CN102827934A - Construction method of immunosensor for measuring DNA (Deoxyribose Nucleic Acid) mark of melamine - Google Patents
Construction method of immunosensor for measuring DNA (Deoxyribose Nucleic Acid) mark of melamine Download PDFInfo
- Publication number
- CN102827934A CN102827934A CN2012103094641A CN201210309464A CN102827934A CN 102827934 A CN102827934 A CN 102827934A CN 2012103094641 A CN2012103094641 A CN 2012103094641A CN 201210309464 A CN201210309464 A CN 201210309464A CN 102827934 A CN102827934 A CN 102827934A
- Authority
- CN
- China
- Prior art keywords
- dna
- melamine
- trimeric cyanamide
- damping fluid
- ultrafiltration
- 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.)
- Granted
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a construction method of an immunosensor for measuring the DNA (Deoxyribose Nucleic Acid) mark of melamine, belonging to the field of immune PCRs (Polymerase Chain Reactions). The method comprises the following steps of: preparing a DNA-melamine antibody conjugate; coating a PCR tube with a melamine coating antigen; and constructing a melamine immunosensor. The invention provides a DNA-melamine antibody conjugate which is subjected to immunological recognition and fluorescent quantitation PCR amplification of DNA, and an amplified fluorescence signal is used for detecting melamine. The method can be used for detecting melamine at ultrahigh sensitivity, and has the advantages of low detection limit, high detection sensitivity and high specificity; and an effective method is provided for the detection of a trace quantity of melamine medicaments.
Description
Technical field
A kind of construction process of immunosensor of the dna marker of measuring trimeric cyanamide belongs to the immuno-PCR field.
Background technology
(Melamine MEL) is commonly called as melamine, extract of protein to trimeric cyanamide, is a kind of three type of tremnbling nitrogen heterocyclic ring organic cpds, is used as industrial chemicals, extensively applies to industries such as timber, plastics, coating, papermaking, weaving, leather, electric, medicine.Trimeric cyanamide is harmful to health, is not useable for food-processing or food additives.Yet because trimeric cyanamide has very high nitrogen content; Foodstuffs industry nitrogen determination commonly used is measured the nitrogen total amount and is estimated Protein content; Therefore trimeric cyanamide is often by the additive of illegal businessman as food and feed, to promote the protein contnt index in the food inspection.On October 8th, 2008; The Ministry of Health, the Ministry of Industry and Information Technology, the Ministry of Agriculture, State Administration for Industry and Commerce and State Administration for Quality Supervision and Inspection and Quarantine unite the issue bulletin; Formulate the temporary control and education value of trimeric cyanamide in breast and milk-product: the value of limiting the quantity of of trimeric cyanamide is 1.0mg/kg in the baby formula milk powder, and the value of limiting the quantity of of trimeric cyanamide is 2.5mg/kg (L) in liquid state milk (comprising raw dairy), milk powder, other prescription emulsifiable powder.The value of limiting the quantity of that contains trimeric cyanamide in breast other food more than 15% is higher than with the product of upper limit amount value and must not sells without exception for 2.5mg/kg (L).At present, the gold standard that is used for melamine residual thing detection by quantitative is chromatogram/mass spectroscopy, but it needs complicated sample pre-treatment process and expensive testing cost.ELISA method and immuno-chromatographic test paper strip, required detection time is short relatively, but its detectability is limited.Therefore, need a kind of simple, quick, sensitive, economic trimeric cyanamide detection method of exploitation.
Based on the enzyme linked immunological ELISA method and the immunochromatography technique of antigen antibody reaction, respectively with horseradish peroxidase (HRP) and Radioactive colloidal gold as the signal tagged molecule, receive the restriction of its sensitivity etc., detection level is limited.
Real time fluorescent quantitative poly Kettenreaktion (RT-qPCR) is a kind of method that DNA is increased, simultaneously the accurate content of the DNA of denier in the quantitative sample of sensitive.Therefore,, as the tagged molecule that detects, can bring more superiority with DNA based on RT-qPCR superpower amplification effect and quantitative analysis, as: detectability is low, highly sensitive, specificity is good, simple to operate and high throughput analysis etc.
Antigen-antibody immunity recognition principle combines the amplification analysis of DNA, through the fluorescent signal that DNA cloning produces, the content of indirect measurement target compound to be checked.The great advantage of present method is to realize that trimeric cyanamide flies to restrain the detection level of level, is a kind of method that realizes that at present the trimeric cyanamide detection sensitivity is the highest.
Summary of the invention
The object of the present invention is to provide a kind of immunosensor of dna marker, through amplification and the quantitative effect of immunity identification and PT-qPCR, the fluorescent signal that produces through DNA cloning carries out indirect detection to trimeric cyanamide.
Technical scheme of the present invention: a kind of construction process of immunosensor of the dna marker of measuring trimeric cyanamide comprises: the preparation of DNA-melamine antibody conjugate, trimeric cyanamide envelope antigen encapsulate PCR pipe, the structure of trimeric cyanamide transmitter; Concrete steps are:
(1) preparation of DNA-melamine antibody conjugate
2.5mg the two exclusive-OR function coupling agent 4-(N-maleimide methyl) of group hexanaphthenes-1-carboxylic acid sulfonic group succinimide ester sodium salt (Sulfo-SMCC) is dissolved in 200 μ L ultrapure waters; Get 2 μ LSulfo-SMCC solution and be diluted to 200 μ L with the 100mM PBS damping fluid that pH7.4 contains 150mM NaCl; The melamine antibody of getting 200 μ L 6mg/mL then mixes with 200 μ L Sulfo-SMCC diluents; Making its final volume is 400 μ L; In room temperature oscillatory reaction 30min, using molecular weight cut-off is that 3000 ultrafiltration pipe carries out ultrafiltration to reactant, to remove unnecessary coupling agent; The ultrafiltration trapped substance is with the 100mM PBS damping fluid dissolving that contains 5mM EDTA and recover its original volume 400 μ L, gets the above-mentioned solvent soln of 200 μ L and is used for step reaction down; The ssDNA of 200 μ L 4nmol 89bp is joined in the solvent soln of 200 μ L; Room temperature oscillatory reaction 30min, the reaction mixture is with the ultrafiltration pipe ultrafiltration of molecular weight cut-off 50000, to remove unconjugated DNA; This step ultrafiltration twice is to guarantee that DNA is removed fully; Last trapped substance contains the 100mM PBS damping fluid dissolving of 5mM EDTA again with 400 μ L, obtain the good DNA-melamine antibody conjugate of coupling;
The ssDNA of said 89bp is:
5’-SH-GGGAAAATGC?AAGAAGAAGT?CATTAGTCCT?AGACAACGTT?ACTATAACGT?GAATGTAATG?AACCTACAAG?ACCTTCCAGA?TTTTTCGGC-3’；
(2) the trimeric cyanamide envelope antigen encapsulates the PCR pipe
At first the glutaraldehyde solution with the effective 50 μ L 0.8% of PCR encapsulates 5h at 37 ℃, then will with ultrapure water
PCR pipe washing three times, each 5min; Trimeric cyanamide envelope antigen with 50 μ L, 8 μ g/mL encapsulates the PCR pipe; Encapsulate 2h at 37 ℃; With pH7.2, contain the PBST damping fluid washing of 0.05% Tween-20,10mM PBS; Again with pH7.2, contain 0.4% gelatin, 10mM PBS sealing damping fluid in 37 ℃ of sealing 2h, sealing finishes the back with above-mentioned PBST damping fluid washes clean; Above washing step all washs 3 times, each 3min;
(3) structure of trimeric cyanamide transmitter
In the PCR pipe that step (2) encapsulates; The trimeric cyanamide standard substance that add ten times of gradient dilutions of 25 μ L 0.001pg/g～10pg/g; Add 25 μ L step (1) synthetic DNA-melamine antibody conjugates again, behind 37 ℃ of reaction 30min, with PBST damping fluid washing 3 times; Each 3min pats the PCR pipe clean at last;
Upstream primer and downstream primer are joined in the SsoFast EvaGreen premixed liquid (moisten inferior biotechnology Development Co., Ltd available from Nanjing; No. 86, yulan road, Nanjing); Make the ultimate density of upstream primer and downstream primer be 20nM; With the abundant mixing of premixed liquid, add the premixed liquid that 50 μ L contain upstream primer and downstream primer in each PCR pipe, measure with quantitative real time PCR Instrument CFX-96 at last.Amplification condition is: 95 ℃ of preparatory sex change 30s at first, and 95 ℃ of sex change 5s, 57 ℃ of annealing and extend 30s then are total up to 39 circulations; Be warming up to 95 ℃ from 65 ℃ again, per 0.5 ℃ is read the first order fluorescence value, obtains the DNA melting curve;
Upstream primer: 5 '-GGGAAAATGCAAGAAGAAGTCAT-3 ',
Downstream primer: 5 '-GCCGAAAAATCTGGAAGGTC-3 '.
Synthesizing of said trimeric cyanamide envelope antigen: adopt mixed anhydride method to be coupled to carrier proteins OVA haptin MEL-ACA (hexosamine) and go up the preparation coating antigen.20mg haptin MEL-ACA is added 1mL N, dinethylformamide (DMF) dissolving, 4 ℃ add 20 μ L tri-n-butylamines and 16 μ L isobutyl chlorocarbonates down, and reaction 2h obtains A liquid under 4 ℃ of stirrings; Take by weighing 100mg OVA be dissolved in the 8mL borate buffer solution (0.2mol/L, pH9.0) in, be B liquid; Under the room temperature magnetic agitation, B liquid slowly is added drop-wise in the A liquid, and under agitation condition, continues reaction 3h, during add the phosphate buffered saline buffer of 2mL at twice altogether, promptly obtain the artificial antigen mixed solution; The artificial antigen mixed solution is moved in the dialysis tubing; Phosphate buffered saline buffer with 0.01mol/L, pH7.2 was dialysed 3-4 days; Change 2 hypophosphite damping fluids every day; Promptly obtain artificial antigen: trimeric cyanamide-bovine serum albumin (this compound method with reference to the applicant's patented claim: a kind of compound method of artificial antigen of melamine, publication number are CN101402683A).Dialysis finishes back centrifuging and taking supernatant, is sub-packed in the 0.5mL centrifuge tube, places-20 ℃ of preservations for use.
The preparation of said melamine antibody: with above-mentioned trimeric cyanamide-bovine serum albumin as immunogen; Mix the fully emulsified back injection 6-8 female BALB/C mice in all ages with the equivalent Freund's complete adjuvant; Take the subcutaneous multi-point injection of nape portion, immunizing dose is 100 μ g/, and every interval 3 all booster immunizations once; Treat to adopt abdominal injection to impact immunity after serum titer reaches requirement, dashing and exempting from dosage is 50 μ g/; Pick out the mouse that is used for cytogamy in merging the immunity of making a spurt of first three sky, merge the same day, aseptic taking-up spleen; The preparation splenocyte suspension also is collected in the 50mL centrifuge tube; The mixed that splenocyte and myeloma cell SP20 are pressed 5-10 ︰ 1 is carried out cytogamy according to ordinary method in the 50mL fusion pipe, cell is cultivated with the HAT nutrient solution; The every hole 100 μ L of 96 porocyte culture plates put 37 ℃ of 5%CO
2Cultivate in the incubator; Adopt limiting dilution assay that the positive hybridoma cell that screens is carried out subclone; With the cell enlarged culturing of test positive or carry out subclone next time; Carry out subclone continuously three times, and for the third time all clones of subclone the hole is all positive can set up cell strain; Select healthy BALB/C mice for use; The abdominal injection Yellow Protopet 2A, every mouse 0.5mL is after 7-10 days; 3 times hybridoma cell strain is washed in the abdominal cavity inoculation with the RPMI-1640 basal liquid; Obviously expand like belly, can gather ascites when skin has tight sense when touching with hand, the centrifugal 20min of ascites 12000rpm that gathers; Adopt sad-ammonium sulfate precipitation method to carry out the purifying of ascites, promptly obtain the trimeric cyanamide monoclonal antibody behind the purifying, place-20 ℃ subsequent use.
Beneficial effect of the present invention: the invention provides a kind of immunosensor of dna marker, through amplification and the quantitative effect of immunity identification and PT-qPCR, the fluorescent signal that produces through DNA cloning carries out indirect detection to trimeric cyanamide.
Description of drawings
Fig. 1 trimeric cyanamide typical curve.
The amplification curve that DNA cloning obtains in Fig. 2 DNA-melamine antibody mixture.
The melting curve that double-stranded DNA fusion after Fig. 3 amplification obtains.
Embodiment
Embodiment 1
(1) preparation of DNA-melamine antibody conjugate
2.5mg the two exclusive-OR function coupling agent 4-(N-maleimide methyl) of group hexanaphthenes-1-carboxylic acid sulfonic group succinimide ester sodium salt (Sulfo-SMCC) is dissolved in 200 μ L ultrapure waters; Get 2 μ LSulfo-SMCC solution and be diluted to 200 μ L with the 100mM PBS damping fluid that pH7.4 contains 150mM NaCl; The melamine antibody of getting 200 μ L 6mg/mL then mixes with 200 μ L Sulfo-SMCC of above-mentioned dilution; Making its final volume is 400 μ L; In room temperature oscillatory reaction 30min, using molecular weight cut-off is that 3000 ultrafiltration pipe carries out ultrafiltration to reactant, to remove unnecessary coupling agent; The ultrafiltration trapped substance is with the 100mM PBS damping fluid dissolving that contains 5mM EDTA and recover its original volume 400 μ L; Get the above-mentioned solvent soln of 200 μ L and be used for step reaction down; The ssDNA of 200 μ L 4nmol 89bp is joined in the lysate of 200 μ L; Room temperature oscillatory reaction 30min, the reaction mixture carries out ultrafiltration with the ultrafiltration pipe of molecular weight cut-off 50000, to remove unconjugated DNA; This step ultrafiltration twice is to guarantee that DNA is removed fully; Last trapped substance contains the 100mM PBS damping fluid dissolving of 5mM EDTA again with 400 μ L, obtain the good DNA-melamine antibody conjugate of coupling;
The ssDNA of said 89bp is:
5’-SH-GGGAAAATGC?AAGAAGAAGT?CATTAGTCCT?AGACAACGTT?ACTATAACGT?GAATGTAATG?AACCTACAAG?ACCTTCCAGA?TTTTTCGGC-3’；
(2) the trimeric cyanamide envelope antigen encapsulates the PCR pipe
At first the glutaraldehyde solution with the effective 50 μ L 0.8% of PCR encapsulates 5h at 37 ℃, with ultrapure water the PCR pipe is washed three times each 5min then; Trimeric cyanamide envelope antigen with 50 μ L, 8 μ g/mL encapsulates the PCR pipe; Encapsulate 2h at 37 ℃; With pH7.2, contain the PBST damping fluid washing of 0.05% Tween-20,10mM PBS; Again with pH7.2, contain 0.4% gelatin, 10mM PBS sealing damping fluid in 37 ℃ of sealing 2h, sealing finishes the back with above-mentioned PBST damping fluid washes clean; Above washing step all washs 3 times, each 3min;
(3) structure of trimeric cyanamide transmitter
In the PCR pipe that step (2) encapsulates; The trimeric cyanamide standard substance that add ten times of gradient dilutions of 25 μ L 0.001pg/g～10pg/g; Add 25 μ L step (1) synthetic DNA-melamine antibody conjugates again, behind 37 ℃ of reaction 30min, with PBST damping fluid washing 3 times; Each 3min pats the PCR pipe clean at last;
Upstream primer and downstream primer are joined in the SsoFast EvaGreen premixed liquid; Make the ultimate density of upstream primer and downstream primer be 20nM; With the abundant mixing of premixed liquid; Add the premixed liquid that 50 μ L contain upstream primer and downstream primer in each PCR pipe, measure with quantitative real time PCR Instrument CFX-96 at last.Amplification condition is: 95 ℃ of preparatory sex change 30s at first, and 95 ℃ of sex change 5s, 57 ℃ of annealing and extend 30s then are total up to 39 circulations; Be warming up to 95 ℃ from 65 ℃ again, per 0.5 ℃ is read the first order fluorescence value, obtains the DNA melting curve, is used for the specificity of validating DNA amplification;
Upstream primer: 5 '-GGGAAAATGCAAGAAGAAGTCAT-3 ',
Downstream primer: 5 '-GCCGAAAAATCTGGAAGGTC-3 '.
(4) detection sensitivity research
The DNA cloning curve that amplification obtains according to RT-qPCR; Exponential phase at amplification curve is chosen appropriate threshold; Obtain Ct value corresponding under each melamine concentration; Concentration with trimeric cyanamide is X-coordinate, and the Ct value is made a typical curve for ordinate zou, calculates melamine detection according to typical curve and is limited to 0.3fg/g.
(5) The specificity
Replace trimeric cyanamide to carry out The specificity with cynnematin as detecting target compound; The concentration of the cynnematin that adds is identical with the adding concentration of above-mentioned trimeric cyanamide; Working method is consistent with the working method that trimeric cyanamide detects, and obtains to detect the DNA cloning curve of cynnematin; Amplification curve under each cynnematin concentration of gained is positioned at same position with the amplification curve that does not add the cynnematin blank, does not discern cynnematin thereby draw the DNA-antibody coupling matter that detects trimeric cyanamide, and the specificity of this method is good.
(6) sample adds recovery research
With the centrifugal 20min of liquid state milk 12000r/min that does not contain trimeric cyanamide; Getting supernatant dilution twice uses; Add the trimeric cyanamide standard substance of 0.001pg/g, 0.01pg/g, 0.1pg/g, 0.5pg/g, 1pg/g level respectively; With the interpolation recovery of this method bioassay standard article, the recovery scope that finally obtains can be used for the mensuration of actual sample 104%～113%.
Claims (1)
1. the construction process of the immunosensor of a dna marker of measuring trimeric cyanamide, it is characterized in that comprising: the preparation of DNA-melamine antibody conjugate, trimeric cyanamide envelope antigen encapsulate PCR pipe, the structure of trimeric cyanamide immunosensor; Concrete steps are:
(1) preparation of DNA-melamine antibody conjugate
2.5mg the two exclusive-OR function coupling agent 4-(N-maleimide methyl) of group hexanaphthenes-1-carboxylic acid sulfonic group succinimide ester sodium salt Sulfo-SMCC is dissolved in 200 μ L ultrapure waters; Get 2 μ L Sulfo-SMCC solution and be diluted to 200 μ L with the 100mM PBS damping fluid that pH7.4 contains 150mM NaCl; The melamine antibody of getting 200 μ L 6mg/mL then mixes with 200 μ L Sulfo-SMCC diluents; Making its final volume is 400 μ L; In room temperature oscillatory reaction 30min, using molecular weight cut-off is that 3000 ultrafiltration pipe carries out ultrafiltration to reactant, to remove unnecessary coupling agent; The ultrafiltration trapped substance is with the 100mM PBS damping fluid dissolving that contains 5mM EDTA and recover its original volume 400 μ L, gets the above-mentioned solvent soln of 200 μ L and is used for step reaction down; The ssDNA of 200 μ L 4nmol 89bp is joined in the solvent soln of 200 μ L; Room temperature oscillatory reaction 30min, the reaction mixture is with the ultrafiltration pipe ultrafiltration of molecular weight cut-off 50000, to remove unconjugated DNA; This step ultrafiltration twice is to guarantee that DNA is removed fully; Last trapped substance contains the 100mM PBS damping fluid dissolving of 5mM EDTA again with 400 μ L, obtain the good DNA-melamine antibody conjugate of coupling;
The ssDNA of said 89bp is:
5’-SH-GGGAAAATGC?AAGAAGAAGT?CATTAGTCCT?AGACAACGTT?ACTATAACGT?GAATGTAATG?AACCTACAAG?ACCTTCCAGA?TTTTTCGGC-3’；
(2) the trimeric cyanamide envelope antigen encapsulates the PCR pipe
At first the glutaraldehyde solution with the effective 50 μ L 0.8% of PCR encapsulates 5h at 37 ℃, with ultrapure water the PCR pipe is washed three times each 5min then; Trimeric cyanamide envelope antigen with 50 μ L, 8 μ g/mL encapsulates the PCR pipe; Encapsulate 2h at 37 ℃; With pH7.2, contain the PBST damping fluid washing of 0.05% Tween-20,10mM PBS; Again with pH7.2, contain 0.4% gelatin, 10mM PBS sealing damping fluid in 37 ℃ of sealing 2h, sealing finishes the back with above-mentioned PBST damping fluid washes clean; Above washing step all washs 3 times, each 3min;
(3) structure of trimeric cyanamide transmitter
In the PCR pipe that step (2) encapsulates; The trimeric cyanamide standard substance that add ten times of gradient dilutions of 25 μ L 0.001pg/g～10pg/g; Add 25 μ L step (1) synthetic DNA-melamine antibody conjugates again, behind 37 ℃ of reaction 30min, with PBST damping fluid washing 3 times; Each 3min pats the PCR pipe clean at last;
Upstream primer and downstream primer are joined in the SsoFast EvaGreen premixed liquid; Make the ultimate density of upstream primer and downstream primer be 20nM; With the abundant mixing of premixed liquid; Add the premixed liquid that 50 μ L contain upstream primer and downstream primer in each PCR pipe, measure with quantitative real time PCR Instrument CFX-96 at last; Amplification condition is: 95 ℃ of preparatory sex change 30s at first, and 95 ℃ of sex change 5s, 57 ℃ of annealing and extend 30s then are total up to 39 circulations; Be warming up to 95 ℃ from 65 ℃ again, per 0.5 ℃ is read the first order fluorescence value, obtains the DNA melting curve;
Upstream primer: 5 '-GGGAAAATGCAAGAAGAAGTCAT-3 ',
Downstream primer: 5 '-GCCGAAAAATCTGGAAGGTC-3 '.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210309464 CN102827934B (en) | 2012-08-28 | 2012-08-28 | Construction method of immunosensor for measuring DNA (Deoxyribose Nucleic Acid) mark of melamine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210309464 CN102827934B (en) | 2012-08-28 | 2012-08-28 | Construction method of immunosensor for measuring DNA (Deoxyribose Nucleic Acid) mark of melamine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102827934A true CN102827934A (en) | 2012-12-19 |
| CN102827934B CN102827934B (en) | 2013-07-17 |
Family
ID=47331241
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201210309464 Active CN102827934B (en) | 2012-08-28 | 2012-08-28 | Construction method of immunosensor for measuring DNA (Deoxyribose Nucleic Acid) mark of melamine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102827934B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103698506A (en) * | 2013-12-31 | 2014-04-02 | 杭州爱贝亚检测技术有限公司 | Method for detecting content of pollutants in sample by utilizing direct immuno-PCR (polymerase chain reaction) assay |
| CN104193825A (en) * | 2014-08-28 | 2014-12-10 | 上海昶达生物科技有限公司 | Novel IgM-IgG polymer and polymerization method thereof |
| CN105510591A (en) * | 2014-09-22 | 2016-04-20 | 程永升 | Detection kit and detection method using antibody to modify immuno-PCR reaction |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102608073A (en) * | 2012-03-09 | 2012-07-25 | 西南大学 | Quick detection method for melamine in dairy products |
-
2012
- 2012-08-28 CN CN 201210309464 patent/CN102827934B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102608073A (en) * | 2012-03-09 | 2012-07-25 | 西南大学 | Quick detection method for melamine in dairy products |
Non-Patent Citations (2)
| Title |
|---|
| SUN,FENGXIA ET AL: "analytical methods and recent developments in the detection of melamine", 《TRENDS IN ANALYTICAL CHEMISTRY》 * |
| 张强等: "三聚氰胺DNA适体的筛选研究", 《长江大学学报（自然科学版）》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103698506A (en) * | 2013-12-31 | 2014-04-02 | 杭州爱贝亚检测技术有限公司 | Method for detecting content of pollutants in sample by utilizing direct immuno-PCR (polymerase chain reaction) assay |
| CN103698506B (en) * | 2013-12-31 | 2015-07-29 | 杭州爱贝亚检测技术有限公司 | Direct immunization PCR is utilized to detect the method for pollutant load in sample |
| CN104193825A (en) * | 2014-08-28 | 2014-12-10 | 上海昶达生物科技有限公司 | Novel IgM-IgG polymer and polymerization method thereof |
| CN105510591A (en) * | 2014-09-22 | 2016-04-20 | 程永升 | Detection kit and detection method using antibody to modify immuno-PCR reaction |
| CN105510591B (en) * | 2014-09-22 | 2019-02-12 | 程永升 | A kind of detection kit and detection method reacted using antibody modification immuno-PCR |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102827934B (en) | 2013-07-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103575889B (en) | A kind of test strips and method detecting vancomycin | |
| CN101863981A (en) | Preparation method of anti-bisphenol A monoclonal antibody | |
| CN103613563B (en) | Preparation method of benzothiostrobin hapten, artificial antigen and specific antibody and application thereof | |
| CN102703453A (en) | DNA aptamer specifically recognizing streptomycin and application of DNA aptamer | |
| CN104151416A (en) | Anti-human sST2 monoclonal antibody and application thereof | |
| CN101125889A (en) | Microcystin monoclonal antibody and its preparation method and application | |
| CN102827934B (en) | Construction method of immunosensor for measuring DNA (Deoxyribose Nucleic Acid) mark of melamine | |
| CN102127523A (en) | Hybridoma cell line and application thereof | |
| CN101251537A (en) | Kit for testing neutralizing antibody racing ELISA in human and animal rabies | |
| CN101936997B (en) | Human anti-rabies virus IgG antibody ELISA test kit | |
| CN102399753B (en) | Specific monoclonal antibody of tilletia controversa kuhn and immunofluorescent detection method | |
| CN102435729A (en) | One-step enzyme-linked immunoassay method for quickly detecting melamine and kit thereof | |
| CN102659693A (en) | 3-methylquinoxaline-2-carboxylic acid artificial antigen and antibody obtained by the 3-methylquinoxaline-2-carboxylic acid artificial antigen | |
| CN103421072A (en) | Dexamethasone semi-antigen, preparation method and applications thereof | |
| CN104387431A (en) | Preparation method of high-specificity ribavirin artificial antigens | |
| CN103698536A (en) | Oncoprotein P185 detection kit | |
| CN103254312A (en) | Mouse monoclonal antibody and its preparation method and application thereof | |
| CN103645322B (en) | A kind of chemical luminescence ELISA detection kit of streptomysin | |
| CN105372415A (en) | Method for labeling antibody with DNA | |
| CN102368070A (en) | Kit for detecting content of MR-1S (Myofibrillogenesis Regulator-1S) in human plasma and preparation method thereof | |
| CN107677807A (en) | A kind of kitasamycin magnetic immunochemiluminescence detection kit | |
| CN101921337B (en) | Antibody against lactate dehydrogenase of plasmodium vivax, related preparation method, hybridoma cell strain and application | |
| CN102690788A (en) | Zearalenone anti-idiotypic antibody, preparation method thereof, and application thereof | |
| CN105273021A (en) | Erythromycin hapten, erythromycin artificial antigen, erythromycin antibody, preparation methods of erythromycin hapten and erythromycin artificial antigen, and uses of erythromycin hapten and erythromycin antibody | |
| CN103698506A (en) | Method for detecting content of pollutants in sample by utilizing direct immuno-PCR (polymerase chain reaction) assay |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CP02 | Change in the address of a patent holder |
Address after: 214016 Jiangsu city of Wuxi Province District Liangxi No. 898 South Road 7 layer beam Creek area of food science and Technology Park Patentee after: Jiangnan University Address before: Food College of Jiangnan University No. 1800 Li Lake Avenue 214122 in Jiangsu province Wuxi City Binhu District Patentee before: Jiangnan University |
|
| CP02 | Change in the address of a patent holder |