CN103033623A - Human M2 type pyruvate kinase chemiluminescence immune assay kit and preparation method - Google Patents
Human M2 type pyruvate kinase chemiluminescence immune assay kit and preparation method Download PDFInfo
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Abstract
The invention relates to a human M2 type pyruvate kinase chemiluminescence immune assay kit and a preparation method. The kit comprises the following components: an M2 type pyruvate kinase standard substance, magnetic particles coated by M2 type pyruvate kinase monoclonal antibodies, M2 type pyruvate kinase monoclonal antibodies marked by horse radish peroxidase, chemiluminiscence substrates A and B, a white opaque microwell plate, and a washing liquid. The kit provided by the invention can accurately and quantitively detect the content of M2-PK in blood serum of a patient and is of significant guiding meaning for detection of malignant tumors such as lung cancer. Compared with the conventional ELISA (Enzyme-Llinked Immuno Sorbent Aassay) method, the kit provided by the invention is high in sensitivity, good in specificity, wide in linear range, high in stability, reliability and accuracy, and is safe, environment-friendly, and simple and convenient to operate.
Description
Technical field
The present invention relates to immunoassay and medical test technical field, concrete, the present invention relates to a kind of people M2 type pyruvate kinase chemical luminescence immune analysis reagent box and preparation method thereof.
Background technology
Cancer is main one of the disease that causes death that threatens at present human health, and cancer has surpassed the primary disease that cardiovascular and cerebrovascular disease becomes the cause of death, and people ' s health and life security in serious harm, and the trend that rises is year by year arranged.Optimal treatment period of cancer, this moment can be by operation or drug therapy with its thorough removing before cancer metastasis.Therefore the early detection early treatment is most important.Most cancer early stage patients there is no any symptom, are difficult to be found.Therefore only have and set up accurately and reliably early detection method, could find early lesion by the health check-up examination, just might in time take the measure of effectively treating, immuno analytical method provides possibility for this early screening.Recent study finds that M2 type pyruvate kinase (M2Pyruvate Kinase, M2-PK) is with a wide range of applications in the cancer diagnosis such as lung cancer, dynamic monitoring, anticancer therapeutic evaluation and prognosis are judged.
M2 type pyruvate kinase is a kind of newfound tumor markers, inquires into it in the effect of the aspects such as pulmonary cancer diagnosis, dynamic monitoring/anti-lung cancer therapeutic evaluation and prognosis evaluation as the new specific marker thing of lung cancer, has become the focus of domestic and international lung cancer research.Pyruvate kinase is a key enzyme of glycolytic pathway, also plays a decisive role in the building-up process of nucleoside triphosphate.PK has two kinds of structural genes (L gene, M gene) and four kinds of isodynamic enzymes to be respectively L-type, R type, M1 type, M2 type, and the distribution of these 4 kinds of isodynamic enzymes has tissue specificity, and often the active tetramer form with enzyme exists.The tissue specificity isodynamic enzyme (such as the M1-PK in muscle and the brain) that often shows as first PK when tumour occurs is expressed minimizing, the up-regulated of M2-PK isodynamic enzyme occurs thereupon, enzyme is transformed into the dimerization build from traditional tetramer type, is overexpression in tumour cell.So claim that this M2-PK is tumour M2-PK or TU M2-PK, M2-PK can be monitored in body fluid in a large number.When occuring, tumour often follows the increase of dimerization build M2-PK content, in the solid tumor of enough oxygen supplies (such as lung cancer) is arranged, the glutamic acid glycolysis provides a large amount of pyruvic acid, the pyruvic acid that produces in these glutamic acid glycolysis and the glycolysis is used for synthesizing lactic acid, glutamic acid and fatty acid, thereby discharge the hydrogen that produces under the effect of glyceraldehyde 3 phosphate dehydrogenasa in the glycolytic cycle, to guarantee the supply of energy in the tumour cell, as seen in tumour metabolism, bringing into play extremely important effect.Oremek G etc. are object with normal healthy controls 195 examples, patients with lung cancer 140 examples of the different pathological types of newly making a definite diagnosis, and detect the level of TU M2-PK in the blood plasma.Studies show that the horizontal contrast group of the TU M2-PK of tumor patient obviously raises, and specificity being 95%, is 78% in small-cell carcinoma of the lung, and gland cancer and non-small cell lung cancer are respectively 73% and 85%.
At present the detection method of M2-PK mainly contained ELISA method and colloidal gold method, set up a kind of enzyme-linked immunoassay method that can detect M2-PK in patient's blood plasma such as German SCHEBO BIOTEO company.The method has the advantages such as simple and efficient and pollution-free, but compares the shortcoming such as have the insufficient sensitivity height, the range of linearity is narrow with chemiluminescence.
Tumor Hispital Attached to Fudan Univ discloses " a kind of kit that detects knubble type M 2 pyruvate kinase and preparation method thereof ", this kit adopts the collaurum determination techniques, measuring the coated anti-human knubble type M 2 pyruvate kinase antibody in district, the coated anti-mouse IgG of reference region or antigen liquid; Knubble type M 2 pyruvate kinase is carried out semi-quantitative analysis, finish test in 10 minutes.Colloidal gold kit is easy to carry and preserves, and is easy and simple to handle, and fast, but accuracy is relatively poor, can't carry out accurate quantification and measure.The patent of invention that Anqun Bioengineering Co., Ltd., Shenzhen discloses a kind of " human tumor M 2-type pyruvate kinase antigen determinant polypeptide, antibody and the application on diagnostic kit thereof ", this invention mainly are preparation human tumor M 2-type pyruvate kinase antigen determinant polypeptide and polyclonal antibody.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, provide a kind of highly sensitive, detection time is short, and specificity is good, reliable and stable people M2 type pyruvate kinase chemical luminescence immune analysis reagent box.
Technical scheme of the present invention is summarized as follows:
A kind of people M2 type pyruvate kinase chemical luminescence immune analysis reagent box comprises following component:
1. M2 type pyruvate kinase standard items;
2. be coated with the magnetic particle of M2 type pyruvate kinase monoclonal antibody;
3. the M2 type pyruvate kinase monoclonal antibody of horseradish peroxidase-labeled;
4. chemical luminous substrate A and B;
5. White-opalescent microwell plate;
6. cleansing solution;
Wherein, the described magnetic particle that is coated with M2 type pyruvate kinase monoclonal antibody prepares through following steps:
1. get the 2.0ml mass content and be 2.5% magnetic particle suspension liquid, magnetic sheet divides the abandonment supernatant, is that 7.0 phosphate buffer is washed the magnetic particle once with 0.02mol/L pH value; Liquid in the described magnetic particle suspension liquid is 0.05mol/L, and the pH value is 7.4 phosphate buffer;
2. be that 7.0 phosphate buffer mixes with the magnetic particle that 1. step obtains with 0.02mol/L pH value, making cumulative volume is 2.0～5.0ml;
3. add 2.0mg M2 type pyruvate kinase monoclonal antibody, homogenize is 10～20 minutes on shaking table;
4. add 4.0～10.0mg carbodiimide, homogenize is 16～20 hours on shaking table;
5. be 7.4 phosphate buffer washing 2 times with 0.02mol/L pH value;
6. add the Tris-EDTA damping fluid, regulate cumulative volume to 2.0ml, place 2～4 ℃ of preservations.
The M2 type pyruvate kinase monoclonal antibody of described horseradish peroxidase-labeled is with following method preparation:
1. get 2.0mg M2 type pyruvate kinase monoclonal antibody, adding 0.5～1.0ml concentration is 0.02mol/L, and the pH value is 7.4 phosphate buffer dissolving, 2～4 ℃ of preservations;
2. get the horseradish peroxidase of 10.0mg, add the 2.0ml deionized water dissolving, take out 0.5～1.0ml, adding 0.2ml concentration is the NaIO of 0.1mol/L
4Aqueous solution, the lucifuge reaction is 0.5～1.5 hour under the room temperature;
3. 1. step is mixed with the liquid that 2. obtains, the lucifuge reaction is 4～6 hours under the room temperature;
4. adding 0.1ml concentration in the solution that 3. obtains to step is the NaBH of 3mg/ml
4Aqueous solution, the lucifuge reaction is 1～2 hour under the room temperature;
5. be 7.4 phosphate buffer dialysis 16～24 hours with 0.02mol/L pH value, carry out again secondarily purifiedly with HPLC, collect protein peak, add behind the equal-volume glycerine in-20 ℃ of lower freezing preservations.
Described chemical luminous substrate A makes with following method:
1. prepare 0.02mol/L pH value and be 8.0 Tris-HCl damping fluid;
2. 5～10mmol luminol is joined the 1000ml step 1. in the described damping fluid, mixing, 2～4 ℃ keep in Dark Place.
Described chemical luminous substrate B makes with following method:
1. prepare 0.02mol/L pH value and be 8.0 Tris-HCl damping fluid;
2. 0.6～1.0mmol tetraphenylboron sodium, the 1.5mmol potassium ferricyanide, 1.0mmol cinnamic acid, 10.0mmol hydrogen peroxide are joined the 1000ml step 1. in the described damping fluid, mixing, 2～4 ℃ of preservations.
Described cleansing solution is made by following method:
Get 6.05g trishydroxymethylaminomethane, 8.5g NaCl, 1.0ml Tween-20, add deionized water to 1L, transfer pH to 7.5 with HCl after the dissolving.
The preparation of M2 type pyruvate kinase standard items
With 0.02mol/L, the pH value be 7.4 phosphate buffer and hyclone by volume the ratio of 4:1 be hybridly prepared into basic damping fluid, with basic damping fluid with M2 type pyruvate kinase antigen be diluted to that concentration is 0, the standard items of 2.5U/mL, 6.4U/mL, 16U/mL, 40U/mL, 100U/mL.
Advantage of the present invention
Kit of the present invention can accurate quantitative analysis detects the content of M2-PK among the patients serum, detection for malignant tumours such as lung cancer has important directive significance, compare with traditional ELISA method, the present invention is highly sensitive, specificity is good, and the range of linearity is wide, and stability, reliability and accuracy are high, safety and environmental protection, easy and simple to handle.
Description of drawings
Fig. 1 M2-PK kit (embodiment 1) typical curve.
Embodiment
The present invention is further illustrated below in conjunction with specific embodiment.
Embodiment 1
People M2 type pyruvate kinase chemical luminescence immune analysis reagent box comprises following component:
1. M2 type pyruvate kinase standard items;
2. be coated with the magnetic particle of M2 type pyruvate kinase monoclonal antibody;
3. the M2 type pyruvate kinase monoclonal antibody of horseradish peroxidase-labeled;
4. chemical luminous substrate A and B;
5. White-opalescent microwell plate;
6. cleansing solution;
Wherein, the described magnetic particle that is coated with M2 type pyruvate kinase monoclonal antibody prepares through following steps:
1. get the 2.0ml mass content and be 2.5% magnetic particle suspension liquid, magnetic sheet divides the abandonment supernatant, is that 7.0 phosphate buffer is washed the magnetic particle once with 0.02mol/L pH value; Liquid in the described magnetic particle suspension liquid is 0.05mol/L, and the pH value is 7.4 phosphate buffer;
2. to be 7.0 phosphate buffer with 0.02mol/L pH value with magnetic particle that 1. step obtains mix, and to make cumulative volume be 2.0ml;
3. add 2.0mg M2 type pyruvate kinase monoclonal antibody, homogenize is 10 minutes on shaking table;
4. add the 4.0mg carbodiimide, homogenize is 16 hours on shaking table;
5. be 7.4 phosphate buffer washing 2 times with 0.02mol/L pH value;
6. add Tris-EDTA damping fluid (Tris0.05mol/L, EDTA0.005mol/L, BSA0.1%, Proclin3001.0mL/L, pH=7.5), regulate cumulative volume to 2.0ml, place 2～4 ℃ of preservations.
The M2 type pyruvate kinase monoclonal antibody of described horseradish peroxidase-labeled is with following method preparation:
1. get 2.0mg M2 type pyruvate kinase monoclonal antibody, adding 0.5ml concentration is 0.02mol/L, and the pH value is 7.4 phosphate buffer dissolving, 2～4 ℃ of preservations;
2. get the horseradish peroxidase of 10.0mg, add the 2.0ml deionized water dissolving, take out 0.5ml, adding 0.2ml concentration is the NaIO of 0.1mol/L
4Aqueous solution, the lucifuge reaction is 0.5 hour under the room temperature;
3. 1. step is mixed with the liquid that 2. obtains, the lucifuge reaction is 4 hours under the room temperature;
4. adding 0.1ml concentration in the solution that 3. obtains to step is the NaBH of 3mg/ml
4Aqueous solution, the lucifuge reaction is 1 hour under the room temperature;
5. use 0.02mol/L, the pH value is 7.4 phosphate buffer dialysis 16 hours, carries out secondarily purifiedly with HPLC again, collects protein peak, adds behind the equal-volume glycerine in-20 ℃ of lower freezing preservations;
Described chemical luminous substrate A makes with following method:
1. prepare 0.02mol/L pH value and be 8.0 Tris-HCl damping fluid;
2. the 5mmol luminol is joined the 1000ml step 1. in the described damping fluid, mixing, 2～4 ℃ keep in Dark Place.
Described chemical luminous substrate B makes with following method:
1. prepare 0.02mol/L pH value and be 8.0 Tris-HCl damping fluid;
2. 0.6mmol tetraphenylboron sodium, the 1.5mmol potassium ferricyanide, 1.0mmol cinnamic acid, 10.0mmol hydrogen peroxide are joined the 1000ml step 1. in the described damping fluid, mixing, 2～4 ℃ of preservations.
Described cleansing solution is made by following method:
Get 6.05g trishydroxymethylaminomethane, 8.5g NaCl, 1.0ml Tween-20, add deionized water to 1L, transfer pH to 7.5 with HCl after the dissolving.
The preparation of M2 type pyruvate kinase standard items
With 0.02mol/L, the pH value be 7.4 phosphate buffer and hyclone by volume the ratio of 4:1 be hybridly prepared into basic damping fluid, with basic damping fluid with M2 type pyruvate kinase antigen be diluted to that concentration is 0, the standard items of 2.5U/mL, 6.4U/mL, 16U/mL, 40U/mL, 100U/mL.
Embodiment 2
People M2 type pyruvate kinase chemical luminescence immune analysis reagent box comprises following component:
1. M2 type pyruvate kinase standard items;
2. be coated with the magnetic particle of M2 type pyruvate kinase monoclonal antibody;
3. the M2 type pyruvate kinase monoclonal antibody of horseradish peroxidase-labeled;
4. chemical luminous substrate A and B;
5. White-opalescent microwell plate;
6. cleansing solution;
Wherein, the described magnetic particle that is coated with M2 type pyruvate kinase monoclonal antibody prepares through following steps:
1. get the 2.0ml mass content and be 2.5% magnetic particle suspension liquid, magnetic sheet divides the abandonment supernatant, is that 7.0 phosphate buffer is washed the magnetic particle once with 0.02mol/L pH value; Liquid in the described magnetic particle suspension liquid is 0.05mol/L, and the pH value is 7.4 phosphate buffer;
2. to be 7.0 phosphate buffer with 0.02mol/L pH value with magnetic particle that 1. step obtains mix, and to make cumulative volume be 3.0ml;
3. add 2.0mg M2 type pyruvate kinase monoclonal antibody, homogenize is 15 minutes on shaking table;
4. add the 7.0mg carbodiimide, homogenize is 18 hours on shaking table;
5. be 7.4 phosphate buffer washing 2 times with 0.02mol/L pH value;
6. add Tris-EDTA damping fluid (Tris0.05mol/L, EDTA0.005mol/L, BSA0.1%, Proclin3001.0mL/L, pH=7.5), regulate cumulative volume to 2.0ml, place 2～4 ℃ of preservations;
The M2 type pyruvate kinase monoclonal antibody of described horseradish peroxidase-labeled is with following method preparation:
1. get 2.0mg M2 type pyruvate kinase monoclonal antibody, adding 0.8ml concentration is 0.02mol/L, and the pH value is 7.4 phosphate buffer dissolving, 2～4 ℃ of preservations;
2. get the horseradish peroxidase of 10.0mg, add the 2.0ml deionized water, take out 0.8ml after the dissolving, adding 0.2ml concentration is the NaIO of 0.1mol/L
4Solution, lucifuge reaction 1.0 hours under room temperature after the sealing;
3. 1. step is mixed with the liquid that 2. obtains, the lucifuge reaction is 5 hours under the room temperature;
4. adding 0.1ml concentration in the solution that 3. obtains to step is the NaBH of 3mg/ml
4Solution, the lucifuge reaction is 1.5 hours under the room temperature;
5. use 0.02mol/L, the pH value is 7.4 phosphate buffer dialysis 20 hours, carries out secondarily purifiedly with HPLC again, collects protein peak, adds behind the equal-volume glycerine in-20 ℃ of lower freezing preservations;
Described chemical luminous substrate A makes with following method:
1. prepare 0.02mol/L pH value and be 8.0 Tris-HCl damping fluid;
2. the 8mmol luminol is joined the 1000ml step 1. in the described damping fluid, mixing, 2～4 ℃ keep in Dark Place; The preparation of chemical luminous substrate B working fluid:
1. prepare 0.02mol/L pH value and be 8.0 Tris-HCl damping fluid;
2. 0.8mmol tetraphenylboron sodium, the 1.50mmol potassium ferricyanide, 1.0mmol cinnamic acid, 10.0mmol hydrogen peroxide are joined the 1000ml step 1. in the described damping fluid, mixing, 2～4 ℃ of preservations.
The preparation of cleansing solution and M2 type pyruvate kinase standard items is with embodiment 1.
Embodiment 3:
People M2 type pyruvate kinase chemical luminescence immune analysis reagent box comprises following component:
1. M2 type pyruvate kinase standard items;
2. be coated with the magnetic particle of M2 type pyruvate kinase monoclonal antibody;
3. the M2 type pyruvate kinase monoclonal antibody of horseradish peroxidase-labeled;
4. chemical luminous substrate A and B;
5. White-opalescent microwell plate;
6. cleansing solution;
Wherein, the described magnetic particle that is coated with M2 type pyruvate kinase monoclonal antibody prepares through following steps:
1. get the 2.0ml mass content and be 2.5% magnetic particle suspension liquid, magnetic sheet divides the abandonment supernatant, is that 7.0 phosphate buffer is washed the magnetic particle once with 0.02mol/L pH value; Liquid in the described magnetic particle suspension liquid is 0.05mol/L, and the pH value is 7.4 phosphate buffer;
2. mix with the magnetic particle that 0.02mol/L pH value is 7.0 phosphate buffer with 1. step obtains that to make cumulative volume be 5.0ml;
3. add 2.0mg M2 type pyruvate kinase monoclonal antibody, homogenize is 20 minutes on shaking table;
4. add the 10.0mg carbodiimide, homogenize is 20 hours on shaking table;
5. be 7.4 phosphate buffer washing 2 times with 0.02mol/L pH value;
6. add Tris-EDTA damping fluid (Tris0.05mol/L, EDTA0.005mol/L, BSA0.1%, Proclin3001.0mL/L, pH=7.5), regulate cumulative volume to 2.0ml, place 2～4 ℃ of preservations stand-by.
The M2 type pyruvate kinase monoclonal antibody of described horseradish peroxidase-labeled is with following method preparation:
1. get 2.0mg M2 type pyruvate kinase monoclonal antibody, adding 1.0ml concentration is 0.02mol/L, and the pH value is 7.4 phosphate buffer dissolving, 2～4 ℃ of preservations;
2. get the horseradish peroxidase of 10.0mg, add the 2.0ml deionized water, take out 1.0ml after the dissolving, adding 0.2ml concentration is the NaIO of 0.1mol/L
4Solution, lucifuge reaction 1.5 hours under room temperature after the sealing;
3. 1. step is mixed with the liquid that 2. obtains, the lucifuge reaction is 6 hours under the room temperature;
4. adding 0.1ml concentration in the solution that 3. obtains to step is the NaBH of 3mg/ml
4Aqueous solution, the lucifuge reaction is 2 hours under the room temperature;
5. be 7.4 phosphate buffer dialysis 24 hours with the 0.02mol/LpH value, carry out again secondarily purifiedly with HPLC, collect protein peak, add behind the equal-volume glycerine in-20 ℃ of lower freezing preservations;
The preparation of chemical luminous substrate A working fluid:
1. prepare 0.02mol/L pH value and be 8.0 Tris-HCl damping fluid;
2. the 10mmol luminol is joined the 1000ml step 1. in the described damping fluid, mixing, 2～4 ℃ keep in Dark Place;
The preparation of chemical luminous substrate B working fluid:
1. prepare 0.02mol/L pH value and be 8.0 Tris-HCl damping fluid;
2. 1.0mmol tetraphenylboron sodium, the 1.50mmol potassium ferricyanide, 1.0mmol cinnamic acid, 10.0mmol hydrogen peroxide are joined the 1000ml step 1. in the described damping fluid, mixing, 2～4 ℃ of preservations.
The preparation of cleansing solution and M2 type pyruvate kinase standard items is with embodiment 1.
Embodiment 4
A kind of people M2 type pyruvate kinase chemical luminescence immune analysis reagent box experimental implementation program (take embodiment 1 kit as example) is as follows:
1. will test required reagent and human serum sample and place room temperature, balance just can be carried out experimental implementation more than 20 minutes;
2. the opaque microwell plate of extracting waste (96 hole) is numbered, and all M2 type pyruvate kinase standard items and human serum sample all do diplopore and repeat;
3. the standard items and the sample 50 μ l that get each concentration add respectively in the hole of corresponding numbering;
4. with M2 type pyruvate kinase monoclonal anti body and function label damping fluid (the 0.05mol/L phosphate buffer of horseradish peroxidase-labeled, pH=7.4, NaCl8.5g/L, BSA0.5%, Proclin3001.0mL/L, Tween-200.5mL/L) press the 1:5000 dilution, again it is got 50 μ l and join respectively each hole;
The magnetic particle that 5. will be coated with M2 type pyruvate kinase monoclonal antibody dilutes to get the magnetic particle suspension liquid with phosphate-BSA damping fluid (0.1mol/L phosphate buffer, pH=7.4, BSA0.2%, Proclin3001.0mL/L) by 1:150; Every hole adds 50 μ l magnetic particle suspension liquids, abundant mixing, room temperature vibration 40 minutes;
6. place it in absorption magnetic particle on the magnetic sheet, suck supernatant;
7. remove magnetic sheet, every hole all adds 300 μ l cleansing solutions;
8. after vibrating 10 seconds, place it in absorption magnetic particle on the magnetic sheet, suck supernatant;
9. come again step 7., 8.;
10. with chemical luminous substrate A with after B working fluid equal-volume mixes, every hole adds 100 μ l mixed liquors;
Measure the relative light unit (RLU) in every hole with Chemiluminescence Apparatus, detection time 0.2 second/hole;
Embodiment 5
The methodology of kit of the present invention is identified
Three kinds of kits among the embodiment 1～3 are identified that prove that through many experiments kit of the present invention is in the mensuration of M2-PK concentration, the methodology index of detection is as follows according to calibrating procedure conventional in this area:
1. sensing range: 0～100U/ml;
2. sensitivity: minimum detects and is limited to 0.15U/ml;
3. precision: the variation within batch coefficient is less than 10%, and interassay coefficient of variation is less than 15%;
4. accuracy: determination of recovery rates is between 90～110%;
5. specificity: with the cross reacting rate of analog M1-PK less than 1.0%;
6. stable: after 6 days, each component is still stable in 37 ℃ of placements for each reagent component.
Embodiment 6
The experimental data of using kit of the present invention that the human serum sample is detected
According to the operation steps of embodiment 4, use the kit of embodiment 1,83 routine human blood samples are measured, by 24 routine non-small cell lung cancer samples, and 59 routine Healthy People samples compositions, measurement result is as shown in the table:
The testing result of table 1. pair 83 routine blood samples
Use kit of the present invention that 83 routine human serum samples are detected, the result shows in the 24 routine non-small cell lung cancer patient blood samples, 21 routine M2-PK concentration values are arranged greater than critical value 15U/mL; In the 59 routine Healthy Human Serum samples, the M2-PK concentration value all below 15U/mL, illustrates that the mensuration specificity of this kit is good.
Kit of the present invention has that method of operating is fast and convenient, sensitivity and the advantage such as stability is high, sensing range is wide.
Claims (5)
1. people M2 type pyruvate kinase chemical luminescence immune analysis reagent box, its feature comprises following component:
1. M2 type pyruvate kinase standard items;
2. be coated with the magnetic particle of M2 type pyruvate kinase monoclonal antibody;
3. the M2 type pyruvate kinase monoclonal antibody of horseradish peroxidase-labeled;
4. chemical luminous substrate A and B;
5. White-opalescent microwell plate;
6. cleansing solution;
Wherein, the described magnetic particle that is coated with M2 type pyruvate kinase monoclonal antibody prepares through following steps:
1. get the 2.0ml mass content and be 2.5% magnetic particle suspension liquid, magnetic sheet divides the abandonment supernatant, is that 7.0 phosphate buffer is washed the magnetic particle once with 0.02mol/L pH value; Liquid in the described magnetic particle suspension liquid is 0.05mol/L, and the pH value is 7.4 phosphate buffer;
2. to be 7.0 phosphate buffer with 0.02mol/L pH value with magnetic particle that 1. step obtains mix, and to make cumulative volume be 2.0～5.0ml;
3. add 2.0mg M2 type pyruvate kinase monoclonal antibody, homogenize is 10～20 minutes on shaking table;
4. add 4.0～10.0mg carbodiimide, homogenize is 16～20 hours on shaking table;
5. be 7.4 phosphate buffer washing 2 times with 0.02mol/L pH value;
6. add the Tris-EDTA damping fluid, regulate cumulative volume to 2.0ml, place 2～4 ℃ of preservations.
2. a kind of people M2 type pyruvate kinase chemical luminescence immune analysis reagent box according to claim 1, the M2 type pyruvate kinase monoclonal antibody that it is characterized in that described horseradish peroxidase-labeled are with following method preparation:
1. get 2.0mg M2 type pyruvate kinase monoclonal antibody, adding 0.5～1.0ml concentration is 0.02mol/L, and the pH value is 7.4 phosphate buffer dissolving, 2～4 ℃ of preservations;
2. get the horseradish peroxidase of 10.0mg, add the 2.0ml deionized water dissolving, take out 0.5～1.0ml, adding 0.2ml concentration is the NaIO of 0.1mol/L
4Aqueous solution, the lucifuge reaction is 0.5～1.5 hour under the room temperature;
3. 1. step is mixed with the liquid that 2. obtains, the lucifuge reaction is 4～6 hours under the room temperature;
4. adding 0.1ml concentration in the solution that 3. obtains to step is the NaBH of 3mg/ml
4Aqueous solution, the lucifuge reaction is 1～2 hour under the room temperature;
5. be 7.4 phosphate buffer dialysis 16～24 hours with 0.02mol/L pH value, carry out again secondarily purifiedly with HPLC, collect protein peak, add behind the equal-volume glycerine in-20 ℃ of lower freezing preservations.
3. a kind of people M2 type pyruvate kinase chemical luminescence immune analysis reagent box according to claim 1 is characterized in that described chemical luminous substrate A makes with following method:
1. prepare 0.02mol/L pH value and be 8.0 Tris-HCl damping fluid;
2. 5～10mmol luminol is joined the 1000ml step 1. in the described damping fluid, mixing, 2～4 ℃ keep in Dark Place.
4. a kind of people M2 type pyruvate kinase chemical luminescence immune analysis reagent box according to claim 1 is characterized in that described chemical luminous substrate B makes with following method:
1. prepare 0.02mol/L pH value and be 8.0 Tris-HCl damping fluid;
2. 0.6～1.0mmol tetraphenylboron sodium, the 1.5mmol potassium ferricyanide, 1.0mmol cinnamic acid, 10.0mmol hydrogen peroxide are joined the 1000ml step 1. in the described damping fluid, mixing, 2～4 ℃ of preservations.
5. a kind of people M2 type pyruvate kinase chemical luminescence immune analysis reagent box according to claim 1 is characterized in that described cleansing solution made by following method:
Get 6.05g trishydroxymethylaminomethane, 8.5g NaCl, 1.0ml Tween-20, add deionized water to 1L, transfer pH to 7.5 with HCl after the dissolving.
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| CN104007260A (en) * | 2014-06-13 | 2014-08-27 | 天津康尔克生物科技有限公司 | Lipoprotein-related phospholipase A2 enzyme-linked immunosorbent assay (ELISA) kit and preparation method thereof |
| CN105403706A (en) * | 2015-04-30 | 2016-03-16 | 北京丹大生物技术有限公司 | Heat shock protein 90 (HSP90) chemiluminescence detection kit |
| CN106405094A (en) * | 2016-09-29 | 2017-02-15 | 广州华弘生物科技有限公司 | Chemiluminiscence immune kit used for detecting TU M2-PK (Tumor M2-Pyruvate Kinase) |
| CN106546744A (en) * | 2015-09-17 | 2017-03-29 | 上海透景生命科技股份有限公司 | By the method and corresponding reagent box of fecal hemoglobin, transferrins and PKM2 joint-detection assessing colorectal cancers |
| CN109576375A (en) * | 2019-02-12 | 2019-04-05 | 中国医科大学附属第医院 | One kind biological sample marker relevant to the preoperative malignant and benign lesion diagnosis of thyroid nodule and its application |
Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19945947A1 (en) * | 1999-09-24 | 2001-03-29 | Schebo Tech Gmbh | Detection of the pyruvate kinase isoenzyme |
| WO2002103035A1 (en) * | 2001-06-14 | 2002-12-27 | The University Of Western Australia | Improved method for assessing liver disease |
| CN1719256A (en) * | 2005-07-15 | 2006-01-11 | 北京倍爱康生物技术股份有限公司 | Magnetic separation enzymatic chemical luminous immune detection method of human thyroglobulin antibody |
| CN1887901A (en) * | 2006-06-23 | 2007-01-03 | 深圳市安群生物工程有限公司 | Human tumor M2-type pyruvate kinase antigen determinant polypeptide and antibody and their application in diagnositic kit |
| CN1924580A (en) * | 2005-08-30 | 2007-03-07 | 郑州安图绿科生物工程有限公司 | Chemical luminescent analysis reagent kid for quantitatively detecting trilute |
| AU2007251933A1 (en) * | 2006-05-19 | 2007-11-29 | F. Hoffman-La Roche Ag | Use of protein S100A 12 as a marker for colorectal cancer |
| CN101226195A (en) * | 2008-02-03 | 2008-07-23 | 复旦大学附属肿瘤医院 | Kit for detecting knubble type M2 pyruvate kinase and preparation method thereof |
| CN101324579A (en) * | 2007-06-13 | 2008-12-17 | 清华大学 | Magnetic microparticle chemiluminescence enzyme immune analytic reagent kit for detecting saccharide antigen and its use method |
| CN101341410A (en) * | 2005-12-21 | 2009-01-07 | 霍夫曼—拉罗奇有限公司 | Method of assessing colorectal cancer by measuring hemoglobin and M2-PK in a stool sample |
| CN101377491A (en) * | 2007-08-31 | 2009-03-04 | 北京科美东雅生物技术有限公司 | Magnetic granule competing method chemiluminescence immune analysis determination reagent kit for detecting hormone and preparing method thereof |
| CN101545912A (en) * | 2008-03-25 | 2009-09-30 | 北京科美东雅生物技术有限公司 | Trypsin micropore plate type magnetic granule chemoluminescence immunoassay measuring kit and preparation method thereof |
| WO2010043927A1 (en) * | 2008-10-13 | 2010-04-22 | Xeptagen Spa | Method for the preparation of immunoconjugates and use thereof |
| US20110224101A1 (en) * | 2009-11-10 | 2011-09-15 | Xuefeng Ling | Tumor associated proteome and peptidome analyses for multiclass cancer discrimination |
| CN102520195A (en) * | 2011-12-30 | 2012-06-27 | 天津市协和医药科技集团有限公司 | Chromogranin A chemiluminescence immunoassay reagent kit and preparation method thereof |
-
2012
- 2012-12-10 CN CN2012105337667A patent/CN103033623A/en active Pending
Patent Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19945947A1 (en) * | 1999-09-24 | 2001-03-29 | Schebo Tech Gmbh | Detection of the pyruvate kinase isoenzyme |
| WO2002103035A1 (en) * | 2001-06-14 | 2002-12-27 | The University Of Western Australia | Improved method for assessing liver disease |
| CN1719256A (en) * | 2005-07-15 | 2006-01-11 | 北京倍爱康生物技术股份有限公司 | Magnetic separation enzymatic chemical luminous immune detection method of human thyroglobulin antibody |
| CN1924580A (en) * | 2005-08-30 | 2007-03-07 | 郑州安图绿科生物工程有限公司 | Chemical luminescent analysis reagent kid for quantitatively detecting trilute |
| CN101341410A (en) * | 2005-12-21 | 2009-01-07 | 霍夫曼—拉罗奇有限公司 | Method of assessing colorectal cancer by measuring hemoglobin and M2-PK in a stool sample |
| AU2007251933A1 (en) * | 2006-05-19 | 2007-11-29 | F. Hoffman-La Roche Ag | Use of protein S100A 12 as a marker for colorectal cancer |
| CN1887901A (en) * | 2006-06-23 | 2007-01-03 | 深圳市安群生物工程有限公司 | Human tumor M2-type pyruvate kinase antigen determinant polypeptide and antibody and their application in diagnositic kit |
| CN101324579A (en) * | 2007-06-13 | 2008-12-17 | 清华大学 | Magnetic microparticle chemiluminescence enzyme immune analytic reagent kit for detecting saccharide antigen and its use method |
| CN101377491A (en) * | 2007-08-31 | 2009-03-04 | 北京科美东雅生物技术有限公司 | Magnetic granule competing method chemiluminescence immune analysis determination reagent kit for detecting hormone and preparing method thereof |
| CN101226195A (en) * | 2008-02-03 | 2008-07-23 | 复旦大学附属肿瘤医院 | Kit for detecting knubble type M2 pyruvate kinase and preparation method thereof |
| CN101545912A (en) * | 2008-03-25 | 2009-09-30 | 北京科美东雅生物技术有限公司 | Trypsin micropore plate type magnetic granule chemoluminescence immunoassay measuring kit and preparation method thereof |
| WO2010043927A1 (en) * | 2008-10-13 | 2010-04-22 | Xeptagen Spa | Method for the preparation of immunoconjugates and use thereof |
| US20110224101A1 (en) * | 2009-11-10 | 2011-09-15 | Xuefeng Ling | Tumor associated proteome and peptidome analyses for multiclass cancer discrimination |
| CN102520195A (en) * | 2011-12-30 | 2012-06-27 | 天津市协和医药科技集团有限公司 | Chromogranin A chemiluminescence immunoassay reagent kit and preparation method thereof |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104007260A (en) * | 2014-06-13 | 2014-08-27 | 天津康尔克生物科技有限公司 | Lipoprotein-related phospholipase A2 enzyme-linked immunosorbent assay (ELISA) kit and preparation method thereof |
| CN105403706A (en) * | 2015-04-30 | 2016-03-16 | 北京丹大生物技术有限公司 | Heat shock protein 90 (HSP90) chemiluminescence detection kit |
| CN106546744A (en) * | 2015-09-17 | 2017-03-29 | 上海透景生命科技股份有限公司 | By the method and corresponding reagent box of fecal hemoglobin, transferrins and PKM2 joint-detection assessing colorectal cancers |
| CN106405094A (en) * | 2016-09-29 | 2017-02-15 | 广州华弘生物科技有限公司 | Chemiluminiscence immune kit used for detecting TU M2-PK (Tumor M2-Pyruvate Kinase) |
| CN106405094B (en) * | 2016-09-29 | 2018-03-06 | 广州华弘生物科技有限公司 | For detecting the chemiluminescence immunoassay kit of knubble type M 2 pyruvate kinase |
| CN109576375A (en) * | 2019-02-12 | 2019-04-05 | 中国医科大学附属第医院 | One kind biological sample marker relevant to the preoperative malignant and benign lesion diagnosis of thyroid nodule and its application |
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