CN113495140A - Helicobacter pylori IgG antibody quantitative kit and use method thereof - Google Patents
Helicobacter pylori IgG antibody quantitative kit and use method thereof Download PDFInfo
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- CN113495140A CN113495140A CN202010205986.1A CN202010205986A CN113495140A CN 113495140 A CN113495140 A CN 113495140A CN 202010205986 A CN202010205986 A CN 202010205986A CN 113495140 A CN113495140 A CN 113495140A
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- helicobacter pylori
- enzyme
- buffer solution
- igg antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
Abstract
The invention provides a helicobacter pylori IgG antibody quantitative kit and a using method thereof. A quantitative reagent kit for the pylorus spirobacterium IgG antibody is composed of a coated plate for coating the antigen of pylorus spirobacterium, an enzyme conjugate containing enzyme for labelling mouse, a reaction liquid and a calibrating substance of pylorus spirobacterium IgG. The kit is prepared based on an enzyme-linked immunosorbent assay, is used in combination with an enzyme-linked immunosorbent assay, and has the advantages of simple operation and high detection speed. By adding the color-changing agent into the system diluent, the design of adding the sample and changing the color improves the detection efficiency and reduces the operation error rate. Protection with casein and other protective proteins in enzyme conjugate dilutions increases the reagent lifetime.
Description
Technical Field
The invention relates to the technical field of immunological detection, in particular to a helicobacter pylori IgG antibody quantitative kit and a using method thereof. The invention also relates to a system reaction liquid for preventing the sample from being added in a missing way or in a wrong way, which is used in the method and is realized by the color change effect of the system reaction liquid after sample addition because the sample adding amount is extremely low.
Background
Helicobacter pylori is a spiral, micro-anaerobic, very demanding bacterium for growing conditions and is currently the only microorganism species known to be able to survive in the human stomach. The worldwide health organization international cancer research institution publishes the list of carcinogens for preliminary reference, helicobacter pylori (infection) is in a list of carcinogens, and the poor prognosis of helicobacter pylori disease is gastric cancer. There are many methods for detecting helicobacter pylori infection, such as biopsy, isolated culture of helicobacter pylori, rapid urease test, urea breath test, urinary ammonia excretion test, polymerase chain reaction, and the like. The method has high detection cost and long detection time.
After the helicobacter pylori is infected, the immune system of a human body can generate corresponding IgA, IgM and IgG antibodies, and whether markers exist or not can be detected through peripheral blood serum, so that whether infection exists or not can be inferred. Serological tests have become the method of choice for the detection of H.pylori infection because of their ease of handling, short detection times and low detection costs compared to more invasive diagnostic tests. Serological positive detection indicates the presence of H.pylori antibodies, confirming both the possibility of past infection and the potential of present infection. The existing products in the market are all colloidal gold qualitative detection reagents, and the sensitivity and specificity of the reagents are limited by a detection platform.
Disclosure of Invention
In order to solve the problems, the invention provides a helicobacter pylori IgG antibody quantitative kit and a using method thereof.
The object of the invention is achieved in the following way: a quantitative reagent kit for the pylorus spirobacterium IgG antibody is composed of a coated plate for coating the antigen of pylorus spirobacterium, an enzyme conjugate containing enzyme for labelling mouse, a reaction liquid and a calibrating substance of pylorus spirobacterium IgG.
The system reaction liquid comprises buffer solution, protective protein, preservative, color developing agent and enzyme protective agent.
The buffer solution is Tris-NaCl buffer solution or PBS buffer solution, the protective protein is casein, the preservative is at least one of Proclin 300, NaN3 and Kathon, the color developing agent is bromocresol purple, and the enzyme protective agent is Tween 20.
The enzyme conjugate diluent in the enzyme conjugate comprises a buffer solution, a protective protein and a preservative, wherein the buffer solution is Tris-NaCl buffer solution or PBS buffer solution, the protective protein comprises at least two proteins, one protein is Casein, the other protein is at least one of calf serum, bovine serum albumin and Casein, and the preservative is at least one of Proclin 300, NaN3 and Kathon.
The enzyme-labeled mouse anti-human antibody is mixed with the enzyme conjugate diluent according to the volume ratio of 1 (4800-5200) to obtain the enzyme conjugate.
The HRP-labeled mouse anti-human enzyme conjugate is an enzyme HRP-labeled mouse anti-human enzyme conjugate.
The diluent of the helicobacter pylori IgG calibration product comprises a HEPES buffer solution, protective protein, nystatin and a preservative; the protective protein is at least one of calf serum, bovine serum albumin and Casein, and the preservative is at least one of Proclin 300, NaN3 and Kathon.
The helicobacter pylori IgG calibrator comprises helicobacter pylori IgG with the concentration of 0U/ml, 10U/ml, 25U/ml, 50U/ml, 100U/ml and 150U/ml respectively.
The application method of the helicobacter pylori IgG antibody quantitative kit,
comprises the following steps of (a) carrying out,
(1) sample adding: taking out a certain amount of numbered coating holes on the coating plate, sequentially adding 50 mul of calibrator and sample to be tested, then respectively adding 50 mul of system reaction solution, and then adding 50 mul of enzyme conjugate;
(2) and (3) incubation: placing the coated plate after sample adding at the ambient temperature of 37 ℃ for 60 minutes;
(3) washing: throwing off liquid in the coating holes, filling the coating holes with diluted washing liquid, standing for 20 seconds, throwing off, repeatedly washing for 5 times in such a way, throwing off the liquid each time, and finally patting on absorbent paper to be dry;
(4) color development: adding 100 mul of substrate solution into each coating hole, and reacting for 20 minutes at 18-23 ℃ in a dark place;
(5) and (4) terminating: the reaction was stopped by adding 50. mu.l of a stop solution to each coated well.
The kit is prepared based on an enzyme-linked immunosorbent assay, is used in combination with an enzyme-linked immunosorbent assay, and has the advantages of simple operation and high detection speed. By adding the color-changing agent into the system diluent, the design of adding the sample and changing the color improves the detection efficiency and reduces the operation error rate. Protection with casein and other protective proteins in enzyme conjugate dilutions increases the reagent lifetime.
The action principle is as follows:
the method uses the gene recombinant antigens of the helicobacter pylori structural region and the non-structural region and the horseradish peroxidase-labeled anti-human IgG as main raw materials, and applies the indirect enzyme-linked immunosorbent assay principle to detect the helicobacter pylori IgG antibody contained in a serum or plasma sample. Preparing a coating plate by using a helicobacter pylori antigen, adding a helicobacter pylori IgG antibody contained in a sample to be detected into the coating plate to be combined with the sample, adding horseradish peroxidase-labeled anti-human IgG, and finally forming an antigen-antibody-enzyme-labeled antibody compound through immunoreaction, wherein the compound catalyzes a luminescent substrate to develop color, and the color development absorbance intensity is in direct proportion to the content of the helicobacter pylori antibody.
Drawings
FIG. 1 is a regression plot.
FIG. 2 is a reference diagram showing the correlation between the kit of the present application and the existing product.
Detailed Description
A quantitative reagent kit for the pylorus spirobacterium IgG antibody is composed of a coated plate for coating the antigen of pylorus spirobacterium, an enzyme conjugate containing enzyme for labelling mouse, a reaction liquid and a calibrating substance of pylorus spirobacterium IgG.
The system reaction liquid comprises buffer solution, protective protein, preservative, color developing agent and enzyme protective agent.
The buffer solution is Tris-NaCl buffer solution or PBS buffer solution, the protective protein is casein, the preservative is at least one of Proclin 300, NaN3 and Kathon, the color developing agent is bromocresol purple, and the enzyme protective agent is Tween 20.
The enzyme conjugate diluent in the enzyme conjugate comprises a buffer solution, a protective protein and a preservative, wherein the buffer solution is Tris-NaCl buffer solution or PBS buffer solution, the protective protein comprises at least two proteins, one protein is Casein, the other protein is at least one of calf serum, bovine serum albumin and Casein, and the preservative is at least one of Proclin 300, NaN3 and Kathon.
The enzyme-labeled mouse anti-human antibody is mixed with the enzyme conjugate diluent according to the volume ratio of 1 (4800-5200) to obtain the enzyme conjugate.
The HRP-labeled mouse anti-human enzyme conjugate is an enzyme HRP-labeled mouse anti-human enzyme conjugate.
The diluent of the helicobacter pylori IgG calibration product comprises a HEPES buffer solution, protective protein, nystatin and a preservative; the protective protein is at least one of calf serum, bovine serum albumin and Casein, and the preservative is at least one of Proclin 300, NaN3 and Kathon.
The helicobacter pylori IgG calibrator comprises helicobacter pylori IgG with the concentration of 0U/ml, 10U/ml, 25U/ml, 50U/ml, 100U/ml and 150U/ml respectively.
The application method of the helicobacter pylori IgG antibody quantitative kit,
comprises the following steps of (a) carrying out,
(1) sample adding: taking out a certain amount of numbered coating holes on the coating plate, sequentially adding 50 mul of calibrator and sample to be tested, then respectively adding 50 mul of system reaction solution, and then adding 50 mul of enzyme conjugate;
(2) and (3) incubation: placing the coated plate after sample adding at the ambient temperature of 37 ℃ for 60 minutes;
(3) washing: throwing off liquid in the coating holes, filling the coating holes with diluted washing liquid, standing for 20 seconds, throwing off, repeatedly washing for 5 times in such a way, throwing off the liquid each time, and finally patting on absorbent paper to be dry;
(4) color development: adding 100 mul of substrate solution into each coating hole, and reacting for 20 minutes at 18-23 ℃ in a dark place;
(5) and (4) terminating: the reaction was stopped by adding 50. mu.l of a stop solution to each coated well. The present invention is described in more detail below with reference to specific examples to facilitate understanding by those skilled in the art. If not specifically stated, the materials and the detecting instruments used in the invention are conventional materials in the industry, and are common products sold in the market. The detection method used is a conventional method used in the industry.
Quantitative detection kit for preparing helicobacter pylori IgG
1. Preparation of coated plate
Adding helicobacter pylori antigen (purchased from Hangzhou Hongtong biology, model number HP33) into 0.05M PBS buffer solution with pH of 7.4 according to the concentration of 1ug/ml to prepare coating solution, and mixing at 18-23 deg.C. .
② adding the prepared coating solution into a blank enzyme label plate (purchased from Xiamen Yunpeng biology) according to 100 mul/hole, and placing for 16-24hr at 2-8 ℃ in a refrigerator.
And thirdly, throwing away the coating liquid in the ELISA plate, and washing the plate for 2 times by using washing liquid after patting the plate dry.
And fourthly, sealing within 2 minutes after the plate washing, adding 1% peptone water of sealing liquid according to 150 mul/hole, and placing for 16-24 hours at the temperature of 2-8 ℃ in a refrigerator.
Fifthly, throwing the confining liquid of the ELISA plate out, and then placing the ELISA plate in a drying oven for drying (the humidity is less than or equal to 30%) for 4-8 hr.
The preparation is finished.
2. Preparation of reaction solution of the inventive System
Preparing tris-NaCl buffer solution, and mixing the raw materials in the following table 1 to prepare tris-NaCl aqueous solution;
TABLE 1
| Name of article | Raw material requirements | Content (wt.) |
| Purified water | Analytical purity | 988ml |
| Tris | Analytical purity | 3.03g |
| NaCl | Analytical purity | 8.2g |
Then, pH was adjusted to 6.0 using 6M hydrochloric acid to prepare a tris-NaCl buffer.
Second step of preparing reaction solution of system
The raw materials in the following table 2 were mixed to prepare the system reaction solution.
TABLE 2
| Name of article | Raw material requirements | Content (wt.) |
| tris-NaCl buffer | 988ml | |
| Casein protein | Analytical purity | 10.00 |
| Tween | ||
| 20 | Analytical purity | 1.04g |
| Bromocresol purple | Analytical purity | 0.020g |
| Proclin300 | Analytical purity | 1ml |
3. Preparation of enzyme conjugates of the invention the first step of preparation of a tris-NaCl buffer
Preparing tris-NaCl buffer solution, and mixing the raw materials in the following table 3 to prepare tris-NaCl aqueous solution;
TABLE 3
| Name of article | Raw material requirements | Content (wt.) |
| Purified water | Analytical purity | 988ml |
| Tris | Analytical purity | 6.06g |
| NaCl | Analytical purity | 8.2g |
Then, pH was adjusted to 7.4 using 6M hydrochloric acid to prepare a tris-NaCl buffer.
Second step preparing enzyme diluent
Each liter of enzyme conjugate diluent was formulated using the raw material mix in table 4 below.
TABLE 4
The prepared enzyme conjugate diluent and an HRP-labeled mouse anti-human antibody (purchased from Hangzhou Boyue biology, model M1006) are mixed according to the weight ratio of 5000: 1 volume ratio were mixed to prepare an enzyme conjugate.
4. Preparation of the calibrator of the invention
Firstly, preparing HEPES buffer solution, and mixing the raw materials in the following table 5 to prepare HEPES solution;
TABLE 5
| Name of article | Content (wt.) | Raw material requirements |
| Purified water | 993ml | Analytical purity |
| HEPES | 6.5g | Analytical purity |
| Nacl | 8.0g | Analytical purity |
Then, the pH value of the HEPES solution is adjusted to 7.2 by using 1M sodium hydroxide to prepare a HEPES buffer solution;
preparing a calibrator diluent by mixing the raw materials in the following table 6;
TABLE 6
| Name of article | Content (wt.) | Raw material requirements |
| HEPES buffer solution | 983ml | |
| BSA | 30g | Analytical purity |
| Nystatin | 0.05g | |
| Proclin300 | 1ml | Analytical purity |
A sample (Boyue organism H1045) containing high-value helicobacter pylori IgG is diluted by the diluent to prepare six concentrations of calibrators of 0U/ml, 10U/ml, 25U/ml, 50U/ml, 100U/ml and 150U/ml respectively. The manufacturer and model of nystatin are respectively source leaf organism-S17029.
4. Other general reagent components: the substrate, stop solution and washing solution are purchased from Zhengzhou Danuo organisms.
5. Procedure of the test
(I) [ main Components ] are shown in Table 7
TABLE 7
(II) [ inspection method ]
Preparation before experiment
1. All reagents and samples were returned to room temperature at 18-25 ℃ for at least 30 minutes.
2. The thermostat or water bath was adjusted to a reaction temperature of 37 ℃.
3. Each bottle of washing solution was diluted with 1000ml of purified water and shaken up for use.
Experimental procedure
1. Sample adding: taking out a certain amount of the numbered coating holes on the coating plate. Mu.l of calibrator and sample to be tested were added in sequence by a micropipette, and then 50. mu.l of enzyme conjugate was added.
2. And (3) incubation: the coated plate after loading was placed at 37 ℃ for 60 minutes.
3. Washing: throwing off liquid in the coating holes, filling the coating holes with diluted washing liquid, standing for 20 seconds, throwing off, washing for 5 times repeatedly (throwing off liquid every time), and finally patting on absorbent paper to be dry.
4. Color development: adding 100 mul of substrate solution into each coating hole, and reacting for 20 minutes at room temperature of 18-23 ℃ in the dark.
5. And (4) terminating: the reaction was stopped by adding 50. mu.l of a stop solution to each coated well.
(III) calculation of results
(1) Detection by a microplate reader: the microplate reader wavelength is 450nm, and the reference wavelength is 630 nm. The OD of each well was measured within 10 minutes after the termination of the reaction.
(2) Calculating: the kit recommends a point-to-point regression fitting mode, namely, a point-to-point standard curve is established by taking the concentration value of a series of calibration substances as an abscissa (X axis) and the OD value of a standard substance as an ordinate (Y axis), the content of 25 hydroxy vitamin in a sample to be detected is calculated, and the sample is judged to be positive if the content is more than 20U/ml. Exemplary data are calculated as in table 8 below, and a point-to-point standard curve, such as the curve described above, is created with reference to fig. 1.
TABLE 8
| Name of article | Concentration (U/ml) | OD |
| Standard substance | ||
| 1 | 0 | 0.012 |
| |
10 | 0.237 |
| |
25 | 0.668 |
| Standard substance 4 | 50 | 1.253 |
| Standard substance 5 | 100 | 1.873 |
| Standard substance 6 | 150 | 2.893 |
6. Clinical performance evaluation and invention advantages of the kit
177 physical examination population samples are examined in parallel by an enzyme-linked immunosorbent assay (ELISA) method by adopting the kit and a helicobacter pylori IgG detection kit produced by Monobind (Mb) company produced by roche company, the concentration range is 0.5-140U/ml, the detection results are analyzed in clinical relevance, the detection results are shown in the following table 9, and a relevance reference graph is prepared according to data in the table and is shown in the figure 2.
TABLE 9
As can be seen from the correlation reference chart of FIG. 2, the kit prepared by the invention has good correlation with the existing products, so that the quantitative detection kit provided by the invention supplements the detection method for helicobacter pylori, makes up the defects of timeliness and cost of the existing detection kit on the market, and simultaneously keeps good detection effect. By adding the color-changing agent into the system diluent, the design of adding the sample and changing the color improves the detection efficiency and reduces the operation error rate.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the overall concept of the present invention, and these should also be considered as the protection scope of the present invention.
Claims (9)
1. A helicobacter pylori IgG antibody quantitative kit is characterized in that: comprises an envelope plate for enveloping helicobacter pylori antigens, four components of enzyme conjugate containing enzyme-labeled mouse antihuman, system reaction liquid and helicobacter pylori IgG calibration products.
2. The helicobacter pylori IgG antibody quantification kit according to claim 1, wherein: the system reaction liquid comprises buffer solution, protective protein, preservative, color developing agent and enzyme protective agent.
3. The helicobacter pylori IgG antibody quantification kit according to claim 2, wherein: the buffer solution is Tris-NaCl buffer solution or PBS buffer solution, the protective protein is casein, the preservative is at least one of Proclin 300, NaN3 and Kathon, the color developing agent is bromocresol purple, and the enzyme protective agent is Tween 20.
4. The helicobacter pylori IgG antibody quantification kit according to claim 1, wherein: the enzyme conjugate diluent in the enzyme conjugate comprises a buffer solution, a protective protein and a preservative, wherein the buffer solution is Tris-NaCl buffer solution or PBS buffer solution, the protective protein comprises at least two proteins, one protein is Casein, the other protein is at least one of calf serum, bovine serum albumin and Casein, and the preservative is at least one of Proclin 300, NaN3 and Kathon.
5. The helicobacter pylori IgG antibody quantification kit according to claim 1, wherein: the enzyme-labeled mouse anti-human antibody is mixed with the enzyme conjugate diluent according to the volume ratio of 1 (4800-5200) to obtain the enzyme conjugate.
6. The helicobacter pylori IgG antibody quantification kit according to claim 1, wherein: the HRP-labeled mouse anti-human enzyme conjugate is an enzyme HRP-labeled mouse anti-human enzyme conjugate.
7. The helicobacter pylori IgG antibody quantification kit according to claim 1, wherein: the diluent of the helicobacter pylori IgG calibration product comprises a HEPES buffer solution, protective protein, nystatin and a preservative; the protective protein is at least one of calf serum, bovine serum albumin and Casein, and the preservative is at least one of Proclin 300, NaN3 and Kathon.
8. The helicobacter pylori IgG antibody quantification kit according to claim 1, wherein: the helicobacter pylori IgG calibrator comprises helicobacter pylori IgG with the concentration of 0U/ml, 10U/ml, 25U/ml, 50U/ml, 100U/ml and 150U/ml respectively.
9. The use of the kit for the quantification of H.pylori IgG antibodies according to any one of claims 1 to 8, wherein:
comprises the following steps of (a) carrying out,
(1) sample adding: taking out a certain amount of numbered coating holes on the coating plate, sequentially adding 50 mul of calibration material and a sample to be detected, then respectively adding 50 mul of system reaction liquid, and then adding 50 mul of enzyme conjugate;
(2) and (3) incubation: placing the coated plate after sample adding at the ambient temperature of 37 ℃ for 60 minutes;
(3) washing: throwing off liquid in the coating holes, filling the coating holes with diluted washing liquid, standing for 20 seconds, throwing off, repeatedly washing for 5 times in such a way, throwing off the liquid each time, and finally patting on absorbent paper to be dry;
(4) color development: adding 100 mul of substrate solution into each coating hole, and carrying out a light-shielding reaction for 20 minutes at 18-23 ℃;
(5) and (4) terminating: adding 50 mu l of stop solution into each coating hole to stop the reaction.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102175849A (en) * | 2010-12-22 | 2011-09-07 | 江苏出入境检验检疫局动植物与食品检测中心 | Kit for quickly detecting swine fever antibody and preparation method thereof |
| CN105445463A (en) * | 2014-09-25 | 2016-03-30 | 苏州新波生物技术有限公司 | Hepatitis E virus IgG antibody detection kit and preparation method and application thereof |
| CN106802345A (en) * | 2017-01-13 | 2017-06-06 | 广州华弘生物科技有限公司 | A kind of helicobacter pylori IgG antibody enzyme-linked immunologic diagnosis kit |
| CN108226494A (en) * | 2018-01-17 | 2018-06-29 | 郑州中道生物技术有限公司 | Porcine reproductive and respiratory syndrome virus ELISA antibody assay kits |
-
2020
- 2020-03-20 CN CN202010205986.1A patent/CN113495140A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102175849A (en) * | 2010-12-22 | 2011-09-07 | 江苏出入境检验检疫局动植物与食品检测中心 | Kit for quickly detecting swine fever antibody and preparation method thereof |
| CN105445463A (en) * | 2014-09-25 | 2016-03-30 | 苏州新波生物技术有限公司 | Hepatitis E virus IgG antibody detection kit and preparation method and application thereof |
| CN106802345A (en) * | 2017-01-13 | 2017-06-06 | 广州华弘生物科技有限公司 | A kind of helicobacter pylori IgG antibody enzyme-linked immunologic diagnosis kit |
| CN108226494A (en) * | 2018-01-17 | 2018-06-29 | 郑州中道生物技术有限公司 | Porcine reproductive and respiratory syndrome virus ELISA antibody assay kits |
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