Standard product stabilizer for liquid type ELISA (enzyme-linked immunosorbent assay) and preparation method thereof
By using a combination of phosphate buffer and inert protein in liquid-type ELISA standards, the problem of poor stability of low-abundance standard proteins in the prior art is solved, and the adjustment of long-term preservation and spiking recovery is achieved.
Patent Information
- Application Number
- CN202311224611.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-21
- Publication Date
- 2025-03-07
AI Technical Summary
The existing liquid-type ELISA standard stabilizers cannot effectively stabilize low-abundance standard proteins, making it difficult to store for a long time in a liquid state with pg/mL concentration, and the spiking recovery rate is difficult to adjust.
The liquid-type ELISA standard stabilizer including phosphate buffer and inert protein is used to reduce the decomposition of PMSF through a certain concentration of phosphate buffer, and provide synergistic stabilization protection through inert protein to achieve long-term preservation of standard proteins.
It has achieved stable storage of ELISA standards for more than 2 years in a liquid state with pg/mL concentration, and the protein activity remains greater than 98%, and the spiking recovery rate can be adjusted within a certain range.
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Abstract
Description
Technical Field
[0001] The present application relates to the technical field of a liquid ELISA standard stabilizer, and more specifically, to a liquid ELISA standard stabilizer and a preparation method thereof. Background Art
[0002] ELISA is a standard method for accurate protein quantification. Standards are the core components of ELISA test kits. There are two methods for preparing standards in commercial kits: freeze-drying and liquid stabilizer. The advantage of freeze-drying is that all types of standards can be prepared. The disadvantage is that they need to be dissolved and then graded diluted to prepare the standard curve before use. The workload is large, the steps are numerous, and it is easy to produce dissolution errors and random errors, resulting in experimental failure. Liquid standard stabilizers have completed the graded dilution of standards when the manufacturer manufactures the kit, avoiding dissolution errors and random errors. The disadvantage of commercially available liquid standard stabilizers is that they can only stabilize standard proteins with an abundance of more than nanograms per milliliter. For the vast majority of standard proteins with a low abundance of several picograms or tens of picograms per milliliter in serum samples, the stability is poor. Therefore, for example, the standard preparation methods of ELISA test kits of companies such as RD, Thermo, and Abcam all use freeze-drying.
[0003] Proteolytic enzymes are the main reason for the loss of protein activity in buffer solutions. PMSF (phenylmethylsulfonyl fluoride) is a broad-spectrum protease inhibitor that can effectively inhibit the hydrolytic activity of a variety of proteases. PMSF is usually not stable in aqueous solutions and is easily decomposed.
[0004] Therefore, a method for preparing an ELISA standard that can be stored for a long time in a liquid state at a concentration of pg / mL and whose spike recovery rate can be adjusted within a certain range is desired. Summary of the invention
[0005] Technical issues
[0006] In view of the deficiencies in the prior art, the present application provides a liquid ELISA standard stabilizer and a preparation method. The ELISA standard can be stored for a long time in a liquid state at a concentration of pg / mL. The preparation method of the ELISA standard can adjust the spiked recovery rate of the ELISA standard within a certain range.
[0007] Technical Solution
[0008] In order to achieve the above advantages of assisting the collection of sampled blood, the specific technical solutions adopted in this application are as follows:
[0009] The present application provides a liquid ELISA standard stabilizer, comprising a phosphate buffer and an inert protein;
[0010] After a large number of experimental studies, it was found that a certain concentration of phosphate buffer can reduce the decomposition of PMSF to a certain extent, thereby maintaining the effect of inhibiting protease activity. A certain concentration of inert protein has a synergistic stabilizing and protective effect on the standard protein in the solution. Therefore, the liquid ELISA standard stabilizer includes phosphate buffer and inert protein. Experimental measurements show that the liquid ELISA standard stabilizer can be stably stored at 4 degrees Celsius for more than 2 years in a pg / mL concentration liquid state.
[0011] Preferably, the phosphate buffer is NP-40 phosphate buffer.
[0012] Preferably, the inert protein is BSA.
[0013] The present application also provides a method for preparing a liquid ELISA standard stabilizer, comprising the following steps:
[0014] Step 1, sequentially mixing 2%-20% by mass of phosphate buffer, 2%-20% by mass of trehalose, 1%-10% by mass of threonine, 5-50 mM of PMSF, and 0.01%-1% by volume of a preservative to prepare a 0.001M-0.1M PBS phosphate buffer at a pH of 7.0-7.4 as a liquid stabilizer base solution;
[0015] Step 2, adding 1% to 15% by mass of inert protein to the liquid stabilizer base liquid to obtain a stabilizer inactivation pre-liquid;
[0016] Step 3, inactivate the stabilizer pre-inactivation liquid at 37-80° C. for 10-120 minutes to obtain a liquid ELISA standard stabilizer.
[0017] Furthermore, in step 1, the phosphate buffer is selected from NP-40 phosphate buffer.
[0018] Furthermore, step one is as follows: 5% by mass of NP-40 phosphate buffer, 5% of trehalose, 3% of threonine, 10 mM of PMSF, and 0.1% by volume of Proclin950 preservative are sequentially mixed to prepare 0.01 M PBS phosphate buffer at pH 7.2 as a liquid stabilizer base solution.
[0019] Furthermore, in step 2, the inert protein is BSA.
[0020] Furthermore, the concentration of the inert protein is determined based on the regression concentration of the spiked recovery of the corresponding standard protein.
[0021] Furthermore, step three is as follows: at 56° C., the stabilizer pre-inactivation liquid is inactivated for 30 minutes to obtain a liquid ELISA standard stabilizer.
[0022] Beneficial Effects
[0023] Compared with the prior art, this application has the following beneficial effects:
[0024] 1. The liquid ELISA standard stabilizer includes phosphate buffer and inert protein. A certain concentration of phosphate buffer can reduce the decomposition of PMSF to a certain extent, thereby maintaining the effect of inhibiting protease activity. A certain concentration of inert protein has a synergistic stabilizing and protective effect on the standard protein in the solution. Therefore, the experiment measured that the liquid ELISA standard stabilizer is stably stored at 4 degrees Celsius for more than 2 years in a pg / mL concentration liquid state, and the protein activity remains greater than 98%.
[0025] 2. The preparation method of the liquid ELISA standard stabilizer can control the spiked recovery rate of different index products within an excellent range of 80%-120% by adjusting the concentration of the corresponding components. DETAILED DESCRIPTION
[0026] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.
[0027] Taking human interleukin 6 standard protein as an example, the preparation method of the liquid ELISA standard stabilizer of the present invention is used to prepare the liquid ELISA standard stabilizer, as shown in the following example.
[0028] Embodiment 1
[0029] Step 1, select 5% NP-40 phosphate buffer by mass, 10mM PMSF, 3% threonine, 5% trehalose, and 0.1% Proclin950 by volume, prepare 0.01M PBS phosphate buffer at pH 7.2, and obtain a liquid stabilizer base solution;
[0030] Step 2, adding 1% BSA to the liquid stabilizer base solution to obtain a stabilizer pre-inactivation solution;
[0031] Step 3: Inactivate the stabilizer pre-inactivation liquid in a 56 degree Celsius water bath for 30 minutes to obtain a liquid ELISA standard stabilizer.
[0032] It should be noted that in order to ensure the consistency of the control standard protein, the standard protein used as the control in the stability experiment was pre-diluted to 10 ng / mL with 1% BSA in 0.01M PBS, packaged in 2.0mL polypropylene cryovials at 0.2mL / tube, and stored at -80°C. This ensures that the control standard protein used in each experiment has undergone one freeze-thaw cycle.
[0033] The human interleukin 6 protein was diluted to 1 pg / mL and 50 pg / mL using the liquid ELISA standard stabilizer of this example, and the obtained human interleukin 6 standard was dispensed into 2.0 mL polypropylene cryovials at 0.2 mL / tube.
[0034] Then the obtained human interleukin 6 standard was divided into two parts, one was stored at -80°C, and the other was stored at 37°C for 28 days. On the 7th, 14th, 21st and 28th days, the standard stored at 37°C was compared with the standard stored at -80°C, and the OD value was read and compared with the change of OD value by sandwich ELISA method.
[0035] Embodiment 2
[0036] Step 1, select 5% NP-40 phosphate buffer by mass, 10mM PMSF, 10% threonine, 5% trehalose, and 0.1% Proclin950 by volume to prepare 0.01M PBS phosphate buffer at pH 7.2 to obtain a liquid stabilizer base solution;
[0037] Step 2, adding 1% BSA to the liquid stabilizer base solution to obtain a stabilizer pre-inactivation solution;
[0038] Step 3: Inactivate the stabilizer pre-inactivation liquid in a 56 degree Celsius water bath for 30 minutes to obtain a liquid ELISA standard stabilizer.
[0039] The standard protein is the same as that used in Example 1 and will not be described again here.
[0040] The human interleukin 6 protein was diluted to 1 pg / mL and 50 pg / mL using the liquid ELISA standard stabilizer of this example, and the obtained human interleukin 6 standard was dispensed into 2.0 mL polypropylene cryovials at 0.2 mL / tube.
[0041] Then the obtained human interleukin 6 standard was divided into two parts, one was stored at -80°C, and the other was stored at 37°C for 28 days. On the 7th, 14th, 21st and 28th days, the standard stored at 37°C was compared with the standard stored at -80°C, and the OD value was read and compared with the change of OD value by sandwich ELISA method.
[0042] Embodiment 3
[0043] Step 1, select 5% NP-40 phosphate buffer by mass, 30mM PMSF, 3% threonine, 5% trehalose, and 0.1% Proclin950 by volume, prepare 0.01M PBS phosphate buffer at pH 7.2, and obtain a liquid stabilizer base solution;
[0044] Step 2, adding 1% BSA to the liquid stabilizer base solution to obtain a stabilizer pre-inactivation solution;
[0045] Step 3: Inactivate the stabilizer pre-inactivation liquid in a 56 degree Celsius water bath for 30 minutes to obtain a liquid ELISA standard stabilizer.
[0046] The standard protein is the same as that used in Example 1 and will not be described again here.
[0047] The human interleukin 6 protein was diluted to 1 pg / mL and 50 pg / mL using the liquid ELISA standard stabilizer of this example, and the obtained human interleukin 6 standard was dispensed into 2.0 mL polypropylene cryovials at 0.2 mL / tube.
[0048] Then the obtained human interleukin 6 standard was divided into two parts, one was stored at -80°C, and the other was stored at 37°C for 28 days. On the 7th, 14th, 21st and 28th days, the standard stored at 37°C was compared with the standard stored at -80°C, and the OD value was read and compared with the change of OD value by sandwich ELISA method.
[0049] Embodiment 4
[0050] Step 1, select 5% NP-40 phosphate buffer by mass, 30mM PMSF, 10% threonine, 5% trehalose, and 0.1% Proclin950 by volume to prepare 0.01M PBS phosphate buffer at pH 7.2 to obtain a liquid stabilizer base solution;
[0051] Step 2, adding 1% BSA to the liquid stabilizer base solution to obtain a stabilizer pre-inactivation solution;
[0052] Step 3: Inactivate the stabilizer pre-inactivation liquid in a 56 degree Celsius water bath for 30 minutes to obtain a liquid ELISA standard stabilizer.
[0053] The standard protein is the same as that used in Example 1 and will not be described again here.
[0054] The human interleukin 6 protein was diluted to 1 pg / mL and 50 pg / mL using the liquid ELISA standard stabilizer of this example, and the obtained human interleukin 6 standard was dispensed into 2.0 mL polypropylene cryovials at 0.2 mL / tube.
[0055] Then the obtained human interleukin 6 standard was divided into two parts, one was stored at -80°C, and the other was stored at 37°C for 28 days. On the 7th, 14th, 21st and 28th days, the standard stored at 37°C was compared with the standard stored at -80°C, and the OD value was read and compared with the change of OD value by sandwich ELISA method.
[0056] Embodiment 5
[0057] Step 1, select 10% NP-40 phosphate buffer by mass, 10mM PMSF, 3% threonine, 5% trehalose, and 0.1% Proclin950 by volume, prepare 0.01M PBS phosphate buffer at pH 7.2, and obtain a liquid stabilizer base solution;
[0058] Step 2, adding 1% BSA to the liquid stabilizer base solution to obtain a stabilizer pre-inactivation solution;
[0059] Step 3: Inactivate the stabilizer pre-inactivation liquid in a 56 degree Celsius water bath for 30 minutes to obtain a liquid ELISA standard stabilizer.
[0060] The standard protein is the same as that used in Example 1 and will not be described again here.
[0061] The human interleukin 6 protein was diluted to 1 pg / mL and 50 pg / mL using the liquid ELISA standard stabilizer of this example, and the obtained human interleukin 6 standard was dispensed into 2.0 mL polypropylene cryovials at 0.2 mL / tube.
[0062] Then the obtained human interleukin 6 standard was divided into two parts, one was stored at -80°C, and the other was stored at 37°C for 28 days. On the 7th, 14th, 21st and 28th days, the standard stored at 37°C was compared with the standard stored at -80°C, and the OD value was read and compared with the change of OD value by sandwich ELISA method.
[0063] Embodiment 6
[0064] Step 1, select 10% NP-40 phosphate buffer by mass, 10mM PMSF, 10% threonine, 5% trehalose, and 0.1% Proclin950 by volume, prepare 0.01M PBS phosphate buffer at pH 7.2, and obtain a liquid stabilizer base solution;
[0065] Step 2, adding 1% BSA to the liquid stabilizer base solution to obtain a stabilizer pre-inactivation solution;
[0066] Step 3: Inactivate the stabilizer pre-inactivation liquid in a 56 degree Celsius water bath for 30 minutes to obtain a liquid ELISA standard stabilizer.
[0067] The standard protein is the same as that used in Example 1 and will not be described again here.
[0068] The human interleukin 6 protein was diluted to 1 pg / mL and 50 pg / mL using the liquid ELISA standard stabilizer of this example, and the obtained human interleukin 6 standard was dispensed into 2.0 mL polypropylene cryovials at 0.2 mL / tube.
[0069] Then the obtained human interleukin 6 standard was divided into two parts, one was stored at -80°C, and the other was stored at 37°C for 28 days. On the 7th, 14th, 21st and 28th days, the standard stored at 37°C was compared with the standard stored at -80°C, and the OD value was read and compared with the change of OD value by sandwich ELISA method.
[0070] Embodiment 7
[0071] Step 1, select 10% NP-40 phosphate buffer by mass, 30mM PMSF, 3% threonine, 5% trehalose, and 0.1% Proclin950 by volume, prepare 0.01M PBS phosphate buffer at pH 7.2, and obtain a liquid stabilizer base solution;
[0072] Step 2, adding 1% BSA to the liquid stabilizer base solution to obtain a stabilizer pre-inactivation solution;
[0073] Step 3: Inactivate the stabilizer pre-inactivation liquid in a 56 degree Celsius water bath for 30 minutes to obtain a liquid ELISA standard stabilizer.
[0074] The standard protein is the same as that used in Example 1 and will not be described again here.
[0075] The human interleukin 6 protein was diluted to 1 pg / mL and 50 pg / mL using the liquid ELISA standard stabilizer of this example, and the obtained human interleukin 6 standard was dispensed into 2.0 mL polypropylene cryovials at 0.2 mL / tube.
[0076] Then the obtained human interleukin 6 standard was divided into two parts, one was stored at -80°C, and the other was stored at 37°C for 28 days. On the 7th, 14th, 21st and 28th days, the standard stored at 37°C was compared with the standard stored at -80°C, and the OD value was read and compared with the change of OD value by sandwich ELISA method.
[0077] Embodiment 8
[0078] Step 1, select 10% NP-40 phosphate buffer by mass, 30mM PMSF, 10% threonine, 5% trehalose, and 0.1% Proclin950 by volume, prepare 0.01M PBS phosphate buffer at pH 7.2, and obtain a liquid stabilizer base solution;
[0079] Step 2, adding 1% BSA to the liquid stabilizer base solution to obtain a stabilizer pre-inactivation solution;
[0080] Step 3: Inactivate the stabilizer pre-inactivation liquid in a 56 degree Celsius water bath for 30 minutes to obtain a liquid ELISA standard stabilizer.
[0081] The standard protein is the same as that used in Example 1 and will not be described again here.
[0082] The human interleukin 6 protein was diluted to 1 pg / mL and 50 pg / mL using the liquid ELISA standard stabilizer of this example, and the obtained human interleukin 6 standard was dispensed into 2.0 mL polypropylene cryovials at 0.2 mL / tube.
[0083] Then the obtained human interleukin 6 standard was divided into two parts, one was stored at -80°C, and the other was stored at 37°C for 28 days. On the 7th, 14th, 21st and 28th days, the standard stored at 37°C was compared with the standard stored at -80°C, and the OD value was read and compared with the change of OD value by sandwich ELISA method.
[0084] Comparative Example 1
[0085] The human interleukin-6 protein was diluted to 1 pg / mL and 50 pg / mL using the EL0002 protein stabilizer produced by Huzhou Yingchuang Biotechnology Co., Ltd. and packaged in 2 mL polypropylene cryovials at 0.2 mL / tube.
[0086] Then the obtained human interleukin 6 standard was divided into two parts, one was stored at -80°C, and the other was stored at 37°C for 28 days. On the 7th, 14th, 21st and 28th days, the standard stored at 37°C was compared with the standard stored at -80°C, and the OD value was read and compared with the change of OD value by sandwich ELISA method.
[0087] The formulas of Examples 1 to 8 and Comparative Example 1 are as follows:
[0088]
[0089]
[0090] The absorbance values (OD values) at a wavelength of 450nm-630nm of Examples 1 to 8 and Comparative Example 1 were read on an ELISA reader. The differences between the standards prepared by the liquid ELISA standard stabilizers of Examples 1 to 8 and the control standard protein, and the differences between the Yingchuang EL0002 standard of Comparative Example 1 and the control standard protein were compared, and the stability of the standard protein by the liquid ELISA standard stabilizer was evaluated by the change in the absorbance value.
[0091] The detection antibodies, HRP and other reagents used in the experiment were stored at -20 degrees Celsius after packaging to ensure that all reagents used in each experiment were frozen and thawed once.
[0092] The results of Human IL-61pg / mL stability are as follows:
[0093]
[0094] The results of Human IL-650pg / mL stability are as follows:
[0095]
[0096]
[0097] The present invention also uses the formulation of Example 3 to test the stability of Human IFN gamma and Human TNF alpha ELISA standards, and the results are shown in the following table:
[0098]
[0099] In summary, the liquid ELISA standard stabilizer of the present invention can be stably stored for 14 days at 37°C at a protein concentration of 1 pg / mL, and can be stably stored for 21 days at 37°C at a protein concentration of 50 pg / mL. According to the Arrhenius formula, one day of storage at 37°C is equivalent to 47 days of storage at 4°C. The liquid ELISA standard stabilizer can be stably stored for 22 months at 4°C at a protein concentration of 1 pg / mL.
[0100] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.
Claims
1. A liquid ELISA standard stabilizer, characterized in that: Includes phosphate buffer and inert protein.
2. A liquid ELISA standard stabilizer according to claim 1, characterized in that, The phosphate buffer includes NP-40 phosphate buffer.
3. A liquid ELISA standard stabilizer according to claim 1 or 2, characterized in that, The inert protein includes BSA.
4. A method for preparing a liquid ELISA standard stabilizer, characterized in that: The following steps are involved: Step 1, sequentially mixing 2%-20% by mass of phosphate buffer, 2%-20% by mass of trehalose, 1%-10% by mass of threonine, 5-50 mM of PMSF, and 0.01%-1% by volume of a preservative to prepare a 0.001M-0.1M PBS phosphate buffer at a pH of 7.0-7.4 as a liquid stabilizer base solution; Step 2, adding 1% to 15% by mass of inert protein to the liquid stabilizer base liquid to obtain a stabilizer inactivation pre-liquid; Step 3, inactivate the stabilizer pre-inactivation liquid at 37-80° C. for 10-120 minutes to obtain a liquid ELISA standard stabilizer.
5. The method for preparing a liquid ELISA standard stabilizer according to claim 4, characterized in that: In step 1, the phosphate buffer is selected from NP-40 phosphate buffer.
6. A method for preparing a liquid ELISA standard stabilizer according to claim 4 or 5, characterized in that: Step 1 is as follows: 5% by mass of NP-40 phosphate buffer, 5% of trehalose, 3% of threonine, 10mM of PMSF, and 0.1% by volume of Proclin950 preservative are mixed in sequence to prepare 0.01M PBS phosphate buffer at pH 7.2 as a liquid stabilizer base solution.
7. The method for preparing a liquid ELISA standard stabilizer according to claim 6, characterized in that: The inert protein in step 2 is BSA.
8. The method for preparing a liquid ELISA standard stabilizer according to claim 7, characterized in that: The concentration of the inert protein was determined based on the regression concentration of the spiked recovery of the corresponding standard protein.
9. The method for preparing a liquid ELISA standard stabilizer according to claim 8, characterized in that: Step three is as follows: at 56° C., the stabilizer pre-inactivation liquid is inactivated for 30 minutes to obtain a liquid ELISA standard stabilizer.