CN110346575A - A kind of homocysteine fluorescence immune chromatography detection kit and its detection method - Google Patents
A kind of homocysteine fluorescence immune chromatography detection kit and its detection method Download PDFInfo
- Publication number
- CN110346575A CN110346575A CN201810318417.0A CN201810318417A CN110346575A CN 110346575 A CN110346575 A CN 110346575A CN 201810318417 A CN201810318417 A CN 201810318417A CN 110346575 A CN110346575 A CN 110346575A
- Authority
- CN
- China
- Prior art keywords
- homocysteine
- reagent
- immune chromatography
- fluorescence immune
- stabilizer
- 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
Links
- FFFHZYDWPBMWHY-VKHMYHEASA-N L-homocysteine Chemical compound OC(=O)[C@@H](N)CCS FFFHZYDWPBMWHY-VKHMYHEASA-N 0.000 title claims abstract description 56
- 238000001514 detection method Methods 0.000 title claims abstract description 49
- 238000004587 chromatography analysis Methods 0.000 title claims abstract description 30
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 34
- 238000012360 testing method Methods 0.000 claims abstract description 21
- 239000000872 buffer Substances 0.000 claims abstract description 13
- MEFKEPWMEQBLKI-AIRLBKTGSA-N S-adenosyl-L-methioninate Chemical compound O[C@@H]1[C@H](O)[C@@H](C[S+](CC[C@H](N)C([O-])=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 MEFKEPWMEQBLKI-AIRLBKTGSA-N 0.000 claims abstract description 12
- 239000003381 stabilizer Substances 0.000 claims abstract description 12
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims abstract description 11
- ZJUKTBDSGOFHSH-WFMPWKQPSA-N S-Adenosylhomocysteine Chemical compound O[C@@H]1[C@H](O)[C@@H](CSCC[C@H](N)C(O)=O)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZJUKTBDSGOFHSH-WFMPWKQPSA-N 0.000 claims abstract description 9
- 239000004005 microsphere Substances 0.000 claims abstract description 9
- 239000004793 Polystyrene Substances 0.000 claims abstract description 7
- 229920002223 polystyrene Polymers 0.000 claims abstract description 7
- 230000006287 biotinylation Effects 0.000 claims abstract description 6
- 238000007413 biotinylation Methods 0.000 claims abstract description 6
- 235000018417 cysteine Nutrition 0.000 claims description 14
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 14
- 108090001008 Avidin Proteins 0.000 claims description 8
- 125000002124 5'-adenosyl group Chemical group N1=CN=C2N(C=NC2=C1N)[C@H]1[C@H](O)[C@H](O)[C@H](O1)C* 0.000 claims description 7
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 6
- 229940098773 bovine serum albumin Drugs 0.000 claims description 6
- 230000004048 modification Effects 0.000 claims description 6
- 238000012986 modification Methods 0.000 claims description 6
- 239000000020 Nitrocellulose Substances 0.000 claims description 5
- 229920001220 nitrocellulos Polymers 0.000 claims description 5
- 238000004445 quantitative analysis Methods 0.000 claims description 5
- 238000003018 immunoassay Methods 0.000 claims description 4
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 claims description 4
- TZRXHJWUDPFEEY-UHFFFAOYSA-N Pentaerythritol Tetranitrate Chemical compound [O-][N+](=O)OCC(CO[N+]([O-])=O)(CO[N+]([O-])=O)CO[N+]([O-])=O TZRXHJWUDPFEEY-UHFFFAOYSA-N 0.000 claims description 3
- CHHHXKFHOYLYRE-UHFFFAOYSA-M 2,4-Hexadienoic acid, potassium salt (1:1), (2E,4E)- Chemical compound [K+].CC=CC=CC([O-])=O CHHHXKFHOYLYRE-UHFFFAOYSA-M 0.000 claims description 2
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 claims description 2
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 2
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 claims description 2
- -1 PC300 Chemical compound 0.000 claims description 2
- 239000002202 Polyethylene glycol Substances 0.000 claims description 2
- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 2
- 239000007983 Tris buffer Substances 0.000 claims description 2
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 claims description 2
- 229910052744 lithium Inorganic materials 0.000 claims description 2
- 229920001223 polyethylene glycol Polymers 0.000 claims description 2
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 2
- 235000010241 potassium sorbate Nutrition 0.000 claims description 2
- 239000004302 potassium sorbate Substances 0.000 claims description 2
- 229940069338 potassium sorbate Drugs 0.000 claims description 2
- 235000010232 propyl p-hydroxybenzoate Nutrition 0.000 claims description 2
- 239000004405 propyl p-hydroxybenzoate Substances 0.000 claims description 2
- 229960003415 propylparaben Drugs 0.000 claims description 2
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 claims description 2
- 235000010234 sodium benzoate Nutrition 0.000 claims description 2
- 239000004299 sodium benzoate Substances 0.000 claims description 2
- 229960003885 sodium benzoate Drugs 0.000 claims description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 2
- 102000004357 Transferases Human genes 0.000 claims 1
- 108090000992 Transferases Proteins 0.000 claims 1
- 230000015572 biosynthetic process Effects 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 18
- 230000002860 competitive effect Effects 0.000 abstract description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 14
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 12
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 6
- 208000024172 Cardiovascular disease Diseases 0.000 description 6
- 229960005305 adenosine Drugs 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 4
- 208000026106 cerebrovascular disease Diseases 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 230000002526 effect on cardiovascular system Effects 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 229930182817 methionine Natural products 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 2
- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108010020869 Homocysteine S-Methyltransferase Proteins 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 230000001351 cycling effect Effects 0.000 description 2
- 229960003067 cystine Drugs 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000002875 fluorescence polarization Methods 0.000 description 2
- 238000003317 immunochromatography Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 208000019553 vascular disease Diseases 0.000 description 2
- QWWVBNODQCWBAZ-WHFBIAKZSA-N (2r)-2-amino-3-[(2r)-2-carboxy-2-(methylamino)ethyl]sulfanylpropanoic acid Chemical compound CN[C@H](C(O)=O)CSC[C@H](N)C(O)=O QWWVBNODQCWBAZ-WHFBIAKZSA-N 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- NBSCHQHZLSJFNQ-GASJEMHNSA-N D-Glucose 6-phosphate Chemical compound OC1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H](O)[C@H]1O NBSCHQHZLSJFNQ-GASJEMHNSA-N 0.000 description 1
- 208000005189 Embolism Diseases 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- VFRROHXSMXFLSN-UHFFFAOYSA-N Glc6P Natural products OP(=O)(O)OCC(O)C(O)C(O)C(O)C=O VFRROHXSMXFLSN-UHFFFAOYSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- VHJLVAABSRFDPM-IMJSIDKUSA-N L-1,4-dithiothreitol Chemical compound SC[C@H](O)[C@@H](O)CS VHJLVAABSRFDPM-IMJSIDKUSA-N 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 208000035868 Vascular inflammations Diseases 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 230000009137 competitive binding Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000012631 diagnostic technique Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 208000030613 peripheral artery disease Diseases 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000036632 reaction speed Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
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
- G01N33/53—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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a kind of homocysteine fluorescence immune chromatography detection kit and its application method based on competition law.The kit includes reagent I, reagent II and fluorescence immune chromatography test paper bar, and reagent I contains dithiothreitol (DTT) (DTT), adenosylmethionine, homocysteine methyltransgerase, buffer, stabilizer；Reagent II contains biotinylation polystyrene fluorescent microsphere, buffer, stabilizer；Test strips by sample pad, the bonding pad, the NC film comprising detection line and nature controlling line, blotting paper and the PVC offset plate that are coated with biotinylated antibody.Adenosyl homocysteine is detected using competitive mode fluorescence immune chromatography method to achieve the purpose that detect homocysteine, and the detection method is easy to operate, can detect great amount of samples simultaneously, and it is few to be disturbed factor, low in cost.
Description
Technical field
The present invention relates to a kind of homocysteine fluorescence immune chromatography detection kit and its detection method, belong to quickly
Vitro diagnostic techniques field.
Background technique
Homocysteine (Homocysteine, hereinafter referred to as Hcy) is in important during methionine metabolism
Between product, under disease conditions, there is obstacle in the metabolism of enzyme, and Hcy concentration in blood increases.Du Vigneaud is first within 1932
Secondary to refer to Hcy, Carson and Neil in 1932 has found that homocysteine urinates disease in congenital dysnoesia children, and finds
These patients are susceptible to suffer from thrombotic disease, McCully in 1969 write articles for the first time propose Hcy level may be with Atherosclerosis
Change related viewpoint, Wilcken in 1976 et al. proposes that Hcy is an independent danger of cardiovascular disease by epidemiological survey
Dangerous factor.Since the nineties, there are numerous studies to be unfolded around the relationship of Hcy and cardiovascular disease, present more generally accepted viewpoint
Be the hyperhomocysteinemiainjury as caused by the metabolic disorder of Hcy be cardiovascular and cerebrovascular disease and vessel embolism morbidity
The safe range there is no so-called Hcy is thought in independent risk factor, epidemiological study, when the plasma concentration of Hcy is greater than 6.3
When μm ol/L, will increase sharply the danger for suffering from heart disease.Boushey et al. divides 4000 vascular diseases clinical patients
Analysis discovery, the total Hcy of blood plasma 5 μm of ol/L of horizontal every raising, the danger level of coronary artery disease increase by 1.6 times, cranial vascular disease danger
Dangerous degree increases by 1.8 times, and peripheral artery disease danger level increases by 6.8 times, increases to cardiovascular and cerebrovascular disease risk and cholesterol
0.5mmol/L is suitable to the danger level of cardiovascular and cerebrovascular disease.Therefore, measure blood in Hcy concentration to monitoring the state of an illness especially
Early stage vascular inflammation, observation therapeutic effect, progress clinical research, discussion pathogenesis are of great significance.
For Hcy as a small molecule, detection is more difficult, gradually develops to the detection of Hcy in blood from the eighties
After getting up, successively there is a variety of detection methods, including radiation enzyme process, gas-chromatography, liquid chromatography (HPLC), Amino acid score
Analyzer method, Enzymatic cycling and fluorescence polarization immunoassay (FPIA).Most widely used is HPLC method and FPIA, HPLC efficient liquid phase
Chromatography is using liquid as mobile phase, and HPLC uses high pressure transfusion system, by single solvent or different proportion with opposed polarity
The mobile phases such as mixed solvent, buffer be pumped into the chromatographic column equipped with stationary phase, after each ingredient is separated in column, into detection
Device is detected, to realize the analysis to sample.FPIA measurement Hcy is one of the most widely used method in recent years, by
Mohammed is put forward for the first time in nineteen ninety-five, in the method, reducing agent dithiothreitol is firstly added, while adenosine is added, in S- gland
Under the action of glycosides-L homocysteine hydrolase, SAH is converted by Hcy, then, the SAH and SAH that fluorescent marker is added are mono-
It is anti-, make the SAH of SAH and fluorescent marker and monoclonal antibody competitive binding, finally measures the fluorescence polarization degree of immune conjugate, can calculate
By the concentration for the SAH that Hcy conversion generates.Both methods is with higher sensitive and precision, but the cost of the two is relatively
Greatly.Enzymatic cycling is the newer detection method released recently, was proposed in 1961 by Lowry OH et al., is initially used to detect
Glucose 6-phosphate.In circulation enzyme reaction, two kinds of toolenzymes recycle substrate, are continuously generated determinand, and substrate is kept
Css generates pseudo first order reaction with sufficiently low concentration, thus the Css of substrate and the speed linearity totally measured
Correlation can determine the amount of substrate by measurement reaction speed.It recycles enzymatic determination sometimes referred to as " amplification " to measure, because of method
Increase the measurement sensitivity of analyte by 100 to 1000 times.This method is easy to operate, cost is relatively low, but needs by complete
The large-scale instruments such as automatic biochemistry analyzer, cannot achieve quick diagnosis.In conclusion development is simple, quick, inexpensive and quasi-
Really reliable novel homotype cysteine detection technique has great importance, and the following classification diagnosis and treatment and cardiovascular and cerebrovascular disease
The urgent need of early screening.
Summary of the invention
The technical problem to be solved by the present invention is developing a kind of simple, quickly, inexpensive sample process joint competition method
Fluorescence immune chromatography detects the kit of homotype cysteine, to meet above-mentioned application demand.
For achieving the above object, the technical solution adopted by the present invention is that:
A kind of conversion of homocysteine and fluorescence immune chromatography quantitative detecting method are established, in the effect of dithiothreitol (DTT)
Under, oxidized form homocysteine is converted into reduced form homocysteine, using adenosylmethionine as substrate, in homotype half
Under the action of cystine transmethylase, homocysteine and adenosylmethionine are separately converted to add methyllanthionine and adenosine same
Type cysteine.Product is mixed with the fluorescent microsphere that Avidin is modified and is loaded in test strips, passes through competition law fluorescence immunoassay layer
Test strips are analysed, and using the fluorescence signal value of Portable fluorescence immune quantitative analyzer test strip detection line, quantitative homotype
The concentration level of cysteine.
A kind of homocysteine fluorescence immune chromatography detection kit, it is characterised in that: comprising reagent I, reagent II and
Three parts of fluorescence immune chromatography test paper bar, wherein reagent I contains dithiothreitol (DTT), adenosylmethionine, homocysteine
Transmethylase, buffer, stabilizer；Reagent II contains Avidin modification polystyrene fluorescent microsphere, buffer, stabilizer；
Fluorescence immune chromatography test paper bar includes five: sample pad, bonding pad, nitrocellulose filter, blotting paper and PVC offset plate, adenosine are same
The biotinylated antibody of type cysteine is fixed on bonding pad, and adenosyl homocysteine-is fixed on nitrocellulose filter
Bovine serum albumin(BSA) conjugate and secondary antibody are turned by competition law quantitative detection by homocysteine as detection line and nature controlling line
Change the adenosyl homocysteine formed.
A kind of homocysteine fluorescence immune chromatography detection kit, it is characterised in that:
(1) content of each component is in every liter of reagent I solution:
(2) content of each component is in every liter of reagent II solution:
0.05~5g/L of polystyrene fluorescent microsphere of Avidin modification
5~100mM of buffer
Stabilizer 0.01~10%
(3) content of fluorescence immune chromatography test paper bar each component per cm is:
1~4 μ L/cm of biotinylation adenosyl homocysteine antibody
0.5~2 μ L/cm of adenosyl homocysteine-bovine serum albumin(BSA) conjugate
0.5~2 μ L/cm of secondary antibody
A kind of homocysteine fluorescence immune chromatography detection kit, it is characterised in that preferred condition are as follows:
(1) content of each component is in every liter of reagent I solution:
(2) content of each component is in every liter of reagent II solution:
The polystyrene fluorescent microsphere 0.1-1g/L of Avidin modification
Buffer 10-15mM
Stabilizer 0.1-0.3%
(3) content of fluorescence immune chromatography test paper bar each component per cm is:
Biotinylation adenosyl homocysteine antibody 2-3 μ L/cm
Adenosyl homocysteine-bovine serum albumin(BSA) conjugate 1-1.5 μ L/cm
Secondary antibody 0.8-1 μ L/cm
The stabilizer be Sodium azide, nitrine lithium, PC300, sodium benzoate, potassium sorbate, propylparaben,
One or more of polyethylene glycol, polyvinyl alcohol, disodium ethylene diamine tetraacetate.
The buffer is one or more of PBS, PB, Tris.
A kind of homocysteine fluorescence immune chromatography detection kit, detection method are as follows: by sample (10-
50 μ L) it is mixed with reagent I (10-50 μ L), it reacts 10-30 minutes, acquired solution is mixed with reagent II (900-980 μ L), takes 100
The μ L mixing sample is loaded onto test strips, reads the fluorescence letter of detection line after 5-15 minutes on fluorescence immunoassay quantitative analysis instrument
Number value, obtain corresponding homocysteine sample content.
The beneficial effects of the present invention are: establishing the homocysteine detection reagent based on competition law fluorescence immune chromatography
Detection can be realized by Portable fluorescence immunochromatography quantitative analysis instrument without large-scale biochemical instrument in box, is convenient for half Guang of homotype
The bedside of propylhomoserin quickly detects.The Indirect Detecting Method high specificity of this homocysteine, not will receive adenosine in sample
The interference of the homotypes cysteine knot structure analog such as methionine, cysteine does not need large-scale Biochemical Analyzer, operation letter
Just, at low cost, convenient for being promoted in clinical and community family.
Detailed description of the invention
The following further describes the present invention with reference to the drawings.
Fig. 1 is kit working principle diagram prepared by the present invention.
Fig. 2 is that kit prepared by the present invention detects linear areal map.
Fig. 3 is kit detection specificity figure prepared by the present invention.
Fig. 4 is kit detection stability diagram prepared by the present invention.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, below in conjunction with the attached drawing in the present invention, to this
Technical solution in invention is clearly and completely described, it is clear that and described embodiments are some of the embodiments of the present invention,
Instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not making creative labor
Every other embodiment obtained under the premise of dynamic, shall fall within the protection scope of the present invention.
Embodiment 1: kit preparation and detection method:
The kit of the competition law fluorescence immune chromatography test paper of homocysteine in preparation detection human serum:
Reagent I: in the PBS buffer solution of 1L 10mmol/L, 5mmol/L adenosylmethionine, 50KU/L homotype half are added
Cystine transmethylase, 10mmol/L dithiothreitol (DTT), 0.1% Sodium azide stir evenly i.e. reagent I.
Reagent II: in the PBS buffer solution of 1L 10mmol/L, 100mg Avidin fluorescent microsphere, 0.1% nitrine are added
Sodium stirs evenly i.e. reagent II.
Test strips: its structure is as shown in Figure 1.There is 0.8 μ L biotinylation adenosine in the test strips of every 4mm, on bonding pad
Homocysteine antibody has 0.4 μ L adenosyl homocysteine-bovine serum albumin(BSA) conjugate on nitrocellulose filter,
0.32 μ L secondary antibody.
Detection method is as follows:
By taking fluorescence immune chromatography quantitative analysis instrument as an example: sample size is 20 μ L, and reagent I is 20 μ L, and reagent II is 960 μ L.
It after sample is mixed with reagent I, reacts 20 minutes, reagent II is added, takes in 100 μ L sample applications to test strips, glimmering after 8 minutes
The fluorescence signal value that detection line is read on light immune quantitative analyzer, obtains corresponding homocysteine sample content.
1. linear detection range measures:
With homocysteine (1,5,10,15,30,40,50 μm of ol/L) titer of calf serum compound concentration gradient,
Each pattern detection three times, takes its average value, and standardization fluorescence signal value is fitted with homotype semicystinol concentration investigating value, is obtained
To equation of linear regression: y=-0.00928x+0.92122, r2=0.99238, P < 0.05, this method are dense in 1-50 μm of ol/L
It is linear preferable in degree range, as shown in Fig. 2.
2. anti-interference is tested
There is adenosylmethionine due to containing cysteine and adenosylmethionine in serum, while in the reaction of R1 reagent
It participates in, therefore the specificity of homocysteine competitive mode fluorescence immune chromatography Test paper is verified.By homotype half
Cystine, adenosylmethionine and cysteine are configured to the ladder of 1.0,5.0,10.0,20.0,30.0,40.0,50.0 μm of ol/L
Solution is spent, detects the relationship that its fluorescence signal value of standardizing changes with concentration, as shown in Fig. 3, kit of the present invention is by adenosine
The interference of the homotypes cysteine knot structure analog such as methionine and cysteine is smaller, has detection specificity well.
3. detecting Precision Experiment
Prepare low value, 5,15 and 40 μm of ol/L of homocysteine titer of intermediate value and high level, each pattern detection 15
It is secondary, calculate its coefficient of variation.The result shows that the present invention detects repeatability preferably, the coefficient of variation (CV value) of level 1 (7 μm of ol/L)
It is 8.51%, the CV value of level 2 (15 μm of ol/L) is 4.00%, and the CV value of level 3 (40 μm of ol/L) is 4.47%, meets detection
It is required that.
4. stability experiment
A collection of homocysteine fluorescence immune chromatography detection kit is prepared, 4 DEG C are stored 1 year, at interval of a monthly test
Same concentration homocysteine sample is surveyed, observes the variation of its fluorescence signal value of standardizing, as shown in Fig. 4.As can be seen that
Kit of the present invention detected value in 1 year storage process is held essentially constant, and performance is stablized, and is laid the foundation for practical application.
The present invention utilizes homocysteine methyltransgerase, using adenosylmethionine as methyl donor, half Guang of homotype
Propylhomoserin is converted into adenosyl homocysteine as substrate, by adenosylmethionine, and homocysteine is converted into methionine,
Gained adenosyl homocysteine is directly proportional to homotype semicystinol concentration investigating.The detection range of linearity of the kit is 1-50 μ
Mol/L, the present invention is small by the interference of the analogues such as adenosylmethionine and cysteine, improves homocysteine inspection
The accuracy and sensitivity of survey.This kit need to only complete detection, operation on Portable fluorescence immunochromatography quantitative analysis instrument
Simply, price is more cheap relative to other homotype cysteine detecting methods, is conducive to the marketing of kit.
It is enlightenment with the embodiment of aforementioned present invention kit, through the above description, relevant staff is complete
Various changes and amendments can be carried out without departing from the scope of the technological thought of the present invention'.This invention it is technical
Range is not limited to the contents of the specification, it is necessary to which the technical scope thereof is determined according to the scope of the claim.
Claims (6)
1. a kind of homocysteine fluorescence immune chromatography detection kit, it is characterised in that: comprising reagent I, reagent II and glimmering
Three parts of light immuno-chromatographic test paper strip, wherein reagent I contains dithiothreitol (DTT), adenosylmethionine, homocysteine first
Based transferase, buffer, stabilizer；Reagent II contains Avidin modification polystyrene fluorescent microsphere, buffer, stabilizer；It is glimmering
Light immuno-chromatographic test paper strip includes five: sample pad, bonding pad, nitrocellulose filter, blotting paper and PVC offset plate, adenyhomotype
The biotinylated antibody of cysteine is fixed on bonding pad, and adenosyl homocysteine-ox is fixed on nitrocellulose filter
Seralbumin conjugate and secondary antibody are converted by competition law quantitative detection by homocysteine as detection line and nature controlling line
The adenosyl homocysteine of formation.
2. a kind of homocysteine fluorescence immune chromatography detection kit according to claim 1, it is characterised in that:
The content of each component is in every liter of reagent I solution:
The content of each component is in every liter of reagent II solution:
0.05~5g/L of polystyrene fluorescent microsphere of Avidin modification
5~100mM of buffer
Stabilizer 0.01~10%
The content of fluorescence immune chromatography test paper bar each component per cm is:
1~4 μ L/cm of biotinylation adenosyl homocysteine antibody
0.5~2 μ L/cm of adenosyl homocysteine-bovine serum albumin(BSA) conjugate
0.5~2 μ L/cm of secondary antibody
3. a kind of homocysteine fluorescence immune chromatography detection kit according to claim 2, which is characterized in that
The content of each component is in every liter of reagent I solution:
The content of each component is in every liter of reagent II solution:
The polystyrene fluorescent microsphere 0.1-1g/L of Avidin modification
Buffer 10-15mM
Stabilizer 0.1-0.3%
The content of fluorescence immune chromatography test paper bar each component per cm is:
Biotinylation adenosyl homocysteine antibody 2-3 μ L/cm
Adenosyl homocysteine-bovine serum albumin(BSA) conjugate 1-1.5 μ L/cm
Secondary antibody 0.8-1 μ L/cm
4. a kind of homocysteine fluorescence immune chromatography detection kit according to claim 2, it is characterised in that: institute
The stabilizer stated be Sodium azide, nitrine lithium, PC300, sodium benzoate, potassium sorbate, propylparaben, polyethylene glycol,
One or more of polyvinyl alcohol, disodium ethylene diamine tetraacetate.
5. a kind of homocysteine fluorescence immune chromatography detection kit according to claim 2, it is characterised in that: institute
Stating buffer is one or more of PBS, PB, Tris.
6. a kind of detection method of homocysteine fluorescence immune chromatography detection kit according to claim 1 are as follows:
Sample (10-50 μ L) is mixed with reagent I (10-50 μ L), is reacted 10-30 minutes, acquired solution and reagent II (900-980 μ L)
Mixing takes the 100 μ L mixing samples to be loaded onto test strips, reads and detects on fluorescence immunoassay quantitative analysis instrument after 5-15 minute
The fluorescence signal value of line obtains corresponding homocysteine sample content.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810318417.0A CN110346575A (en) | 2018-04-04 | 2018-04-04 | A kind of homocysteine fluorescence immune chromatography detection kit and its detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810318417.0A CN110346575A (en) | 2018-04-04 | 2018-04-04 | A kind of homocysteine fluorescence immune chromatography detection kit and its detection method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110346575A true CN110346575A (en) | 2019-10-18 |
Family
ID=68173254
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810318417.0A Pending CN110346575A (en) | 2018-04-04 | 2018-04-04 | A kind of homocysteine fluorescence immune chromatography detection kit and its detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110346575A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113848330A (en) * | 2021-11-30 | 2021-12-28 | 山东子峰生物技术有限公司 | Detection method of homocysteine and vitamin B12, detection test strip and application thereof |
| CN114577875A (en) * | 2022-03-21 | 2022-06-03 | 上海健康医学院 | Electrochemical immunodetection method for total homocysteine |
Citations (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050009128A1 (en) * | 2003-07-10 | 2005-01-13 | Chong-Sheng Yuan | Methods and compositions for assaying homocysteine |
| CN1773283A (en) * | 2005-11-11 | 2006-05-17 | 袁洪 | Integrated testing method for protein marker rolated to predicting cardiocerebrovascular diseases accuring |
| US20070259376A1 (en) * | 2005-12-22 | 2007-11-08 | Toru Yoshimura | Homocysteine immunoassay |
| CN101246177A (en) * | 2007-02-14 | 2008-08-20 | 北京华安佛医药研究中心有限公司 | Homo-type cysteine immunity colloidal gold detecting test paper and method for producing the same |
| KR20090012382A (en) * | 2007-07-30 | 2009-02-04 | 한국과학기술연구원 | Protein marker adenosylhomocysteinase for colon cancer diagnosis and diagnosis kit for colon cancer using antibodies against the same |
| CN101592666A (en) * | 2008-05-30 | 2009-12-02 | 北京华安佛医药研究中心有限公司 | Homocysteine-folic acid Immune colloidal gold by chromatography combined detection card and preparation method thereof |
| CN202149903U (en) * | 2011-05-19 | 2012-02-22 | 博阳生物科技（上海）有限公司 | Time-resolved fluorescence immune chromatography quantitative detection test strip for C-reactive protein |
| CN102841207A (en) * | 2011-06-24 | 2012-12-26 | 北京乐普医疗科技有限责任公司 | Fluorescent immune chromatographic test strip for quantitively detecting troponin I and preparation method thereof |
| CN102967713A (en) * | 2012-11-29 | 2013-03-13 | 深圳市伯劳特生物制品有限公司 | Homocysteine detection kit and preparation method thereof |
| CN103172941A (en) * | 2011-12-26 | 2013-06-26 | 苏州新波生物技术有限公司 | Polystyrene fluorescent nanometer particle as well as preparation method and applications thereof |
| CN104630324A (en) * | 2015-02-28 | 2015-05-20 | 北京爱必信生物技术有限公司 | Improved homocysteine detection reagent and method |
| CN104991076A (en) * | 2015-07-06 | 2015-10-21 | 同昕生物技术（北京）有限公司 | Lateral chromatography system and application thereof |
| CN205538994U (en) * | 2016-04-26 | 2016-08-31 | 河南美凯生物科技有限公司 | Highly sensitive time -resolved fluorescence immunity chromatography detect reagent device |
| CN106053830A (en) * | 2016-05-31 | 2016-10-26 | 安徽伊普诺康生物技术股份有限公司 | Kit for determining homocysteine and preparation method thereof |
| US20160349251A1 (en) * | 2015-05-25 | 2016-12-01 | Xiujuan Hao | Use of fluorescence for the quick and easy determination of s-adenosylmethionine, s-adenosylhomocysteine and homocysteine |
| CN106198995A (en) * | 2016-06-24 | 2016-12-07 | 常州英赞美科生物科技有限公司 | A kind of homotype semicystionl diagnostic kit and the assay method of homotype semicystinol concentration investigating |
-
2018
- 2018-04-04 CN CN201810318417.0A patent/CN110346575A/en active Pending
Patent Citations (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050009128A1 (en) * | 2003-07-10 | 2005-01-13 | Chong-Sheng Yuan | Methods and compositions for assaying homocysteine |
| CN1773283A (en) * | 2005-11-11 | 2006-05-17 | 袁洪 | Integrated testing method for protein marker rolated to predicting cardiocerebrovascular diseases accuring |
| US20070259376A1 (en) * | 2005-12-22 | 2007-11-08 | Toru Yoshimura | Homocysteine immunoassay |
| CN101246177A (en) * | 2007-02-14 | 2008-08-20 | 北京华安佛医药研究中心有限公司 | Homo-type cysteine immunity colloidal gold detecting test paper and method for producing the same |
| KR20090012382A (en) * | 2007-07-30 | 2009-02-04 | 한국과학기술연구원 | Protein marker adenosylhomocysteinase for colon cancer diagnosis and diagnosis kit for colon cancer using antibodies against the same |
| CN101592666A (en) * | 2008-05-30 | 2009-12-02 | 北京华安佛医药研究中心有限公司 | Homocysteine-folic acid Immune colloidal gold by chromatography combined detection card and preparation method thereof |
| CN202149903U (en) * | 2011-05-19 | 2012-02-22 | 博阳生物科技（上海）有限公司 | Time-resolved fluorescence immune chromatography quantitative detection test strip for C-reactive protein |
| CN102841207A (en) * | 2011-06-24 | 2012-12-26 | 北京乐普医疗科技有限责任公司 | Fluorescent immune chromatographic test strip for quantitively detecting troponin I and preparation method thereof |
| CN103172941A (en) * | 2011-12-26 | 2013-06-26 | 苏州新波生物技术有限公司 | Polystyrene fluorescent nanometer particle as well as preparation method and applications thereof |
| CN102967713A (en) * | 2012-11-29 | 2013-03-13 | 深圳市伯劳特生物制品有限公司 | Homocysteine detection kit and preparation method thereof |
| CN104630324A (en) * | 2015-02-28 | 2015-05-20 | 北京爱必信生物技术有限公司 | Improved homocysteine detection reagent and method |
| US20160349251A1 (en) * | 2015-05-25 | 2016-12-01 | Xiujuan Hao | Use of fluorescence for the quick and easy determination of s-adenosylmethionine, s-adenosylhomocysteine and homocysteine |
| CN104991076A (en) * | 2015-07-06 | 2015-10-21 | 同昕生物技术（北京）有限公司 | Lateral chromatography system and application thereof |
| CN205538994U (en) * | 2016-04-26 | 2016-08-31 | 河南美凯生物科技有限公司 | Highly sensitive time -resolved fluorescence immunity chromatography detect reagent device |
| CN106053830A (en) * | 2016-05-31 | 2016-10-26 | 安徽伊普诺康生物技术股份有限公司 | Kit for determining homocysteine and preparation method thereof |
| CN106198995A (en) * | 2016-06-24 | 2016-12-07 | 常州英赞美科生物科技有限公司 | A kind of homotype semicystionl diagnostic kit and the assay method of homotype semicystinol concentration investigating |
Non-Patent Citations (2)
| Title |
|---|
| JAMES G DONNELLY等: "Evaluation of the Abbott IMx TM ¯uorescence polarization immunoassay and the Bio-Rad enzyme immunoassay for homocysteine: comparison with high-performance liquid chromatography", 《ANN CLIN BIOCHEM》, vol. 37 * |
| 樊兵;: "酶比色法与HPLC法测定血清同型半胱氨酸的相关性研究", 中国医药导刊, no. 06 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113848330A (en) * | 2021-11-30 | 2021-12-28 | 山东子峰生物技术有限公司 | Detection method of homocysteine and vitamin B12, detection test strip and application thereof |
| CN114577875A (en) * | 2022-03-21 | 2022-06-03 | 上海健康医学院 | Electrochemical immunodetection method for total homocysteine |
| CN114577875B (en) * | 2022-03-21 | 2024-01-19 | 上海健康医学院 | Electrochemical immunodetection method for total homocysteine |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zuk et al. | Enzyme immunochromatography--a quantitative immunoassay requiring no instrumentation. | |
| CN102628864B (en) | Kit for determining heart-type fatty acid binding protein in serum or urine by latex enhanced turbidimetric immunoassay | |
| US4604365A (en) | Immunoprecipitation assay | |
| CN109541241B (en) | A kind of assay kit of lipoprotein (a) | |
| US5705353A (en) | Method of reducing interferences in assays | |
| CN105891508A (en) | TRF (time-resolved fluorescence) immunochromatography reagent for rapidly and quantitatively detecting H-FABP (heart fatty acid-binding protein) and preparation method | |
| CN104345149A (en) | Immunochromatography test strip for detecting glycosylated hemoglubin and preparation method thereof | |
| JPH0629852B2 (en) | Quantitative analysis method of test substance in liquid sample using biased dry analysis element | |
| CN106370860A (en) | Kit and test paper strip for serum immunoglobulin E colloidal gold chromatography quantitative detection | |
| Dati et al. | Proposals from the IFCC Committee on Standardization of Markers of Cardiac Damage (C-SMCD): strategies and concepts on standardization of cardiac marker assays | |
| CN110346575A (en) | A kind of homocysteine fluorescence immune chromatography detection kit and its detection method | |
| CN104569431B (en) | Homogenous phase fluorescence immunoassay reagent group for fast and quantitatively detecting troponin I and preparation method thereof | |
| CN106324240A (en) | Enzyme-linked immunoassay kit for detecting chlorpyrifos and application of kit | |
| CN109298178A (en) | Cardiac myosin binding protein C(cMyBP-C based on immunomagnetic beads) time-resolved fluoroimmunoassay kit | |
| CN101315379A (en) | Reagent kit for detecting Ractopamine and application thereof | |
| JP6574839B2 (en) | Accurate assay measurement of hydrophobic hapten analytes | |
| CN102967713A (en) | Homocysteine detection kit and preparation method thereof | |
| CN105131105A (en) | Cortisol immunogen, derivative, antibody, detection reagent and preparation method | |
| CN104569430B (en) | A kind of homogeneous fluorescent immunoreagent group of Quantitative detection H-FABP and preparation method thereof | |
| CN106501526A (en) | A kind of combination directing agent optimizes latex coupled antibody detection prealbumin（PA）Test kit | |
| CN110596378A (en) | Multichannel universal chromatography method for detecting small molecules, test strip and kit | |
| JPH08304406A (en) | Calibrator matrix | |
| CN107860930A (en) | The immunoturbidimetry detection reagent and method of a kind of cardic fatty acid binding protein | |
| US20140011190A1 (en) | Method for performing a rapid test | |
| CN108593905A (en) | A kind of digoxin immune detection reagent and its preparation and detection method |
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 | ||
| DD01 | Delivery of document by public notice | ||
| DD01 | Delivery of document by public notice |
Addressee: Geng Xin Document name: Notice of First Examination Opinion |
|
| DD01 | Delivery of document by public notice |
Addressee: Geng Xin Document name: Deemed withdrawal notice |