Conjugate for improving T3 micromolecule detection sensitivity and coupling method thereof, ELISA plate and kit

Abstract

The invention provides a conjugate for improving T3 micromolecule detection sensitivity, a coupling method thereof, an elisa plate and a kit. Compared with a traditional method that micromolecules are coupled with carrier protein and then coated for detection, the method has the advantages that the carrier protein is coated in the microwell plate in advance, after T3 is coupled in situ, ELISA detection is directly carried out, a complex purification method is not needed, T3 epitopes are not hindered by the carrier protein by using the coupling method, and the T3 micromolecule ELISA detection sensitivity can be greatly improved. The ELISA plate and the kit prepared by the coupling method are suitable for detection of animal serum, plasma and other samples in scientific research laboratories, and solve the problems of insufficient sensitivity, large intra-batch and inter-batch difference, low dilution and standard addition recovery rate and difficulty in large-scale industrial production of the traditional micromolecule T3 ELISA detection kit.

Description

Technical Field

[0001] The invention relates to the technical field of immunological detection, and in particular to a conjugate for improving the detection sensitivity of T3 small molecules, a conjugation method thereof, an ELISA plate and a kit. Background Art

[0002] Triiodothyronine, also known as T3 (Triiodothyronine), is one of the two main hormones released by the thyroid gland into the blood. The thyroid gland also produces tetraiodothyronine, known as T4. T4 (Thyroxine) and T3 work together and are often referred to as "thyroid hormones." T3 in serum is mainly involved in the metabolism of the three major nutrients (sugar, lipids and proteins) in the body to maintain the function of the thyroid gland. T3 is the active form of thyroid hormone, which means it affects cells in the body, while T4 is the inactive form of thyroid hormone. The liver and kidneys convert most of the T4 released by the thyroid gland into T3. The medical team will usually order a T3 test to help diagnose hyperthyroidism or determine the severity of hyperthyroidism.

[0003] The conventional small molecule T3 immunoassay method is to couple first and then coat, and use the principle of ELISA competition method for detection, which requires a tedious purification process after coupling. In addition, due to the random spatial distribution of antigens when the conjugate is coated, the small molecule T3 epitope is spatially shielded and cannot bind to the detection antibody, resulting in low detection sensitivity. Summary of the invention

[0004] In view of this, the present invention proposes a conjugate and a conjugation method, an ELISA plate and a kit for improving the detection sensitivity of T3 small molecules. The conjugation method can reduce the cumbersome conjugation and purification process, and the T3 epitope is not spatially hindered by the carrier protein, which can effectively improve the ELISA detection sensitivity.

[0005] The technical solution of the present invention is achieved in this way:

[0006] The present invention provides a coupling method for improving the detection sensitivity of T3 small molecules, which comprises the following steps:

[0007] (1) Pre-coated with carrier protein;

[0008] (2) Activate T3 small molecules;

[0009] (3) The activated T3 small molecule in step (2) is co-incubated with the coated carrier protein in step (1) for in situ coupling to form a stable carrier protein-T3 conjugate for ELISA detection.

[0010] On the basis of the above technical solution, preferably, the carrier protein is at least one of BSA, OVA, KLH and Tg carrier protein.

[0011] More preferably, the carrier protein is BSA.

[0012] On the basis of the above technical solution, preferably, the carrier protein is pre-coated with ELISA coating solution, and the coating concentration is 1 μg / mL to 4 μg / mL.

[0013] On the basis of the above technical solution, preferably, the T3 small molecule is activated by the EDC / NHS method, and the molar ratio of the T3 small molecule:EDC:NHS is 5:10:4.

[0014] Further preferably, the step of activating the T3 small molecule is: weighing 0.39g of T3 small molecule and adding it to 10ml4M NH4OH solution to completely dissolve it, adding PBS buffer to adjust the pH to 7.4±0.1, and fixing the volume to 39mL; weighing 0.23g EDC and 0.056g NHS respectively, adding them together to the T3 solution, incubating at room temperature for 2h, to obtain the activated T3 small molecule.

[0015] Based on the above technical solution, preferably, the concentration of the activated T3 small molecule in step (3) is 0.05 μg / mL to 0.8 μg / mL.

[0016] Based on the above technical solution, preferably, the incubation temperature in step (3) is 2° C.-8° C., and the incubation time is 1 h to 6 h.

[0017] The present invention also provides a carrier protein-T3 conjugate, which is prepared by the conjugation method.

[0018] The present invention also provides a pre-coated ELISA plate, which contains the carrier protein-T3 conjugate. The preparation method thereof is specifically as follows: pre-coating the carrier protein on the ELISA plate, activating the T3 small molecule and adding it to the ELISA plate coated with the carrier protein, incubating to form the carrier protein-T3 conjugate, washing away the unreacted small molecules in the ELISA plate, adding an ELISA plate protective agent, and drying to obtain the pre-coated ELISA plate.

[0019] On the basis of the above technical solution, preferably, a method for preparing a pre-coated ELISA plate is specifically as follows:

[0020] (1) Dilute the carrier protein to 4 μg / mL with coating solution, add 100 μl to each well of the ELISA plate, and incubate at 2-8°C overnight;

[0021] (2) Weigh 0.39 g of T3 small molecule and add it to 10 ml of 4 M NH4OH solution to completely dissolve it. Add PBS buffer to adjust the pH to 7.4 ± 0.1 and make the volume to 39 mL. Weigh 0.23 g of EDC and 0.056 g of NHS respectively and add them to the T3 solution. Incubate at room temperature for 2 h to obtain an activated T3 solution.

[0022] (3) Empty the liquid in the plate and add 100 μl of activated T3 solution (T3:EDC:NHS molar ratio is 5:10:4) to each well at a concentration of 0.2 μg / mL. Incubate at 2-8°C for 4-6 h.

[0023] (3) Protection: Add 200 μl of protective agent to each well, incubate at 2-8°C overnight, and then empty the liquid in the plate to prepare a pre-coated ELISA plate.

[0024] More preferably, the ELISA plate protective agent is a 0.01M PBS buffer solution containing 0.1% BSA, 5% sucrose, 5% sorbitol, and 0.1% ProClin300, and its pH is 7.4.

[0025] The present invention also provides a T3 small molecule detection ELISA kit, which comprises the pre-coated ELISA plate, T3 standard, biotinylated antibody, streptavidin-coupled HRP enzyme, TMB substrate, diluent, washing solution and stop solution.

[0026] On the basis of the above technical scheme, preferably, the preparation method of the biotinylated antibody is specifically as follows: the pH value of the T3-specific antibody is adjusted to 7.6±0.1 using a carbonate buffer, the pH-adjusted T3-specific antibody is mixed with biotin at a molar ratio of 1:15-25, the reaction is protected from light and placed at room temperature, and the biotinylated antibody is obtained after ultrafiltration.

[0027] Further preferably, the preparation method of the biotinylated antibody is specifically as follows: 500 μg of T3-specific antibody is placed in a penicillin bottle, the pH is adjusted to 7.6±0.1 with a carbonate buffer, 11 μg of biotin is added to the antibody solution at a molar ratio of 1:20 between the antibody and biotin, and the mixture is thoroughly mixed; the mixture is reacted in a 37°C incubator in the dark for 30 minutes, 50 μl of 1M Tris (pH 8.7) is added to terminate the reaction, and the mixture is placed at room temperature for 10 minutes after mixing; the antibody labeling solution after the reaction is ultrafiltered using a 10kd ultrafiltration tube to obtain a biotinylated antibody.

[0028] The present invention also provides the use of the pre-coated ELISA plate in preparing a kit for auxiliary diagnosis of hyperthyroidism.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The present invention first pre-coats the carrier protein BSA on the ELISA plate, and then couples the EDC-activated T3 with the BSA protein in situ in the micropores of the ELISA plate to generate a BSA-T3 conjugate, and then washes away the unreacted substances in the micropores of the microplate. No additional purification and blocking process is required, and the T3 antibody binding epitopes are all exposed to the outside, which can maximize the sensitivity of T3 small molecule ELISA detection. Therefore, the present invention provides a coupling method that can effectively improve the sensitivity of T3 small molecule ELISA detection, which is simple and has high coupling efficiency.

[0031] (2) Compared with the existing T3 small molecule competitive ELISA kit, the present invention proposes for the first time a method for preparing a kit for in situ coupling of small molecules, which increases the utilization efficiency of T3 small molecules and effectively solves the problems of insufficient sensitivity, large CV, and inability to achieve industrialized stable mass production of traditional T3 small molecule ELISA kits. At the same time, it provides new development ideas for other small molecule immunoassays. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0033] Figure 1 is a flow chart of the in situ coupling method of T3 small molecule in Example 4;

[0034] Figure 2 This is a detection flow chart of the T3 small molecule ELISA detection kit in Example 6. DETAILED DESCRIPTION

[0035] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] In the following examples, unless otherwise specified, all raw material components are commercially available products known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means known to those skilled in the art. Unless otherwise specified, the professional and scientific terms used in the present invention have the same meanings as those familiar to those skilled in the art.

[0037] In the present invention, T3-specific antibody was purchased from Hytest, catalog number 2T7. BSA protein was purchased from Beijing Solebow Technology Co., Ltd., catalog numbers A8010; OVA protein, KLH protein, and Tg protein were purchased from Sigma. T3 small molecule was purchased from Shanghai Dibai Company, catalog number H648002. EDC (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide) was purchased from ThermoFisher, catalog number PG82079, and NHS (N-hydroxysuccinimide), catalog number 24500. High adsorption ELISA coating solution was purchased from Wuhan Elerite Biotechnology Co., Ltd., catalog number E-ELIR-001. ANS (8-anilino-1-naphthalenesulfonic acid) was purchased from Merck, catalog number A1028.

[0038] Example 1

[0039] This embodiment provides a method for biotin-labeling an antibody, which specifically comprises the following steps:

[0040] Take 500 μg of T3 specific antibody and place it in a vial. Adjust the pH to 7.6±0.1 with carbonate buffer. Add 11 μg of biotin to the antibody solution at a molar ratio of 1:20 between antibody and biotin. Mix thoroughly. Incubate in a 37°C incubator in the dark for 30 minutes. Terminate the reaction with 50 μl of 1M Tris (pH 8.7). Mix well and place at room temperature for 10 minutes. Ultrafilter the antibody labeling solution after the reaction with a 10kd ultrafiltration tube to obtain the biotinylated antibody.

[0041] Example 2

[0042] This embodiment provides a method for biotin-labeling an antibody, which specifically comprises the following steps:

[0043] Take 500μg T3 specific antibody and place it in a vial. Adjust the pH to 7.6±0.1 with carbonate buffer. Add 11μg biotin to the antibody solution at a molar ratio of 1:15 between antibody and biotin. Mix thoroughly. Incubate in a 37℃ incubator in the dark for 30min. Terminate the reaction with 50μl 1M Tris (pH 8.7). Mix well and place at room temperature for 10min. Ultrafilter the antibody labeling solution using a 10kd ultrafiltration tube to obtain biotinylated antibody.

[0044] Example 3

[0045] This embodiment provides a method for biotin-labeling an antibody, which specifically comprises the following steps:

[0046] Take 500μg T3 specific antibody and place it in a vial. Adjust the pH to 7.6±0.1 with carbonate buffer. Add 11μg biotin to the antibody solution at a molar ratio of 1:25 between antibody and biotin. Mix thoroughly. Incubate in a 37℃ incubator in the dark for 30min. Terminate the reaction with 50μl 1M Tris (pH 8.7). Mix well and place at room temperature for 10min. Ultrafilter the antibody labeling solution using a 10kd ultrafiltration tube to obtain biotinylated antibody.

[0047] Example 4

[0048] This embodiment provides a T3 small molecule in situ coupling method and a pre-coated ELISA plate production method, which specifically includes the following steps:

[0049] Step (1): Use high adsorption ELISA coating solution to prepare BSA carrier protein coating working solution with a concentration of 5 μg / mL. Take 100 μl of BSA carrier protein coating working solution and add it to the ELISA plate, and incubate it at 4°C overnight.

[0050] Step (2): Weigh 0.39 g of T3 small molecule and add it to 10 ml of 4M NH4OH solution to completely dissolve it, add PBS buffer to adjust the pH to 7.4±0.1, and make the volume to 39 mL; weigh 0.23 g of EDC and 0.056 g of NHS respectively, add them to the T3 solution, incubate at room temperature for 2 hours, and obtain an activated T3 solution. The molar ratio of T3:EDC:NHS in the solution is 5:10:4.

[0051] Step (3): According to the experimental scheme in Table 1, the activated T3 solution was added to the ELISA plate coated with BSA protein in step (1), and incubated at 2-8°C for 4 hours. After washing the plate, 200 μl of protection solution was added to each well, and the liquid in the plate was emptied after incubation at 4°C for 18 hours, and vacuum dried for 1 hour to obtain a pre-coated ELISA plate.

[0052] The ELISA plate protective agent is a 0.01M PBS buffer solution containing 0.1% BSA, 5% sucrose, 5% sorbitol, and 0.1% ProClin300, and its pH is 7.4.

[0053] Table 1 Experimental design for in situ coupling of activated T3 with different concentrations and BSA-coated ELISA plate

[0054]

[0055] Step (4): using the ELISA plate prepared in step (3), after adding 50 μl of the standard working solution to each well, immediately add 50 μl of the biotinylated antibody prepared in Example 1 to each well, and incubate at 37°C for 45 min; after washing the plate, add 100 μl of SA-HRP to each well, and incubate at 37°C for 30 min; after washing the plate again, add a single-component TMB solution, incubate at 37°C for 15-25 min, and then add the stop solution; finally, read the absorbance value at a wavelength of 450 nm.

[0056] The detection results of the above different experimental schemes are shown in Table 2. As the concentration of activated T3 increases, the OD value also increases. When the detection-antibody ratio is 1 / 5k, the OD value range is 0.358-3.479, and when the detection-antibody ratio is 1 / 1w, the OD value range is 0.088-2.682, indicating that the above-mentioned T3 small molecule in situ coupling method can effectively detect T3, and the coupling efficiency and coupling utilization rate are high. When the concentration of activated T3 is 0.05μg / mL, the detection still has a good effect; the concentration of activated T3 is 0.1-0.4μg / mL, and the detection-antibody ratio is more appropriate between 1 / 5k-1 / 1w.

[0057] Table 2 ELISA test results of different concentrations of activated T3 and BSA coated ELISA plate in situ coupling

[0058]

[0059] Example 5

[0060] This example compares the effects of different carrier proteins, and specifically includes the following steps:

[0061] Step (1): prepare different concentrations of bovine serum albumin (BSA) using a highly adsorbable ELISA coating solution, and also include hen egg white albumin (OVA), keyhole limpet hemocyanin (KLH), and bovine thyroglobulin (Tg) carrier protein coating working solutions, wherein the concentrations are 1, 2, and 4 μg / mL, respectively. Take 100 μl of the carrier protein coating working solution and add it to the ELISA plate, and incubate it at 4°C overnight.

[0062] Step (2): Weigh 0.39 g of T3 small molecule and add it to 10 ml of 4M NH4OH solution to completely dissolve it, add PBS buffer to adjust the pH to 7.4±0.1, and make the volume to 39 mL; weigh 0.23 g of EDC and 0.056 g of NHS respectively, add them to the T3 solution, incubate at room temperature for 2 hours, and obtain an activated T3 solution. The molar ratio of T3:EDC:NHS in the solution is 5:10:4.

[0063] Step (3): According to the experimental scheme in Table 3, the activated T3 solution was added to the ELISA plate coated with the carrier protein in step (1), and incubated at 2-8°C for 4 hours. After washing the plate, 200 μl of protection solution was added to each well, and the plate was incubated at 4°C for 18 hours, and then the liquid in the plate was emptied and vacuum dried for 1 hour to obtain a pre-coated ELISA plate.

[0064] Table 3 Experimental design for in situ coupling of different carrier proteins and their coating concentrations with activated T3

[0065]

[0066] Step (4): using the ELISA plate prepared in step (3), after adding 50 μl of the standard working solution to each well, immediately add 50 μl of the biotinylated antibody prepared in Example 1 to each well, and incubate at 37°C for 45 min; after washing the plate, add 100 μl of SA-HRP to each well, and incubate at 37°C for 30 min; after washing the plate again, add a single-component TMB solution, incubate at 37°C for 15-25 min, and then add the stop solution; finally, read the absorbance value at a wavelength of 450 nm.

[0067] The test results of the above different experimental schemes are shown in Table 4. When the carrier protein is BSA, the OD value range is 0.088-3.120; when the carrier protein is BSA, the OD value range is 0.088-3.120; when the carrier protein is OVA, the OD value range is 0.257-2.514; when the carrier protein is KLH, the OD value range is 0.268-2.368; when the carrier protein is Tg, the OD value range is 0.071-1.624; the OD value of the carrier protein increases with the increase of the coating concentration, indicating that the in situ coupling of the T3 small molecule using different carrier proteins shows a good coupling effect, further indicating that the in situ coupling method can be used on different carrier proteins, and then used for ELISA detection. Therefore, the coating concentration of the carrier protein is 1-4μg / mL. For KLH and Tg proteins, when the concentration is greater than 2μg / mL, continuing to increase its concentration has no significant effect on the measured OD value.

[0068] Table 4 ELISA test results of different carrier proteins and their coating concentrations coupled with activated T3 in situ

[0069]

[0070] Example 6

[0071] 1. This embodiment provides a T3 small molecule kit, which contains a pre-coated ELISA plate, a T3 standard, a biotinylated antibody, a streptavidin-coupled HRP enzyme (SA-HRP), a TMB substrate, a diluent, a washing solution, and a stop solution.

[0072] The diluent was 0.01 M PBS buffer containing 0.01% ANS, 0.1% BSA, 0.05% Tween-20, and 0.1% Proclin 300, with a pH of 7.4.

[0073] The preparation method of pre-coated ELISA plate is as follows:

[0074] (1) Pre-coating with carrier protein: dilute BSA protein to 4 μg / mL with coating solution, add 100 μl to each well of the ELISA plate, and place at 2-8°C overnight;

[0075] (2) In situ coupling: Empty the liquid in the plate and add 100 μl of activated T3 (T3:EDC:NHS molar ratio is 5:10:4) to each well at a concentration of 0.2 μg / mL. Incubate at 2-8°C for 4-6 h.

[0076] (3) Protection: Add 200 μl of protective agent to each well, incubate at 2-8°C overnight, and then empty the liquid in the plate to prepare a pre-coated ELISA plate.

[0077] 2. Test steps of the kit:

[0078] Set standard wells, blank wells and sample wells respectively. Add 50μL of the diluted standard (T3 standard concentration is 100pg / mL) to the standard wells, 50μL of the diluent to the blank wells, and 50μL of the diluted sample to the remaining wells. Add 50μL of the pre-prepared biotinylated antibody working solution to each well. After the ELISA plate is coated, incubate at 37℃ for 45min. Empty the liquid in the plate and pat dry on a clean absorbent paper. Add 350μL of washing solution to each well, soak for 1min, absorb or shake off the liquid in the ELISA plate, and pat dry. Repeat this plate washing step 3 times. Add 100μL of SA-HRP enzyme conjugate working solution to each well, and incubate at 37℃ for 30min after the ELISA plate is coated. Empty the liquid in the plate and wash the plate 5 times. Add 90μL of single-component TMB solution to each well, coat the ELISA plate, and incubate at 37℃ in the dark for 15min. Add 50μL of stop solution to each well to terminate the reaction. The absorbance (OD value) was immediately measured using an enzyme reader at a wavelength of 450 nm.

[0079] 3. Kit performance test verification: including human serum plasma test verification, stability test verification, sensitivity test verification, dilution recovery and addition recovery test verification, intra-batch and inter-batch precision test verification and specificity test verification.

[0080] (1) Human serum plasma test verification. According to the scheme coating and test conditions in Table 5 below, a human serum plasma test verification scheme was designed, and serum plasma samples were diluted 50 times with diluent before testing.

[0081] Table 5 Human serum plasma test validation experimental design

[0082]

[0083]

[0084] The results obtained according to the above experimental scheme are shown in Table 6. The detection OD value range of human serum is 0.550-2.611; the detection OD value range of human plasma is 0.600-1.072, indicating that the human serum and plasma detection results of the kit are good. The experimental detection and calculation show that the human serum detection value is 661.87pg / mL~1987.62pg / mL, and the plasma detection value is 740.78pg / mL~1887.71pg / mL. The sample values ​​meet the clinical requirements, which shows that the kit is suitable for the detection of human serum and plasma in clinical practice.

[0085] Table 6 Human serum plasma test validation experimental results

[0086]

[0087] (2) Stability test verification. After the pre-coated ELISA plate, biotinylated antibody, and standard sample are packaged, they are placed at 37°C and 2-8°C for 7 days (7 days at 37°C is equivalent to 1 year at 2-8°C), and single-factor and overall stability tests at 37°C and 2-8°C are compared.

[0088] The results are shown in Table 7. The efficiency of the pre-coated ELISA plate is 104.71%, the efficiency of the labeled antibody is 96.78%, the efficiency of the standard is 97.63%, and the overall efficiency is 95.04%. The above results show that the stability of the kit meets the requirements of the range of 90%-110%, indicating that the kit is stored at 2-8°C and has a shelf life of about 1 year.

[0089] Table 7 Stability test results of each component of the kit

[0090] ELISA plate Labeled Antibodies Standards overall Effective rate (37℃ / 2-8℃) 104.71％ 96.78％ 97.63％ 95.04％

[0091] (3) Sensitivity test verification and comparison. The sensitivity of the test kit was tested according to the coupling and coating schemes shown in Table 8. Two parallel standard curves were set up, and 16 0 concentration negative wells were set up. After calculating the mean value M and standard deviation SD, the sensitivity was calculated by substituting them into the formula M-2SD.

[0092] Table 8 Experimental design scheme for comparison of coupling utilization loss rate and sensitivity between in situ coupling and T3 coating after coupling

[0093]

[0094] The results obtained according to the above experimental scheme are shown in Table 9. When the method of in situ coupling of T3 and BSA is used, the OD value range is 0.144-2.650, while when the method of coupling and then coating of T3 and BSA is used, the OD value range is 0.065-1.321, which is significantly lower than the OD value of the in situ coupling method, indicating that the T3 epitope is not blocked by the carrier protein after the in situ coupling of T3 and BSA, and the OD value is higher than that of the coupling and then coating under the same conditions, that is, the utilization efficiency of T3 is higher. By calculating the sensitivity, the sensitivity of the in situ coupling of T3 and BSA is 0.29pg / mL, while the sensitivity of the coupling and then coating method is 3.24pg / mL. It can be seen that the sensitivity of ELISA detection by the in situ coupling method has increased by more than 10 times.

[0095] T3 loss rate calculation formula:

[0096]

[0097] By calculating the T3 utilization loss rate of in situ coupling and direct coupling (coating after coupling), the results showed that direct coupling lost 52.36% of T3 utilization compared with in situ coupling, indicating that in situ coupling can effectively improve the utilization of T3 molecules.

[0098] Table 9 Comparison of the coupling utilization loss rate and sensitivity of in-situ coupling and T3 coating after coupling

[0099]

[0100] (4) Dilution recovery and surface recovery test verification.

[0101] Dilution recovery rate verification. Human serum and human EDTA plasma samples were diluted 2 times, 4 times, 8 times and 16 times for dilution recovery experiments, and the dilution recovery rate detection range and average dilution recovery rate were obtained. The test results are shown in Table 10. When the sample dilution ratio is 1:2, the dilution recovery rate of human serum ranges from 87% to 102%, and the average dilution recovery rate is 94%; the dilution recovery rate of human EDTA plasma ranges from 97% to 110%, and the average dilution recovery rate is 103%. When the sample dilution ratio is 1:4, the dilution recovery rate of human serum ranges from 91% to 105%, and the average dilution recovery rate is 98%; the dilution recovery rate of human EDTA plasma ranges from 95% to 108%, and the average dilution recovery rate is 101%. When the sample dilution ratio is 1:8, the dilution recovery rate of human serum ranges from 90% to 106%, and the average dilution recovery rate is 98%; the dilution recovery rate of human EDTA plasma ranges from 88% to 92%, and the average dilution recovery rate is 90%. When the sample dilution ratio is 1:16, the dilution recovery rate of human serum ranges from 88%-103%, and the average dilution recovery rate is 95%; the dilution recovery rate of human EDTA plasma ranges from 92%-106%, and the average dilution recovery rate is 89%. The above shows that the kit meets the industry standard of 80%-120%.

[0102] Table 10 Sample dilution recovery test results

[0103]

[0104] Verification of spike recovery rate. T3 of known concentration (100 pg / mL) was added to 8 human serum plasma samples (10-fold dilution) of different concentrations (respectively: 1850.67 pg / mL, 1768.23 pg / mL, 1660.57 pg / mL, 1625.20 pg / mL, 1287.45 pg / mL, 1023.80 pg / mL, 860.57 pg / mL, 781.12 pg / mL) to perform recovery experiments, and obtain the spike recovery detection range and average spike recovery rate. The test results are shown in Table 11. The spike recovery rate of human serum is 88%-105%, and the average spike recovery rate is 96%. The spike recovery rate of human plasma is 92%-114%, and the average spike recovery rate is 103%, indicating that the spike recovery test results of the kit meet the industry standard of 80%-120%.

[0105] Table 11 Spiked recovery test results

[0106] Sample Type Spiked recovery rate (%) Average spike recovery (%) Human serum (n=8) 88-105 96 Human EDTA plasma (n=8) 92-114 103

[0107] (5) Intra-batch and inter-batch difference detection and verification

[0108] Intra-batch precision test: Low concentration (769.54 pg / mL), medium concentration (1275.03 pg / mL), and high concentration (1921.84 pg / mL) human serum plasma samples were tested 20 times on one plate respectively.

[0109] Inter-batch precision test: Low-concentration (769.54 pg / mL), medium-concentration (1275.03 pg / mL), and high-concentration (1921.84 pg / mL) samples were tested 20 times on 3 plates respectively.

[0110] The test results are shown in Table 12. For the intra-batch precision test, the coefficients of variation of human serum samples 1#, 2#, and 3# were 5.89%, 4.96%, and 2.83%, respectively, and the coefficients of variation of human plasma samples 1#, 2#, and 3# were 3.98%, 2.78%, and 3.03%, respectively; for the inter-batch precision test, the coefficients of variation of human serum samples 1#, 2#, and 3# were 3.68%, 6.31%, and 2.78%, respectively, and the coefficients of variation of human plasma samples 1#, 2#, and 3# were 2.91%, 3.65%, and 3.59%, respectively. These results show that the intra-batch difference of the kit is <10%, and the inter-batch difference is <10%, which are in line with industry standards.

[0111] Table 12 Intra-batch and inter-batch difference test results

[0112]

[0113] (6) Specific detection verification. The verified structural analogs are: L-T4, D-T4, L-Rt3, L-T2, and the concentration of these substances is 200pg / mL. The detection is carried out according to the above-mentioned "Human serum plasma detection verification" coating and detection method.

[0114] The results are shown in Table 13. The cross-reaction rate of L-T4 is 1.71%, the cross-reaction rate of D-T4 is 1.10%, the cross-reaction rate of L-rT3 is 0.16%, and the cross-reaction rate of L-T2 is 3.66%. These results show that the cross-reaction rate of the kit is <15%, which meets the requirements.

[0115] Table 13 Specificity test results

[0116]

[0117]

[0118] This embodiment is also compared with a commercially available test kit, which was purchased from Wuhan Cloud Clone Technology Co., Ltd., numbered CEA453Ge. The sensitivity of the sample was compared, and the sensitivity detection was to set 2 parallel standard curves, and 16 0 concentration negative holes were set at the same time. After calculating the mean value M and standard deviation SD of the 0 concentration hole, the sensitivity was calculated by entering the formula M-2SD. The results show that human serum plasma samples meet clinical requirements. In addition, the sensitivity test result of the commercially available test kit is 53.8pg / mL, and the sensitivity of the test kit of the in situ coupling method is 0.37pg / mL. It can be seen that the sensitivity of the test kit of the present invention has increased by 2 orders of magnitude.

[0119] In summary, the in situ coupling small molecule method of the present invention can significantly improve the detection sensitivity of T3 small molecule ELISA, while greatly ensuring the stability of the kit and the intra-batch and inter-batch precision, while also ensuring its specific detection, laying a solid foundation for the industrial production of this target, and also providing a new development solution for other small molecule detections.

[0120] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A coupling method for improving the sensitivity of T3 small molecule detection, characterized in that: The coupling method comprises the following steps: (1) Pre-coated with carrier protein; (2) Activate T3 small molecules; (3) The activated T3 small molecule in step (2) is co-incubated with the coated carrier protein in step (1) for in situ coupling to form a stable carrier protein-T3 conjugate for ELISA detection.

2. The coupling method according to claim 1, wherein: The carrier protein is at least one of BSA, OVA, KLH and Tg carrier proteins.

3. The coupling method according to claim 1, wherein: The carrier protein is pre-coated with ELISA coating solution, and the coating concentration is 1 μg / mL to 4 μg / mL.

4. The coupling method according to claim 1, characterized in that: The concentration of the activated T3 small molecule in step (3) is 0.05 μg / mL to 0.8 μg / mL.

5. The coupling method according to claim 1, characterized in that: The incubation temperature in step (3) is 2°C-8°C, and the incubation time is 1h-6h.

6. A carrier protein-T3 conjugate, characterized in that: The conjugate is prepared by the conjugation method according to any one of claims 1 to 5.

7. A pre-coated ELISA plate, characterized in that: The pre-coated ELISA plate contains the carrier protein-T3 conjugate according to claim 6, and its preparation method is specifically as follows: pre-coating the carrier protein on the ELISA plate, activating the T3 small molecule and adding it to the ELISA plate coated with the carrier protein, incubating to form the carrier protein-T3 conjugate, washing away the unreacted small molecules in the ELISA plate, adding an ELISA plate protective agent, and drying to obtain the pre-coated ELISA plate.

8. A T3 small molecule detection ELISA kit, characterized in that: The kit comprises the pre-coated ELISA plate according to claim 7, T3 standard, biotinylated antibody, streptavidin-coupled HRP enzyme, TMB substrate, diluent, washing solution and stop solution.

9. The T3 small molecule detection ELISA kit according to claim 8, characterized in that: The preparation method of the biotinylated antibody is specifically as follows: adjusting the pH value of the T3-specific antibody to 7.6±0.1 using a carbonate buffer, mixing the pH-adjusted T3-specific antibody with biotin at a molar ratio of 1:15-25, allowing the mixture to react in a dark environment and then stand at room temperature, and obtaining the biotinylated antibody after ultrafiltration.

10. Use of the pre-coated ELISA plate according to claim 7 in preparing a kit for auxiliary diagnosis of hyperthyroidism.

Images