CN115598344A - Detection kit for fecal pancreatic elastase 1 and preparation method thereof - Google Patents
Detection kit for fecal pancreatic elastase 1 and preparation method thereof Download PDFInfo
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- CN115598344A CN115598344A CN202211400478.4A CN202211400478A CN115598344A CN 115598344 A CN115598344 A CN 115598344A CN 202211400478 A CN202211400478 A CN 202211400478A CN 115598344 A CN115598344 A CN 115598344A
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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
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/966—Elastase
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a detection kit for fecal pancreatic elastase 1 and a preparation method thereof, belonging to the technical field of medical inspection and in-vitro diagnosis. The kit comprises sodium hexametaphosphate and potassium citrate as components of an analysis buffer solution, so that the whole reaction system is more stable, the intra-batch difference and inter-batch difference of the fecal trypsin 1 detection kit are reduced, the sensitivity of the kit is improved to a certain extent, and the kit has a better clinical application prospect.
Description
Technical Field
The invention belongs to the technical field of medical examination and in-vitro diagnosis, relates to a kit for determining pancreatic elastase 1 in human excrement, and particularly relates to a kit for determining pancreatic elastase 1 in human excrement by using a chemiluminescence method and application thereof.
Background
The Fecal pancreatic elastase 1 (Fecal elastase 1, FE-1) is a proteolytic enzyme secreted by pancreatic acinus, is closely related to pancreatic exocrine function, has been widely applied as a non-invasive index of pancreatic function, can monitor moderate and severe hyposecretion of pancreas, and therefore has clinical significance for detecting the content of the Fecal pancreatic elastase 1.
The early pancreatic exocrine function test is a gold standard for detecting pancreatic exocrine insufficiency, but the test process is complex and the cost is high, so the early pancreatic exocrine function test is basically abandoned in clinic at present. Indirect pancreatic exocrine function tests are also rarely used, with the best current method being the fecal pancreatic elastase assay. There are various methods for detecting fecal pancreatic elastase 1, such as latex-enhanced immunoturbidimetry, immunochromatography, chemiluminescence, and the like.
Chinese patent 201910850314.3 discloses that monoclonal antibodies are combined into a latex enhanced immunoturbidimetry for detecting pancreatic elastase 1 in human excrement and diagnosing chronic pancreatitis or pancreatic exocrine dysfunction, and the detection sensitivity and specificity are obviously improved by adopting a monoclonal antibody for specifically recognizing pancreatic elastase 1.
Chinese patent 201811024574.7 discloses a kit for quantitatively determining trypsin 1 in excrement, which comprises an R1 reagent, an R2 reagent and a trypsin 1 antigen calibrator solution; the R1 reagent comprises electrolyte, stabilizer, surfactant, preservative and buffer; the R2 reagent comprises latex particles coated with polyclonal antibody of anti-human trypsin 1, electrolyte, stabilizer, surfactant, preservative and buffer solution; the pancreatic elastase 1 antigen calibrator solution comprises pancreatic elastase 1 and a stabilizer. The kit is a device for determining the pancreatic elastase 1 in the excrement by using a latex enhanced immunoturbidimetry method, is stable and accurate, saves time and labor, can realize the effect of clinical detection and analysis of a full-automatic rapid determination sample, improves the detection sensitivity by more than 10 times compared with the traditional immunoturbidimetry reagent, and effectively protects an active region combining an antibody and an antigen and improves the detection sensitivity by coupling a polyclonal antibody on the surface of latex particles by a chemical crosslinking method.
However, when the prior art detects the fecal pancreatic elastase 1, the following defects are found to exist: if the quantitative determination cannot be carried out accurately, the detection result is unstable, and the like. Therefore, it is necessary to improve the method for detecting fecal pancreatic elastase 1.
Disclosure of Invention
In order to solve the problems, the invention provides a detection kit for detecting the fecal pancreatic elastase 1 by a chemiluminescence method, and a preparation method and a use method thereof, and solves the problems that the method for detecting the fecal pancreatic elastase 1 in the prior art is low in sensitivity, easy to interfere by the environment and the like.
The terms:
in the present invention, "EDC" is one of carbodiimides, i.e., 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride. Is a water-soluble carbodiimide, is used as a carboxyl activating reagent in amide synthesis, and is also used for activating phosphate groups, crosslinking proteins and nucleic acids and preparing immune conjugates. The pH range of the coupling agent is 4.0-6.0 when the coupling agent is used, and the coupling agent is usually used together with N-hydroxysuccinimide (NHS) or N-hydroxythiosuccinimide to improve the coupling efficiency. Carboxylic acids are esterified with alcohols in organic chemistry using EDC and the catalyst 4-Dimethylaminopyridine (DMAP).
In the invention, "Sulfo-NHS", namely N-hydroxy thiosuccinimide, is commonly used as a polypeptide condensing agent and can form a stable active ester intermediate; can be used for preparing hydrophilic active ester, such as protein cross-linking agent.
In the present invention, "PBS", i.e., PBS buffer, contains Na as a main component 2 HPO 4 、KH 2 PO 4 NaCl and KCl, generally act as solvents to solubilize the protective agent. Due to Na 2 HPO 4 And KH 2 PO 4 There is a second order dissociation, the buffered pH range is wide, and NaCl and KCl mainly function to increase the salt ion concentration.
In the present invention, "HEPES" means 4-hydroxyethylpiperazine ethanesulfonic acid. 4-hydroxyethylpiperazine ethanesulfonic acid is of the molecular formula C 8 H 18 N 2 O 4 Chemical of SAnd (4) quality.
In the present invention, "Triton X-100" means polyethylene glycol octylphenyl ether. The polyethylene glycol octyl phenyl ether is an organic high molecular compound with a structural formula of C 14 H 22 O(C 2 H 4 O) n 。
On one hand, the invention provides application of sodium hexametaphosphate and potassium citrate in preparation of a stool pancreatic elastase 1 detection kit.
The kit comprises luminous microspheres coupled with an anti-coproagulase 1 antibody and avidin marked photosensitive microspheres; biotin is marked on the anti-fecal pancreatic elastase 1 antibody; the kit also comprises sodium hexametaphosphate and potassium citrate.
The use concentration of the sodium hexametaphosphate is 5-10g/L; the use concentration of the potassium citrate is 1-6g/L.
Preferably, the use concentration of the sodium hexametaphosphate is 8g/L; the use concentration of the potassium citrate is 4g/L.
In another aspect, the invention provides a kit for detecting fecal pancreatic elastase 1.
The kit comprises luminous microspheres coupled with anti-fecal pancreatic elastase 1 antibodies and avidin-labeled photosensitive microspheres; biotin is marked on the anti-coproagulase elastase 1 antibody; the kit also comprises sodium hexametaphosphate and potassium citrate.
The use concentration of the sodium hexametaphosphate is 5-10g/L; the use concentration of the potassium citrate is 1-6g/L.
Preferably, the use concentration of the sodium hexametaphosphate is 5-8g/L, 6-8g/L, 5-9g/L, 6-9g/L and 8-9g/L.
Preferably, the use concentration of the potassium citrate is 1-5g/L, 1-3g/L, 1-4g/L, 3-5g/L and 4-5g/L.
In some embodiments, the sodium hexametaphosphate is used in a concentration of 5g/L; the use concentration of the potassium citrate is 5g/L.
In some embodiments, the sodium hexametaphosphate is used at a concentration of 8g/L; the use concentration of the potassium citrate is 4g/L.
The sodium hexametaphosphate and potassium citrate were used as components of the assay buffer.
The analysis buffer solution also comprises HEPES, triton X-100, casein and dextran.
The assay buffer is used for dilution of the sample and drug.
The preparation method of the luminous microsphere coupled with the anti-coproagulase elastase 1 antibody comprises the following steps: the anti-fecal trypsin 1 monoclonal antibody is mixed with the activated luminescent microspheres for reaction to couple the antibody to the microspheres, and freshly prepared carbodiimide is added while mixing the antibody and the activated microspheres.
The activation method of the luminescent microsphere comprises the following steps: the luminescent microspheres were activated in PBS buffer by using prepared carbodiimide and Sulfo-NHS solutions.
The luminous microspheres are carboxyl modified luminous microspheres, and the luminous microspheres are coated with dimethylthiophene, anthracene and rubrene and have europium chelate.
The carbodiimide is 50mg/mL.
The carbodiimide is freshly prepared.
The coupling method is shaking for 2 hours at room temperature.
The mass ratio of the luminescent microspheres to the fecal trypsin elastase 1 antibody is 10-50:1.
in the preparation step of the biotin-labeled anti-fecal trypsin 1 antibody, the molecular ratio of biotin to antibody is 10-50:1.
the molecular ratio of biotin to antibody was 30.
The photosensitive microsphere is provided with phthalocyanine dye.
The dosage ratio of the photosensitive microspheres to the avidin is 10:1.
preferably, the sample tested is a stool sample; the fecal sample is diluted with assay buffer.
The kit also comprises a positive quality control product and a negative quality control product.
On the other hand, the invention provides a preparation method of the fecal trypsin elastase 1 detection kit.
The method is used for preparing the kit.
The method comprises the following steps:
(1) Coupling an anti-fecal pancreatic elastase 1 antibody with a luminescent microsphere;
(2) Biotin-labeled anti-fecal pancreatic elastase 2 antibody;
(3) Marking photosensitive microspheres with avidin;
(4) An assay buffer was prepared containing sodium hexametaphosphate and potassium citrate.
The invention has the beneficial effects that:
the analytical buffer solution in the fecal pancreatic elastase 1 detection kit is optimized, the overall reaction system is more stable by adding the sodium hexametaphosphate and the potassium citrate, the intra-batch difference and the inter-batch difference of the fecal pancreatic elastase 1 detection kit are reduced, and the sensitivity of the kit is improved to a certain extent.
Detailed Description
The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples, unless otherwise specified, and experimental methods not specified in specific conditions in the examples, are generally commercially available according to conventional conditions, and materials, reagents, and the like used in the following examples, unless otherwise specified.
Basic Experimental example
1. The luminescent microspheres are linked with the antibody
(1) Preparing antibodies and microspheres: 0.1mg of fecal trypsin elastase 1 monoclonal antibody 1 (YuBo Bio, MAB483Hu 22) was added to the ultrafiltration tube, centrifuged for 8min, washed repeatedly 6-8 times with PBS buffer (pH 8.0), and then diluted to 2mg/mL for use. The luminescent microspheres (Shenzhen Elvingtai Biotech Co., ltd.) were resuspended at 20mg/mL with PBS buffer (pH8.0).
(2) Activated luminescent microspheres
1mg of luminescent microspheres was measured, 1mL of PBS (pH 7.4) was added, vortexed, and the suspension was added to a 1.5mL centrifuge tube, centrifuged at 12000g, carefully aspirated and the supernatant discarded. Add 100. Mu.L of washing buffer PBS (p H7.4), suspend 0.05% Tween-20, shake and sonicate followed by centrifugation at 12000g, carefully aspirate and discard the supernatant. 1mL of PBS buffer (pH 6.2) was added, followed by 10. Mu.L of freshly prepared EDC (environmental protection technology, inc., 50mg/mL, jiangsu Ren), 10. Mu.L of freshly prepared 50mg/mL Sulfo-NHS was added, and the mixture was shaken at room temperature for 20 minutes after shaking at high speed for 30 seconds. 12000g were centrifuged, carefully aspirated and the supernatant discarded. The luminescent microspheres were suspended by adding 1ml of phosphate buffer (pH 7.2) or borate buffer (pH 7.8).
(3) Antibody coupled luminescent microsphere
100 mu g of fecal trypsin elastase 1 monoclonal antibody 1 is added into 1mL of the activated luminescent microspheres, and the mixture is shaken for 2 hours at room temperature. 12000g were centrifuged, carefully aspirated and the supernatant discarded. Wash once with 500. Mu.L PBS buffer, centrifuge at 12000g, carefully aspirate and discard the supernatant. The luminescent microspheres were suspended by adding 250 μ L of PBS buffer (1% bsa, p Η 7.4), shaken at room temperature for 30 min, centrifuged at 12000g, carefully aspirated and the supernatant discarded. The luminescent microspheres were washed by adding 600 μ L of PBS buffer (0.1% BSA,0.02% Tween-20, p H7.4), centrifuged at 12000g, and the supernatant carefully aspirated and discarded. Finally, the luminescent microspheres were suspended in 150. Mu.L of PBS buffer (0.1% BSA,0.02% Tween-20, containing 0.05% Proclin-300, p H7.4) and stored at 4 ℃ in the dark until use.
2. Biotin to antibody linkage
(1) Antibody preparation: 1mg of antitoxic trypsin antibody 2 (Baiolabor, K13480) was added to the ultrafiltration tube, centrifuged, and washed repeatedly 6 times with buffer (pH 8.0) PBS or carbonate buffer, and then the antibody was diluted to 5mg/mL for use.
(2) Biotinylation: mu.L of the above antibody solution was added to 10. Mu.L of 2mg/mL biotin (in DMSO) and incubated with shaking at room temperature for 4h.
(3) Antibody washing: and ultra-filtering with an ultra-filtering tube to remove excessive biotin.
3. Avidin-labeled photosensitive microsphere
Adding 1mg of photosensitive microspheres into a centrifuge tube, adding 1.25 mu L of Tween-20 with the mass concentration of 10%, 0.1mg of avidin (avastin, A103033) and 10 mu L of sodium cyanoborohydride (0.4M), supplementing the volume to 200 mu L by using 0.1M of 2- (N-morpholine) ethanesulfonic acid MES buffer solution with the pH value of 6.0 or 0.1M Hepes buffer solution, and carrying out dark oscillation reaction at 37 ℃ for 48 hours; adding 10 mu L0.3M carboxymethyl amino semi-hydrochloride (CMO) solution with pH value of 5.0 to seal the non-binding sites, incubating for 1 hour at 37 ℃ in a dark place, centrifuging, separating to obtain the photosensitive microspheres connected with the avidin, and diluting for later use.
Example 1A kit for detecting fecal pancreatic elastase 1
Measuring the components according to the dosage:
| HEPES | 0.5g |
| Triton X-100 | 0.5mL |
| casein (Aladdin, C288596) | 0.1g |
| Dextran (Supelco, 00895) | 150mg |
| Sodium hexametaphosphate | 0.8g |
| Potassium citrate | 0.4g |
Adding 90mL of water to dissolve, adjusting pH to 7-8, and adding water to 100mL to obtain analysis buffer solution.
The components prepared in the basic experimental example were diluted:
(1) Diluting the concentration of the luminescent microspheres coupled with the fecal trypsin 1 antibody to 50 mug/mL by using an analysis buffer;
(2) Diluting the biotinylated antibody with assay buffer, adjusting the antibody concentration to 0.5mg/mL;
(3) The conjugated avidin-labeled photosensitive microspheres were diluted to 80. Mu.g/mL using assay buffer.
The detection method comprises the following steps:
0.1g fecal sample is mixed in 1mL analysis buffer, 20 μ L of each well of 96-well plate is added, 40 μ L biotinylated antibody and 40 μ L luminescent microsphere coupled with fecal pancreatic elastase 1 antibody are respectively added, 100 μ L of avidin-coupled photosensitive microsphere is added after 0.5 hour incubation in dark at room temperature, the machine is read after 0.5 hour incubation in dark at room temperature, the standard curve of the kit is measured by using the conventional method of standard (fecal pancreatic elastase, HZbscience, ZY483Hu 013), and the detection result is calculated according to the curve.
Example 2 kit accuracy test
The control samples were each measured using the kit provided in example 1, and the concentration of fecal pancreatic elastase (HZbscience, ZY483Hu 013) in the control samples was: the CV values of the quality control test values were calculated by a conventional method using 20 duplicate wells for each of the low value (1. Mu.g/mL), the median value (10. Mu.g/mL), and the high value (30. Mu.g/mL), and the results were as follows:
| in-batch CV/%) | Inter-batch CV/% | |
| Low value quality control product | 1.35 | 1.83 |
| Median quality control product | 0.92 | 1.26 |
| High-value quality control product | 1.49 | 2.17 |
The above results show that the kit provided in example 1 has better accuracy.
Example 3 interference assay with kit
The kit of detection example 1 detects the accuracy of the specimen in the presence of interfering substances (hemolysis, hyperlipidemia, hyperbilirubin).
Referring to the test method of example 1, hemoglobin solution, triglyceride solution, and bilirubin solution were added at 1mg/mL in the sample test.
The results show that the kit of example 1 is not interfered by hemoglobin, triglyceride and bilirubin when detecting a fecal sample.
EXAMPLE 4 sample testing
Stool samples of 10 healthy volunteers were tested using the kit of example 1, and the test calculation results were: 238.9mg/g, 351.4mg/g, 329.7mg/g, 288.4mg/g, 413.9mg/g, 257.1mg/g, 262.3mg/g, 248.6mg/g, 277.8mg/g and 316.4mg/g, which all meet the standard of healthy people.
Example 6
The difference from example 1 is that: the dosage of the sodium hexametaphosphate is 0.5g; the amount of potassium citrate is 0.5g. And (3) accuracy detection results:
| in-batch CV/%) | Inter-batch CV/%) | |
| Low value quality control product | 1.42 | 1.57 |
| Median quality control product | 1.04 | 2.25 |
| High-value quality control product | 1.17 | 1.82 |
Example 7
The difference from example 1 is that: the dosage of the sodium hexametaphosphate is 0.8g; the amount of potassium citrate is 0.6g.
Example 8
The difference from example 1 is that: the dosage of the sodium hexametaphosphate is 1.0g; the amount of potassium citrate is 0.4g.
Example 9
The difference from example 1 is that: the using amount of the sodium hexametaphosphate is 0.6g; the amount of potassium citrate is 0.3g.
Comparative example
A comparative example was set up with reference to example 1, as follows:
| comparative example | Differences from example 1 |
| Comparative example 1 | Sodium hexametaphosphate was not included in the assay buffer |
| Comparative example 2 | Potassium citrate was not included in the assay buffer |
| Comparative example 3 | Sodium hexametaphosphate and potassium citrate were not included in the assay buffer |
| Comparative example 4 | The concentration of sodium hexametaphosphate is 20g/L, and the concentration of potassium citrate is 10g/L |
Comparative example a low-value quality control material and a high-value quality control material were tested by the test method of example 2. The detection result of the accuracy of the low-value quality control product (1 mu g/mL) is as follows:
the detection result of the high-value quality control product (30 mu g/mL) accuracy is as follows:
| comparative example | In-batch CV/%) | Inter-batch CV/%) |
| Comparative example 1 | 2.36 | 4.24 |
| Comparative example 2 | 3.43 | 4.72 |
| Comparative example 3 | 2.88 | 4.43 |
| Comparative example 4 | 3.07 | 4.91 |
Finally, it should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered in the claims of the present invention.
Claims (15)
1. The application of sodium hexametaphosphate and potassium citrate in preparing the stool trypsin 1 detection kit.
2. The use according to claim 1, wherein the sodium hexametaphosphate is used in a concentration of 5 to 10g/L; the use concentration of the potassium citrate is 1-6g/L.
3. The use according to claim 2, wherein the sodium hexametaphosphate is used in a concentration of 8g/L; the use concentration of the potassium citrate is 4g/L.
4. A fecal pancreatic elastase 1 detection kit is characterized by comprising luminescent microspheres coupled with anti-fecal pancreatic elastase 1 antibodies and avidin-labeled photosensitive microspheres; biotin is marked on the anti-coproagulase elastase 1 antibody; the kit also comprises sodium hexametaphosphate and potassium citrate.
5. The kit according to claim 4, wherein the sodium hexametaphosphate is used in a concentration of 5 to 10g/L; the use concentration of the potassium citrate is 1-6g/L; the sodium hexametaphosphate and potassium citrate were used to formulate assay buffers.
6. The kit according to claim 5, wherein the sodium hexametaphosphate is used at a concentration of 8g/L; the use concentration of the potassium citrate is 4g/L.
7. The kit of claim 6, wherein the assay buffer further comprises HEPES, triton X-100, casein or dextran.
8. The kit of claim 7, wherein the mass ratio of the luminescent microspheres to the coproagulant trypsin 1 antibody is 10-50:1.
9. the kit according to claim 4, wherein in the step of preparing the biotin-labeled anti-coproagulase elastase 1 antibody, the molecular ratio of biotin to antibody is 10-50:1.
10. the kit according to claim 9, characterized in that the molecular ratio of biotin to antibody is 30.
11. The kit of claim 4, wherein said photosensitive microspheres carry a phthalocyanine dye.
12. The kit of claim 11, wherein the ratio of the photosensitive microspheres to the avidin is 10:1.
13. the kit of claim 12, further comprising a positive control and a negative control.
14. A method for preparing a fecal trypsin elastase 1 detection kit, characterized by preparing the kit of any one of claims 4-13.
15. The method of claim 14, comprising the steps of:
(1) Coupling an anti-fecal pancreatic elastase 1 antibody with a luminescent microsphere;
(2) Labeling an anti-coproagulase elastase 2 antibody with biotin;
(3) Marking photosensitive microspheres with avidin;
(4) An assay buffer was prepared containing sodium hexametaphosphate and potassium citrate.
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| CN109470872A (en) * | 2018-12-07 | 2019-03-15 | 迈克生物股份有限公司 | Magnetic particle buffer |
| CN110763841A (en) * | 2019-11-07 | 2020-02-07 | 浙江爱康生物科技有限公司 | Plasminogen assay kit and preparation method thereof |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011127467A1 (en) * | 2010-04-09 | 2011-10-13 | Companion Diagnostics, Inc. | Devices, systems, and methods for biomarker stabilization |
| US20120164674A1 (en) * | 2010-10-28 | 2012-06-28 | Selinfreund Richard H | Devices and washes for biomarker stabilization |
| CN104360072A (en) * | 2014-12-05 | 2015-02-18 | 重庆中元生物技术有限公司 | D-dimmer latex-reinforced immunonephelometry detection kit cooperatively adopting ion stabilizer and suspension stabilizer |
| CN109470872A (en) * | 2018-12-07 | 2019-03-15 | 迈克生物股份有限公司 | Magnetic particle buffer |
| CN110763841A (en) * | 2019-11-07 | 2020-02-07 | 浙江爱康生物科技有限公司 | Plasminogen assay kit and preparation method thereof |
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