An antibody protectant for pre-coating antibody enzyme labeled plates

Abstract

The application discloses an antibody protective agent for pre-coated antibody enzyme labeled plates, and the protective agent comprises the following components: 1-4% tetrahydropyrimidine and 4-8% mannitol in terms of mass percentage. The antibody protective agent can effectively improve the storage period and stability of the pre-coated enzyme labeled plate after being opened based on the specific combination of tetrahydropyrimidine and mannitol, and can promote the pre-coated enzyme labeled plate using the antibody protective agent to be stably stored for one month at 25 DEG C after being opened, and the real-time stability at 2-8 DEG C can be as long as 12 months.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of in vitro diagnostic reagents, in particular to an antibody protective agent for pre-coated antibody enzyme labeled plates. BACKGROUND

[0002] Enzyme-linked immunosorbent assay (ELISA) is one of the most commonly used enzyme immunoassay techniques. Its principle is to adsorb known antigens or antibodies on the surface of a solid carrier, add the test substance to generate specific antigen-antibody binding reactions, and then perform color development under enzyme catalysis. Therefore, it is mainly used for qualitative or quantitative detection.

[0003] Pre-coated plates are the reaction carriers of ELISA. The common preparation steps are as follows: coating (overnight at 2-8℃ or 2 hours at 37℃), washing (2-3 times), blocking (2-4 hours at room temperature), washing (2-3 times), drying (3-4 hours at room temperature), and vacuum packaging. In this process, reagents such as coating solution, blocking solution, and washing solution are used. The solid carrier used for coating is generally a polystyrene enzyme labeled plate. Generally speaking, under the condition that the vacuum packaging is sealed and dry, the pre-coated enzyme labeled plate can be stored at 2-8℃ for 1-2 years. However, after opening, the stability of the pre-coated enzyme labeled plate will be greatly reduced, and it usually cannot be stored at room temperature and can only be stored at low temperature for no more than one month. The most common enzyme labeled plate on the market is 96-well. If the sample quantity is small, the remaining wells need to be stored at low temperature for future use. However, even if it is placed in a low-temperature environment after opening, the performance of the enzyme labeled plate will be affected. The most common case is that the detection value decreases due to the inactivation of the pre-coated antibody in the enzyme labeled plate, thereby reducing the precision and accuracy and affecting the detection results. SUMMARY

[0004] The present application aims to solve at least one of the above technical problems in the prior art. To this end, the purpose of the present application is to provide an antibody protective agent for pre-coated antibody enzyme labeled plates. The antibody protective agent is based on a specific composition of ingredients, which can effectively improve the shelf life and stability of the pre-coated enzyme labeled plate after opening. It can be stably stored for one month at 25℃ after opening, and the real-time stability at 2-8℃ can be as long as 12 months for efficient storage.

[0005] In a first aspect of the present application, a protective agent is provided, which comprises the following components:

[0006] 1-4% tetrahydroimidazole and 4-8% mannitol by mass percentage.

[0007] In some embodiments of the present application, the protective agent further comprises at least one of serum or serum albumin, a preservative, and a solvent.

[0008] In some embodiments of the present application, the serum or serum albumin includes, but is not limited to, BSA.

[0009] In some embodiments of the present application, the preservative includes, but is not limited to, Proclin 300.

[0010] In some embodiments of the present application, the solvent includes, but is not limited to, buffer and water.

[0011] In some embodiments of the present application, the buffer includes phosphate buffer.

[0012] In some embodiments of the present application, the protective agent includes the following components:

[0013] 1%-4% of tetrahydroprimidine, 1%-4% of BSA, 4%-8% of mannitol, 0.05%-0.1% of Proclin 300, 0.02M-0.05M of phosphate buffer, by mass percentage.

[0014] In some embodiments of the present application, the protective agent includes or consists of the following components:

[0015] 1% of tetrahydroprimidine, 2% of BSA, 5% of mannitol, 0.1% of Proclin 300, 0.05M of phosphate buffer, by mass percentage.

[0016] In some embodiments of the present application, the pH of the protective agent is 7.3-7.5.

[0017] In some embodiments of the present application, the preparation method of the protective agent is:

[0018] Mixing tetrahydroprimidine, BSA, mannitol, Proclin 300 and phosphate buffer to obtain.

[0019] In some embodiments of the present application, the mixing ratio of tetrahydroprimidine, BSA, mannitol, Proclin 300 and phosphate buffer is as described in the above aspects.

[0020] The second aspect of the present application provides the use of the protective agent of the first aspect of the present application in the storage or protection of antibodies.

[0021] In the present application, the type of antibody is not limited, including but not limited to any antibody commonly used in the art or can be used in ELISA.

[0022] In some embodiments of the present application, the antibody is human IgG.

[0023] In a third aspect of the present application, the protective agent of the first aspect of the present application is used in enzyme-linked immunoassay.

[0024] In some embodiments of the present application, the enzyme-linked immunoassay does not involve diagnosis of a disease.

[0025] In a fourth aspect of the present application, a pre-coated enzyme plate is provided, which is pre-coated with an antibody and blocked by the protective agent of the first aspect of the present application.

[0026] In some embodiments of the present application, the type of the antibody pre-coated in the pre-coated enzyme plate is not limited, including but not limited to any antibody commonly used in the art or that can be used in ELISA.

[0027] In some embodiments of the present application, the time of blocking and the amount of the protective agent used are not specifically limited, and can be reasonably adjusted according to the actual situation of the pre-coated enzyme plate to achieve the effect of protecting the antibody according to the needs of the art.

[0028] In a fifth aspect of the present application, the pre-coated enzyme plate of the fourth aspect of the present application is used in enzyme-linked immunoassay.

[0029] In some embodiments of the present application, the enzyme-linked immunoassay does not involve diagnosis of a disease.

[0030] In a sixth aspect of the present application, an enzyme-linked immunoassay method is provided, comprising the following steps:

[0031] The reaction container coated with an antibody is blocked by the protective agent of the first aspect of the present application, and the enzyme-linked immunoassay is performed by adding a sample to be detected.

[0032] Alternatively, the pre-coated enzyme plate of the fourth aspect of the present application is used as a reaction container, and the enzyme-linked immunoassay is performed by adding a sample to be detected.

[0033] In some embodiments of the present application, the enzyme-linked immunoassay method does not involve diagnosis of a disease.

[0034] In some embodiments of the present application, the specific steps of the enzyme-linked immunoassay can be performed according to the conventional operation in the art, and can be reasonably adjusted according to the detection object and needs, but do not affect the aforementioned steps.

[0035] In a seventh aspect of the present application, a detection product is provided, which comprises the protective agent of the first aspect of the present application or the pre-coated enzyme plate of the fourth aspect of the present application.

[0036] In some embodiments of the present application, the detection product bag further comprises other auxiliary agents.

[0037] In some embodiments of the present application, the adjuvant includes, but is not limited to, buffer, standard, enzyme, etc.

[0038] In some embodiments of the present application, the detection product includes a detection kit or a detection set.

[0039] The beneficial effects of the present application are:

[0040] 1. The present application first attempts to combine tetrahydropyrimidine and mannitol for antibody protection, and finds that it has excellent protective effect on antibodies, which can be stably stored for one month at 25℃ after opening the pre-coated enzyme-labeled plate, and can be stably stored for up to 12 months at 2-8℃.

[0041] 2. The components in the present application have significant synergistic effect, and through comparison of different experimental groups, it can be found that the combination of tetrahydropyrimidine and mannitol has significantly stronger effect, and cannot be replaced by conventional substances. DETAILED DESCRIPTION

[0042] The content of the present application is further described in detail through specific examples. The raw materials, reagents or devices used in the examples and comparative examples are commercially available or can be obtained by the prior art method unless otherwise specified. Unless otherwise specified, the test or test method is the conventional method in the art.

[0043] Example 1

[0044] The present example provides an antibody protective agent for pre-coated antibody enzyme-labeled plate, which includes the following components:

[0045] 1% tetrahydropyrimidine, 2% BSA, 5% mannitol, 0.1% Proclin 300, and 0.05M phosphate buffer by mass / volume ratio. The pH is 7.3-7.5.

[0046] The preparation method is:

[0047] Mix tetrahydropyrimidine, BSA, mannitol, Proclin 300 and phosphate buffer according to the above ratio to obtain.

[0048] Example 2

[0049] The present example provides an antibody protective agent for pre-coated antibody enzyme-labeled plate, which includes the following components:

[0050] 2% tetrahydropyrimidine, 4% BSA, 4% mannitol, 0.05% Proclin 300, and 0.02M phosphate buffer by mass / volume ratio. The pH is 7.3-7.5.

[0051] The preparation method is the same as that of Example 1.

[0052] Example 3

[0053] The present example provides an antibody protective agent for pre-coating an antibody ELISA plate, comprising the following components:

[0054] 4% tetrahydroprimidine, 1% BSA, 8% mannitol, 0.1% Proclin 300, 0.05M phosphate buffer, by mass volume ratio. The pH is 7.3-7.5.

[0055] The preparation method is the same as that of Example 1.

[0056] Comparative Example 1

[0057] The present comparative example provides an antibody protective agent for pre-coating an antibody ELISA plate, comprising the following components:

[0058] 1% tetrahydroprimidine, 2% BSA, 5% sorbitol, 0.1% Proclin 300, 0.05M phosphate buffer, by mass volume ratio. The pH is 7.3-7.5.

[0059] The difference from Example 1 is that sorbitol is used instead of mannitol.

[0060] The preparation method is:

[0061] Mix the above components in the above proportions to obtain.

[0062] Comparative Example 2

[0063] The present comparative example provides an antibody protective agent for pre-coating an antibody ELISA plate, comprising the following components:

[0064] 1% tetrahydroprimidine, 2% BSA, 5% polyethylene glycol 8000, 0.1% Proclin 300, 0.05M phosphate buffer, by mass volume ratio. The pH is 7.3-7.5.

[0065] The difference from Example 1 is that polyethylene glycol 8000 is used instead of mannitol.

[0066] The preparation method is:

[0067] Mix the above components in the above proportions to obtain.

[0068] Comparative Example 3

[0069] The present comparative example provides an antibody protective agent for pre-coating an antibody ELISA plate, comprising the following components:

[0070] 2% BSA, 5% mannitol, 0.1% Proclin 300, 0.05M phosphate buffer by mass / volume. The pH is 7.3-7.5.

[0071] The difference from Example 1 is that tetrahydropyrimidine is not used.

[0072] The preparation method is:

[0073] The above components are mixed in the above proportions to obtain.

[0074] Comparative Example 4

[0075] This comparative example provides an antibody protective agent for pre-coating an antibody enzyme plate, comprising the following components:

[0076] 2% BSA, 5% sorbitol, 0.1% Proclin 300, 0.05M phosphate buffer by mass / volume. The pH is 7.3-7.5.

[0077] The difference from Example 1 is that tetrahydropyrimidine is not used and sorbitol is used instead of mannitol.

[0078] The preparation method is:

[0079] The above components are mixed in the above proportions to obtain.

[0080] Comparative Example 5

[0081] This comparative example provides an antibody protective agent for pre-coating an antibody enzyme plate, comprising the following components:

[0082] 2% BSA, 5% polyethylene glycol 8000, 0.1% Proclin 300, 0.05M phosphate buffer by mass / volume. The pH is 7.3-7.5.

[0083] The difference from Example 1 is that tetrahydropyrimidine is not used and polyethylene glycol 8000 is used instead of mannitol.

[0084] The preparation method is:

[0085] The above components are mixed in the above proportions to obtain.

[0086] Comparative Example 6

[0087] This comparative example provides an antibody protective agent for pre-coating an antibody enzyme plate, comprising the following components:

[0088] 1% tetrahydropyrimidine, 2% BSA, 0.1% Proclin 300, 0.05M phosphate buffer by mass / volume. The pH is 7.3-7.5.

[0089] The difference from Example 1 is that mannitol is not used.

[0090] The preparation method is:

[0091] The above components are mixed in the above proportions to obtain.

[0092] Test Example

[0093] To verify the use effect of each example and comparative example, the antibody protectant in each example and comparative example is used as a blocking agent to coat an enzyme-labeled plate.

[0094] The specific steps are as follows:

[0095] (1) Human IgG is used as a coating antibody (coating concentration 5 μg / mL), and the enzyme-labeled plate is coated with carbonate buffer solution at 100 μL / well at 2-8°C overnight.

[0096] (2) PBST solution is used as a cleaning solution to clean the plate in a plate cleaner for 2 times.

[0097] (3) The antibody protectant in each example and comparative example is used to block at 37°C for 2 hours.

[0098] (4) PBST solution is used as a cleaning solution to clean the plate in a plate cleaner for 2 times.

[0099] (5) Dry at 25°C and humidity below 30% for 3 hours.

[0100] (6) The pre-coated enzyme-labeled plate is vacuum sealed with an aluminum foil bag and stored at 2-8°C.

[0101] Among them, the coating solution containing human IgG antibody is added to rows A, B, C, and D of each enzyme-labeled plate as test wells, and the coating solution without human IgG antibody is added to rows E, F, G, and H as blank control wells.

[0102] After the above enzyme-labeled plate is stored at 2-8°C overnight, the seal is opened and placed at room temperature, and detected once a week.

[0103] At the same time, another batch is prepared according to the above method and placed at 2-8°C, and detected once a month. Humidity is not limited.

[0104] The detection method is:

[0105] HRP labeled anti-human IgG was diluted with PBS buffer containing 2% BSA at a dilution of 1 : 10000, and added to the pre-coated enzyme plate wells at an amount of 100 μL per well, and incubated at room temperature for 1 hour. 7 times of washing (washing solution is PBST solution) were performed using a plate washer. Then 100 μL of color developing solution (TMB color developing solution) was added to each well, and color developed for 5 minutes in the dark. Then 100 μL of stop solution (diluted sulfuric acid) was added to each well, and the absorbance of OD450-OD630 was measured using an enzyme labeler.

[0106] The results are shown in the following table.

[0107] Table 1 Stability test results of the examples and comparative examples opened at room temperature

[0108]

[0109]

[0110] Table 2 Stability test results of Example 1 opened at 2-8°C

[0111]

[0112] Table 3 Stability test results of comparative examples opened at 2-8°C

[0113]

[0114]

[0115] Based on the above results, the signal attenuation percentage of the examples and comparative examples was further calculated according to the following formula:

[0116] The results are shown in the following table.

[0117] Table 4 Signal attenuation percentage of the examples and comparative examples

[0118]

[0119] Wherein, Example A refers to the attenuation percentage of Example 1 detected at room temperature to the fourth week, and detected at 2-8°C to the twelfth month;

[0120] Example B refers to the attenuation percentage of Example 1 detected at room temperature to the fourth week, and detected at 2-8°C to the fourth month.

[0121] From the above results, the protection effect of using the specific combination of tetrahydropyrimidine and mannitol is much higher than using tetrahydropyrimidine or mannitol alone or other similar substances in the art, which indicates that there is a special synergistic effect between tetrahydropyrimidine and mannitol, thereby further cooperating with other combinations to jointly play a role and greatly improve the stability of the pre-coated enzyme-labeled plate after being opened.

[0122] The same experimental verification was performed on examples 2 and 3, and it was found that the protection effect was equivalent to that of example 1.

[0123] The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. A pre-coated enzyme assay plate, characterized in that, The pre-coated enzyme-labeled plate is pre-coated with antibodies and blocked by a protective agent; The protective agent is composed of the following components: 1%-4% tetrahydro pyrimidine, 1%-4% BSA, 4%-8% mannitol, 0.05%-0.1% Proclin 300 and 0.02M-0.05M phosphate buffer by mass percentage.

2. The pre-coated enzyme-labeled plate of claim 1 is applied in enzyme-linked immunoassay. wherein The enzyme-linked immunoassay is not related to the diagnosis of diseases.

3. An enzyme-linked immunoassay method, comprising the following steps: The pre-coated enzyme-labeled plate of claim 1 is used as a reaction container for enzyme-linked immunoassay by adding a detection object; wherein The enzyme-linked immunoassay method is not related to the diagnosis of diseases.

4. A test product, characterized in that The detection product comprises the pre-coated enzyme-labeled plate of claim 1; and the detection product comprises a detection kit.

Images