CN113493514B - Enzyme conjugate of anti-triiodothyronine monoclonal antibody, total triiodothyronine quantitative detection kit and use method thereof - Google Patents
Enzyme conjugate of anti-triiodothyronine monoclonal antibody, total triiodothyronine quantitative detection kit and use method thereof Download PDFInfo
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Abstract
The application provides an enzyme conjugate of anti-triiodothyronine monoclonal antibody, a total triiodothyronine quantitative detection kit and a use method thereof, wherein the enzyme conjugate diluent provided by the application can enhance the stability of the enzyme conjugate, inhibit the damage of a dissociation agent to the enzyme conjugate, realize that the enzyme conjugate and the dissociation agent can coexist in the same reagent component, and simultaneously meet the conditions of total triiodothyronine dissociation, new and old sample interference and HAMA interference in the detection of the total triiodothyronine, so that the total triiodothyronine detection kit provided by the application can achieve faster and more convenient operation and obtain more accurate detection results.
Description
Technical Field
The application relates to the technical field of immunological detection, in particular to an enzyme conjugate of anti-triiodothyronine monoclonal antibody, a total triiodothyronine quantitative detection kit and a use method thereof.
Background
The molecular weight of triiodothyronine (T3) is 651, about 25% is directly produced by thyroid gland, 75% is formed by thyroxine (T4) in peripheral tissues through the action of 5' -deiodinase, and the biological activity of the triiodothyronine is 3-5 times that of thyroxine which is the most bioactive known at present. Therefore, the detection of triiodothyronine can effectively evaluate the thyroid functional state.
The existing detection method for the total triiodothyronine (TT 3) comprises a liquid chromatography-tandem mass spectrometry method and an immunological method. The immunological method is a mainstream method for clinical application at present because of rapid detection and low cost. The platform also forms the product forms of chemiluminescence, enzyme-linked immunity, immunofluorescence, immunoturbidimetry, fluorescence immunity and the like.
Among products using immunological methods, the methods currently used are: immobilized anti-triiodothyronine (T3) antibody, enzyme-labeled triiodothyronine (T3) antigen, and pre-treating the sample to dissociate total triiodothyronine (TT 3) from the binding protein into a free state. Then, the enzyme-labeled triiodothyronine (T3) antigen and the sample treated to present the free-form triiodothyronine (T3) antigen compete for binding to the immobilized anti-triiodothyronine (T3) antibody, and the competition method detection is carried out. For example: the method is used by companies such as Beijing-family Dongya, beijing Yuan De organism, beijing Meikang organism and the like, and the method needs to independently perform one-step sample pretreatment, and is important that the method is easy to cause the error of the detection result of a sample with abnormal binding protein in a certain proportion. The reason is that these sample binding proteins have the opportunity to form new binding with the enzyme-labeled triiodothyronine (T3) antigen after dissociation, thereby resulting in interference with the outcome of the competition reaction. Or can only be operated in a two-step process, avoiding binding of the binding protein to the enzyme-labeled antigen.
In addition, some products on the market adopt another method, namely a method for immobilizing the triiodothyronine (T3) antigen and enzymatically labeling an anti-triiodothyronine (T3) antibody, adding a dissociating agent component into a reaction reagent to dissociate the total triiodothyronine (TT 3) from a binding protein into a free state, and then performing competition detection on the immobilized triiodothyronine (T3) antigen and the sample treated to present the free-state triiodothyronine (T3) antigen in competition for the anti-triiodothyronine (T3) antibody labeled by the binding enzyme. Although this method can avoid the effect of interfering with competition reaction by forming new binding with triiodothyronine (T3) antigen in matrix solution after dissociation of binding protein in sample, it has been possible to use only product form of enzyme dilution and enzyme concentrate since physical and chemical properties of dissociating agent have some degradation effect on enzyme, separating enzyme conjugate (existing in enzyme concentrate) and dissociating agent (existing in enzyme dilution) in different reagent components, thus causing complicated operation, such as working solution of enzyme conjugate prior to use or two-step sample addition. For example: this approach is used by the Zhengzhou Anji biotechnology Co., ltd.
Disclosure of Invention
In order to solve the problems, the application provides an enzyme conjugate of anti-triiodothyronine monoclonal antibody, a total triiodothyronine quantitative detection kit and a use method thereof, and the enzyme conjugate diluent provided by the application can enhance the stability of the enzyme conjugate, inhibit the damage of a dissociation agent to the enzyme conjugate, realize that the enzyme conjugate and the dissociation agent can coexist in the same reagent component, and simultaneously meet the conditions of total triiodothyronine dissociation, new and old sample interference and HAMA interference in the detection of the total triiodothyronine, so that the total triiodothyronine detection kit provided by the application achieves faster and more convenient operation and more accurate detection result.
The object of the application is achieved in the following way: an enzyme conjugate of anti-triiodothyronine monoclonal antibody, comprising enzyme conjugate diluent and enzyme-labeled triiodothyronine monoclonal antibody, wherein the volume ratio of the enzyme conjugate diluent to the enzyme-labeled triiodothyronine monoclonal antibody is 4500-5500:1, a step of; enzyme conjugate dilutions including BSA, tryptone, aminopyrine.
The enzyme-labeled triiodothyronine monoclonal antibody may be a monoclonal antibody containing HRP-labeled anti-triiodothyronine.
Specifically, enzyme conjugate dilutions include barbital sodium buffer, ANS, BSA, tryptone, aminopyrine, IPBC, HBR, proclin, 300.
The barbital sodium buffer is a barbital sodium buffer with pH value of 6.8-8.
The enzyme conjugate diluent contains 25-35g BSA, 7-12g tryptone and 0.8-1.2g aminopyrine in each liter.
The enzyme conjugate diluent contains 978ml of barbital sodium buffer, ANS 3g, BSA 30g, tryptone 10g, aminopyrine 1g, IPBC 1g, HBR 50mg and Proclin300 ml per liter, wherein the barbital sodium buffer is prepared into 1L of barbital sodium water solution per 10.3g of barbital sodium, and then 6M hydrochloric acid is added to prepare the barbital sodium buffer with the pH value of 7.4.
The volume ratio of the enzyme conjugate diluent to the enzyme-labeled triiodothyronine monoclonal antibody is 5000:1.
a total triiodothyronine quantitative detection kit comprises a coated plate coated with triiodothyronine, an enzyme conjugate of enzyme-labeled anti-triiodothyronine monoclonal antibody and a triiodothyronine calibrator, wherein the enzyme conjugate is prepared by adding the enzyme-labeled triiodothyronine monoclonal antibody into an enzyme conjugate diluent.
The preparation method of the coated plate coated with the triiodothyronine comprises the following steps: (1) Adding T3-BSA into tris-HCl buffer solution to prepare coating solution, and fully and uniformly mixing;
(2) Adding the prepared coating liquid into a blank ELISA plate, and standing at 2-8deg.C for 16-24hr;
(3) Removing the coating liquid in the ELISA plate, and washing the plate at least 2 times by using washing liquid after the surface of the ELISA plate is free of liquid;
(4) Sealing within 2 minutes after washing the plate, adding sealing liquid into each hole, and then standing at 2-8 ℃ for 16-24 hours;
(5) And (3) throwing the sealing liquid of the ELISA plate out, and then placing the ELISA plate in a drying device for drying (humidity is less than or equal to 30%) for 4-8hr.
The preparation method of the triiodothyronine calibrator comprises the following steps: (1) Preparing a calibrator dilution using HEPES buffer, BSA, nystatin and Proclin300 at pH 6.8-7.5;
(2) And diluting the triiodothyronine by using a calibrator diluent to prepare the triiodothyronine calibrator with different concentrations.
Six concentrations of triiodothyronine calibrator were made in step (2) of 0ng/ml,0.5ng/ml,1ng/ml,2ng/ml,4ng/ml,8 ng/ml.
The application method of the total triiodothyronine quantitative detection kit comprises the following steps of (1) sample adding: taking out a certain amount of coating holes numbered on the coating plate, sequentially adding 50 μl of calibrator and sample to be tested by using a micropipette, and adding 50 μl of enzyme conjugate;
(2) Incubation: placing the coated plate after sample addition at 37 ℃ for 60 minutes;
(3) Washing: throwing away the liquid in the coating holes, filling the coating holes with diluted washing liquid, standing for 20 seconds, throwing away, repeatedly washing for 5 times, throwing away the liquid each time, and finally beating on water absorbing paper;
(4) And (3) color development: 100 mu l of each coating Kong Jiade substance liquid reacts for 20 minutes at 18-23 ℃ in a dark place;
(5) And (5) terminating: the reaction was stopped by adding 50. Mu.l of stop solution to each coated well.
The kit is prepared based on an enzyme immune method, is matched with an enzyme-labeled instrument for use, and has the advantages of simple operation and high detection speed. Compared with the total triiodothyronine (TT 3) detection kit sold in the market, the kit adopts a reaction mode of immobilized triiodothyronine (T3) antigen and enzyme-labeled anti-triiodothyronine (T3) antibody; and optimizing the formula of the enzyme conjugate, adding aminopyrine, tryptone and BSA, and effectively protecting the enzyme-labeled antibody from being influenced by a dissociating agent. The reaction principle is not influenced by binding proteins after sample dissociation due to the immobilization of the competing triiodothyronine antigen. In addition, the effective substances are added into the enzyme conjugate, so that the dissociation of total triiodothyronine in a sample can be simultaneously satisfied, the interference between new and old samples and the HAMA interference can be reduced, and the enzyme conjugate can be directly used without being prepared in an existing way.
The action principle is as follows:
the total triiodothyronine is combined with the binding protein through chemical bonds in a natural state, and the binding force is stable and has a corresponding combination proportion. When total triiodothyronine is dissociated from binding protein using a dissociating agent, the binding protein will form a new free binding relationship with the matrix environment due to its physicochemical properties. In the past, the reaction principle of immobilized antibodies and enzyme-labeled antigens is adopted, and if a method of synchronous reaction of dissociation and competition is used, a certain binding interference can be caused on the enzyme-labeled antigens by binding proteins in a sample. Or to avoid this risk, the marketed products often employ a two-step procedure, in which the sample is dissociated in a first step, the dissociated antigen is bound to the immobilized antibody, and then the unreacted antigen and matrix solution (including binding proteins) are washed away, and in which enzyme-bound antigen is added in a second step to bind to the immobilized antibody that does not participate in the first step. The above problems are solved by avoiding contact of the binding protein with the enzyme-labeled antigen by a two-step reaction.
The application exchanges the positions of antigen and antibody, solidifies the antigen and marks the antibody. The antigen loses the condition of combining with the binding protein after being immobilized, and the corresponding antigenic determinant is also completely reserved, so that the antigen has the characteristics of natural antigen when competing with the antigen in the sample, and has good competing reaction effect and obvious gradient.
The blocking agent in the enzyme conjugate is an effective component for eliminating the interference of the HAMA sample, and the main action principle is that the Fc epitope of the HAMA sample xenophilic antibody is actively affinitized, so that the Fc epitope loses the capability of combining with the triiodothyronine antibody; the addition of aminopyrine, tryptone and BSA in the enzyme conjugate effectively protects the enzyme-labeled antibody from the influence of the dissociating agent.
Drawings
Fig. 1 is a regression graph.
FIG. 2 is a reference diagram of the correlation of the kit of the present application with existing products.
Detailed Description
An enzyme conjugate of anti-triiodothyronine monoclonal antibody, comprising enzyme conjugate diluent and enzyme-labeled triiodothyronine monoclonal antibody, wherein the volume ratio of the enzyme conjugate diluent to the enzyme-labeled triiodothyronine monoclonal antibody is 4500-5500:1, a step of; enzyme conjugate dilutions including BSA, tryptone, aminopyrine.
The enzyme-labeled triiodothyronine monoclonal antibody may be a monoclonal antibody containing HRP-labeled anti-triiodothyronine.
Specifically, enzyme conjugate dilutions include barbital sodium buffer, ANS, BSA, tryptone, aminopyrine, IPBC, HBR, proclin, 300.
The barbital sodium buffer is a barbital sodium buffer with pH value of 6.8-8.
The enzyme conjugate diluent contains 25-35g BSA, 7-12g tryptone and 0.8-1.2g aminopyrine in each liter.
The enzyme conjugate diluent contains 978ml of barbital sodium buffer, ANS 3g, BSA 30g, tryptone 10g, aminopyrine 1g, IPBC 1g, HBR 50mg and Proclin300 ml per liter, wherein the barbital sodium buffer is prepared into 1L of barbital sodium water solution per 10.3g of barbital sodium, and then 6M hydrochloric acid is added to prepare the barbital sodium buffer with the pH value of 7.4.
The volume ratio of the enzyme conjugate diluent to the enzyme-labeled triiodothyronine monoclonal antibody is 5000:1.
a total triiodothyronine quantitative detection kit comprises a coated plate coated with triiodothyronine, an enzyme conjugate of enzyme-labeled anti-triiodothyronine monoclonal antibody and a triiodothyronine calibrator, wherein the enzyme conjugate is prepared by adding the enzyme-labeled triiodothyronine monoclonal antibody into an enzyme conjugate diluent.
The preparation method of the coated plate coated with the triiodothyronine comprises the following steps: (1) Adding T3-BSA into tris-HCl buffer solution to prepare coating solution, and fully and uniformly mixing;
(2) Adding the prepared coating liquid into a blank ELISA plate, and standing at 2-8deg.C for 16-24hr;
(3) Removing the coating liquid in the ELISA plate, and washing the plate at least 2 times by using washing liquid after the surface of the ELISA plate is free of liquid;
(4) Sealing within 2 minutes after washing the plate, adding sealing liquid into each hole, and then standing at 2-8 ℃ for 16-24 hours;
(5) And (3) throwing the sealing liquid of the ELISA plate out, and then placing the ELISA plate in a drying device for drying (humidity is less than or equal to 30%) for 4-8hr.
The preparation method of the triiodothyronine calibrator comprises the following steps: (1) Preparing a calibrator dilution using HEPES buffer, BSA, nystatin and Proclin300 at pH 6.8-7.5;
(2) And diluting the triiodothyronine by using a calibrator diluent to prepare the triiodothyronine calibrator with different concentrations.
Six concentrations of triiodothyronine calibrator were made in step (2) of 0ng/ml,0.5ng/ml,1ng/ml,2ng/ml,4ng/ml,8 ng/ml.
The application method of the total triiodothyronine quantitative detection kit comprises the following steps of (1) sample adding: taking out a certain amount of coating holes numbered on the coating plate, sequentially adding 50 μl of calibrator and sample to be tested by using a micropipette, and adding 50 μl of enzyme conjugate;
(2) Incubation: placing the coated plate after sample addition at 37 ℃ for 60 minutes;
(3) Washing: throwing away the liquid in the coating holes, filling the coating holes with diluted washing liquid, standing for 20 seconds, throwing away, repeatedly washing for 5 times, throwing away the liquid each time, and finally beating on water absorbing paper;
(4) And (3) color development: 100 mu l of each coating Kong Jiade substance liquid reacts for 20 minutes at 18-23 ℃ in a dark place;
(5) And (5) terminating: the reaction was stopped by adding 50. Mu.l of stop solution to each coated well.
The present application will be described in more detail with reference to specific examples, which will be understood by those skilled in the art. If no special description exists, the materials and the detection instrument used in the application are all conventional materials products in the industry, and are all commonly sold in the market. The detection method is a conventional use method in the industry.
Preparation of improved Total triiodothyronine (TT 4) detection kit
1. Preparation of coated sheet
(1) T3-BSA (purchased from Danoea, model D1001) was added to a tris-HCl buffer solution of 0.1M PH7.4 at a concentration of 2.5ug/ml to prepare a coating solution, and the coating solution was thoroughly mixed at a temperature of 18-23 ℃.
(2) Adding the prepared coating solution into a blank ELISA plate (purchased from Xiamen Yunpeng living beings) at a volume of 100 μl/hole, and standing at 2-8deg.C in a refrigerator for 16-24hr.
(3) And (5) removing the coating liquid in the ELISA plate, and washing the plate for 2 times by using washing liquid after the plate is patted dry.
(4) After washing the plate, the plate is closed within 2 minutes, 150 μl/hole of a closing liquid, 1% gelatin, is added, and the plate is placed in a refrigerator at 2-8deg.C for 16-24hr.
(5) And (3) throwing out the sealing liquid of the ELISA plate, and then placing the ELISA plate in a drying box for drying (humidity is less than or equal to 30%) for 4-8hr.
The preparation is completed.
2. Preparation of enzyme conjugates of the application
Firstly, preparing a barbital sodium buffer solution, and mixing the raw materials in the table 1 to prepare a barbital sodium aqueous solution;
TABLE 1
| Name of product | Content of | Raw material requirements |
| Purified water | 996ml | Analytical grade |
| Barbital sodium | 10.3g | Analytical grade |
The pH of the aqueous solution of barbital sodium was then adjusted to 7.4 using 6M hydrochloric acid to prepare a barbital sodium buffer.
Preparing enzyme diluent in the second step
The enzyme dilutions were prepared using the raw materials in table 2 below.
TABLE 2
The enzyme dilutions were prepared and a monoclonal antibody (model 17C6, purchased from biontix) containing HRP enzyme-labelled triiodothyronine was added at 5000:1 volume ratio to prepare the enzyme conjugate.
3. Preparation of the calibration Material of the application
Preparing HEPES buffer solution, and mixing the raw materials in the following table 3 to prepare HEPES solution;
TABLE 3 Table 3
| Name of product | Content of | Raw material requirements |
| Purified water | 993ml | Analytical grade |
| HEPES | 6.5g | Analytical grade |
| Nacl | 8.0g | Analytical grade |
Then adjusting the pH of the HEPES solution to 7.2 by using 1M sodium hydroxide to prepare HEPES buffer solution
Preparing a calibrator diluent in the second step, and mixing the raw materials in the following table 4 to prepare the calibrator diluent;
TABLE 4 Table 4
| Name of product | Content of | Raw material requirements |
| HEPES buffer | 983ml | |
| BSA | 30g | Analytical grade |
| Nystatin | 0.05g | |
| Proclin300 | 1ml | Analytical grade |
Triiodothyronine (T3) (sigma T2877) was diluted with this dilution to prepare six concentrations of calibrator at 0ng/ml,0.5ng/ml,1ng/ml,2ng/ml,4ng/ml,8ng/ml, respectively. The manufacturer and model of the nystatin are respectively the source leaf organism and S17029.
4. Other general reagent components: substrates, stop liquor, wash liquor were purchased from the organism dano in zhengzhou.
5. Test procedure
(one) [ Main component ] Table 5 below
TABLE 5
(II) [ test method ]
Preparation before experiments
1. All reagents and samples were returned to room temperature 18-25 ℃ for at least 30 minutes.
2. The reaction temperature was adjusted to 37℃with an incubator or water bath.
3. Each bottle of washing liquid is diluted by 1000ml of purified water and is shaken uniformly for standby.
Experimental procedure
1. Sample adding: a certain amount of the numbered coated wells on the coated plate were removed. Mu.l of calibrator and sample to be tested were added sequentially with a micropipette, followed by 50. Mu.l of enzyme conjugate.
2. Incubation: the coated plate after the sample addition was placed at 37℃for 60 minutes.
3. Washing: and (3) throwing away the liquid in the coating holes, filling the coating holes with diluted washing liquid, standing for 20 seconds, throwing away, repeatedly washing for 5 times, throwing away the liquid each time, and finally beating on absorbent paper.
4. Color development: 100 mu l of each coating Kong Jiade substance liquid is reacted for 20 minutes at the room temperature of 18-23 ℃ in a dark place.
5. And (3) terminating: the reaction was stopped by adding 50. Mu.l of stop solution to each coated well.
(III) calculation of results
(1) And (3) detecting by an enzyme-labeled instrument: the microplate reader wavelength was selected to be 450nm and the reference wavelength was selected to be 630nm. The OD value of each well was determined within 10 minutes after termination of the reaction.
(2) Calculating: the kit recommends a point-to-point regression fit mode, namely, a point-to-point standard curve is established by taking a serial calibrator concentration value as an abscissa (X axis) and a standard OD value as an ordinate (Y axis), and the content of the total triiodothyronine in the sample to be detected is calculated. Normal human reference interval (this range is calculated statistically for the normal population tested by the kit): 0.80-11ng/ml. Example data are calculated as in table 6 below, such that an established point-to-point standard curve such as the curve is referenced in fig. 1.
TABLE 6
| Name of product | Concentration (ng/ml) | OD value |
| Standard 1 | 0 | 2.949 |
| Standard substance 2 | 0.5 | 2.278 |
| Standard substance 3 | 1 | 1.735 |
| Standard substance 4 | 2 | 1.053 |
| Standard substance 5 | 4 | 0.532 |
| Standard 6 | 8 | 0.201 |
| Sample 1 | 1.59 | 1.333 |
8. Clinical performance evaluation and application advantages of the kit
The kit is adopted to carry out parallel examination on 168 physical examination crowd samples with concentration ranging from 0.3 ng/ml to 7.0ng/ml by an electrochemiluminescence method with the total triiodothyronine assay kit produced by roche company, the detection results are subjected to clinical correlation analysis, the detection results are shown in the following table 7, and a correlation reference graph is prepared according to the data in the table and is shown in figure 2.
TABLE 7
According to the correlation reference diagram of FIG. 2, the prepared kit has good correlation with the existing product, so that the application solves the operation complexity of the two-step reaction of the existing product on the market, and simultaneously maintains a good detection effect. Most commercial products of domestic enterprises are subjected to two-step reaction, the dissociation process is completed independently in the first step, the competition reaction is realized in the second step, the plates are required to be washed twice, and the reaction time is long. The detection kit provided by the application can realize one-step reaction, improves the detection efficiency, reduces the operation error rate, and simultaneously reduces one reagent component.
The foregoing is merely a preferred embodiment of the present application, and it should be noted that it will be apparent to those skilled in the art that several changes and modifications can be made without departing from the general inventive concept, and these should also be regarded as the scope of the application.
Claims (5)
1. A total triiodothyronine quantitative determination kit is characterized in that: the kit comprises a coating plate coated with triiodothyronine, an enzyme conjugate of enzyme-labeled anti-triiodothyronine monoclonal antibody and a triiodothyronine calibrator, wherein the enzyme conjugate is prepared by adding enzyme-labeled triiodothyronine monoclonal antibody into enzyme conjugate diluent; the enzyme conjugate of the enzyme-labeled anti-triiodothyronine monoclonal antibody comprises an enzyme conjugate diluent and an enzyme-labeled triiodothyronine monoclonal antibody, wherein the volume ratio of the enzyme conjugate diluent to the enzyme-labeled triiodothyronine monoclonal antibody is 4500-5500:1, a step of; the enzyme conjugate diluent contains 978ml of barbital sodium buffer, ANS 3g, BSA 30g, tryptone 10g, aminopyrine 1g, IPBC 1g, HBR 50mg and Proclin300 ml per liter, wherein the barbital sodium buffer is prepared into 1L of barbital sodium water solution per 10.3g of barbital sodium, and then 6M hydrochloric acid is added to prepare the barbital sodium buffer with the pH value of 7.4.
2. The kit for quantitative detection of total triiodothyronine according to claim 1, characterized in that: the volume ratio of the enzyme conjugate diluent to the enzyme-labeled triiodothyronine monoclonal antibody is 5000:1.
3. the kit for quantitative detection of total triiodothyronine according to claim 1, characterized in that: the preparation method of the coated plate coated with the triiodothyronine comprises the following steps: (1) Adding T3-BSA into tris-HCl buffer solution to prepare coating solution, and fully and uniformly mixing;
(2) Adding the prepared coating liquid into a blank ELISA plate, and standing at 2-8deg.C for 16-24hr;
(3) Removing the coating liquid in the ELISA plate, and washing the plate at least 2 times by using washing liquid after the surface of the ELISA plate is free of liquid;
(4) Sealing within 2 minutes after washing the plate, adding sealing liquid into each hole, and then standing at 2-8 ℃ for 16-24 hours;
(5) And (3) throwing out the sealing liquid of the ELISA plate, and then placing the ELISA plate in a drying device for drying for 4-8hr, wherein the humidity of the drying device is less than or equal to 30%.
4. The kit for quantitative detection of total triiodothyronine according to claim 1, characterized in that: the preparation method of the triiodothyronine calibrator comprises the following steps: (1) Preparing a calibrator dilution using HEPES buffer, BSA, nystatin and Proclin300 at pH 6.8-7.5;
(2) And diluting the triiodothyronine by using a calibrator diluent to prepare the triiodothyronine calibrator with different concentrations.
5. The method for using the total triiodothyronine quantitative detection kit according to claim 1, characterized in that: not used for disease diagnosis and treatment purposes, comprising the steps of (1) loading: taking out a certain amount of coating holes numbered on the coating plate, sequentially adding 50 mu l of calibrator and sample to be tested by using a micropipette, and then adding 50 mu l of enzyme conjugate;
(2) Incubation: placing the coated plate after sample addition at 37 ℃ for 60 minutes;
(3) Washing: throwing away the liquid in the coating holes, filling the coating holes with diluted washing liquid, standing for 20 seconds, throwing away, repeatedly washing for 5 times, throwing away the liquid each time, and finally beating on water absorbing paper;
(4) And (3) color development: each coating Kong Jiade material liquid is 100 μl, and the reaction is carried out at 18-23 ℃ in a dark place for 20 minutes;
(5) And (5) terminating: and adding 50 mu l of stop solution into each coating hole, and stopping the reaction.
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| CN202010205997.XA CN113493514B (en) | 2020-03-20 | 2020-03-20 | Enzyme conjugate of anti-triiodothyronine monoclonal antibody, total triiodothyronine quantitative detection kit and use method thereof |
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| CN202010205997.XA CN113493514B (en) | 2020-03-20 | 2020-03-20 | Enzyme conjugate of anti-triiodothyronine monoclonal antibody, total triiodothyronine quantitative detection kit and use method thereof |
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