent is 0.1% - 1%, preferably 0.4%.

[0011] In the present invention, the pH of the preservation solution is 7.00 - 8.00, preferably 7.4.

[0012] In the present invention, the buffer salt is selected from at least one of Tris, MES, PB, and HEPES; the concentration of the buffer salt in the preservation solution is 10 - 100 mmol / L, preferably 50 mmol / L.

[0013] In the present invention, the protein is selected from at least one of bovine serum albumin, casein, and gelatin; the concentration of the protein in the preservation solution is 2 - 20 g / L, preferably 10 g / L.

[0014] In the present invention, the surfactant is selected from at least one of Tween 20, Tween 80, Triton x - 100, and Triton x - 405; the concentration of the surfactant in the preservation solution is 0.5 - 5 g / L, preferably 1 g / L.

[0015] In the present invention, the salt ions are selected from at least one of sodium chloride, magnesium chloride, zinc chloride, and potassium chloride, preferably a combination of sodium chloride, magnesium chloride, and zinc chloride. In the specific embodiments of the present invention, the salt ions are composed of sodium chloride, magnesium chloride, and zinc chloride, and the concentrations of each component in the preservation solution are: 0.1 - 0.3 mol / L sodium chloride, 1 - 25 mmol / L magnesium chloride, and 0.2 - 5 mmol / L zinc chloride.

[0016] In the present invention, the preservative is selected from at least one of sodium azide, PC300, chloramphenicol, and gentamicin, preferably PC300; the concentration of the preservative is 0.5 - 5 g / L, preferably 1.0 g / L.

[0017] The present invention also provides a method for preparing the preservation solution, comprising:

[0018] Adding a buffer salt to water, mixing evenly, and adjusting the pH to 7.00 - 8.00; then adding an ionic salt, a protein, a surfactant, a preservative, and a protective agent, mixing evenly, and adjusting the pH to 7.00 - 8.00; making up the volume and filtering.

[0019] In the present invention, the filtering is through a filter membrane with a pore size of 0.10 - 0.50 μm, specifically 0.1 μm, 0.22 μm, or 0.5 μm.

[0020] The present invention provides a preservation solution for alkaline phosphatase-labeled conjugates and a preparation method thereof. The preservation solution consists of water and the following components: 10 - 100 mmol / L buffer salt, 2 - 20 g / L protein, 0.5 - 5 g / L surfactant, 0.1 - 0.5 mol / L salt ions, 0.5 - 5 g / L preservative, and 0.1% - 1% protective agent; the protective agent is diglycine. Compared with the prior art, the advantages of the present invention are as follows

[0021] 1. The preservation solution of the present invention can not only significantly improve the thermal stability of alkaline phosphatase conjugates labeled with different antibodies (such as NT-proBNP, PG I, TGAb, TPO) after 7 days of acceleration at 37°C, but also improve the test signal-to-noise ratio of NT-proBNP and PG I, and reduce the cost of reagent preparation;

[0022] 2. The present invention uses diglycine as a protective agent in the preservation solution for alkaline phosphatase conjugates, without the need to add stabilizers such as IgG, sugars, lipids, alcohols, etc.; the components are simple, the preparation is convenient, and it is convenient to achieve mass production. Specific embodiments

[0023] The present invention provides a preservation solution for alkaline phosphatase-labeled conjugates and a preparation method thereof. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate modifications and combinations to the methods and applications in this article without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0024] In the present invention, "BSA" is the abbreviation of "bovine serum albumin".

[0025] Unless otherwise specified, the test materials used in the present invention are all ordinary commercially available products and can be purchased in the market.

[0026] The following combines examples to further elaborate on the present invention:

[0027] Example 1

[0028] For the preservation solution of alkaline phosphatase-labeled conjugates, based on the volume of the preservation solution, the components and their contents are as follows: 50 mmol / L Tris, 10 g / L BSA, 0.15 mol / L NaCl, 1 g / L Tween 20, 1 g / L PC300, 1 mmol / L MgCl2, 0.2 mmol / L ZnCl2, 4 g of diglycine.

[0029] The preparation steps are as follows:

[0030] 1. After adding 6.06 g of Tris buffer salt into a clean container, add 800 mL of pure water and mix well. Adjust the pH to 7.00 - 8.00 with 1 M HCl;

[0031] 2. Add 9 g of NaCl, 0.09 g of MgCl2, and 0.027 g of ZnCl2 to the above solution and stir to mix evenly;

[0032] 3. Add 10 g of BSA and stir to mix evenly;

[0033] 4. Add 1 g of surfactant Tw-20 and stir to mix evenly;

[0034] 5. Add 1 g of preservative PC300 and stir to mix evenly;

[0035] 6. Add the protective agent diglycine, stir to mix evenly, adjust the pH to 7.40 with 5 M HCl or 5 M NaOH, make up the volume to 1000 mL with pure water, and filter through a 0.22 - μm filter to obtain the preservation solution.

[0036] Example 2

[0037] The preservation solution for alkaline phosphatase-labeled conjugate, based on the volume of the preservation solution, the components and their contents are as follows: 10 mmol / L Tris, 2 g / L BSA, 0.1 mol / L NaCl, 0.5 g / L Tween 20, 0.5 g / L PC300, 5 mmol / L MgCl2, 1 mmol / L ZnCl2, 1 g of diglycine.

[0038] The preparation steps are as follows:

[0039] 1. After adding 1.2100 g of Tris buffer salt into a clean container, add 800 mL of pure water and mix well. Adjust the pH to 7.00 - 8.00 with 1 M HCl;

[0040] 2. Add 6.0000 g of NaCl, 0.4500 g of MgCl2, and 0.1350 g of ZnCl2 to the above solution and stir to mix evenly;

[0041] 1. Add 2.0000 g of BSA and stir to mix evenly;

[0042] 2. Add 0.5000 g of surfactant Tween 20 and stir to mix evenly;

[0043] 3. Add 0.5000 g of preservative PC300 and stir to mix evenly;

[0044] 4. Add 1.0000 g of diglycine as a preservative, stir to mix evenly, adjust the pH to 7.40 with 5M HCl or 5M NaOH, make up the volume to 1000 mL with pure water, and filter through a 0.22 μm filter to obtain the storage solution.

[0045] Example 3

[0046] The storage solution for alkaline phosphatase-labeled conjugate, based on the volume of the storage solution, the components and their contents are as follows: 50 mmol / L Tris, 15 g / L BSA, 0.2 mol / L NaCl, 2 g / L Tween 20, 2 g / L PC300, 10 mmol / L MgCl2, 5 mmol / L ZnCl2, 10 g of diglycine.

[0047] The preparation steps are as follows:

[0048] 1. Add 6.0600 g of Tris buffer salt to a clean container, then add 800 mL of pure water and mix well, adjust the pH to 7.00 - 8.00 with 1M HCl;

[0049] 2. Add 12.0000 g of NaCl, 0.9000 g of MgCl2, and 0.675 g of ZnCl2 to the above solution, and stir to mix evenly;

[0050] 3. Add 15.0000 g of BSA and stir to mix evenly;

[0051] 4. Add 2.0000 g of Tween 20 as a surfactant and stir to mix evenly;

[0052] 5. Add 2 g of PC300 as a preservative and stir to mix evenly;

[0053] 6. Add 10 g of diglycine as a preservative, stir to mix evenly, adjust the pH to 7.40 with 5M HCl or 5M NaOH, make up the volume to 1000 mL with pure water, and filter through a 0.22 μm filter to obtain the storage solution.

[0054] Comparative Example 1

[0055] The storage solution for alkaline phosphatase-labeled conjugate, based on the volume of the storage solution, the components and their contents are as follows:

[0056] 50 mmol / L Tris, 10 g / L BSA, 0.15 mol / L NaCl, 1.0000 g / L Tween 20, 1.0000 g / L PC300, 1 mmol / L MgCl2, 2 mmol / L ZnCl2, 50 g / L glycerol, 10 g / L glucose, 1 g / L chitosan, 1 g / L polyethylene glycol 4000, 5 g / L mouse IgG.

[0057] Test Example

[0058] The enzyme-labeled reagent is prepared by adding amino-terminal pro-brain natriuretic peptide (NT-proBNP) or pepsinogen I (PG I) and an alkaline phosphatase-labeled conjugate to the above-mentioned preservation solution.

[0059] The reagents of the above-mentioned Examples 1 to 3 and Comparative Example 1 were each divided into three portions. One portion was used for signal-to-noise ratio testing, one portion was placed at 37 °C for accelerated aging for 7 days, and one portion was placed at 2 - 8 °C for storage. After 7 days of placement, using the reagent placed at 2 - 8 °C as a control, the same samples were synchronously tested with the two enzyme-labeled reagents.

[0060] The signal-to-noise ratio test results are as follows:

[0061] Table 1 Signal-to-noise ratio test results of NT-proBNP and PG I enzyme-labeled conjugates

[0062]

[0063] The results of accelerated aging at 37 °C for 7 days are as follows:

[0064] Table 2 Stability results of NT-proBNP and PG I enzyme-labeled conjugates after accelerated aging at 37 °C for 7 days

[0065]

[0066]

[0067] It can be seen from the detection results that the thermal stability of the alkaline phosphatase conjugate solution prepared with the preservation solution of the present invention is significantly improved and the signal-to-noise ratio is better; after accelerated aging at 37 °C for 7 days, the decrease in luminescence values of NT-proBNP and PG I is within 3%; the content of the protective agent has little effect on the stability.

[0068] It can be seen from Comparative Experimental Example 1 that in the preservation solution containing alcohols, sugars, lipids, and murine IgG, after accelerated aging at 37 °C for 7 days, the decrease in luminescence values of the alkaline phosphatase conjugate is all above 15%;

[0069] In Table 1, NT-proBNP and PG I were replaced with TGAb and TPO, and the luminescence values after 7 days of accelerated aging were detected by the above method. The test results are as follows:

[0070] Table 3 Stability results of TGAb and TPO enzyme-labeled conjugates after accelerated aging at 37 °C for 7 days

[0071]

[0072]

[0073] It can be seen from the detection results that the decrease in luminescence values for Examples 1 - 3 is also within 4%; for Comparative Experiment Example 1, the decrease in luminescence values is above 15% for all.

[0074] Considering the structural characteristics of diglycine, the effects of diglycine and glycine on the thermal stability of the enzyme-labeled working solution were investigated separately.

[0075] Table 4

[0076]

[0077]

[0078] It can be seen from the detection results that when diglycine is added to the enzyme-labeled working solution, the decrease in luminescence values is within 5% for all; compared with adding glycine, the decrease in luminescence values is above 15% for all. Adding glycine to the enzyme-labeled working solution has no significant protective effect. Moreover, based on the pH of the solution, the protective effect of diglycine is not the protective effect as an amino acid protectant after hydrolysis into glycine.

[0079] Therefore, the preservation solution of the present invention can not only significantly improve the thermal stability of different antibody alkaline phosphatase conjugates accelerated at 37°C for 7 days, but also improve the test signal-to-noise ratio of some test items.

[0080] In addition, the following tests were also carried out on the basis of the formulation of Example 1 of the present invention:

[0081] When the buffer salt was replaced with other types, such as MES, HEPES, PB, etc., the effect of the preservation solution was relatively close to that of Example 1, with no obvious difference.

[0082] When the ionic salt NaCl was replaced with KCl, the effect of the preservation solution was relatively close to that of Example 1, with no obvious difference.

[0083] When the surfactant was replaced with Tween 80, Triton x-100, Triton x-405, the effect of the preservation solution was relatively close to that of Example 1, with no obvious difference.

[0084] When the preservative was replaced with sodium azide, chloramphenicol, gentamicin, the effect of the preservation solution was relatively close to that of Example 1, with no obvious difference.

[0085] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the principle of the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A preservation solution for alkaline phosphatase-labeled conjugate, characterized in that, It consists of water and the following components: 10 - 100 mmol / L buffer salt, 2 - 20 g / L protein, 0.5 - 5 g / L surfactant, 0.1 - 0.5 mol / L salt ions, 0.5 - 5 g / L preservative, and 0.1% - 1% protective agent; The protective agent is diglycine.

2. The preservation solution according to claim 1, characterized in that, Its pH is 7.00 - 8.

00.

3. The preservation solution according to claim 1, characterized in that, The buffer salt is selected from at least one of Tris, MES, PB, and HEPES.

4. The preservation solution according to claim 1, characterized in that, The protein is selected from at least one of bovine serum albumin, casein, and gelatin protein.

5. The preservation solution according to claim 1, characterized in that, The surfactant is selected from at least one of Tween 20, Tween 80, Triton x - 100, and Triton x - 405.

6. The preservation solution according to claim 1, characterized in that, The salt ions are selected from at least one of sodium chloride, magnesium chloride, zinc chloride, and potassium chloride.

7. The preservation solution according to claim 6, characterized in that, The salt ions consist of sodium chloride, magnesium chloride, and zinc chloride, and each component is composed of 0.1 - 0.3 mol / L sodium chloride, 1 - 25 mmol / L magnesium chloride, and 0.2 - 5 mmol / L zinc chloride.

8. The preservation solution according to claim 1, characterized in that, The preservative is selected from at least one of sodium azide, PC300, chloramphenicol, and gentamicin.

9. A preparation method of the preservation solution according to any one of claims 1 to 8, characterized in that, It includes: Add the buffer salt to water, mix well, and adjust the pH to 7.00 - 8.00; then add the ionic salt, protein, surfactant, preservative, and protective agent, mix well, and adjust the pH to 7.00 - 8.00; make up the volume and filter.

10. The preparation method according to claim 9, characterized in that, The filtration is through a 0.10 - 0.50 μm filter membrane.

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