CN111879946A - Multi-index rapid detection fluorescence immunochromatography kit - Google Patents
Multi-index rapid detection fluorescence immunochromatography kit Download PDFInfo
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- G—PHYSICS
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- 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
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or 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
- G01N33/573—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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
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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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4737—C-reactive protein
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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
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/908—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
Abstract
The invention relates to a multi-index rapid detection fluorescence immunochromatographic kit, which comprises a coating layer coated with a fluorescent microsphere labeled antibody on a fluorescent pad in a detection card, wherein the antibody is a monoclonal antibody of C-reactive protein, myeloperoxidase and heart fatty acid binding protein; the invention optimizes the parameters such as the proportion of the activating agent and the like, and meets the requirements of more sensitive, quicker and more accurate detection of the three markers at the same time.
Description
Technical Field
The invention relates to the field of biotechnology detection, in particular to a fluorescence immunochromatographic kit which can detect 3 markers more quickly and conveniently.
Background
The fluorescence immunochromatography detection reagent belongs to a point-of-care testing (POCT) detection product, has the characteristics of convenience and rapidness in detection, low comprehensive cost and the like, and is widely applied clinically. However, the complexity of human body determines that a single biomarker is difficult to timely and accurately reflect the physical condition of a subject comprehensively, and multiple detections and comprehensive judgment are often required at the same time. The large outpatient quantity is a characteristic of the medical and health system in China, is also a cause for the contradiction between doctors and patients, and is under great working pressure in both doctors and auxiliary departments. The multiple markers are combined into one kit for detection, so that a lot of repeated labor is undoubtedly reduced, the inspection efficiency is greatly improved, and the diagnosis and treatment efficiency of clinicians is accelerated.
The multi-index joint detection is not only beneficial to relieving the doctor-patient contradiction, but also beneficial to protecting the life health of the patient. Particularly in the field of cardiovascular diseases, the acute and severe mortality rate and disability rate of myocardial infarction and cerebral infarction exceed 40 percent, seriously threaten the life safety of patients, and bring huge economic burden and mental pain to families and even society. How to diagnose the morbidity risk of a patient as soon as possible before an emergency happens is the key to reducing or even avoiding tragedies by taking effective treatment measures. The multi-index joint detection kit is a good assistant for improving the clinical diagnosis and treatment efficiency and the patient prognosis level, but the simultaneous detection of a plurality of markers has certain technical difficulty, different markers have different properties and characteristics, the kit is suitable for different systems, interference can be formed among the markers, and particularly, the kit is a fluorescence detection kit with higher sensitivity.
Disclosure of Invention
The invention aims to solve the technical problem that a multi-index combined detection product with higher sensitivity and accuracy and an early warning function is lacked in the field of cardiovascular diseases at present.
In order to solve the technical problems, the invention provides a fluorescence immunochromatographic assay kit which can simultaneously detect three markers, namely C-reactive protein, myeloperoxidase and cardiac fatty acid binding protein in blood under the condition of ensuring sensitivity and accuracy, and the specific technical scheme is as follows:
the fluorescence immunochromatographic kit comprises a detection card, wherein the detection card comprises a sample pad, a fluorescence pad, a detection pad with a detection line and a quality control line, a sample suction pad and a bottom plate. Wherein the fluorescent pad is coated with a coating of a fluorescent microsphere labeled antibody, and the antibody is a monoclonal antibody of C-reactive protein, myeloperoxidase and cardiac fatty acid binding protein; the activator used in the antibody marking process is EDC and NHS, the dosage ratio of the fluorescent microsphere and the two activators is 1 (14-16) to (14-16); the detection lines are provided with 3 detection lines which are respectively coatings formed by paired antibodies or antigens capable of being specifically combined with the fluorescent microsphere labeled antibodies in the coatings.
The detection card also comprises a hydrophobic shell, and the shell is provided with a sample adding hole and a detection window. The test strip composed of the sample pad, the fluorescence pad, the detection pad, the sample suction pad and the bottom plate is fixed in the shell. The sample adding hole is positioned above the sample pad, and the inspection window is positioned above the detection pad.
The C-reactive protein (CRP) and the Myeloperoxidase (MPO) are early warning markers of cardiovascular and cerebrovascular diseases, and the risk of cardiovascular and cerebrovascular acute and severe diseases can be evaluated through content change of the CRP and the MPO. The cardiac fatty acid binding protein (hFABP) is a cardiac muscle damage marker with strong specificity and high sensitivity, and can detect the cardiac muscle damage condition in asymptomatic or slight angina pectoris symptoms. The three markers are detected in a combined manner, so that the risk of malignant events such as myocardial infarction and the like of a subject can be evaluated more quickly and accurately, and precious time is won for treatment.
The principle and structure of fluorescence immunochromatography detection products are known technologies, and detection of different detection objects is mainly realized by adjusting a process part, wherein selection of parameters such as reagent types, concentration, pH value, proportion and the like is particularly important, and as the process steps and related reagents are various, the process screening optimization work is difficult to obtain by a one-to-one practical method, and the experiment scheme and the results thereof need to be deeply analyzed and tried and found on the basis. Especially for products with multiple indexes detected simultaneously, the property difference of the marker antibody makes the products have buffer systems and dosage ratios suitable for each matter. The method realizes that the three do not interfere with each other and the content of the product can be accurately detected, and is finished by simple adjustment or limited trial and error of the technology which can not detect the product according to the common single index. Many chemical interactions are unknown to our current technology, and the experimental process often shows the conditions of feasible theory but unsatisfactory results.
Such as the selection of the amount of activator of the present invention. Usually, the dosage of the fluorescent microspheres is obviously more than that of the activating agent, and the fluorescent signal intensity meeting the detection requirement of the three markers in the product of the invention cannot be obtained within the range of the common mixture ratio. Through a large number of experiments, the fluorescent microsphere and the two activators are used in a ratio of 1 (14-16) to (14-16), and the fluorescent signal is very strong. The activators EDC and NHS refer to carbodiimide and N-hydroxysuccinimide, respectively, preferably in a 1:1 ratio.
In a preferred embodiment, the fluorescent microspheres of the present invention and the two activators are used in an amount of 1:15: 15.
Because the dosage of the activating agent is larger, the adding mode of the activating agent is as follows: adding a certain amount of activation buffer solution into the washed fluorescent microspheres, and then adding specific amounts of EDC and NHS instead of the activator solution.
The activation time of the marking process is 25-45 minutes, namely the reaction time after the two activators are added is 25-45 minutes, the coupling rate is low when the time is less than the time, the fluorescence signal is weak, and the microspheres can be aggregated to different degrees when the time is more than the time. The activation time is more preferably 30 minutes, and the detection effect, the detection efficiency and the detection cost are considered at the same time.
In another preferred embodiment, the ratio of the amount of fluorescent microspheres to C-reactive protein, myeloperoxidase and cardiac fatty acid binding protein triabodies added in the labeling process of the present invention is 100:13:8:10, i.e., the amount of antibodies to C-reactive protein, myeloperoxidase and cardiac fatty acid binding protein required to be added to 100. mu.L (1mg/mL) of microspheres is 13. mu.g, 8. mu.g and 10. mu.g, respectively. The fluorescent microspheres prepared according to the ratio cannot aggregate and have strongest fluorescent signals.
The fluorescent microsphere marked antibody on the fluorescent pad also comprises a chicken IgY antibody.
Preferably, the quality control line is formed by coating antibodies including, but not limited to, goat anti-mouse IgG, goat anti-chicken IgY, or goat anti-rabbit IgG; the detection pad is a nitrocellulose membrane, and is preferably a porous structure membrane with the pore diameter of 5-12 um; the material of the sample loading pad and the fluorescence pad is glass cellulose membrane or non-woven fabric, the material of the sample absorbing pad is water absorbing filter paper, and the material of the bottom plate is plastic.
The fluorescent microsphere is selected from modified polystyrene microsphere, and lanthanide chelate is filled in the fluorescent microsphere, wherein the lanthanide chelate is selected from one of europium (Eu), terbium (Tb), samarium (Sm), neodymium (Nd) or dysprosium (Dy). The fluorescent microsphere detection technology and the immunochromatography detection technology are known, and the fluorescent immunochromatography kit can be prepared by adopting a known method or a known method for fine adjustment on the basis of the technical scheme.
The invention provides a more convenient, rapid and accurate detection reagent product, which assists clinical diagnosis and treatment of high-risk people with cardiovascular and cerebrovascular diseases in time and efficiently, reduces the occurrence of tragedy of non-death or disability, is also beneficial to reducing medical insurance expenditure, and has important clinical significance and great social benefit for early discovery and early treatment of critical severe diseases such as myocardial infarction and the like.
The inventors further verified the technical effects of the present invention through experiments.
Restated again: the following experiments are only exemplary experiments among many experiments in the development process of the present invention, and do not cover and exhaust all the experiments performed by the present inventors, and are only intended to illustrate the effects of different parameters on the release effect of the microspheres and the fluorescence signal.
Selection experiment of the amount of activator
The experimental method comprises the following steps: reagent cards were prepared according to the procedure of labeling antibody with fluorescent microspheres as reported in example 1 and the amounts of microspheres and activators as shown in Table 1. The reagent card is put into a dry type fluorescence immunoassay analyzer for detection, and the signal intensity is observed.
Table 1 activator dose investigation
The results show that when the amount of activator is less than the amount of microspheres, the fluorescence signal is very weak; the improvement is seen with increasing activator levels, with the fluorescent microspheres having a very strong side 5 signal with EDC and NHS levels of 1:15:15, and the side 5 signal being strongest compared to the side 4 and side 7 with the same EDC and NHS1:1 and a slightly reduced amount of EDC, and side 6.
Detailed Description
Example 1
Labeling antibody with fluorescent microsphere part (1000 parts)
1) Weighing 500 mu L (1mg/mL) of fluorescent microsphere solution in a centrifuge tube, carrying out 12000r/min, centrifuging for 15min, and discarding the supernatant;
2) adding 5ml of activating buffer solution (0.03mol/L, MES solution with pH 6.0), 12000r/min, centrifuging for 15min, and discarding the supernatant; cleaning is repeated for one time; then 5ml of activation buffer solution is added;
3) respectively adding specific amounts of EDC and NHS, and oscillating and reacting for a certain time at normal temperature in a dark place;
4) centrifuging the activated solution at 12000r/min for 15min, and removing the supernatant;
5) adding 5ml coupling buffer solution (PB buffer solution of 0.01mol/L, pH 7.0.0), 12000r/min, centrifuging for 15min, and discarding the supernatant; cleaning is repeated for one time; then adding 4.0ml of coupling buffer solution;
6) adding a specific amount of antibody, supplementing a proper amount of coupling buffer solution, adjusting the final volume to 5ml, and oscillating at normal temperature and in dark place for reaction for 15 min.
A multi-index rapid detection fluorescence immunochromatographic kit comprises a detection card, wherein the detection card comprises a sample pad, a fluorescence pad, a detection pad with a detection line and a quality control line, a sample sucking pad and a bottom plate; wherein the fluorescent pad is coated with a coating of a fluorescent microsphere labeled antibody, and the antibody is a monoclonal antibody of C-reactive protein, myeloperoxidase and cardiac fatty acid binding protein; the dosage of the activating agent used in the antibody marking process is 7.5mg of EDC and NHS, and the activating time is 30 minutes; the detection lines are provided with 3 detection lines which are respectively coatings formed by paired antibodies capable of being specifically combined with the three antibodies marked by the fluorescent microspheres in the coatings. The quality control line is coated with a goat anti-chicken IgY polyclonal antibody. And preparing the reagent card according to the content of the steps of the fluorescent microsphere labeled antibody part and other conventional steps and parameters of the fluorescence immunochromatography reagent card.
Example 2
The difference from the embodiment 1 is that: the dosage of the activating agents EDC and NHS is 7.0mg and 8.0mg respectively, and the activating time is 25 minutes.
Example 3
The difference from the embodiment 1 is that: the dosage of the activating agents EDC and NHS is 8.0mg and 7.0mg respectively, and the activating time is 45 minutes.
Example 4
The difference from the embodiment 1 is that: the dosage of the activating agents EDC and NHS is 7.0mg, and the activating time is 35 minutes.
Example 5
The difference from the embodiment 1 is that: the amounts of C-reactive protein, myeloperoxidase and cardiac fatty acid binding protein antibody added were 65. mu.g, 40. mu.g and 50. mu.g, respectively.
The present invention has been described in detail in order to enable those skilled in the art to understand the invention and to practice it, and it is not intended to limit the scope of the invention, and all equivalent changes and modifications made according to the spirit of the present invention should be covered by the present invention.
Claims (8)
1. The utility model provides a many indexes short-term test fluorescence immunochromatography kit, this kit contains the detection card, the detection card includes sample pad, fluorescence pad, has detection line and quality control line's detection pad, inhale a kind pad and bottom plate, its characterized in that: the fluorescent pad is coated with a coating of a fluorescent microsphere labeled antibody, and the antibody is a monoclonal antibody of C-reactive protein, myeloperoxidase and heart fatty acid binding protein; the activator used in the antibody marking process is EDC and NHS, and the dosage ratio of the fluorescent microsphere to the two activators is as follows: 1, (14-16) and (14-16); the detection lines are provided with 3 detection lines which are respectively coatings formed by paired antibodies or antigens capable of being specifically combined with the fluorescent microsphere labeled antibodies in the coatings.
2. The kit of claim 1, wherein: the dosage ratio of the two activators is 1: 1.
3. The kit of claim 1, wherein: the dosage ratio of the fluorescent microsphere to the two activators is 1:15: 15.
4. The kit of claim 1, wherein: the activation time of the marking process is 25-45 minutes.
5. The kit of claim 4, wherein: the activation time of the labelling process was 30 minutes.
6. The kit of claim 1, wherein: the adding amount ratio of the fluorescent microspheres to the C-reactive protein, the myeloperoxidase and the cardiac fatty acid binding protein triabody in the labeling process is 100:13:8: 10.
7. The kit according to any one of claims 1 to 5, characterized in that: the fluorescent microspheres mark antibodies, and the antibodies also comprise chicken IgY antibodies.
8. The kit of claim 7, wherein: the quality control line is formed by coating goat anti-mouse IgG, goat anti-chicken IgY or goat anti-rabbit IgG antibody.
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| CN202010735295.2A CN111879946A (en) | 2020-07-28 | 2020-07-28 | Multi-index rapid detection fluorescence immunochromatography kit |
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Citations (6)
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| CN110554197A (en) * | 2018-06-01 | 2019-12-10 | 常州博闻迪医药股份有限公司 | Fluorescence immunochromatography joint detection kit and preparation method thereof |
| CN111273017A (en) * | 2020-03-02 | 2020-06-12 | 江苏扬新生物医药有限公司 | Fluorescence immunochromatography kit for rapidly detecting novel coronavirus |
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2020
- 2020-07-28 CN CN202010735295.2A patent/CN111879946A/en active Pending
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| US20120021944A1 (en) * | 2010-07-23 | 2012-01-26 | Harold Baker | Co-Coupling To Control Reactivity Of Reagents In Immunoassays |
| CN105203754A (en) * | 2014-08-18 | 2015-12-30 | 董俊 | Method and kit for fast detection of moraxella catarrhalis based on magnetic resolution and quantum dot labelling |
| CN105277713A (en) * | 2014-08-18 | 2016-01-27 | 董俊 | Rapid detection method and kit for streptococcus pneumoniae based on magnetic separation and quantum dot labeling |
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