ELISA kit based on CHR detection

Abstract

The utility model relates to an enzyme-linked immunosorbent assay (ELISA) kit based on CHR detection. The ELISA kit comprises a kit body, an elisa plate is mounted on one side in the kit body; micropores are formed in the elisa plate; a coating liquid container, a confining liquid container, a washing liquid and a detection antibody are arranged on the other side of the combination body; a substrate developing solution; and a signal detector. The double-antibody sandwich ELISA has the advantages that the double-antibody sandwich ELISA has the advantages of high specificity and sensitivity, and is suitable for detection of macromolecular antigens which have a plurality of epitopes and can be recognized by two antibodies at the same time. Two antibodies are used, one antibody is used as a capture antibody fixed antigen, and the other antibody is used as a detection antibody to generate a signal, so that the detection accuracy and repeatability can be remarkably improved, a more accurate sepsis biomarker monitoring means can be provided by accurately monitoring the CHR level, a doctor can be helped to formulate a more effective treatment scheme, and the clinical application prospect is wide. Therefore, the treatment effect and prognosis of the sepsis patient are improved.

Description

Technical Field

[0001] The utility model relates to the technical field of medical devices, in particular to an ELISA kit based on CHR detection. Background Art

[0002] Sepsis, a severe multi-organ dysfunction syndrome, causes millions of deaths worldwide each year and is a major challenge in the medical field. Its complex pathogenesis, including systemic inflammatory response, immune dysfunction and abnormal coagulation function, makes sepsis difficult to diagnose and treat effectively. Currently, the diagnosis of sepsis mainly relies on traditional biomarkers such as C-reactive protein (CRP) and procalcitonin (PCT). However, these markers have obvious deficiencies in sensitivity and specificity, and it is difficult to meet the clinical needs for early diagnosis and accurate monitoring.

[0003] In recent years, CHR (chromofungin), a peptide derived from chromaffin A (CgA), has been considered as a new potential biomarker for sepsis detection due to its effects on cardiovascular protection, anti-inflammation, immunomodulation and regulation of vascular endothelial function. These physiological and pathological effects of CHR, especially its ability to reduce cardiac ischemia / reperfusion injury, inhibit proinflammatory macrophage function and stabilize vascular endothelium, demonstrate its important value in the early diagnosis of sepsis, disease monitoring and treatment effect evaluation.

[0004] Therefore, the development of an ELISA kit based on CHR detection is not only an important supplement to the existing sepsis diagnostic technology, but also an innovation in medical research. This kit can provide a more accurate means of monitoring sepsis biomarkers, which can help doctors develop more effective treatment plans, thereby improving the treatment effect and prognosis of sepsis patients. By accurately monitoring CHR levels, this ELISA kit demonstrates its necessity, innovation and practicality in the early diagnosis and disease management of sepsis, and provides a new direction and strategy for the treatment of sepsis.

[0005] Chinese patent document 201420625757.5, application date 2014-10-27, patent name: ELISA kit, discloses an ELISA kit, the technical scheme includes a box body, and an ELISA plate, a standard tube, an antibody tube, a diluent bottle and a substrate bottle in the box body, the ELISA plate includes a plate body and a plate hole on the plate body. The bottom of the plate hole is coated with a capture antibody, and a filter element is inserted at the upper opening, the filter element includes a filter cover and a filter membrane, the filter cover is provided with a membrane hole, and the membrane hole is provided with a filter membrane.

[0006] An ELISA kit in the above patent document solves the problem that the operation of the ELISA kit is complicated, time-consuming and labor-intensive when adding samples to the ELISA plate. However, there is no corresponding disclosure of an ELISA kit that can provide a more accurate means of monitoring sepsis biomarkers by accurately monitoring CHR levels, which helps doctors formulate more effective treatment plans, thereby improving the treatment effect and prognosis of sepsis patients.

[0007] In summary, there is an urgent need for an ELISA kit that can provide a more accurate means of monitoring sepsis biomarkers by accurately monitoring CHR levels, which can help doctors formulate more effective treatment plans, thereby improving the treatment effect and prognosis of sepsis patients. However, there is no report on such an ELISA kit. Summary of the invention

[0008] The purpose of the utility model is to provide an ELISA kit that can provide a more accurate means of monitoring sepsis biomarkers by accurately monitoring CHR levels, which helps doctors formulate more effective treatment plans, thereby improving the treatment effect and prognosis of sepsis patients.

[0009] In order to achieve the above purpose, the technical solution adopted by the utility model is:

[0010] The utility model has the advantages that:

[0011] 1. The utility model is an ELISA kit based on CHR detection, which uses a double antibody sandwich format. The double antibody sandwich ELISA has the advantages of high specificity and sensitivity and is suitable for the detection of macromolecular antigens with multiple epitopes that can be recognized by two antibodies at the same time. By using two antibodies, one as a capture antibody to fix the antigen and the other as a detection antibody to generate a signal, the accuracy and repeatability of the detection can be significantly improved. By accurately monitoring the CHR level, it can provide a more accurate means of monitoring sepsis biomarkers, which helps doctors develop more effective treatment plans, thereby improving the treatment effect and prognosis of sepsis patients.

[0012] 2. The ELISA plate is equipped with a covering film to prevent external contaminants from entering the microwells and affecting the measurement results. There are 96 microwells on the ELISA plate, which is convenient for simultaneous control experiments, such as establishing a blank control group and a standard control group, to achieve the reliability of data obtained by comparison.

[0013] 3. A blocking solution is provided to block the sites on the ELISA plate that are not occupied by the first antibody, to prevent nonspecific binding and to improve the accuracy of data measurement. An enzyme-containing marker is provided in the second antibody to facilitate detection by the signal detector.

[0014] 4. A buffer solution is provided to wash away unbound substances and reduce background signals; a diluent is provided to dilute the sample to be tested and the standard so that its concentration falls within the detection range; a substrate solution, a stop solution, and a substrate color developing solution are provided to make the labeled enzyme produce color changes for easy reading by an enzyme reader; the stop solution is used to stop the substrate reaction to ensure the accuracy of the measurement. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Attached Figure 1 It is a structural schematic diagram of an ELISA kit based on CHR detection of the utility model.

[0016] Attached Figure 2 It is a partial plan view of the test kit.

[0017] Attached Figure 3 It is a structural diagram of a sandwich structure.

[0018] Attached Figure 4 This is a schematic diagram of the structure of the ELISA plate.

[0019] Attached Figure 5 The utility model is a flowchart of an ELISA kit based on CHR detection. DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with the embodiments and with reference to the accompanying drawings.

[0021] The reference numerals and components involved in the drawings are as follows:

[0022] 1. Box 2. ELISA plate

[0023] 21. Microwell 3. Coating solution

[0024] 4. Blocking solution 41. Primary antibody

[0025] 5. Washing solution 6. Detection antibody

[0026] 7. Substrate color development solution 8. Signal detector

[0027] 9. Target antigen 10. Microplate reader

[0028] 11. Sandwich structure

[0029] In order to make the purpose, technical solution and advantages of the embodiment of the utility model clearer, the technical solution in the embodiment of the utility model will be clearly and completely described below in conjunction with the drawings in the embodiment of the utility model. Obviously, the described embodiment is a part of the embodiment of the utility model, not all of the embodiments. Based on the embodiment of the utility model, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the utility model.

[0030] To facilitate the understanding of the embodiments of the present utility model, the following will further explain and illustrate specific embodiments in conjunction with the accompanying drawings. The embodiments do not constitute a limitation on the embodiments of the present utility model.

[0031] In the description of the embodiments of the present utility model, it should be noted that the terms "first", "second", "one end", "the other end", "upper top surface", "lower bottom surface", etc. indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present utility model and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present utility model; in addition, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be an indirect connection through an intermediate medium, or it can be a connection between the two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present utility model can be understood according to specific circumstances.

[0032] Example 1

[0033] Please refer to Figure 1 and Figure 2 , Figure 1 It is a structural schematic diagram of an ELISA kit based on CHR detection of the utility model. Figure 2It is a partial plan view of the test kit. An ELISA test kit based on CHR detection; the ELISA test kit comprises a box body 1; an ELISA plate 2 is installed on one side of the box body 1; the ELISA plate 2 is provided with micropores 21; a coating liquid 3, a blocking liquid 4, a washing liquid 5, a detection antibody 6 are provided on the other side of the combination; a substrate color developing liquid 7, and a signal detector 8. The target antigen 9 is added to the micropore 21, and the coating liquid 3 contains a first antibody 41. After the coating liquid 3 is injected into the micropore 21, the first antibody 41 is combined with the target antigen 9; and the blocking liquid 4 is added for blocking, and then the washing liquid 5 is added to remove the substances that are not combined between the first antibody 41 and the target antigen 9; and then the detection antibody 6 is added, and the detection antibody 6 is a second antibody labeled with an enzyme, and the second antibody forms a sandwich structure 11 with the target antigen 9 and the first antibody 41 (see Figure 3 ); then the sandwich structure 11 is washed again to remove the unbound detection antibody 6, and a substrate color development solution is added, and finally the signal intensity is measured by the signal detector 8 to quantitatively analyze the content of the target antigen 9.

[0034] The coating solution 3 is a CHR monoclonal antibody, that is, a monoclonal antibody developed according to the CHR sequence, which is the first antibody 41 used to capture the target antigen 9 and needs to be diluted to a certain concentration according to experimental requirements.

[0035] The blocking solution 4 contains 1% BSA or 5% skim milk in 10mM PBST, which is used to block the sites on the ELISA plate 2 that are not occupied by the first antibody 41 to prevent non-specific binding; add 100ul / well of the blocking solution 4 and incubate at 4°C overnight;

[0036] The washing solution 5 is PBST washing buffer, specifically: 10 mM phosphate buffered saline solution containing Tween-20, which is used to wash away unbound substances.

[0037] The detection antibody 6 is a detection antibody 6 (secondary antibody) labeled with an enzyme, and these antibodies also specifically recognize the target antigen 9.

[0038] The enzyme labeled with the substrate color developing solution 7 produces a color change, so as to be read by an ELISA reader 10 .

[0039] The signal detector 8 is an ELISA instrument 10 .

[0040] Please refer to Figure 4 , Figure 4 Schematic diagram of the structure of the ELISA plate 2. The ELISA plate 2 has 96 microwells 21 in total, which are specifically distributed as follows: 12 microwells 21 in each row, and 8 rows in total; the ELISA plate 2 is also equipped with a covering film.

[0041] What needs to be explained in this embodiment is:

[0042] The first antibody 41 is a CHR monoclonal antibody; the second antibody is labeled with an enzyme-containing marker; and the signal detector 8 is an ELISA reader 10. The effect of this design is that since CHR is considered to be a new potential biomarker in sepsis detection, the study of CHR monoclonal antibodies provides a new direction and strategy for the treatment of sepsis; secondly, the second antibody is labeled with an enzyme-containing marker, which has a labeling effect and is convenient for the signal detector 8 to detect.

[0043] The concentration of the first antibody 41 is 10 ug / ml. The first antibody 41 at this concentration has a strong compressive binding property with the target.

[0044] The amount of the first antibody 41 added to the microwell 21 is 100ul / microwell 21, and the first antibody 41 is used after being kept overnight at 4°C. The effect of this design is that the amount of the first antibody 41 and the use under low temperature conditions ensure that it can fully bind to the target antigen 9.

[0045] The amount of the second antibody is 100ul / microwell 21, and after use, the reaction is carried out at 37°C for 1 hour. The effect of this design is that the antibody at this amount and temperature is convenient for specifically recognizing the target antigen 9.

[0046] The second antibody needs to be diluted with washing solution 5 after working, and the dilution factor is 1: 2000. The effect of this design is that the concentration of this dilution factor falls within the detection range.

[0047] The blocking solution 4 is used in an amount of 350-400 μl / well 21 and is used overnight at 37° C. The effect of this design is that the blocking solution 4 under this amount and temperature treatment is used to block the sites on the ELISA plate 2 that are not occupied by the first antibody 41, and the effect of preventing non-specific binding is better.

[0048] In the scheme of the utility model, the first antibody 41 and the second antibody are combined on both sides of the target antigen 9 to form a sandwich structure 11. The basic principle is to use the recognition and binding ability of specific antibodies to the target antigen 9, combined with enzyme labeling technology, to achieve quantitative detection of specific proteins, hormones, pathogens and other molecules. The following are the specific principles and steps of double antibody sandwich ELISA (see Figure 5 ):

[0049] 1. Immobilized capture antibody

[0050] In each microwell 21 of the ELISA plate, a layer of highly specific capture antibodies is first immobilized. These antibodies can specifically bind to the target antigen 9 but do not participate in signal generation.

[0051] 2. Sample addition

[0052] A sample containing the target antigen 9 is added to the treated microwell 21. The target antigen 9 binds to the immobilized capture antibody in the sample.

[0053] 3. Washing

[0054] Remove unbound material and reduce nonspecific binding through washing steps, improving the specificity and sensitivity of the detection.

[0055] 4. Addition of detection antibody 6

[0056] Enzyme-labeled detection antibodies 6 (secondary antibodies) are added. These antibodies also specifically recognize the target antigen 9, but the epitope recognized by the capture antibody is different, forming a "sandwich" structure (capture antibody-antigen-detection antibody 6).

[0057] 5. Wash again

[0058] Removal of unbound detection antibody6 ensures reduction of background signal.

[0059] 7. Signal Detection

[0060] The signal intensity (such as the depth of color) is measured by an instrument such as a spectrophotometer, and the content of the target antigen 9 in the sample is quantitatively analyzed based on this.

[0061] The operation process of an ELISA kit based on CHR detection in the utility model is as follows:

[0062] 1. Coating antibody:

[0063] - Prepare CHR monoclonal antibody and dilute to a certain concentration according to experimental needs.

[0064] - Add 100 μl of coating solution 3 (containing CHR antibody) to each well of 96-well ELISA plate 2 and incubate at 37°C or 4°C overnight.

[0065] - On the next day, discard the liquid in the wells and wash the plate twice with 10mM PBST washing buffer.

[0066] 2. Closure:

[0067] - Prepare 10mM PBST containing 1% BSA or 5% skim milk as blocking solution4.

[0068] - Add 350-400 μl / well of Blocking Buffer 4 and continue to incubate at 37°C overnight.

[0069] 3. Wash the plate:

[0070] - Discard the liquid in the wells and shake dry.

[0071] - Wash the plate 4 times with 10 mM PBST wash buffer.

[0072] 4. Add sample:

[0073] - Set up blank wells, standard wells and sample wells.

[0074] - Add 100 μl of sample diluent to the blank wells and 100 μl of standard or test sample to the other wells.

[0075] - Cover or film the ELISA plate 2 and incubate at 37°C for 60-120 minutes.

[0076] 5. Wash the plate:

[0077] - Discard the liquid in the wells and shake dry.

[0078] - Wash the plate 4 times with 10 mM PBST wash buffer.

[0079] 6. Add detection antibody 6:

[0080] - Dilute the detection antibody 6 to a certain concentration with PBST according to the experimental needs.

[0081] - Add 100 μl / well of detection antibody 6 and react at 37°C for 1 hour.

[0082] 7. Wash the plate:

[0083] - - Discard the liquid in the wells and shake dry.

[0084] - Wash the plate 4 times with 10 mM PBST wash buffer.

[0085] 8. Add secondary antibody:

[0086] - Dilute the secondary antibody to a certain concentration using 10mM PBST according to the experimental needs.

[0087] - Add 100 μl / well secondary antibody and react at 37℃ for 1 hour.

[0088] 9. Wash the plate:

[0089] - Discard the liquid in the wells and shake dry.

[0090] - Wash the plate 4 times with 10 mM PBST wash buffer.

[0091] 10. Reading value of ELISA reader 10:

[0092] - Using 630 nm as the calibration wavelength, measure the absorbance (OD value) of each well in turn at 450 nm using a microplate reader 10.

[0093] - Read within 5 minutes after adding the stop solution.

[0094] 11. Result judgment:

[0095] - The OD value of each standard and sample must be subtracted from the OD value of the zero well. If duplicate wells are set, the average value should be taken.

[0096] - Use professional curve making software to perform four-parameter fitting (4-PL). According to the OD value of the sample, the corresponding fitting concentration is calculated from the standard curve, and then multiplied by the dilution factor to obtain the measured concentration of the sample.

[0097] The utility model is an ELISA kit based on CHR detection, which uses a double antibody sandwich format. The double antibody sandwich ELISA has the advantages of high specificity and sensitivity and is suitable for the detection of macromolecular antigens with multiple epitopes that can be recognized by two antibodies at the same time. By using two antibodies, one as a capture antibody to fix the antigen and the other as a detection antibody 6 to generate a signal, the accuracy and repeatability of the detection can be significantly improved.

[0098] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the principle of the present invention. These improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. An ELISA kit based on CHR detection, characterized in that: The ELISA kit comprises a box body; an ELISA plate is installed on one side of the box body; microwells are arranged on the ELISA plate; a target antigen, a first antibody, a second antibody, and a signal detector are arranged on one side of the box body; the target antigen is added to the microwell, and the first antibody and the second antibody are added to the microwell in sequence, and the first antibody and the second antibody are combined on both sides of the target antigen in sequence to form a sandwich structure; the signal detector detects the signal intensity of the sandwich structure, thereby quantitatively analyzing the content of the target antigen.

2. The ELISA kit according to claim 1, characterized in that There are 96 microwells on the ELISA plate, which are specifically distributed as follows: 12 microwells in each row, and 8 rows in total; the ELISA plate is also equipped with a covering film.

3. The ELISA kit according to claim 1, characterized in that The first antibody is a CHR monoclonal antibody; the second antibody is labeled with an enzyme-containing marker; and the signal detector is an enzyme marker.

4. The ELISA kit according to claim 1, characterized in that The ELISA kit also includes a blocking solution, a washing solution, a substrate color developing solution, and a blocking solution containing 1% BSA or 5% skim milk in 10mM PBST, which is used to block the sites on the ELISA plate not occupied by the first antibody to prevent non-specific binding; the washing solution is a PBST washing buffer, specifically: a 10mM phosphate buffered saline solution containing Tween-20, which is used to wash away unbound substances.

5. The ELISA kit according to claim 4, characterized in that The substrate color developing solution is used to produce color changes on the labeled enzyme, which is convenient for reading by an enzyme marker.

6. The ELISA kit according to claim 1, characterized in that The concentration of the primary antibody is 10 ug / ml.

7. The ELISA kit according to claim 1, characterized in that The amount of the first antibody added to the microwells was 100ul / microwell, and the first antibody was used after being incubated at 4°C overnight; the amount of the second antibody was 100ul / microwell, and the reaction was continued at 37°C for 1 hour after use.

8. The ELISA kit according to claim 1, characterized in that The second antibody needs to be diluted with washing solution after working, and the dilution multiple is 1:2000.

9. The ELISA kit according to claim 4, characterized in that The blocking solution is used in an amount of 350-400 μl / microwell and is used at 37° C. overnight.

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