Application of compound in preparation of enzyme-linked immunosorbent assay (ELISA) detection reagent

Abstract

The invention relates to the technical field of biology, in particular to application of a compound in preparation of an ELISA detection reagent and the ELISA detection reagent. The compound with the structure shown in the formula I is applied to the ELISA detection reagent disclosed by the invention. Experiments show that the detection reagent provided by the invention can obviously reduce false positive, reduce background signals, increase the signal-to-noise ratio and obviously improve the detection accuracy and sensitivity of the kit.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the application of compounds in the preparation of ELISA detection reagents. Background Art

[0002] The basic principle of ELISA indirect detection is: the antigen / antibody is coated on a solid phase carrier. If the sample to be tested contains relevant antibodies / antibodies, they will combine with them to form a solid phase antigen-antibody complex. Then, an enzyme-labeled secondary antibody (such as goat anti-human IgG) will combine with the solid phase immune complex to form a solid phase antigen-antibody-enzyme-labeled secondary antibody complex or a solid phase antibody-antigen-enzyme-labeled antibody complex. After adding the corresponding substrate, the color is developed, and the antibody / antigen content in the sample to be tested is determined based on the intensity of the color development.

[0003] The ELISA indirect method is a commonly used test method for antigen / antibody detection, but its test results are easily affected by the sample to be tested. If the sample contains more interfering substances, the background signal will be high, resulting in false positives and easy misjudgment.

[0004] At present, the main method of reducing false positives in the prior art is to add Tween series substances in ELISA detection. Tween has good water solubility and mild surface activity, which can reduce nonspecific adsorption and enhance the washing effect. It can reduce false positives to a certain extent and improve the accuracy of the test results, but the effect is still not ideal. Therefore, it is urgent to provide a method with better effect to reduce false positives, reduce background signals, increase signal-to-noise ratio, and significantly improve the detection accuracy of the kit, which is also the goal that technicians have been pursuing.

[0005] The poloxamer involved in the present invention is a nonionic polymer. At present, it is mainly used in the field of pharmaceutical preparations, biomedical fields and food fields. In the field of pharmaceutical preparations, poloxamer 188 is one of the very few synthetic emulsifiers used for intravenous emulsions, such as fat emulsion infusion for injection. Poloxamer can also be used as a solubilizer to increase the solubility of poorly soluble drugs such as salicylic acid, diazepam, indomethacin, metronidazole, etc., and improve the dissolution rate and degree of the drug. In the field of biomedical engineering, poloxamer can be used to prepare drug carriers, tissue engineering scaffolds, etc., such as using its temperature sensitivity to prepare in-situ gels to achieve local slow release of drugs, or as a component of scaffold materials to adjust the performance of the scaffold. In the field of food, poloxamer can be used as an emulsifier, such as in products such as ice cream, cream, chocolate, etc., which helps to form a stable emulsion structure, prevent the separation of oil phase and water phase, and improve the stability and taste of the product.

[0006] At present, there is no research or report on using the poloxamer compound of formula I of the present invention to prepare an ELISA detection kit. Summary of the invention

[0007] In view of this, the present invention provides the use of the compound in preparing an ELISA detection reagent. The ELISA detection reagent prepared using the compound can significantly reduce false positives, reduce background signals, increase the signal-to-noise ratio, and significantly improve the detection accuracy and sensitivity of the kit.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] Application of the compound of formula I in the preparation of ELISA detection reagents;

[0010]

[0011] Wherein, X and Z are each independently any integer between 15 and 150;

[0012] Y is any integer between 15 and 67.

[0013] In the present invention, the X is selected from any integer between 80 and 141; the Y is selected from any integer between 27 and 56; and the Z is selected from any integer between 80 and 141.

[0014] In some specific embodiments, in the compound of formula I, the compound is an α-hydrogen-ω-hydroxy polyoxyethylene polyoxypropylene polyoxyethylene block copolymer, including but not limited to poloxamer 188, poloxamer 338, and poloxamer 407.

[0015] In the above compounds, in Poloxamer 188, Poloxamer 338, and Poloxamer 407, the average values ​​of X, Y, and Z are selected from one of the following combinations:

[0016] Combination 1: X is 80, Y is 27, Z is 80;

[0017] Combination 2: X is 141, Y is 44, and Z is 141;

[0018] Combination 3: X is 101, Y is 56, and Z is 101.

[0019] In a specific embodiment of the present invention, the compound of formula I is poloxamer 407 and poloxamer 338, both of which are common commercial products.

[0020] Studies have shown that ELISA test reagents made from compounds of Formula I can significantly reduce false positives, reduce background signals, increase signal-to-noise ratios, and significantly improve the detection accuracy and sensitivity of the kit. After research and comparison, the above effects of Poloxamer 407 are significantly better than those of other compounds in Formula I.

[0021] In the present invention, the mass percentage of the compound of formula I in the ELISA detection reagent is 0.02-1%, preferably 0.1-0.5%, specifically 0.1%, 0.2%, 0.3%, 0.4% or 0.5%.

[0022] In the present invention, the ELISA detection reagent includes a blocking solution and / or a sample diluent.

[0023] In a specific embodiment of the present invention, taking GAD antibody detection as an example, the effect of adding a compound of formula I structure to an ELISA detection reagent on the detection result was investigated. It was found that the sample diluent and blocking solution made using the compound can significantly reduce false positives, reduce background signals, increase signal-to-noise ratio, and significantly improve the detection accuracy and sensitivity of the kit. In the present invention, there are no special requirements for the type of target detected by the ELISA detection reagent, and the antigen and antibody detected based on ELISA can theoretically achieve the above-mentioned equivalent effect. The target includes but is not limited to glutamic acid decarboxylase antibody (GADA), tyrosine phosphatase antibody (IA-2A), zinc transporter 8 antibody (ZnT8A), insulin antibody (IAA), islet cell antibody (ICA), cytotoxin-associated protein A antibody (CagA), vacuolating toxin A antibody (VacA), urease A antibody (UreA), urease B antibody (UreB) at least one.

[0024] The present invention also provides an ELISA detection reagent, which comprises the compound of formula I.

[0025] In some embodiments, the ELISA detection reagent is a blocking solution, which includes 20 mM PBS buffer and the following components by mass fraction:

[0026] 0.1% casein, 2% BSA, 2% trehalose, 0.05% Tween 20 and 0.1% compound of formula I.

[0027] In some specific embodiments, the blocking solution includes 20 mM PBS buffer and the following components by mass fraction:

[0028] 0.1% casein, 2% BSA, 2% trehalose, 0.05% Tween 20, and 0.1% poloxamer 407;

[0029] or 0.1% casein, 2% BSA, 2% trehalose, 0.05% Tween 20 and 0.1% poloxamer 338 (.

[0030] In some embodiments, the ELISA detection reagent is a sample diluent, which includes 20 mM PBS buffer and the following components by mass fraction:

[0031] 0.1% casein, 2% BSA, 0.05% Tween 20 and 0.1% compound of formula I.

[0032] In some specific embodiments, the sample diluent comprises 20 mM PBS buffer and the following components by mass fraction:

[0033] 0.1% casein, 2% BSA, 0.05% Tween 20, and 0.1% poloxamer 407;

[0034] or 0.1% casein, 2% BSA, 0.05% Tween 20 and 0.1% Poloxamer 338.

[0035] In the ELISA detection reagent of the present invention, the dosage unit of Tween 20 is volume percentage, and the percentages "%" of other components are all mass percentages.

[0036] In the ELISA detection reagent of the present invention, the pH of the blocking solution and the sample diluent is 6.5-8.5, preferably 7.0-7.4, specifically 7.0, 7.1, 7.2, 7.3 or 7.4.

[0037] The present invention also provides the use of the ELISA detection reagent in detecting antigens and / or antibodies at non-diagnostic destinations.

[0038] The present invention also provides a method for detecting antigens and / or antibodies for non-diagnostic purposes using the ELISA detection reagent, comprising: diluting the sample using the ELISA detection reagent (sample diluent) described in the present invention; and / or, blocking using the ELISA detection reagent (blocking solution) described in the present invention after spotting and fixation.

[0039] The present invention uses the compound of formula I in the ELISA detection reagent of the present invention. Experiments show that the detection reagent provided by the present invention can significantly reduce false positives, reduce background signals, increase the signal-to-noise ratio, and significantly improve the detection accuracy and sensitivity of the kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 1 and 2 show the test results of sample 1 in the system of comparative example 1 and the test system of the present invention; a is the result of the system of comparative example 1, and b is the result of the poloxamer 407 system of the present invention;

[0041] Figure 2 The test results of sample 2 in the comparative example 1 system and the detection system of the present invention are shown; a is the result of the comparative example 1 system, and b is the result of the poloxamer 407 system of the present invention.

[0042] Figure 3The test results of sample 3 in the comparative example 1 system and the detection system of the present invention are shown; a is the result of the comparative example 1 system, and b is the result of the poloxamer 407 system of the present invention. DETAILED DESCRIPTION

[0043] The present invention provides the application of compounds in the preparation of ELISA detection reagents. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve. It is particularly important to point out that all similar replacements 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 relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0044] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0045] The present invention will be further described below in conjunction with embodiments:

[0046] Example 1: Formula of the blocking solution and sample diluent of the present invention

[0047] Blocking solution formula: 20 mM PBS + 0.1% casein + 2% BSA + 2% trehalose + 0.05% Tween 20 + 0.1% poloxamer 407 (pH 7.2±0.2).

[0048] Sample diluent formula: 20 mM PBS + 0.1% casein + 2% BSA + 0.05% Tween 20 + 0.1% poloxamer 407 (pH 7.2±0.2).

[0049] Except Tween 20, the percentages “%” of other components are all weight percentages.

[0050] Example 2: The formula of the blocking solution and sample diluent of the present invention

[0051] Blocking solution formula: 20 mM PBS + 0.1% casein + 2% BSA + 2% trehalose + 0.05% Tween 20 + 0.1% poloxamer 338 (pH 7.2±0.2).

[0052] Sample diluent formula: 20 mM PBS + 0.1% casein + 2% BSA + 0.05% Tween 20 + 0.1% poloxamer 338 (pH 7.2±0.2).

[0053] Except Tween 20, the percentages “%” of other components are all weight percentages.

[0054] Blocking solution and sample diluent of comparative example 1

[0055] Blocking solution formula: 20 mM PBS + 0.1% casein + 2% BSA + 2% trehalose + 0.05% Tween 20 (pH 7.2±0.2).

[0056] Sample diluent formula: 20 mM PBS + 0.1% casein + 2% BSA + 0.05% Tween 20 (pH 7.2 ± 0.2).

[0057] Blocking solution and sample diluent of comparative example 2

[0058] Blocking solution formula: 20 mM PBS + 0.1% casein + 2% BSA + 2% trehalose + 0.15% Tween 20 (pH 7.2±0.2).

[0059] Sample diluent formula: 20 mM PBS + 0.1% casein + 2% BSA + 0.15% Tween 20 (pH 7.2 ± 0.2).

[0060] Test Case

[0061] This example takes GAD antibody detection as an example to examine the effect of adding a compound with a structure of Formula I into an ELISA detection reagent on the detection result.

[0062] The blocking solution and sample diluent of Examples 1-2 and Comparative Examples 1-2 were used respectively, and the other components in the kit were matched: 96-well plate coated with antigen, enzyme-labeled secondary antibody (from Jackson Immuno Research), washing solution, substrate solution, etc. The obtained kits were named Poloxamer 407 system, Poloxamer 338 system, Comparative Example 1 system and Comparative Example 2 system (see Table 1). The above kits were used to detect 30 GAD antibody positive sera and negative sera respectively, and the operation method was as follows:

[0063] The operation method includes: spotting → fixing and sealing → drying → packaging → testing. The specific steps include the following:

[0064] (1) The GAD antigen was spotted and fixed onto an ELISA 96-well plate, and then placed at a certain temperature (2-8°C) for a period of time (18-22 h). After washing with a detergent, the plate was blocked with a blocking solution at room temperature for a period of time (3 h). The blocking solution was discarded, and the plate was dried at a certain temperature (37°C ± 2°C) for a period of time (18-24 h). The plate was packaged and stored for subsequent testing.

[0065] (2) Detection process: Add 100 μL of sample diluent to each well of the ELISA 96-well plate after spotting and fixation, then add 20 μL of the sample to be tested, react at 37°C for 30 min, wash three times, add 100 μL of enzyme-labeled secondary antibody, react at 37°C for 15 min, wash, add 50 μL of luminescent substrate, mix well, and read the value after 2 min.

[0066] The sample diluent and blocking solution were used in accordance with Tables 1 and 2, and the test was performed according to the above-mentioned operation method. The results are shown in Tables 3 and 4.

[0067] Table 1 Comparison of blocking solutions and sample diluents in different systems

[0068]

[0069] Table 2

[0070]

[0071]

[0072] 1) 30 GAD antibody positive sera and 30 negative sera were detected using the poloxamer 407 system of the present invention and the system of comparative example 1, respectively.

[0073] Table 3

[0074]

[0075]

[0076] Table 4

[0077]

[0078] Table 5 Signal-to-noise ratio

[0079]

[0080] It can be seen that after treatment with the Poloxamer 407 system and the Poloxamer 338 system, the signal-to-noise ratio increased significantly, and the effect was significantly better than that of the comparative examples 1 to 2 systems, among which the Poloxamer 407 system had a more obvious effect.

[0081] 2) Stability test of the test kit

[0082] The test kit was placed at 2-8°C, and precision reference materials (i.e., positive serum standards R1 and R2) were used as test samples. The test was performed after 0 months, 3 months, 6 months, 9 months, 12 months, and 13 months according to the above test process. The test was repeated 3 times each time, and the trend significance test of the slope was performed using the t-test. The calculation formula is as follows:

[0083]

[0084] Where:

[0085] S y，x ——Regression standard error (can be directly obtained by Excel's STEYX formula);

[0086] Yi——the mean of the test results at the i-th time point;

[0087] ——Theoretical value obtained by straight line;

[0088] n——number of repeated tests;

[0089] s(b1) – standard deviation of the slope;

[0090] b1——slope (can be directly obtained by Excel's SLOPE formula);

[0091] When |b1| <t 0.95，n-2 ·s(b1), the slope is insignificant, which proves that the kit is stable during this period of time, otherwise it is unstable.

[0092] Table 6

[0093]

[0094] After calculation, the stability results of the test kits prepared by the poloxamer 407 system and the poloxamer 338 system are shown in the table below.

[0095] Table 7

[0096]

[0097] It can be seen from the above figure that |b1| in R1 and R2 of the two systems is less than t 0.95，n-2 ·s(b1), which proves that the test kit is stable for 13 months and meets the product design requirements.

[0098] 3) According to the above method, the system of comparative example 1 and the poloxamer 407 system were used to detect three human serum negative samples 1 to 3 respectively. The detection methods included traditional ELISA, plate luminescent ELISA and microarray luminescent ELISA. The microarray luminescent images are shown in Figures 1 to 3 The test results are shown in Table 8. Among them, samples 1 to 3 were tested with gold standard reagents, and the results were all negative.

[0099] Table 8 ELISA test results

[0100]

[0101] The results show that the use of the detection reagent containing poloxamer 407 in the present application can effectively reduce the impact of interfering substances in human serum samples on the test results, reduce false positives, and make the test results more accurate.

[0102] From the above results, it can be seen that compared with the reagents of Comparative Examples 1 to 2 that do not contain the compound of Formula I, the addition of the compound of Formula I in the sample diluent and the blocking solution of the present invention can effectively reduce nonspecificity, reduce false positives, and increase the signal-to-noise ratio, thereby significantly improving the accuracy and sensitivity of the detection; at the same time, the reagent was tested for stability, and the results showed that the kit had good stability within the 13-month validity period.

[0103] According to the above method, the ELISA kit prepared by using the sample diluent and blocking solution of Examples 1 and 2 of the present invention is used to detect various analytes such as tyrosine phosphatase antibody (IA-2A), zinc transporter 8 antibody (ZnT8A), insulin antibody (IAA), islet cell antibody (ICA), cytotoxin-associated protein A antibody (CagA), vacuolating toxin A antibody (VacA), urease A antibody (UreA), and urease B antibody (UreB). The results show that compared with traditional sample diluents and blocking solutions, the present invention can significantly reduce false positives, reduce background signals, increase signal-to-noise ratio, significantly improve the detection accuracy and sensitivity of the kit, and the kit has good stability. It is shown that the sample diluent and blocking solution containing the structure of formula I provided by the present invention are suitable for ELISA detection of most antigen-antibody systems.

[0104] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of the compound of formula I in the preparation of ELISA detection reagents; in, X and Z are each independently any integer between 15 and 150; Y is any integer between 15 and 67.

2. The use according to claim 1, characterized in that: The X is selected from any integer between 80 and 141; the Y is selected from any integer between 27 and 56; and the Z is selected from any integer between 80 and 141.

3. The use according to claim 1, characterized in that: The mass percentage of the compound of formula I in the ELISA detection reagent is 0.02-1%, preferably 0.1-0.5%.

4. The use according to claim 1, characterized in that: The ELISA detection reagent includes a blocking solution and / or a sample diluent.

5. The use according to any one of claims 1 to 4, characterized in that: The target detected by the ELISA detection reagent includes at least one of glutamate decarboxylase antibody, tyrosine phosphatase antibody, zinc transporter 8 antibody, insulin antibody, islet cell antibody, cytotoxin-associated protein A antibody, vacuolating toxin A antibody, urease A antibody, and urease B antibody.

6. ELISA detection reagent, characterized in that, The invention comprises a compound of formula I structure and an auxiliary agent; the ELISA detection reagent comprises a traditional ELISA reagent, a plate-type luminescent ELISA reagent or a microarray protein chip.

7. The ELISA detection reagent according to claim 6, characterized in that The ELISA detection reagent is a blocking solution, which includes 10mM to 100mM PBS buffer and the following components in mass fractions: 0.1-2 wt% casein, 1-20 wt% BSA, 0.5-5 wt% trehalose, 0.05-2 vol% Tween 20 and 0.02-1 wt% compound of formula I.

8. The ELISA detection reagent according to claim 6, characterized in that The ELISA detection reagent is a sample diluent, which includes 10mM to 100mM PBS buffer and components of the following concentrations: 0.1-2 wt% casein, 1-20 wt% BSA, 0.05-2 vol% Tween 20 and 0.02-1 wt% compound of formula I.

9. The ELISA detection reagent according to any one of claims 6 to 8, wherein the pH value thereof is 6.5 to 8.

5.

10. Use of the ELISA detection reagent according to any one of claims 6 to 9 in detecting antigens and / or antibodies for non-diagnostic purposes.

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