β-Estradiol small molecule sandwich ELISA detection kit and preparation method thereof

Abstract

The present invention provides a β-Estradiol antigen epitope, a β-Estradiol antigen, a β-Estradiol specific coating antibody, and a β-Estradiol specific detection antibody. The present invention also provides an ELISA detection kit, which includes the β-Estradiol specific coating antibody, the β-Estradiol specific detection antibody, a standard, Streptavidin-HRP, a single-component TMB colorimetric solution, a stop solution, and a diluent. The kit is suitable for the detection of samples such as animal serum and plasma in scientific research laboratories, and solves the problems of insufficient sensitivity, large intra-batch and inter-batch differences, poor dilution and spike recovery, and difficulty in large-scale industrial production of traditional small molecule β-Estradiol competitive ELISA detection kits.

Description

Technical Field

[0001] The patent of the present invention relates to the field of immunological detection technology, and in particular to a β-Estradiol small molecule sandwich ELISA detection kit and a preparation method thereof. Background Art

[0002] β-Estradiol is a steroid hormone, mainly secreted by ovarian follicles, corpus luteum and placenta during pregnancy. It is the most important and biologically active hormone among estrogens. It is an important indicator in gonadal hormone detection and the most important component of physiological effects. Its main function is to promote the development of female reproductive organs, maintain secondary sexual characteristics and reproductive function. Elevated levels of this indicator are common in multiple pregnancies, diabetic pregnant women, liver cirrhosis, ovarian cancer, male breast development, and female precocious puberty. Reduced levels of this indicator are common in pregnancy-induced hypertension, hypercortisolism, glucose patients, and menopausal syndrome.

[0003] Currently, commonly used β-Estradiol detection methods include: high-performance liquid chromatography, chemiluminescent magnetic beads, and ELISA competition. High-performance liquid chromatography has high detection costs and low detection throughput, and is not suitable for the detection of large quantities of scientific research samples; the chemiluminescent magnetic beads method is not easy to operate and the reaction mechanism is similar to the ELISA competition method, so the detection results vary greatly; the ELISA competition method has problems such as insufficient detection sensitivity, large intra-batch and inter-batch differences, poor dilution and spike recovery, and difficulty in large-scale industrial production. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. Therefore, in a first aspect of the present invention, the present invention provides a β-Estradiol antigenic epitope, which is selected from one of the following structures:

[0005]

[0006]

[0007] In the second aspect of the present invention, the present invention provides a β-Estradiol antigen, which is obtained by coupling the β-Estradiol antigen epitope described in the first aspect of the present invention with OVA or KLH.

[0008] In the third aspect of the present invention, the present invention provides a β-Estradiol-specific coated antibody prepared from the β-Estradiol antigen described in the second aspect of the present invention.

[0009] In a fourth aspect of the present invention, the present invention provides a β-Estradiol specific detection antibody, which is obtained by biotin-labeling the antibody prepared from the β-Estradiol antigen described in the second aspect of the present invention.

[0010] Preferably, the molar ratio of β-Estradiol-specific coating antibody to biotin is 1:10-1:40, and the pH of the labeling buffer is 7.4-8.0.

[0011] In the fifth aspect of the present invention, the present invention provides an ELISA detection kit, which includes the β-Estradiol-specific coating antibody described in the third aspect of the present invention, the β-Estradiol-specific detection antibody described in the fourth aspect of the present invention, a standard, Streptavidin-HRP, a single-component TMB colorimetric solution, a stop solution and a diluent.

[0012] Preferably, the ELISA detection kit uses a double antibody sandwich method to detect β-Estradiol, the double antibody sandwich method uses a first anti-β-Estradiol specific coating antibody as the coating antibody, the first anti-β-Estradiol specific coating antibody is derived from one of the β-Estradiol antigen epitopes described in claim 1, and the double antibody sandwich method uses a second anti-β-Estradiol specific detection antibody as the detection antibody, the second anti-β-Estradiol specific detection antibody is derived from another of the β-Estradiol antigen epitopes described in claim 1.

[0013] Preferably, the concentration of the β-Estradiol-specific coated antibody is 1-6 μg / mL.

[0014] Preferably, the mass concentration of the β-Estradiol-specific detection antibody is 1:2000-1:20000.

[0015] In a sixth aspect of the present invention, the present invention provides a double antibody sandwich method for detecting β-Estradiol, comprising the following steps:

[0016] 1) using the compound of formula 3 or the compound of formula 4 as antigens to immunize animals and obtain hybridoma cells;

[0017] 2) Preparation of monoclonal antibodies through hybridoma cells;

[0018] 3) Identifying the epitope of the monoclonal antibody to obtain a monoclonal antibody with a known epitope, selecting two monoclonal antibodies with different epitopes, reacting one of them with biotin to obtain a biotin-labeled antibody and an unlabeled biotin antibody;

[0019] 4) using an unlabeled biotin antibody as a coating antibody and a labeled biotin antibody as a detection antibody for antibody pairing, and then detecting β-Estradiol based on the antibody pairing data and the sandwich method;

[0020] The compounds of formula 3 and formula 4 are as follows:

[0021]

[0022] The structure of β-Estradiol(17β-OH)-KLH is shown in Formula 4 below:

[0023]

[0024] In the present invention, the stability test verification means that the overall stability of the test kit at 37°C is between 90-110%.

[0025] In the present invention, the mouse serum plasma detection verification refers to the detectable values ​​of mouse serum plasma, and the mice are all SPF grade BALB / c mice.

[0026] In the present invention, the recovery rate and intra-batch and inter-batch difference detection and verification refer to that the dilution recovery rate and the spiked recovery rate meet the range requirements of 80-120%, the intra-batch difference is less than 10%, and the inter-batch difference is less than 15%.

[0027] In the present invention, the specific detection verification refers to no obvious cross reaction with related structural analogs after verification (cross reaction rate <15%), and the structural analogs are 17-epi Estradiol Valerate, 3-Valerate Estradiol, EstradiolBenzoate, and Estriol.

[0028] The working principle of the present invention is as follows: a hapten with a relatively small molecular weight is deeply buried in the antigen binding site after binding with an antibody. According to research and experiments, when a small molecule has 4 or more ring structures, two functional groups (the 3rd OH and the 17β-OH in the present invention) separated by 7-9 carbon atoms on the small molecule are modified to derive intermediates that can be coupled to amino groups, and then coupled with different carrier proteins KLH or OVA, respectively. Subsequently, the two conjugates are used as immunogens to immunize animals respectively to prepare mouse hybridoma monoclonal antibodies for the development of double antibody sandwich kits.

[0029] Beneficial effects of the present invention:

[0030] 1. Provide a β-Estradiol ELISA detection kit with high detection sensitivity, small intra-batch and inter-batch difference, good dilution and spike recovery rate, easy industrial production and good repeatability.

[0031] 2. Compared with the existing β-Estradiol small molecule competitive ELISA kit, the present invention provides for the first time a small molecule kit preparation method based on the double antibody sandwich method, which effectively solves the problems of large sample CV, insufficient sensitivity, and inability to achieve industrialized stable production of the traditional β-Estradiol small molecule competitive ELISA kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The flowchart of the preparation of the β-Estradiol small molecule sandwich ELISA detection kit of Example 4 and Example 5;

[0033] Figure 2 The figure is a flow chart of the preparation of the comparative example β-Estradiol small molecule competitive ELISA detection kit. DETAILED DESCRIPTION

[0034] The present invention is further described below in conjunction with specific examples and drawings, but the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. In the following examples, if no specific conditions are specified, they are carried out according to conventional conditions or conditions recommended by the manufacturer. The methods used are conventional methods known in the art unless otherwise specified, and the consumables and reagents used are commercially available unless otherwise specified. Unless otherwise specified, the professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the content described can also be applied to the present invention. The blocking solution is a PBS buffer containing BSA, trehalose, and Proclin 300. Specifically, it is a 0.01M PBS buffer containing 0.1% BSA, 8% trehalose, and 0.1% Proclin 300, with a pH of 7.4; the detection conditions refer to the diluent being a PBS buffer containing BSA, Tween 20, and Proclin 300. The diluent is specifically 0.01M PBS buffer containing 0.1% BSA, 0.05% Tween 20, and 0.1% Proclin 300, with a pH of 7.4.

[0035] Example 1

[0036] β-Estradiol antigen epitope, characterized in that: the β-Estradiol antigen epitope is as follows:

[0037]

[0038]

[0039] This example provides two modified β-Estradiol derivatives (Elarite Wuhan Biotechnology Co., Ltd., catalog number: E-BC-E001, E-BC-E002), which are then coupled with KLH and OVA to obtain β-Estradiol (3-OH) -OVA conjugate (concentration of 13.7 mg / mL) and β-Estradiol (17β-OH) -KLH conjugate (concentration of 24.5 mg / mL).

[0040] The two synthesized complete antigens (β-Estradiol (3-OH)-OVA and β-Estradiol (17β-OH)-KLH) were used as immunogens to immunize animals and then used for the preparation of mouse hybridoma monoclonal antibodies (synthesized by Wuhan Dai'an Biotechnology).

[0041] The structure of β-Estradiol(3-OH)-OVA is shown in Formula 3 below:

[0042]

[0043] The structure of β-Estradiol(17β-OH)-KLH is shown in Formula 4 below:

[0044]

[0045] Example 2

[0046] This embodiment provides mouse monoclonal antibodies prepared from two immunogens (prepared by Wuhan Dai'an Biological). After testing, there are 3 cell lines prepared by β-Estradiol (3-OH) -OVA, and 5 cell lines prepared by β-Estradiol (17β-OH) -KLH. After preliminary ELISA matching test, the antibody derived from one strain of β-Estradiol (3-OH) -OVA (named 1#Ab, Wuhan Dai'an Biological, item number Cat#: 2547-1) and the antibody derived from two strains of β-Estradiol (17β-OH) -KLH (named 2#Ab, 3#Ab, Wuhan Dai'an Biological, item numbers Cat#: 2547-2, Cat#: 2547-3, respectively) have good specificity and titer, and can be tried for matching test for the development of β-Estradiol double antibody sandwich method kit.

[0047] Example 3

[0048] This embodiment provides a method for biotin-labeling an antibody, which specifically comprises the following steps:

[0049] S1, take 600 μg of the above 1#Ab, 2#Ab, and 3#Ab respectively, then divide them into 200 μg and put them into 2mL vials and number them, adjust the pH value to 7.6±0.1 with carbonate buffer, add 2.2 μg, 4.4 μg, and 8.8 μg of biotin to each numbered antibody solution according to the molar ratio of antibody to biotin of 1:10, 1:20, and 1:40, respectively, and mix them thoroughly;

[0050] S2, stirring the reaction at room temperature for 2 h and then terminating the reaction with ethanolamine;

[0051] S3, the antibody labeling solution after the reaction is completed is ultrafiltered using a 10kd ultrafiltration tube to obtain biotinylated antibodies, and the concentration is 500 μg / mL.

[0052] Example 4

[0053] This embodiment provides a method for preparing a β-Estradiol small molecule sandwich ELISA detection kit, and the screening and pairing of antibodies is the most basic step.

[0054] S1, antibody screening and pairing. 1#Ab, 2#Ab, and 3#Ab in Example 2 were used as coating antibodies, and the biotinylated antibodies 2#Ab-Bio, 3#Ab-Bio, and 1#Ab-Bio in Example 3 were used as detection antibodies, and pairing screening was performed to determine the closest matching conditions. The specific experimental design scheme is shown in Table 1:

[0055] Table 1

[0056]

[0057] S2. Add the prepared coating antibody solution to the wells of the ELISA plate according to the above experimental scheme. After overnight at 2-8°C, add 100 μL of 0.4% BSA solution to each well and incubate at 37°C for 1 hour. Then add 100 μL of 100 pg / mL β-Estradiol standard to the microplate and incubate at 37°C for 1 hour. After washing the plate, add the pre-prepared biotinylated antibody according to the experimental scheme and incubate at 37°C for 1 hour. After washing the plate, add SA-HRP and incubate at 37°C for 0.5 hour. After washing the plate, add the single-component TMB solution, incubate for 15-25 minutes, then add the stop solution, and finally read the absorbance at a wavelength of 450 nm.

[0058] S3, the test results of the above different pairing conditions are shown in Table 2.

[0059] Table 2

[0060]

[0061] From the above test result table, we can know that when 1#Ab is used as coating antibody, 2#Ab-Bio is used as detection antibody, and 3#Ab is used as coating antibody, 1#Ab-Bio is used as detection antibody, all of them show a good concentration gradient. Considering the coating cost and sensitivity, it is obviously more appropriate to use 1#Ab as coating antibody and 2#Ab-Bio as detection antibody. Through analysis, it is determined that the coating concentration of 1#Ab is 1μg / mL, and the mass concentration of 2#Ab-Bio is more appropriate between 1:5000-1:1000.

[0062] Example 5

[0063] This embodiment provides a method for preparing a β-Estradiol small molecule sandwich ELISA detection kit, which is as follows:

[0064] Kit preparation process:

[0065] Coating: Dilute Ab #1 to 1 μg / mL with coating buffer, add 100 μL per well to the ELISA plate, and place at 2-8°C overnight;

[0066] Blocking: shake off the liquid in the coated wells, pat dry on absorbent paper, block with blocking solution, add 200 μL to each well, and then incubate at 37°C for 1 hour;

[0067] Test steps:

[0068] Set up standard wells, blank wells, and sample wells. Add 100 μL of the diluted standard (β-Estradiol) to the standard wells, with a standard concentration of 200 pg / mL, add 100 μL of diluent to the blank wells, and add 100 μL of the sample to be tested to the remaining wells. Cover the ELISA plate with film and incubate at 37°C for 90 minutes.

[0069] Shake off the liquid in the wells without washing. Add 100 μL of biotinylated antibody (2#Ab-Bio) working solution to each well, cover the ELISA plate with a film, and incubate at 37°C for 1 hour.

[0070] Shake off the liquid in the wells and pat dry on clean absorbent paper. Add 350μL of washing solution to each well, soak for 1 minute, absorb or shake off the liquid in the ELISA plate, and pat dry. Repeat this washing step 3 times. Add 100μL of SA-HRP enzyme conjugate working solution to each well, cover the ELISA plate with a film, and incubate at 37℃ for 30 minutes.

[0071] Shake off the liquid in the wells and wash the plate 5 times. Add 90 μL of single-component TMB solution to each well, cover the ELISA plate with a film, and incubate at 37°C in the dark for about 15 minutes.

[0072] Add 50 μL of stop solution to each well to terminate the reaction.

[0073] Immediately measure the optical density (OD value) of each well at a wavelength of 450 nm using a microplate reader;

[0074] Kit performance test verification process:

[0075] It includes the selection of detection conditions, mouse serum plasma detection verification, stability detection verification, sensitivity detection verification, dilution recovery rate and spike recovery rate detection verification, intra-batch and inter-batch difference detection verification, and specificity detection verification.

[0076] S1, selection of blocking and detection conditions. According to the preliminary conditions for coating and detection determined in Example 4, the experimental scheme is set as shown in Table 3 below:

[0077] Table 3

[0078]

[0079] The blocking solution is a PBS buffer containing BSA, trehalose, and Proclin 300. Specifically, it is a 0.01M PBS buffer containing 0.1% BSA, 8% trehalose, and 0.1% Proclin 300, with a pH of 7.4; the detection conditions refer to the diluent being a PBS buffer containing BSA, Tween 20, and Proclin 300. Specifically, the diluent is a 0.01M PBS buffer containing 0.1% BSA, 0.05% Tween 20, and 0.1% Proclin300, with a pH of 7.4.

[0080] According to the above experimental scheme, the test results are shown in Table 4 below.

[0081] Table 4

[0082] OD value 1 2 3 4 5 6 7 8 9 10 A 3.635 3.182 2.731 2.236 1.825 1.233 0.681 0.312 0.173 0.052 B 3.217 2.764 2.233 1.782 1.321 0.833 0.542 0.287 0.139 0.051 C 2.541 2.013 1.498 0.955 0.422 0.296 0.176 0.109 0.074 0.051

[0083] From the above results, we can see that: when 1#Ab is used as the coating antibody and the coating concentration is 1μg / mL, 2#Ab-Bio is used as the detection antibody and the detection ratio is 1 / 7.5K, the standard concentration is selected as 50pg / mL, and the blocking solution is selected as the above blocking solution, the standard curve linearity shows a good gradient.

[0084] S2, mouse serum plasma detection verification. According to the encapsulation and detection conditions in Example 5 and S1, the mouse serum plasma detection verification experimental scheme was designed as follows (the mouse serum and plasma were all normal healthy non-physiological BALB / c mice):

[0085] Table 5

[0086] 1 2 3 4 5 6 A 50pg / mL 50pg / mL 1# Male mouse serum 1#Male mouse plasma 1# Female mouse serum 1#Female mouse plasma B 25pg / mL 25pg / mL 2# Male mouse serum 2#Male mouse plasma 2# Female mouse serum 2# Female mouse plasma C 12.5 pg / mL 12.5 pg / mL 3# Male mouse serum 3#Male mouse plasma 3# Female mouse serum 3# Female mouse plasma D 6.25 pg / mL 6.25 pg / mL 4# Male mouse serum 4#Male mouse plasma 4# Female mouse serum 4# Female mouse plasma E 3.13pg / mL 3.13pg / mL 5# Male mouse serum 5#Male mouse plasma 5# Female mouse serum 5# Female mouse plasma F 1.56 pg / mL 1.56 pg / mL 6# Male mouse serum 6# Male mouse plasma 6# Female mouse serum 6# Female mouse plasma G 0.78pg / mL 0.78pg / mL 7# Male mouse serum 7# Male mouse plasma 7# Female mouse serum 7# Female mouse plasma H 0pg / mL 0pg / mL 8# Male mouse serum 8#Male mouse plasma 8# Female mouse serum 8# Female mouse plasma

[0087] The test results according to the above experimental scheme are as follows in Table 6:

[0088] Table 6

[0089] 1 2 3 4 5 6 A 2.312 2.348 1.573 1.621 2.123 2.154 B 1.796 1.801 1.488 1.52 2.148 2.033 C 1.421 1.409 1.031 1.098 2.398 2.398 D 0.839 0.812 1.354 1.377 2.654 2.655 E 0.533 0.547 1.655 1.604 2.387 2.402 F 0.297 0.288 1.541 1.407 2.779 2.801 G 0.142 0.152 1.211 1.279 2.461 2.387 H 0.056 0.060 1.422 1.506 2.587 2.608

[0090] The above results show that: when the mouse serum plasma stock solution is tested, it shows a good detection effect. The OD value of female mouse serum plasma detection is higher, and further dilution detection can obtain the final concentration.

[0091] S3: 37℃ stability test verification. The antibody-coated ELISA plate, detection antibody, and standard sample were packaged and placed at 37℃ for 7 days (equivalent to 2-8℃ for 1 year) and 2-8℃ for 7 days.

[0092] After 7 days, single factor and overall stability comparison tests were conducted at 37°C and 2-8°C. The test results are shown in Table 7:

[0093] Table 7

[0094] ELISA plate Standards Detection antibodies overall Effectiveness (37℃ / 4℃*100%) 101.30％ 98.70％ 96.40％ 97.90％

[0095] The above results show that the kit is valid for about 1 year when stored at 2-8°C.

[0096] S4, sensitivity detection verification. According to the standard curve setting scheme of S2 in Example 5, three parallel standard curves were set, and 20 replicate wells of β-Estradiol at 0 concentration were set at the same time, and the absorbance value at 450nm was tested. The mean value M and standard deviation SD of the absorbance value were calculated and inserted into the formula M+2SD, and the final calculated value was substituted into the Orijin9 standard curve fitting equation, and the sensitivity was obtained to be 0.29pg / mL.

[0097] S5, verification of spike recovery and dilution recovery.

[0098] Spiked recovery rate test verification. 8 different concentrations of mouse serum plasma samples were added with known concentrations of β-Estradiol, and recovery experiments were performed to obtain the spike recovery rate range and average recovery rate. The test results are shown in Table 8:

[0099] Table 8

[0100] Sample type (BALB / c mice) Spiked recovery rate (%) Average spike recovery (%) Serum (n=8) 89-102 97 EDTA plasma (n=8) 93-114 103

[0101] Dilution recovery rate test verification. The 8 samples were diluted 2 times, 4 times, 8 times, and 16 times to conduct dilution recovery experiments, and the dilution recovery rate range and average dilution recovery rate were obtained. The test results are shown in Table 9 below:

[0102] Table 9

[0103]

[0104] The above results show that the dilution recovery rate and spike recovery rate test results meet the prevailing standard of 80-120%.

[0105] S6, intra-batch and inter-batch difference detection verification.

[0106] Intra-batch precision: Low, medium, and high concentration mouse serum plasma were tested 20 times on one batch of plates. The test results are shown in Table 10:

[0107] Table 10

[0108]

[0109] Inter-batch precision: Low, medium, and high concentration mouse serum plasma were tested 20 times on 3 batches of plates. The test results are shown in Table 11:

[0110] Table 11

[0111]

[0112] The above results show that the intra-batch difference is less than 10%, and the inter-batch difference is less than 10%, which is in line with the existing common standards.

[0113] S7, specific detection verification. The verified structural analogs are 3-Valerate Estradiol, EstradiolBenzoate, and Estriol, and these substances are configured to 500 pg / mL respectively. The detection is carried out according to the coating and detection conditions of S2 in Example 5, and the detection results are shown in Table 12 below:

[0114] Table 12

[0115] Structural analogs 3-Valerate Estradiol EstradiolBenzoate Estriol Detection concentration 37.55 29.2 47.35 Crossover rate (%) 7.51 5.84 9.47

[0116] The above results show that the cross-reaction rate is less than 15%, which meets the requirements.

[0117] Comparative Example

[0118] This comparative example is mainly based on the use of β-Estradiol-KLH as the coating source, 1#Ab-Bio and 2#Ab-Bio as the detection antibodies to establish a competitive detection system, and then compare the sensitivity, intra-batch difference and inter-batch difference, dilution recovery rate and spiked recovery rate of the detection kit with the above-mentioned double antibody sandwich system.

[0119] S1. The conditions of the β-Estradiol competition method system were established. The preparation process of the β-Estradiol small molecule competition ELISA detection kit is as follows: Figure 2 As shown, the details are as follows:

[0120] Kit preparation process:

[0121] Coating: dilute β-Estradiol-KLH to 1 μg / mL, 2 μg / mL, and 4 μg / mL with coating buffer, add 100 μL to each well of the ELISA plate, and then place at 2-8°C overnight.

[0122] Blocking: Shake off the liquid in the coated wells, pat dry on absorbent paper, block with blocking solution, add 200 μL to each well, and then incubate at 37°C for 1 hour.

[0123] Test steps:

[0124] Set up standard wells, blank wells, and sample wells respectively. Add 50 μL of the standard (β-Estradiol) diluted in multiples to the standard wells, with a standard concentration of 2000 pg / mL, add 50 μL of diluent to the blank wells, and add 50 μL of the sample to be tested to the remaining wells. Immediately add 50 μL of the prepared biotinylated antibody (2#Ab-Bio) working solution to each well. Cover the ELISA plate with a film and incubate at 37°C for 45 minutes. The sample addition time should be controlled within 10 minutes.

[0125] Shake off the liquid in the wells and pat dry on clean absorbent paper. Add 350μL of washing solution to each well, soak for 1 minute, absorb or shake off the liquid in the ELISA plate, and pat dry. Repeat this washing step 3 times. Add 100μL of SA-HRP enzyme conjugate working solution to each well, cover the ELISA plate with a film, and incubate at 37℃ for 30 minutes.

[0126] Shake off the liquid in the wells and wash the plate 5 times. Add 90 μL of substrate solution (TMB) to each well, cover the ELISA plate with a film, and incubate at 37°C in the dark for about 15 minutes.

[0127] Add 50 μL of stop solution to each well to terminate the reaction.

[0128] The optical density (OD value) of each well was immediately measured using a microplate reader at a wavelength of 450 nm.

[0129] The coating concentration of β-Estradiol-KLH was established to be 2 μg / mL through the square array, the detection ratios of 1#Ab-Bio and 2#Ab-Bio were 1:8000 and 1:10000 respectively, the concentration of the standard was 500 pg / mL, and the blocking conditions of the ELISA plate and the use conditions of the diluent were the same as in Example 5.

[0130] The performance comparison of the β-Estradiol small molecule competition ELISA detection kit and the β-Estradiol small molecule sandwich ELISA detection kit is as follows: Experiment 1: Sensitivity detection comparison of the β-Estradiol competition kit and the sandwich kit.

[0131] The same healthy negative mouse serum sample was tested repeatedly in 20 wells using the competition kit and sandwich kit, and the average OD value M and standard deviation SD of the 20 wells were calculated. Then, the M value and SD value measured by the competition kit and sandwich kit were substituted into the M-2SD and M+2SD formulas, respectively. The sensitivity of the competition kit was calculated to be 4.1pg / mL and the sensitivity of the sandwich kit was 0.32pg / mL using the origin9 curve fitting equation.

[0132] Experiment 2: Comparison of intra-batch and inter-batch differences between the β-Estradiol competition kit and the sandwich kit.

[0133] The implementation of the intra-batch difference detection comparison is based on the intra-batch and inter-batch differences of S6 in Example 5. The detection results are shown in Table 13.

[0134] Table 13

[0135]

[0136] Experiment 3: Comparison of dilution recovery and spike recovery of β-Estradiol competition kit and sandwich kit.

[0137] The implementation of the comparison of the dilution recovery rate and the spiked recovery rate detection is based on the dilution recovery rate and the spiked recovery rate verification method of S5 in Example 5. The comparative detection results are shown in Tables 14 to 15 below:

[0138] Table 14

[0139]

[0140]

[0141] Table 15

[0142]

[0143] In summary, the newly invented small molecule β-Estradiol sandwich assay kit is significantly superior to the competitive assay kit in terms of sensitivity, dilution recovery rate, spike recovery rate, and intra-batch and inter-batch differences. This not only lays a solid foundation for the industrial production of this target, but also provides a reliable guarantee for the accuracy of the test results for scientific research users.

[0144] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention, which shall all be included in the scope of protection of the present invention.

Claims

1. An ELISA detection kit, characterized in that: The ELISA detection kit includes a β-Estradiol-specific coating antibody, a β-Estradiol-specific detection antibody, a standard substance, Streptavidin-HRP, a single-component TMB colorimetric solution, a stop solution, and a diluent; The ELISA detection kit uses a double antibody sandwich method to detect β-Estradiol, the double antibody sandwich method uses a first anti-β-Estradiol specific antibody as a coating antibody, the first anti-β-Estradiol specific coating antibody is derived from one of Formula 3 and Formula 4, the double antibody sandwich method uses a second anti-β-Estradiol specific antibody as a detection antibody, the second anti-β-Estradiol specific detection antibody is derived from the other of Formula 3 and Formula 4; The compounds of formula 3 and formula 4 are as follows:

2. The ELISA test kit according to claim 1, characterized in that: The concentration of the β-Estradiol specific coating antibody is 1-6 μg / mL.

3. The ELISA test kit according to claim 1, characterized in that: The mass concentration of the β-Estradiol specific detection antibody is 1:2000-1:20000.

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