CN107664700A - Cardiac muscle troponin I and creatine kinase isozyme and myoglobins three-in-one detection reagent box and preparation method thereof - Google Patents

Cardiac muscle troponin I and creatine kinase isozyme and myoglobins three-in-one detection reagent box and preparation method thereof Download PDF

Info

Publication number
CN107664700A
CN107664700A CN201710852525.1A CN201710852525A CN107664700A CN 107664700 A CN107664700 A CN 107664700A CN 201710852525 A CN201710852525 A CN 201710852525A CN 107664700 A CN107664700 A CN 107664700A
Authority
CN
China
Prior art keywords
myoglobins
rare
creatine kinase
cardiac muscle
muscle troponin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201710852525.1A
Other languages
Chinese (zh)
Inventor
王鹏浩
宋璐琳
张培镇
于鸿翔
孙宁宁
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Weihai Niu Pu Bioisystech Co Ltd
Original Assignee
Weihai Niu Pu Bioisystech Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Weihai Niu Pu Bioisystech Co Ltd filed Critical Weihai Niu Pu Bioisystech Co Ltd
Priority to CN201710852525.1A priority Critical patent/CN107664700A/en
Publication of CN107664700A publication Critical patent/CN107664700A/en
Pending legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • 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
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • 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
    • 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
    • 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
    • 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
    • G01N2333/4712—Muscle proteins, e.g. myosin, actin, protein
    • 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 present invention relates to fluorescence immune chromatography technical field in Medical Immunology, specifically a kind of cardiac muscle troponin I and creatine kinase isozyme and myoglobins three-in-one detection reagent box and preparation method, provided with test card, it is characterised in that the test card is provided with by lower from being above sequentially provided with：PVC board, sample pad, pad, nitrocellulose filter and adsorptive pads, the cardiac muscle troponin I and creatine kinase isozyme and the three-in-one monoclonal antibody of myoglobins of rare-earth fluorescent microballoon mark are wherein adsorbed with pad, a diameter of 60 120nm of the rare-earth fluorescent microballoon, the rare earth doped lanthanide series of rare-earth fluorescent microballoon, it is stable under ground state, launch fluorescence of the wave-length coverage in 540 600nm under 340 380nm excitation source effect；The monoclonal antibody is the monoclonal antibody mixed after purification, from the cell strain of monoclonal antibody for 26 different cardiac muscle troponin Is and creatine kinase isozyme and the three-in-one epitope of myoglobins, there is easy to operate, rapid reaction, high sensitivity, high specificity.

Description

Cardiac muscle troponin I and the three-in-one detection examination of creatine kinase isozyme and myoglobins Agent box and preparation method thereof
Technical field：
The present invention relates to fluorescence immune chromatography technical field in Medical Immunology, specifically one kind can be quick and precisely It is three-in-one to cardiac muscle troponin I and creatine kinase isozyme and myoglobins progress quantitative analysis cardiac muscle troponin I And creatine kinase isozyme and myoglobins three-in-one detection reagent box and preparation method thereof.
Background technology：
CTnI is myocardial structural albumen, there is the tissue specificity of height, is a very sensitive and special Acute myocardial Infarct mark, cardiac muscle in cTnI it is very abundant, be released into blood within 4 hours~6 hours after myocardial damage, reach diagnosis signals, send out Make peak occur in 14 hours~36 hours, continue 3 days~7 days or so.Window phase is longer, up to 10 days or so, is advantageous to diagnose The AMI and Unstable angina of delay.CK-MB is creatine kinase (CK) isoenzymes, and CK is that early diagnosis AMI uses are most extensive Myocardial Damage Index, AMI occurs, and CK-MB2 hour~4 hour start to raise, and can exceed normal upper limit within 4 hours~6 hours, Reach peak value within 18 hours~36 hours, last about 2 days~4 days.If CK-MB is significantly raised in serum, involvement of myocardium is prompted, but Its specificity is slightly worse, can also increase in bone lesion and chronic renal insufficiency.Myo is a kind of oxygen combination hemoprotein, Cardiac muscle and skeletal muscle tissue are distributed mainly on, at acute myocardial infarction AMI (AMI), myocardium cell necrosis, Myo is released into blood In.After symptom occurs about 2 hours~3 hours, Myo can exceed normal upper limit in blood, reach within 9 hours~12 hours peak value, and 24 Hour~recover normal after 36 hours.Myo diagnostic sensitivities in acute myocardial infarction breaks out 9 hours are very high, are advantageous to early diagnose, It is earliest acute myocardial infarction mark occur in serum now.Myocardial infarction except Myo feminine genders additionally aid.
CTnI, CK-MB and Myo three are used alone and had some limitations, as cTnl is small in myocardial damage morbidity 6 When within susceptibility it is relatively low, to determine whether early application thrombolytic therapy value it is smaller；Myo specificity is poor, and window phase is too short； CK-MB specificity is also poor.Detect tri- indexs of cTnI, CK-MB and Myo simultaneously, it can be seen that in the difference of AMI morbidities Section, positive rate have different degrees of rise, and the early stage for heart infarction excludes and diagnosis provides timely and effectively foundation.
Immuno analytical method is using the immune response between trace antigen and corresponding antibody with high specificity, to detect such as It is living in the organisms such as hormone, medicine, protein, polypeptide, enzyme, tumor associated antigen, micro-element, virus, bacterium and metallic element Property material.Immuno analytical method includes labelling immunoassay, non-marked immunoassay and instrument immunoassay.This kit utilizes The carboxyl latex microballoon labelling immunoassay technology containing rare earth element be to belong to one kind of labelling immunoassay.
Fluoroimmunoassay (FIA) and radiommunoassay (RIA) since the advent of the world, experienced the development of decades, but It is that people increasingly feel FIA because of naturally local too high, interference detection results；RIA uses isotope marks, has pole to human body It is big to endanger and made troubles to experiment.EIA enzyme immunoassay (EIA) is larger by other influences factor also because enzyme itself is unstable, pushes away Wide application is restricted.At the beginning of the eighties, people begin one's study replaces fluorescent material and isotope-labelled protein with rare earth element Or antibody, TIME RESOLVED TECHNIQUE is incorporated into field of biological detection, establishes new ultramicron time-resolved fluoroimmunoassay point Analysis technology (Time resolved Fluoroimmunoassay, abbreviation TrFIA).The technology uses multidisciplinary advanced technology, collection The characteristics of having tied other immunoassays, in fields such as immunology, molecular biology, cytology and medical science, obtain significant progress And extensive use.
TrFIA make use of trivalent rare earth ion and chelate with unique fluorescent characteristic for tracer replace fluorescent material, Enzyme, isotope, chemiluminescent substance, labelled antibody, antigen, hormone, polypeptide, protein, nucleic acid probe and biological cell, treat anti- Answer system (such as antigen-antibody reaction, nucleic acid probe hybridization, biotin-labeled pentylamine reaction and the killing of target cell pairing effect cell Effect etc.) occur after, with TrFIA detectors determine reaction product in fluorescence intensity.According to product fluorescence intensity and relatively glimmering The ratio of luminous intensity, the concentration of analyte in reaction system is judged, so as to reach quantitative analysis.In common fluoremetry, Due to containing a variety of fluorescent components in test sample, background fluorescence is (caused by the colloidal solid and solvent molecule in sample The non-specific fluorescence that scattering light and Proteins in Serum and other compounds are sent) intensity is big, it is strong to disturb, turn into fluorescence point The bottleneck that analysis method is promoted on a large scale.Why TrFIA can turn into after the new sensitive detection method of EIA, RIA latter, Depend primarily in the unique fluorescence feature of lanthanide series, detection the wavelength resolution used and time-delay technique and dissociation- Enhancing technology.
Lanthanide series (lanthanide, Ln) belongs to rare earth element, in sharing 17, be usually used in TrFIA mainly have europium (Eu), Samarium (Sm), terbium (Tb), dysprosium (Dy).Lanthanide series has unique fluorescence radiation feature, compared with common fluorescent, lanthanide ion chela Compound fluorescence decay time is grown, and is the 10 of conventional fluorescent3-106Times.If the fluorescence decay time of lanthanide ion chelate is in 60- 900 μ s, conventional Eu3+Fluorescence decay time is 714 μ s, and the fluorescence decay time of fluorogen only has in common fluorescent immunoassay 1-100 μ s, the fluorescence decay time of some protein is only 1-10 μ s in sample, therefore utilizes TIME RESOLVED TECHNIQUE, delay one Measured after fixing time, Eu can be obtained3+Specific fluorescence signal.Simultaneously because decay time is grown, Eu3+Label is in measurement Between in can be excited repeatedly, ground state is transitted to by excitation state quickly after exciting every time, just has fluorescence to send, then again can be weighed Newly excite, so it is per second have 1000 times excite so that the relative specific activity of TrFIA fluorescent markers is very high.Lanthanide series is glimmering The maximum of light spectrum is characterized in that exciting light and the Stokes displacements launched between light are larger, Eu3+Excitation wavelength is 337nm, transmitting Wavelength is 615nm, and Stokes displacements are up to 278nm；Eu simultaneously3+The fluorescence light belt being excited is extremely narrow, and the emission peak of fluorescence is very Sharply, instrument adjustment can be made to be determined in extremely narrow wave-length coverage, thus almost completely eliminate the interference of background fluorescence, after And passage time delay and wavelength resolution, strong specificity fluorescent and background fluorescence are distinguished into open (therefore referred to as time resolution), make interference Reach almost nil.
In view of the application of above labeling method and detection technique, this kit has good detection specific, higher Sensitivity, the simplicity of operation and stable fluorescent marker ensure that the accuracy of detection.
The content of the invention：
The present invention is for shortcoming and defect present in prior art, it is proposed that a kind of to utilize the sensitive of fluorescence immune chromatography Property, the high sensitivity, fast and simple realized with reference to fluorescence immune chromatography analyzer can be with the cardiac muscle troponin I of accurate quantitative analysis And creatine kinase isozyme and myoglobins three-in-one detection reagent box and preparation method.
The present invention can be reached by following measures：
A kind of cardiac muscle troponin I and creatine kinase isozyme and myoglobins three-in-one detection reagent box, provided with test paper Card, it is characterised in that the test card is sequentially provided with from the bottom to top：PVC board, sample pad, pad, nitrocellulose filter and suction Water cushion, be wherein adsorbed with pad rare-earth fluorescent microballoon mark cardiac muscle troponin I and creatine kinase isozyme and flesh it is red The three-in-one monoclonal antibody of albumen, a diameter of 60-120nm of the rare-earth fluorescent microballoon, the rare earth doped lanthanum of rare-earth fluorescent microballoon Series elements, it is stable under ground state, launch wave-length coverage in the glimmering of 540-600nm under 340-380nm excitation source effect Light；The monoclonal antibody is the monoclonal antibody mixed after purification, from for 2-6 different cardiac muscle troponin Is And the cell strain of monoclonal antibody of creatine kinase isozyme and the three-in-one epitope of myoglobins.
The diameter of the rare-earth fluorescent microballoon of pad of the present invention is preferably 90-110nm；The rare-earth fluorescent microballoon is excellent Choosing is doped with rare earth lanthanide, for any one or a few of the lanthanide series such as europium (Eu), samarium (Sm), erbium (Er), neodymium (Nd) Mixture；The preferably rare earth doped complex compound of rare-earth fluorescent microballoon；The antibody that rare-earth fluorescent microballoon marks on pad is excellent Choosing derives from the monoclonal cell cell line for 3 different epitopes.
Pad of the present invention is made using following steps：Glass fibre membrane is soaked in 200mM Tris-HCL processing In liquid (X-100 containing 1.5%Triton, 1.5%BSA, pH7.5), 4 DEG C are soaked 4 hours, and it is small to then take out 37 DEG C of oven for drying 4 When, it is standby, it is non-contact with Bio-Jet Quanti300 by glass fibre membrane on Bio-DotXYZ3050 three-dimensional specking platforms The cardiac muscle troponin I and creatine kinase isozyme and myoglobins that the quantitation nozzle that declines marks rare-earth fluorescent microballoon are three-in-one Monoclonal antibody is sprayed onto glass fibre membrane, and 37 DEG C of drying are made after 2 hours.
The cardiac muscle troponin I and creatine kinase isozyme of rare-earth fluorescent microballoon mark in the present invention on pad And the three-in-one monoclonal antibody of myoglobins is made using following steps：
Step 1：The acquisition of cell strain of monoclonal antibody：It is red with reference to cardiac muscle troponin I and creatine kinase isozyme and flesh The peptide sequence of the three-in-one amino acid sequence of albumen, strong artificial synthesized 20 amino acid in site of selection antigenicity or so, crosslinking Onto KLH, the cell strain of monoclonal antibody of specific high-affinity is prepared using the method for preparing monoclonal antibody of standard, by institute Monoclonal antibody corresponding to the cell line of acquisition carries out pairing experiment and affinity determination experiment, determines to capture according to experimental result Antibody and detection antibody；
Step 2：The preparation of monoclonal antibody：Prepared using the ascites production technology of standard and purify the cardiac muscle for detection Troponin I and the three-in-one monoclonal antibody of creatine kinase isozyme and myoglobins, be stored in after packing -20 DEG C it is standby；
Step 3：The aldehyde radical of rare-earth fluorescent microballoon：3mg rare-earth fluorescent microballoons are taken, with 50mM, pH 9.5 carbonate delays Fliud flushing, washed 3 times, centrifugal speed 12000rpm using centrifugal process, the time is 5 minutes, is finally resuspended in 100 μ l above-mentioned carbon In phthalate buffer, the glucan of 300 μ l aldehyde radicals is added, is mixed, at room temperature dark reaction 4 hours, using same centrifugal process In the above-mentioned carbonate buffer solution for washing and being resuspended to 100 μ l, be placed in 4 DEG C it is standby；
Step 4：The cardiac muscle troponin I and creatine kinase isozyme and myoglobins of rare-earth fluorescent microballoon mark are three-in-one The preparation of monoclonal antibody：2mg is three-in-one for the cardiac muscle troponin I and creatine kinase isozyme and myoglobins detected Monoclonal antibody in 4 DEG C of dialysed overnights, is then mixed with above-mentioned carbonate buffer solution with the rare-earth fluorescent microballoon of above-mentioned aldehyde radical, 4 DEG C of reactions are overnight；Then, sodium borohydride is added to final concentration 10mM, and 4 DEG C are reacted 4 hours；Add isometric confining liquid (100mM Tris-HCL, pH7.5, containing 2%BSA, 5% sucrose), 4 DEG C of closings are overnight；Then 100mM Tris-HCL are used, PH7.5 buffer solution is washed 3 times using centrifugal process, is resuspended in 100 μ l 100mM Tris-HCL buffer solutions and (is contained 1.2% NaCL, 0.5%BSA, 0.2%Tween 20), 4 DEG C be kept in dark place it is standby.
It is of the present invention to be coated with detection line and the nitrocellulose filter of nature controlling line is made by following steps：
Step 1：According to foregoing pairing experiment and affinity determination experiment, select for monoclonal corresponding to the antibody of capture Antibody cell strain, prepared according to the ascites production technology of standard and purify the cardiac muscle troponin I and creatine kinase for capture Isodynamic enzyme and the three-in-one monoclonal antibody of myoglobins, be stored in -20 DEG C it is standby；
Step 2：It is with coating dilution that cardiac muscle troponin I and creatine kinase isozyme and myoglobins is three-in-one respectively Monoclonal antibody and goat anti-mouse igg antibody adjust concentration to 1-5mg/ml, film liquid amount be 1-2 μ l/cm, using they as Be sprayed on nitrocellulose filter on parallel with nature controlling line of detection line is coated with, and detection line and nature controlling line are at intervals of 3-7mm, then It is placed in baking oven, 37 DEG C dry 2 hours.
Sample pad of the present invention is made by following steps：Glass fibre membrane is soaked in containing 2.0%Triton X- 100,2%BSA, 0.1M Tris buffer solutions, in pH7.5 treatment fluid, 4 hours are soaked in 4 DEG C, are subsequently placed in baking oven, 37 DEG C drying 2 hours.
Present invention also offers it is a kind of as described above kit realize cardiac muscle troponin I and creatine kinase isozyme and Myoglobins preparation of three-in-one, it is characterised in that comprise the following steps：
Step 1：Detection reagent and sample are balanced to room temperature, test card is taken out, keeps flat；
Step 2：Accurate to draw 25 μ l serum samples, sample is drawn 40 μ l samples, is added in sample aperture when being whole blood, then 100 μ l Sample dilutions are added in the buffering fluid apertures of bottom immediately, Sample dilution is using physiological saline or PBS, 15-30 points In clock result is quantitatively judged with fluorescence immune chromatography analyzer；
Step 3：After the relevant parameter for setting fluorescence immune chromatography analyzer, test card is put into storehouse and detected, Instrument would indicate that the quantified results of sample concentration, and the fluorescence immune chromatography analyzer is a kind of Systems for optical inspection, The detection range three-in-one to cardiac muscle troponin I and creatine kinase isozyme and myoglobins is 0-20ng/ml.
It is same that the present invention provides a kind of cardiac muscle troponin I prepared using rare-earth fluorescent immunochromatography technique and creatine kinase Work enzyme and the three-in-one fast quantification immunochromatographytest test kit of myoglobins, while it is adapted to serum and whole blood sample, and be adapted to Clinically single part detection, relative to cardiac muscle troponin I and the three-in-one qualitative colloid of creatine kinase isozyme and myoglobins Gold reagent, cardiac muscle troponin I and creatine kinase isozyme and the three-in-one content of myoglobins in detection sample, tool can be quantified Have more specific Clinical significance of MG, have easy to operate, rapid reaction, high sensitivity, high specificity, be adapted to Site Detection and The advantages that economical and practical.
Brief description of the drawings：
Accompanying drawing 1 is the structural representation of test card in the present invention.
Accompanying drawing 2 is the accuracy analysis result schematic diagram of embodiment 2 in the present invention.
Subordinate list 3 is the Precision Analyze result data of embodiment 3 in the present invention.
Reference：PVC board 1, sample pad 2, pad 3, nitrocellulose filter 4, adsorptive pads 5.
Embodiment：
The present invention is further illustrated with reference to the accompanying drawings and examples：
As shown in Figure 1, present invention firstly provides a kind of cardiac muscle troponin I and creatine kinase isozyme and flesh red eggs White three-in-one detection reagent box, box is interior to be provided with test card, and the test card is sequentially provided with from the bottom to top：PVC board 1, sample pad 2, Pad 3, nitrocellulose filter 4 and adsorptive pads 5, the myocardium myo calcium of rare-earth fluorescent microballoon mark is wherein adsorbed with pad 3 Protein I and the three-in-one monoclonal antibody of creatine kinase isozyme and myoglobins, a diameter of 60- of the rare-earth fluorescent microballoon 120nm, the rare earth doped lanthanide series of rare-earth fluorescent microballoon is stable under ground state, is issued in 340-380nm excitation source effect Project fluorescence of the wave-length coverage in 540-600nm；The monoclonal antibody is the monoclonal antibody mixed after purification, from pin The cardiac muscle troponin I different to 2-6 and the monoclonal of creatine kinase isozyme and the three-in-one epitope of myoglobins resist Body cell strain；
The diameter of the rare-earth fluorescent microballoon of the pad 3 is preferably 90-110nm；The rare-earth fluorescent microballoon is preferably mixed It is miscellaneous to have rare earth lanthanide, it is any one or a few mixed of the lanthanide series such as europium (Eu), samarium (Sm), erbium (Er), neodymium (Nd) Compound；The preferably rare earth doped complex compound of rare-earth fluorescent microballoon；The antibody that rare-earth fluorescent microballoon marks on pad preferably comes Come from the monoclonal cell cell line for 3 different epitopes.
Embodiment 1：
The each group of test card in cardiac muscle troponin I and creatine kinase isozyme and myoglobins three-in-one detection reagent box It can be made into part by following measures：
1st, the preparation of sample pad 2：
Glass fibre membrane is soaked in containing 2.0%Triton X-100,2%BSA, 0.1M Tris buffer solutions, pH7.5 Treatment fluid in, in 4 DEG C soak 4 hours, be subsequently placed in baking oven, 37 DEG C dry 2 hours.2nd, it is anti-to adsorb fluorescent microsphere mark The preparation of the pad 3 of body：
Glass fibre membrane is soaked in 200mM Tris-HCL treatment fluids (X-100 containing 1.5%Triton, 1.5% BSA, pH7.5), 4 DEG C are soaked 4 hours, then take out 37 DEG C of oven for drying 4 hours, standby.By glass fibre membrane in Bio- On DotXYZ3050 three-dimensional specking platforms, with the non-contact quantitation nozzles that decline of Bio-Jet Quanti300 by rare-earth fluorescent microballoon The three-in-one monoclonal antibody of cardiac muscle troponin I and creatine kinase isozyme and myoglobins of mark is sprayed onto glass fibre membrane, 37 DEG C dry 2 hours, standby；
The aldehyde radical of rare-earth fluorescent nanoparticle：3mg rare-earth fluorescent nanoparticles are taken, with 50mM, pH9.5 carbonate delays Fliud flushing, washed 3 times, centrifugal speed 12000rpm using centrifugal process, the time is 5 minutes, is finally resuspended in 100 μ l above-mentioned carbon In phthalate buffer, the glucan of 300 μ l aldehyde radicals is added, is mixed, at room temperature dark reaction 4 hours, using same centrifugal process In the above-mentioned carbonate buffer solution for washing and being resuspended to 100 μ l, be placed in 4 DEG C it is standby；
Rare-earth fluorescent nanoparticle marks cardiac muscle troponin I and creatine kinase isozyme and the three-in-one Dan Ke of myoglobins The preparation of grand antibody：The cardiac muscle troponin I and creatine kinase isozyme and the three-in-one Dan Ke of myoglobins that 2mg is used to detect Grand antibody, in 4 DEG C of dialysed overnights, is then mixed, 4 DEG C with above-mentioned carbonate buffer solution with the rare-earth fluorescent microballoon of above-mentioned aldehyde radical Reaction is overnight；Then, sodium borohydride is added to final concentration 10mM, and 4 DEG C are reacted 4 hours；Add isometric confining liquid (100mM Tris-HCL, pH7.5, containing 2%BSA, 5% sucrose), 4 DEG C of closings are overnight；Then 100mM Tris-HCL are used, PH7.5 buffer solution is washed 3 times using centrifugal process, is resuspended in 100 μ l 100mM Tris-HCL buffer solutions and (is contained 1.2% NaCL, 0.5%BSA, 0.2%Tween 20), 4 DEG C be kept in dark place it is standby；
3rd, it is coated with the preparation of the nitrocellulose filter 4 of detection line and nature controlling line：
According to foregoing pairing experiment and affinity determination experiment, select thin for monoclonal antibody corresponding to the antibody of capture Born of the same parents' strain, prepared according to the ascites production technology of standard and purify the cardiac muscle troponin I and creatine kinase isozyme for capture And the three-in-one monoclonal antibody of myoglobins, be stored in -20 DEG C it is standby；
Coating dilution is used respectively by cardiac muscle troponin I and the three-in-one monoclonal of creatine kinase isozyme and myoglobins Antibody and goat anti-mouse igg antibody adjust concentration to 1-5mg/ml, film liquid amount be 1-2 μ l/cm, using them as detection line Be sprayed on nitrocellulose filter on parallel with nature controlling line is coated with, and detection line and nature controlling line are subsequently placed in baking at intervals of 3-7mm In case, 37 DEG C dry 2 hours；
The assembling of test card：Paste treated sample pad 2 successively in PVC board 1, be adsorbed with rare-earth fluorescence labeling The pad 3 of antibody, the nitrocellulose filter 4 and adsorptive pads 5 for being coated with detection line and nature controlling line, it is big to obtain test paper after assembling Plate, it is wide to cut into 4mm as requested, and test paper is loaded in plastic clip and forms test card.
The preferably following raw material of equipment and raw material selected in above steps：
Cardiac muscle troponin I and the three-in-one specific pairs antibody of creatine kinase isozyme and myoglobins；Myocardium myo calcium Protein I and the three-in-one quality-control product of creatine kinase isozyme and myoglobins：Landau laboratory diagnosis Co., Ltd of Britain；Rare-earth fluorescent Microballoon：Shanghai Zhen Zhun bio tech ltd；Nitrocellulose (NC) film：Millipore Products；Bovine serum albumin(BSA) (BSA), polyethylene glycol PEG20000, caseinhydrolysate：Sigma products, other common agents are AR.
Embodiment 2：Accuracy test
From above-mentioned test card and fluorescence immune chromatography analyzer (model：NEO-007),
The setting of fluorescence immunity analyzer parameter：After test card technological parameter is set on fluorescence immunity analyzer, take The above-mentioned test card assembled, respectively with 0.2,0.5,1,2,5,20ng/ml cardiac muscle troponin I and creatine kinase isozyme And the three-in-one calibration object of myoglobins, it is measured with test card, obtains the fluorescence intensity level of each calibration object, result is input to In the parameter of analyzer, the setting of the parameter of analyzer is completed.
Predominantly detect material：Clinical sample is obtained by relevant hospital, totally 200 parts of Roche Electrochemiluminescence immunoassay definite values Sample, wherein 100 parts of serum sample, 100 parts of whole blood sample, cardiac muscle troponin I and creatine kinase isozyme and myoglobins Three-in-one content distribution section is between 0-20ng/mL.
Preparation method：
Step 1：Detection reagent and sample are balanced to room temperature, test card is taken out, keeps flat；
Step 2：Accurate to draw 25 μ l serum samples, sample is drawn 50 μ l samples, is added in sample aperture when being whole blood, then 100 μ L Sample dilutions (physiological saline or PBS) are added in the buffering fluid apertures of bottom immediately, 15-30 minutes, interior fluorescence was exempted from Epidemic disease chromatographic analysis instrument quantitatively judges result；
Step 3：Set test card to be put into storehouse after instrument relevant parameter and detected, instrument would indicate that sample is dense The quantified results of degree.
Test result analysis：
After the completion of prepared by clinical sample detection reagent, all clinical samples are detected by preparation method, and analyze inspection Survey result.
Result of the test：
As shown in Figure 2, using the detected value of experimental system as Y-axis, using the test value of contradistinction system as X-axis, scatterplot is drawn Figure, and carry out correlation analysis.Clinical sample detection is respectively less than to 200 parts of clinical definite value pattern detections, sample mean deviation 10%, maximum deviation is less than 25%, R2>0.98, consistency coefficient>0.90.Testing result shows the detection kit prepared Can be good, it is suitable for clinical detection, meets the differentiation needs of the different detection occasions of different clients.
Embodiment 3：Precision test
Using the test card and measuring system of embodiment 2, the test card and fluorescence immune chromatography analyzer of the present invention are entered Row Precision Experiment.
Predominantly detect material：Clinical sample is obtained by relevant hospital, totally 2 parts of Chemiluminescence immunoassay definite value serum samples, Wherein low value definite value sample clinical measures are 0.24ng/ml, and high level definite value sample clinical measures are 1.78ng/ml.
Preparation method：
Using the test card and measuring system of embodiment 2, replication is respectively carried out 20 times to 2 parts of definite value samples.
Test result analysis：
After the completion of prepared by clinical sample detection reagent, clinical sample is detected by preparation method, and analyzes detection knot Fruit.
Result of the test：
As shown in table 1, the low value definite value sample and clinical measures that are 0.24ng/ml to clinical measures are 1.78ng/ Average value, standard deviation and CV, the reality of the acquired results display present invention are calculated after ml high level definite value sample replication 20 times Check system test low value definite value sample CV is 8.13%, and test high level definite value sample CV is 5.61%.Testing result shows to prepare Detection kit it is functional, be suitable for clinical detection, meet the differentiation needs of the different detection occasion of different clients.
It is same that the present invention provides a kind of cardiac muscle troponin I prepared using rare-earth fluorescent immunochromatography technique and creatine kinase Work enzyme and the three-in-one fast quantification immunochromatographytest test kit of myoglobins, while it is adapted to serum and whole blood sample, and be adapted to Clinically single part detection, relative to cardiac muscle troponin I and the three-in-one qualitative colloid of creatine kinase isozyme and myoglobins Gold reagent, cardiac muscle troponin I and creatine kinase isozyme and the three-in-one content of myoglobins in detection sample, tool can be quantified Have more specific Clinical significance of MG, have easy to operate, rapid reaction, high sensitivity, high specificity, be adapted to Site Detection and The advantages that economical and practical.

Claims (7)

1. a kind of cardiac muscle troponin I and creatine kinase isozyme and myoglobins three-in-one detection reagent box, provided with test card, It is characterized in that the test card is provided with by lower from being above sequentially provided with：PVC board, sample pad, pad, nitrocellulose filter and suction Water cushion, be wherein adsorbed with pad rare-earth fluorescent microballoon mark cardiac muscle troponin I and creatine kinase isozyme and flesh it is red The three-in-one monoclonal antibody of albumen, a diameter of 60-120nm of the rare-earth fluorescent microballoon, the rare earth doped lanthanum of rare-earth fluorescent microballoon Series elements, it is stable under ground state, launch wave-length coverage in the glimmering of 540-600nm under 340-380nm excitation source effect Light；The monoclonal antibody is the monoclonal antibody mixed after purification, from for 2-6 different cardiac muscle troponin Is And the cell strain of monoclonal antibody of creatine kinase isozyme and the three-in-one epitope of myoglobins.
2. a kind of cardiac muscle troponin I according to claim 1 and creatine kinase isozyme and the three-in-one inspection of myoglobins Test agent box, it is characterised in that the diameter of the rare-earth fluorescent microballoon of the pad is preferably 90-110nm；The rare-earth fluorescent Microballoon is doped with rare earth lanthanide.
3. a kind of cardiac muscle troponin I according to claim 2 and creatine kinase isozyme and the three-in-one inspection of myoglobins Test agent box, it is characterised in that the rare earth doped complex compound of rare-earth fluorescent microballoon；Rare-earth fluorescent microballoon marks on pad Antibody sources are in the monoclonal cell cell line for 3 different epitopes.
4. a kind of cardiac muscle troponin I according to claim 1 and creatine kinase isozyme and the three-in-one matter of myoglobins Detection kit, it is characterised in that the pad is made using following steps：Glass fibre membrane is soaked in 200mM Tris- In HCL treatment fluids, 4 DEG C are soaked 4 hours, then take out 37 DEG C of oven for drying 4 hours, standby, by glass fibre membrane in Bio- On DotXYZ3050 three-dimensional specking platforms, with the non-contact quantitation nozzles that decline of Bio-Jet Quanti300 by rare-earth fluorescent microballoon The three-in-one monoclonal antibody of cardiac muscle troponin I and creatine kinase isozyme and myoglobins of mark is sprayed onto glass fibre membrane, 37 DEG C drying 2 hours after be made.
5. a kind of cardiac muscle troponin I according to claim 4 and creatine kinase isozyme and the three-in-one matter of myoglobins Detection kit, it is characterised in that the cardiac muscle troponin I and creatine kinase of the rare-earth fluorescent microballoon mark on pad Isodynamic enzyme and the three-in-one monoclonal antibody of myoglobins are made using following steps：
Step 1：The acquisition of cell strain of monoclonal antibody：With reference to cardiac muscle troponin I and creatine kinase isozyme and myoglobins The peptide sequence of three-in-one amino acid sequence, strong artificial synthesized 20 amino acid in site of selection antigenicity or so, is linked to KLH On, the cell strain of monoclonal antibody of specific high-affinity is prepared using the method for preparing monoclonal antibody of standard, will be obtained Cell line corresponding to monoclonal antibody carry out pairing experiment and affinity determination experiment, according to experimental result determine capture antibody With detection antibody；
Step 2：The preparation of monoclonal antibody：Prepared using the ascites production technology of standard and purify the myocardium myo calcium for detection Protein I and the three-in-one monoclonal antibody of creatine kinase isozyme and myoglobins, be stored in after packing -20 DEG C it is standby；
Step 3：The aldehyde radical of rare-earth fluorescent microballoon：3mg rare-earth fluorescent microballoons are taken, with 50mM, pH9.5 carbonate buffer solution, Washed 3 times, centrifugal speed 12000rpm using centrifugal process, the time is 5 minutes, is finally resuspended in 100 μ l above-mentioned carbonate In buffer solution, the glucan of 300 μ l aldehyde radicals is added, is mixed, at room temperature dark reaction 4 hours, is washed using same centrifugal process Be resuspended in 100 μ l above-mentioned carbonate buffer solution, be placed in 4 DEG C it is standby；
Step 4：The cardiac muscle troponin I and creatine kinase isozyme and the three-in-one Dan Ke of myoglobins of rare-earth fluorescent microballoon mark The preparation of grand antibody：The cardiac muscle troponin I and creatine kinase isozyme and the three-in-one Dan Ke of myoglobins that 2mg is used to detect Grand antibody, in 4 DEG C of dialysed overnights, is then mixed, 4 DEG C with above-mentioned carbonate buffer solution with the rare-earth fluorescent microballoon of above-mentioned aldehyde radical Reaction is overnight；Then, sodium borohydride is added to final concentration 10mM, and 4 DEG C are reacted 4 hours；Add isometric confining liquid (100mM Tris-HCL, pH7.5, containing 2%BSA, 5% sucrose), 4 DEG C of closings are overnight；Then 100mM Tris-HCL are used, PH7.5 buffer solution is washed 3 times using centrifugal process, is resuspended in 100 μ l 100mM Tris-HCL buffer solutions, and 4 DEG C of lucifuges are protected Deposit standby.
6. a kind of cardiac muscle troponin I according to claim 1 and creatine kinase isozyme and the three-in-one matter of myoglobins Detection kit, it is characterised in that described to be coated with detection line and the nitrocellulose filter of nature controlling line is made by following steps：
Step 1：According to foregoing pairing experiment and affinity determination experiment, select for monoclonal antibody corresponding to the antibody of capture Cell line, prepared according to the ascites production technology of standard and purify the same work of cardiac muscle troponin I and creatine kinase for capture Enzyme and the three-in-one monoclonal antibody of myoglobins, be stored in -20 DEG C it is standby；
Step 2：Coating dilution is used respectively by cardiac muscle troponin I and the three-in-one Dan Ke of creatine kinase isozyme and myoglobins Grand antibody and goat anti-mouse igg antibody adjust concentration to 1-5mg/ml, film liquid amount be 1-2 μ l/cm, using them as detection Be sprayed on nitrocellulose filter on parallel with nature controlling line of line is coated with, and detection line and nature controlling line are subsequently placed at intervals of 3-7mm In baking oven, 37 DEG C dry 2 hours.
7. a kind of cardiac muscle troponin I according to claim 1 and creatine kinase isozyme and the three-in-one matter of myoglobins Detection kit, it is characterised in that the sample pad is made by following steps：Glass fibre membrane is soaked in containing 2.0% Triton X-100,2%BSA, 0.1M Tris buffer solutions, in pH7.5 treatment fluid, 4 hours are soaked in 4 DEG C, are subsequently placed in In baking oven, 37 DEG C dry 2 hours.
CN201710852525.1A 2017-09-19 2017-09-19 Cardiac muscle troponin I and creatine kinase isozyme and myoglobins three-in-one detection reagent box and preparation method thereof Pending CN107664700A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710852525.1A CN107664700A (en) 2017-09-19 2017-09-19 Cardiac muscle troponin I and creatine kinase isozyme and myoglobins three-in-one detection reagent box and preparation method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710852525.1A CN107664700A (en) 2017-09-19 2017-09-19 Cardiac muscle troponin I and creatine kinase isozyme and myoglobins three-in-one detection reagent box and preparation method thereof

Publications (1)

Publication Number Publication Date
CN107664700A true CN107664700A (en) 2018-02-06

Family

ID=61097964

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710852525.1A Pending CN107664700A (en) 2017-09-19 2017-09-19 Cardiac muscle troponin I and creatine kinase isozyme and myoglobins three-in-one detection reagent box and preparation method thereof

Country Status (1)

Country Link
CN (1) CN107664700A (en)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108303551A (en) * 2018-02-07 2018-07-20 浙江聚康生物工程有限公司 Human cardiac troponin I/three-in-one assay kit of myoglobins/creatine kinase isozyme and preparation method
CN111308085A (en) * 2019-12-30 2020-06-19 菲鹏生物股份有限公司 Detection method and kit for hypersensitive cardiac troponin I
CN111308084A (en) * 2019-12-30 2020-06-19 菲鹏生物股份有限公司 Detection method and kit for hypersensitive cardiac troponin I
CN112305228A (en) * 2019-07-31 2021-02-02 上海奥普生物医药股份有限公司 Myoglobin direct chemiluminescence detection kit, preparation method and application
CN113125743A (en) * 2019-12-30 2021-07-16 广东唯实生物技术有限公司 Detection method and kit for hypersensitive cardiac troponin I
CN113125742A (en) * 2019-12-30 2021-07-16 广东唯实生物技术有限公司 Detection method and kit for hypersensitive cardiac troponin I
CN113433083A (en) * 2021-06-21 2021-09-24 哈尔滨工程大学 Method for detecting ammonia concentration in water by combining active microsphere cavity and phenol red

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104714015A (en) * 2014-10-28 2015-06-17 威海纽普生物技术有限公司 Detection kit and detection method for heart-type fatty acid binding protein
CN104714033A (en) * 2014-11-28 2015-06-17 威海纽普生物技术有限公司 Procalcitonin detection kit and detection method
CN104714025A (en) * 2014-11-28 2015-06-17 威海纽普生物技术有限公司 NT-proBNP detection kit and detection method
CN104730245A (en) * 2014-11-28 2015-06-24 威海纽普生物技术有限公司 D-dimer detection kit and D-dimer detection method
CN104730251A (en) * 2014-11-28 2015-06-24 威海纽普生物技术有限公司 Troponin I detection kit and troponin I detection method

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104714015A (en) * 2014-10-28 2015-06-17 威海纽普生物技术有限公司 Detection kit and detection method for heart-type fatty acid binding protein
CN104714033A (en) * 2014-11-28 2015-06-17 威海纽普生物技术有限公司 Procalcitonin detection kit and detection method
CN104714025A (en) * 2014-11-28 2015-06-17 威海纽普生物技术有限公司 NT-proBNP detection kit and detection method
CN104730245A (en) * 2014-11-28 2015-06-24 威海纽普生物技术有限公司 D-dimer detection kit and D-dimer detection method
CN104730251A (en) * 2014-11-28 2015-06-24 威海纽普生物技术有限公司 Troponin I detection kit and troponin I detection method

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108303551A (en) * 2018-02-07 2018-07-20 浙江聚康生物工程有限公司 Human cardiac troponin I/three-in-one assay kit of myoglobins/creatine kinase isozyme and preparation method
CN112305228A (en) * 2019-07-31 2021-02-02 上海奥普生物医药股份有限公司 Myoglobin direct chemiluminescence detection kit, preparation method and application
CN111308085A (en) * 2019-12-30 2020-06-19 菲鹏生物股份有限公司 Detection method and kit for hypersensitive cardiac troponin I
CN111308084A (en) * 2019-12-30 2020-06-19 菲鹏生物股份有限公司 Detection method and kit for hypersensitive cardiac troponin I
CN111308085B (en) * 2019-12-30 2021-02-23 菲鹏生物股份有限公司 Detection method and kit for hypersensitive cardiac troponin I
CN111308084B (en) * 2019-12-30 2021-04-13 菲鹏生物股份有限公司 Detection method and kit for hypersensitive cardiac troponin I
CN113125743A (en) * 2019-12-30 2021-07-16 广东唯实生物技术有限公司 Detection method and kit for hypersensitive cardiac troponin I
CN113125742A (en) * 2019-12-30 2021-07-16 广东唯实生物技术有限公司 Detection method and kit for hypersensitive cardiac troponin I
CN113125742B (en) * 2019-12-30 2022-12-27 广东唯实生物技术有限公司 Detection method and kit for hypersensitive cardiac troponin I
CN113433083A (en) * 2021-06-21 2021-09-24 哈尔滨工程大学 Method for detecting ammonia concentration in water by combining active microsphere cavity and phenol red

Similar Documents

Publication Publication Date Title
CN104714025B (en) NT-proBNP detection kit and detection method
CN107664700A (en) Cardiac muscle troponin I and creatine kinase isozyme and myoglobins three-in-one detection reagent box and preparation method thereof
CN104714033B (en) Procalcitonin. detection kit and detection method
CN104714015B (en) Detection kit and detection method for heart-type fatty acid binding protein
CN104730251B (en) Troponin I detection kit and detection method
CN104730245B (en) D-dimer detection reagent kit and detection method
CN106706926A (en) Serum amyloid A testing kit and manufacturing method
CN106841631A (en) Cardiac muscle troponin I/N ends Natriuretic Peptide/D dimer is three-in-one to determine kit and preparation method
EP3258270B1 (en) Cardiac troponin i ultra-sensitive detection reagent kit, and ultra-sensitive detection method therefor
CN108254563B (en) Detect time-resolved fluoroimmunoassay chromatograph test strip, the kit and preparation method thereof of cTnI
CN106959372A (en) Serum amyloid A protein and the two-in-one measure kit of C reactive proteins and preparation method
CN106153927A (en) A kind of fast quantification detects time-resolved fluoroimmunoassay chromatography reagent and the preparation method of cTnI, CKMB, Myo simultaneously
CN106248958A (en) The fluorescence immune chromatography reagent of a kind of detection by quantitative cTnI and preparation method
CN105891508A (en) TRF (time-resolved fluorescence) immunochromatography reagent for rapidly and quantitatively detecting H-FABP (heart fatty acid-binding protein) and preparation method
CN106771128A (en) Parathyroid hormone determines kit and preparation method
CN106771239A (en) Serum amyloid A protein/Procalcitonin/C reactive proteins are three-in-one to determine kit and preparation method
CN109975559B (en) Kit and method for time-resolved fluorescence quantitative detection of 25-hydroxy vitamin D
CN106990254A (en) 25 hydroxycholecalciferols determine kit and preparation method
CN107703110A (en) G17 detection kit and preparation method thereof
CN105717303A (en) Method and reagent kit for detecting phosphatidylinositol proteoglycan 3 with fluorescence immunochromatographic method
CN107907676A (en) Alzheimer related neural silk-fibroin detection kit and preparation method thereof
CN106872716A (en) Serum amyloid A protein and two-in-one measure kit and the preparation method of Procalcitonin
CN106443018A (en) Myoglobin monoclonal abzyme marking compound and preparation method thereof and detection test kit
CN109188000A (en) A kind of test strips and preparation method thereof of portable inspectiont human parathyroid hormone
CN107942066A (en) Interleukin 6 detection kit and preparation method thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20180206

RJ01 Rejection of invention patent application after publication