CN102520195A - Chromogranin A chemiluminescence immunoassay reagent kit and preparation method thereof - Google Patents
Chromogranin A chemiluminescence immunoassay reagent kit and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a chromogranin A chemiluminescence immunoassay reagent kit and a preparation method of the reagent kit. The reagent kit comprises the following ingredients: 1) chromogranin A standard products; 2) horseradish peroxidase labeled chromogranin A monoclonal antibodies; 3) chromogranin A monoclonal antibody covered magnetic particles; 4) white non-transparent micro porous plate; 5) cleaning solution and 6) chemiluminescence substrate A and B work solution. The chemiluminescence immunoassay and the nanometer magnetic particle technology are combined, and the chromogranin A chemiluminescence immunoassay reagent kit is provided. The defects of low sensitivity and poor accuracy of the traditional enzyme-linked immunosorbent assay (ELISA) method are overcome, the radioactive pollution of the radioimmunoassay (RIA) technology is avoided, and the chromogranin A chemiluminescence immunoassay reagent kit and the preparation method thereof have the advantages that safety is realized, the environment is protected, the specificity is high, the sensitivity and the accuracy are high, and the like.
Description
Technical field
The present invention relates to the immunoassay medicine technology field, relate to Chromogranin A in a kind of human serum (CgA) chemical luminescence immune analysis reagent box and preparation method thereof particularly.
Background technology
(Chromogranin A CgA) is a kind of acid soluble protein of being made up of 439 amino acid to Chromogranin A, is the main member of chromogranin family.Extensively be present in and secrete in the nerve in the cell, the rising of CgA concentration can be pointed out the tumour in neuroendocrine source in the blood plasma.Secrete the rising that all can occur the CgA level in the diagnosis of tumour in the nerve of nearly all type.Therefore secrete in the diagnosis of tumour in the nerve, CgA is valuable diagnosis marker.If as critical value, CgA is respectively 82.1% and 96.2% to the sensitivity and the specificity of pancreatic neoplasm with 32.7U/L, the sensitivity and the specificity of pheochromocytoma is respectively 88.5% and 96.2%.Therefore, the detection by quantitative of CgA has crucial meaning for the clinical diagnosis of cancer of pancreas and chromaffin cell cancer.
Mensuration reported method to CgA mainly contains enzyme-linked immunosorbent assay (ELISA), immunoradiometric assay (IRMA) etc. at present.Grinding import reagent box that the brightness bio tech ltd provides like Shanghai uses double antibody sandwich method and measures people's chromogranin A (CgA) level in the sample.People's chromogranin A (CgA) antibody sandwich microwell plate with purifying; Process insolubilized antibody, in the micropore that encapsulates monoclonal antibody, add chromogranin A (CgA) successively, the goat anti-human antibody with the HRP mark combines again; Form antibody-antigen-hrp-antibody complex, through adding substrate TMB colour developing after the washing.Measure absorbance (OD value) with ELIASA, through people's chromogranin A (CgA) content in the typical curve calculation sample.China Concord Medical Science University adopts enzyme linked immunosorbent assay analysis method (ELISA) that change of serum C gA level is detected for another example.The result shows that change of serum C gA concentration is relevant with the expression intensity of CgA in the chromaffin cell tumor tissue, is proportionate with tumor weight, and high-caliber change of serum C gA concentration prompting tumour is pernicious probably.The influenced factor of ELISA method is more, is easy to cause distortion as a result, and accuracy is relatively poor.Therefore should technology in scientific research, should have morely, and in Clinical detection, should have and seldom.
France CIS company has developed the kit that a kind of immunoradiometric assay detects CgA, encapsulates test tube with a strain CgA monoclonal antibody, another strain CgA monoclonal antibody with iodine-125 (
125I) mark is through the concentration of CgA in the double antibody sandwich method detection serum.This method is simple to operate, measures the result more accurately and reliably.But because iodine-125 has radioactive contamination, to environmental requirement increasingly high today, radiating immuning analysis technology obviously is not inconsistent the development trend of biological medicine technology at us.
Summary of the invention
The objective of the invention is to overcome the deficiency of prior art, provide a kind of highly sensitive, accuracy is high, do not have alpha-contamination Chromogranin A chemical luminescence immune analysis reagent box again.
Second purpose of the present invention provides a kind of preparation method of Chromogranin A chemical luminescence immune analysis reagent box.
Technical scheme of the present invention is summarized as follows:
A kind of Chromogranin A chemical luminescence immune analysis reagent box comprises following component:
1) Chromogranin A standard items;
2) the Chromogranin A monoclonal antibody of horseradish peroxidase-labeled;
3) the magnetic particle that encapsulates of Chromogranin A monoclonal antibody;
4) White-opalescent microwell plate;
5) cleansing solution;
6) chemical luminous substrate A and B working fluid.
The Chromogranin A monoclonal antibody of said horseradish peroxidase-labeled is to process with following method:
1. take by weighing CgA monoclonal antibody 2.0mg, adding 1～2ml concentration is 0.05mol/L, the carbonate buffer solution dissolving of pH=7.5, and 4 ℃ store for future use;
2. take by weighing the horseradish peroxidase of 10.0mg, add the 1.0ml deionized water, vibration after the dissolving, is taken out 0.14～0.28ml, adds the NaIO of 0.1mol/L
4Solution 0.2ml, the lucifuge reaction is 1～2 hour under the room temperature;
3. the liquid that 1. step is obtained and the liquid mixing of step acquisition 2., the lucifuge reaction is 4～8 hours under the room temperature;
4. adding 200 μ l concentration is the NaBH of 5mg/ml
4The WS, the lucifuge reaction is 1～2 hour under the room temperature;
5. with 0.02M PBS dialysis 12-24 hour, secondarily purified with HPLC, collect protein peak and freezing preservations under-20 ℃.
The magnetic particle that said Chromogranin A monoclonal antibody encapsulates is to process with following method:
1. get 5mL magnetic granular mass content and be 2.5% PBS solution, divide the abandonment supernatant, wash the magnetic particle with deionized water with magnetic sheet;
2. the PBS with 0.02mol/L pH=6.2 washes the magnetic particle;
3. using the PBS adjustment cumulative volume of 0.02mol/L pH=6.2 is 5～10ml;
4. adding CgA monoclonal antibody 5mg is placed on and carries out homogenize 10～20 minutes on the shaking table;
5. add carbodiimide 5～10mg; Room temperature is placed on and carries out homogenize 5～10 hours on the shaking table;
6. the phosphate buffer with 0.1mol/L pH=7.4 washs 2 times;
7. add Tris and EDTA, making the content of Tris is 0.05mol/L, and the content that makes EDTA is 0.004mol/L, transfers pH=7.5, and making AC is 1.0mg/ml, 2-4 ℃ of preservation.
The preparation of said cleansing solution:
Get: trishydroxymethylaminomethane 6.05g; NaCl8.55g; Tween-20 0.5ml; Add water to 1L; Transfer pH to 7.5 ± 0.1 with HCl.
Chemical luminous substrate A working fluid is to process with following method:
(1) the Tris-HCl damping fluid of preparation 0.05mol/L pH=8.0;
(2) in the Tris-HCl damping fluid of the said step of 1000ml (1) preparation, add 3～5mmol luminol, mixing; Let the chemical luminous substrate B working fluid be to process with following method:
(1) the Tris-HCl damping fluid of preparation 0.05mol/L pH=8.0;
(2) in the Tris-HCl damping fluid of the said step of 1000ml (1) preparation, add: 0.5～1.0mmol tetraphenylboron sodium; 1.42mmol the potassium ferricyanide; 0.38mmol cinnamic acid; 7.56mmol hydrogen peroxide, mixing.
A kind of preparation method of Chromogranin A chemical luminescence immune analysis reagent box comprises the steps:
1) prepares the Chromogranin A standard items;
2) the Chromogranin A monoclonal antibody of preparation horseradish peroxidase-labeled;
3) prepare the magnetic particle that the Chromogranin A monoclonal antibody encapsulates;
4) prepare the White-opalescent microwell plate;
5) preparing washing liquid;
6) preparation chemical luminous substrate A and B working fluid.
The step of the Chromogranin A monoclonal antibody of preparation horseradish peroxidase-labeled is:
1. take by weighing CgA monoclonal antibody 2.0mg, adding 1～2ml concentration is 0.05mol/L, the carbonate buffer solution dissolving of pH=7.5, and 4 ℃ store for future use;
2. take by weighing the horseradish peroxidase of 10.0mg, add the 1.0ml deionized water, vibration after the dissolving, is taken out 0.14～0.28ml, adds the NaIO of 0.1mol/L
4Solution 0.2ml, the lucifuge reaction is 1～2 hour under the room temperature;
3. the liquid that 1. step is obtained and the liquid mixing of step acquisition 2., the lucifuge reaction is 4～8 hours under the room temperature;
4. adding 200 μ l concentration is the NaBH of 5mg/ml
4The WS, the lucifuge reaction is 1～2 hour under the room temperature;
5. with 0.02M PBS dialysis 12-24 hour, secondarily purified with HPLC, collect protein peak and freezing preservations under-20 ℃.
The step of the magnetic particle that preparation Chromogranin A monoclonal antibody encapsulates is:
1. get 5mL magnetic granular mass content and be 2.5% PBS solution, divide the abandonment supernatant, wash the magnetic particle with deionized water with magnetic sheet;
2. the PBS with 0.02mol/L pH=6.2 washes the magnetic particle;
3. using the PBS adjustment cumulative volume of 0.02mol/L pH=6.2 is 5～10ml;
4. adding CgA monoclonal antibody 5mg is placed on and carries out homogenize 10～20 minutes on the shaking table;
5. add carbodiimide 5～10mg; Room temperature is placed on and carries out homogenize 5～10 hours on the shaking table;
6. the phosphate buffer with 0.1mol/L pH=7.4 washs 2 times;
7. add Tris and EDTA, making the content of Tris is 0.05mol/L, and the content that makes EDTA is 0.004mol/L, transfers pH=7.5, and making AC is 1.0mg/ml, 2-4 ℃ of preservation.
The step for preparing said cleansing solution is:
Get: trishydroxymethylaminomethane 6.05g; NaCl8.55g; Tween-200.5ml; Add water to 1L; Transfer pH to 7.5 ± 0.1 with HCl.
The step of preparation chemical luminous substrate A working fluid is:
(1) the Tris-HCl damping fluid of preparation 0.05mol/L pH=8.0;
(2) in the Tris-HCl damping fluid of the said step of 1000ml (1) preparation, add 3～5mmol luminol, mixing; The step of said preparation chemical luminous substrate B working fluid is:
(1) the Tris-HCl damping fluid of preparation 0.05mol/L pH=8.0;
(2) in the Tris-HCl damping fluid of the said step of 1000ml (1) preparation, add: 0.5～1.0mmol tetraphenylboron sodium; 1.42mmol the potassium ferricyanide; 0.38mmol cinnamic acid; 7.56mmol hydrogen peroxide, mixing.
The present invention combines chemiluminescence immune assay with nanometer magnetic granule technology, a kind of Chromogranin A chemical luminescence immune analysis reagent box is provided.This method had both overcome the shortcoming that the sensitivity of traditional E LISA method is low, accuracy is relatively poor, did not have the radioactive contamination of RIA technology again, had safety and environmental protection, high specificity, sensitivity and accuracy advantages of higher.
Description of drawings
Fig. 1 CgA chemiluminescence immune assay principle schematic.
Embodiment
Below in conjunction with specific embodiment the present invention is further described:
Embodiment 1
A kind of preparation method of Chromogranin A chemical luminescence immune analysis reagent box and experimental implementation program:
1. the Chromogranin A monoclonal antibody of horseradish peroxidase-labeled (abbreviation enzyme labelled antibody) preparation:
1. take by weighing CgA monoclonal antibody 2.0mg and put into the 5ml vial, add 1.0ml 0.05mol/L, the sodium carbonate buffer dissolving of pH=7.5,4 ℃ store for future use;
2. the horseradish peroxidase that takes by weighing 10.0mg is put into 16 * 100mm glass test tube, adds the 1.0ml deionized water, vibration, treat that it dissolves fully after, take out 0.14ml and put into another 16 * 100mm glass test tube, add the NaIO of 0.1mol/L
4Solution 0.2ml (NaIO
4The necessary fresh of solution), with glass test tube mouth jam-pack, the lucifuge reaction is 1 hour under the room temperature with plug;
3. the liquid that 1. step is obtained and the liquid mixing of step acquisition 2., the lucifuge reaction is 4 hours under the room temperature;
4. adding 200 μ l concentration is the NaBH of 5mg/ml
4The WS, the lucifuge reaction is 1 hour under the room temperature;
5. with 0.02M PBS dialysis 18 hours, secondarily purified with HPLC, collect protein peak and freezing preservations under-20 ℃;
2, the preparation of the magnetic particle that encapsulates of Chromogranin A monoclonal antibody;
1. get 5mL magnetic granular mass content and be in 2.5% the PBS solution, divide the abandonment supernatant, wash the magnetic particle 1 time with deionized water with magnetic sheet;
2. the PBS with 0.02mol/L pH=6.2 washes 1 time;
3. the PBS adjustment cumulative volume with 0.02mol/L pH=6.2 is about 5ml;
4. adding CgA monoclonal antibody 5mg is placed on and carries out homogenize 10 minutes on the shaking table;
5. add carbodiimide (EDAC) 5mg; Room temperature is placed on and carries out homogenize 5 hours on the shaking table;
6. with 0.1mol/L pH=7.4 phosphate buffer washing 2 times;
7. add Tris and EDTA, making the content of Tris is 0.05mol/L, and the content that makes EDTA is 0.004mol/L, transfers pH=7.5, and making AC is 1.0mg/ml, 4 ℃ of preservations.
3, the preparation of cleansing solution:
Get trishydroxymethylaminomethane (TrisBase) 6.05g; NaCl8.55g; Tween-200.5ml; Add water to 1L; Transfer pH to 7.5 ± 0.1, mixing with HCl;
4, the preparation of chemical luminous substrate A working fluid:
(1) the Tris-HCl damping fluid of preparation 0.05mol/L pH=8.0;
(2) in the Tris-HCl damping fluid of the said step of 1000ml (1) preparation, add 3mmol luminol, mixing.
5. the preparation of chemical luminous substrate B working fluid:
(1) the Tris-HCl damping fluid of preparation 0.05mol/L pH=8.0;
(2) in the Tris-HCl damping fluid of the said step of 1000ml (1) preparation, add: the 0.5mmol tetraphenylboron sodium; 1.42mmol the potassium ferricyanide; 0.38mmol cinnamic acid; 7.56mmol hydrogen peroxide, mixing.
6, the preparation of standard items:
0.01M PBS preparation:
①Na
2HPO
4 1.104g/L；
②NaH
2PO
4 0.25g/L；
③NaCl 8.55/L；
4. ProClin300 1.0ml/L transfers PH to 7.4 ± 0.1 with HCl.
Above-mentioned 0.01M PBS and 4: 1 by volume mixed of cow's serum are mixed with basic damping fluid, process the standard items of variable concentrations again with the CgA psma ligand.
7, chemiluminescence immune assay (CLIA) the method experimental implementation program of Chromogranin A (CgA) is following in the human serum:
(1) will test required reagent and sample and place room temperature, balance just can be operated more than 30 minutes at least;
(2) get one 96 hole microwell plate and number, all experiments are all done diplopore and are repeated;
(3) standard items and the sample 50 μ l that get each concentration add in the hole of corresponding numbering;
(4) every hole all adds 50 μ l enzymic-labelled antibodies;
(5) the abundant magnetic particle that encapsulates of mixing Chromogranin A monoclonal antibody, every hole all adds 50 μ l magnetic particles;
(6) abundant mixing, room temperature vibration 0.5 hour;
(7) place it in absorption magnetic particle on the magnetic sheet, inhale and remove supernatant;
(8) remove magnetic sheet, every hole all adds 250 μ l cleansing solutions;
(9) vibration placed it in absorption magnetic particle on the magnetic sheet after 5 seconds, inhaled and removed supernatant;
(10) come again step (8), (9);
(11) every hole adds chemical luminous substrate A working fluid 50 μ l;
(12) every hole adds into chemical luminous substrate B working fluid 50 μ l;
(13) measure the luminous intensity in every hole with Chemiluminescence Apparatus;
(14) read the concentration of sample from typical curve.
Embodiment 2
A kind of preparation method of Chromogranin A chemical luminescence immune analysis reagent box and experimental implementation program:
1. the Chromogranin A monoclonal antibody of horseradish peroxidase-labeled (abbreviation enzyme labelled antibody) preparation:
1. take by weighing CgA monoclonal antibody 2.0mg and put into the 5ml vial, add 1.5ml 0.05mol/L, the sodium carbonate buffer dissolving of pH=7.5,4 ℃ store for future use;
2. the horseradish peroxidase that takes by weighing 10.0mg is put into 16 * 100mm glass test tube, adds the 1.0ml deionized water, vibration, treat that it dissolves fully after, take out 0.21ml and put into another 16 * 100mm glass test tube, add the NaIO of 0.1mol/L
4Solution 0.2ml (NaIO
4The necessary fresh of solution), with glass test tube mouth jam-pack, the lucifuge reaction is 1.5 hours under the room temperature with plug;
3. the liquid that 1. step is obtained and the liquid mixing of step acquisition 2., the lucifuge reaction is 6 hours under the room temperature;
4. adding 200 μ l concentration is the NaBH of 5mg/ml
4The WS, the lucifuge reaction is 1.5 hours under the room temperature;
5. with 0.02M PBS dialysis 24 hours, secondarily purified with HPLC, collect protein peak and freezing preservations under-20 ℃;
2, the preparation of the magnetic particle that encapsulates of Chromogranin A monoclonal antibody;
1. get 5mL magnetic granular mass content and be in 2.5% the PBS solution, divide the abandonment supernatant, wash the magnetic particle 1 time with deionized water with magnetic sheet;
2. the PBS with 0.02mol/L pH=6.2 washes 1 time;
3. use the PBS adjustment cumulative volume of 0.02mol/L pH=6.2 to be 8ml;
4. adding CgA monoclonal antibody 5mg is placed on and carries out homogenize 15 minutes on the shaking table;
5. add carbodiimide (EDAC) 8mg; Room temperature is placed on and carries out homogenize 8 hours on the shaking table;
6. with 0.1mol/L pH=7.4 phosphate buffer washing 2 times;
7. add Tris and EDTA, making the content of Tris is 0.05mol/L, and the content that makes EDTA is 0.004mol/L, transfers pH=7.5, and making AC is 1.0mg/ml, 2 ℃ of preservations.
3, the preparation of cleansing solution (with embodiment 1)
4, the preparation of chemical luminous substrate A working fluid:
(1) the Tris-HCl damping fluid of preparation 0.05mol/L pH=8.0;
(2) in the Tris-HCl damping fluid of the said step of 1000ml (1) preparation, add 3mmol luminol, mixing.
5. the preparation of chemical luminous substrate B working fluid:
(1) the Tris-HCl damping fluid of preparation 0.05mol/L pH=8.0;
(2) in the Tris-HCl damping fluid of the said step of 1000ml (1) preparation, add: the 0.5mmol tetraphenylboron sodium; 1.42mmol the potassium ferricyanide; 0.38mmol cinnamic acid; 7.56mmol hydrogen peroxide, mixing.
6, the preparation of standard items (with embodiment 1)
7, the experimental implementation step of chemiluminescence immune assay (CLIA) method of Chromogranin A (CgA) in the human serum:
(1) will test required reagent and sample and place room temperature, balance just can be operated more than 30 minutes at least;
(2) get one 48 hole microwell plate and number, all experiments are all done diplopore and are repeated;
(3) standard items and the sample 50 μ l that get each concentration add in the hole of corresponding numbering;
(4) every hole all adds 50 μ l enzymic-labelled antibodies;
(5) the abundant magnetic particle that encapsulates of mixing Chromogranin A monoclonal antibody, every hole all adds 50 μ l magnetic particles;
(6) abundant mixing, room temperature vibration 45 minutes;
(7) place it in absorption magnetic particle on the magnetic sheet, inhale and remove supernatant;
(8) remove magnetic sheet, every hole all adds 250 μ l cleansing solutions;
(9) vibration placed it in absorption magnetic particle on the magnetic sheet after 8 seconds, inhaled and removed supernatant;
(10) come again step (8), (9);
(11) every hole adds chemical luminous substrate A working fluid 50 μ l;
(12) every hole adds into chemical luminous substrate B working fluid 50 μ l;
(13) measure the luminous intensity in every hole with Chemiluminescence Apparatus;
(14) read the concentration of sample from typical curve.
Embodiment 3
A kind of preparation method of Chromogranin A chemical luminescence immune analysis reagent box and experimental implementation program:
1. the Chromogranin A monoclonal antibody of horseradish peroxidase-labeled (abbreviation enzyme labelled antibody) preparation:
1. take by weighing CgA monoclonal antibody 2.0mg and put into the 5ml vial, add 2.0ml 0.05mol/L, the sodium carbonate buffer dissolving of pH=7.5,4 ℃ store for future use;
2. the horseradish peroxidase that takes by weighing 10.0mg is put into 16 * 100mm glass test tube, adds the 1.0ml deionized water, vibration, treat that it dissolves fully after, take out 0.28ml and put into another 16 * 100mm glass test tube, add the NaIO of 0.1mol/L
4Solution 0.2ml (NaIO
4The necessary fresh of solution), with glass test tube mouth jam-pack, the lucifuge reaction is 2 hours under the room temperature with plug;
3. the liquid that 1. step is obtained and the liquid mixing of step acquisition 2., the lucifuge reaction is 8 hours under the room temperature;
4. adding 200 μ l concentration is the NaBH of 5mg/ml
4The WS, the lucifuge reaction is 2 hours under the room temperature;
5. with 0.02M PBS dialysis 12 hours, secondarily purified with HPLC, collect protein peak and freezing preservations under-20 ℃;
2, the preparation of the magnetic particle that encapsulates of Chromogranin A monoclonal antibody;
1. get 5mL magnetic granular mass content and be in 2.5% the PBS solution, divide the abandonment supernatant, wash the magnetic particle 1 time with deionized water with magnetic sheet;
2. the PBS with 0.02mol/L pH=6.2 washes 1 time;
3. use the PBS adjustment cumulative volume of 0.02mol/L pH=6.2 to be 10ml;
4. adding CgA monoclonal antibody 5mg is placed on and carries out homogenize 20 minutes on the shaking table;
5. add carbodiimide (EDAC) 10mg; Room temperature is placed on and carries out homogenize 10 hours on the shaking table;
6. with 0.1mol/L pH=7.4 phosphate buffer washing 2 times;
7. add Tris and EDTA, making the content of Tris is 0.05mol/L, and the content that makes EDTA is 0.004mol/L, transfers pH=7.5, and making AC is 1.0mg/ml, 3 ℃ of preservations.
3, the preparation of cleansing solution (with embodiment 1)
4. the preparation of chemical luminous substrate A working fluid:
(1) the Tris-HCl damping fluid of preparation 0.05mol/L pH=8.0;
(2) in the Tris-HCl damping fluid of the said step of 1000ml (1) preparation, add 5mmol luminol, mixing.
5. the preparation of chemical luminous substrate B working fluid:
(1) the Tris-HCl damping fluid of preparation 0.05mol/L pH=8.0;
(2) in the Tris-HCl damping fluid of the said step of 1000ml (1) preparation, add: the 1.0mmol tetraphenylboron sodium; 1.42mmol the potassium ferricyanide; 0.38mmol cinnamic acid; 7.56mmol hydrogen peroxide, mixing.
6, the preparation of standard items (with embodiment 1)
7, the experimental implementation step of the chemical luminescence immune analysis reagent box of Chromogranin A (CgA) in the human serum:
(1) will test required reagent and sample and place room temperature, balance just can be operated more than 30 minutes at least;
(2) get one 384 hole microwell plate and number, all experiments are all done diplopore and are repeated;
(3) standard items and the sample 100 μ l that get each concentration add in the hole of corresponding numbering;
(4) every hole all adds 50 μ l enzymic-labelled antibodies;
(5) the abundant magnetic particle that encapsulates of mixing Chromogranin A monoclonal antibody, every hole all adds 50 μ l magnetic particles;
(6) abundant mixing, room temperature vibration 1 hour;
(7) place it in absorption magnetic particle on the magnetic sheet, inhale and remove supernatant;
(8) remove magnetic sheet, every hole all adds 250 μ l cleansing solutions;
(9) vibration placed it in absorption magnetic particle on the magnetic sheet after 10 seconds, inhaled and removed supernatant;
(10) come again step (8), (9);
(11) every hole adds chemical luminous substrate A working fluid 50 μ l;
(12) every hole adds into chemical luminous substrate B working fluid 50 μ l;
(13) measure the luminous intensity in every hole with Chemiluminescence Apparatus;
(14) read the concentration of sample from typical curve.
This method detection sensitivity can reach 0.8U/mL, does not have obvious cross reaction, and it is 0.8～800U/mL that accuracy 95-105%, typical curve satisfy the required measurement range of clinical samples.
This kit each item technical indicator is analyzed:
This kit detection sensitivity can reach 0.8U/mL, does not have obvious cross reaction with analog, and accuracy can reach 95-105%, and it is 0.8～800U/mL that typical curve satisfies the required measurement range of clinical samples.
We have carried out determination test to a collection of cancer of pancreas, chromaffin cell carninomatosis human serum sample and the normal human serum sample of making a definite diagnosis through hospital respectively with this kit, and the result is following:
| Group | Sample example number | Detect positive routine number | Positive rate |
| The cancer of pancreas group | 52 | 45 | 86.54％ |
| Chromaffin cell cancer group | 63 | 57 | 90.47％ |
| Normal person's group | 80 | 0 | 0 |
Can find out from above result; This kit can reach 86.54% and 90.47% respectively to the positive rate of cancer of pancreas and chromaffin cell cancer; And be 0 to the false positive rate that the normal person detects, so this kit has crucial reference value to the clinical assistant diagnosis of cancer of pancreas and chromaffin cell cancer.
Claims (10)
1. Chromogranin A chemical luminescence immune analysis reagent box is characterized in that comprising following component:
1) Chromogranin A standard items;
2) the Chromogranin A monoclonal antibody of horseradish peroxidase-labeled;
3) the magnetic particle that encapsulates of Chromogranin A monoclonal antibody;
4) White-opalescent microwell plate;
5) cleansing solution;
6) chemical luminous substrate A and B working fluid.
2. a kind of Chromogranin A chemical luminescence immune analysis reagent box according to claim 1, the Chromogranin A monoclonal antibody that it is characterized in that said horseradish peroxidase-labeled is to process with following method:
1. take by weighing CgA monoclonal antibody 2.0mg, adding 1～2ml concentration is 0.05mol/L, the carbonate buffer solution dissolving of pH=7.5, and 4 ℃ store for future use;
2. take by weighing the horseradish peroxidase of 10.0mg, add the 1.0ml deionized water, vibration after the dissolving, is taken out 0.14～0.28ml, adds the NaIO of 0.1mol/L
4Solution 0.2ml, the lucifuge reaction is 1～2 hour under the room temperature;
3. the liquid that 1. step is obtained and the liquid mixing of step acquisition 2., the lucifuge reaction is 4～8 hours under the room temperature;
4. adding 200 μ l concentration is the NaBH of 5mg/ml
4The WS, the lucifuge reaction is 1～2 hour under the room temperature;
5. with 0.02M PBS dialysis 12-24 hour, secondarily purified with HPLC, collect protein peak and freezing preservations under-20 ℃.
3. a kind of Chromogranin A chemical luminescence immune analysis reagent box according to claim 1 is characterized in that the magnetic particle that said Chromogranin A monoclonal antibody encapsulates is to process with following method:
1. get 5mL magnetic granular mass content and be 2.5% PBS solution, divide the abandonment supernatant, wash the magnetic particle with deionized water with magnetic sheet;
2. the PBS with 0.02mol/L pH=6.2 washes the magnetic particle;
3. using the PBS adjustment cumulative volume of 0.02mol/L pH=6.2 is 5～10ml;
4. adding CgA monoclonal antibody 5mg is placed on and carries out homogenize 10～20 minutes on the shaking table;
5. add carbodiimide 5～10mg; Room temperature is placed on and carries out homogenize 5～10 hours on the shaking table;
6. the phosphate buffer with 0.1mol/L pH=7.4 washs 2 times;
7. add Tris and EDTA, making the content of Tris is 0.05mol/L, and the content that makes EDTA is 0.004mol/L, transfers pH=7.5, and making AC is 1.0mg/ml, 2-4 ℃ of preservation.
4. a kind of Chromogranin A chemical luminescence immune analysis reagent box according to claim 1 is characterized in that the preparation of said cleansing solution:
Get: trishydroxymethylaminomethane 6.05g; NaCl8.55g; Tween-20 0.5ml; Add water to 1L; Transfer pH to 7.5 ± 0.1 with HCl.
5. a kind of Chromogranin A chemical luminescence immune analysis reagent box according to claim 1 is characterized in that said chemical luminous substrate A working fluid is to process with following method:
(1) the Tris-HCl damping fluid of preparation 0.05mol/L pH=8.0;
(2) in the Tris-HCl damping fluid of the said step of 1000ml (1) preparation, add 3～5mmol luminol, mixing; Let the said chemical luminous substrate B working fluid be to process with following method:
(1) the Tris-HCl damping fluid of preparation 0.05mol/L pH=8.0;
(2) in the Tris-HCl damping fluid of the said step of 1000ml (1) preparation, add: 0.5～1.0mmol tetraphenylboron sodium; 1.42mmol the potassium ferricyanide; 0.38mmol cinnamic acid; 7.56mmol hydrogen peroxide, mixing.
6. the preparation method of a kind of Chromogranin A chemical luminescence immune analysis reagent box of claim 1 is characterized in that comprising the steps:
1) prepares the Chromogranin A standard items;
2) the Chromogranin A monoclonal antibody of preparation horseradish peroxidase-labeled;
3) prepare the magnetic particle that the Chromogranin A monoclonal antibody encapsulates;
4) prepare the White-opalescent microwell plate;
5) preparing washing liquid;
6) preparation chemical luminous substrate A and B working fluid.
7. the preparation method of a kind of Chromogranin A chemical luminescence immune analysis reagent box according to claim 6 is characterized in that the step of the Chromogranin A monoclonal antibody of said preparation horseradish peroxidase-labeled is:
1. take by weighing CgA monoclonal antibody 2.0mg, adding 1～2ml concentration is 0.05mol/L, the carbonate buffer solution dissolving of pH=7.5, and 4 ℃ store for future use;
2. take by weighing the horseradish peroxidase of 10.0mg, add the 1.0ml deionized water, vibration after the dissolving, is taken out 0.14～0.28ml, adds the NaIO of 0.1mol/L
4Solution 0.2ml, the lucifuge reaction is 1～2 hour under the room temperature;
3. the liquid that 1. step is obtained and the liquid mixing of step acquisition 2., the lucifuge reaction is 4～8 hours under the room temperature;
4. adding 200 μ l concentration is the NaBH of 5mg/ml
4The WS, the lucifuge reaction is 1～2 hour under the room temperature;
5. with 0.02M PBS dialysis 12-24 hour, secondarily purified with HPLC, collect protein peak and freezing preservations under-20 ℃.
8. the preparation method of a kind of Chromogranin A chemical luminescence immune analysis reagent box according to claim 6 is characterized in that the step of the magnetic particle that said preparation Chromogranin A monoclonal antibody encapsulates is:
1. get 5mL magnetic granular mass content and be 2.5% PBS solution, divide the abandonment supernatant, wash the magnetic particle with deionized water with magnetic sheet;
2. the PBS with 0.02mol/L pH=6.2 washes the magnetic particle;
3. using the PBS adjustment cumulative volume of 0.02mol/L pH=6.2 is 5～10ml;
4. adding CgA monoclonal antibody 5mg is placed on and carries out homogenize 10～20 minutes on the shaking table;
5. add carbodiimide 5～10mg; Room temperature is placed on and carries out homogenize 5～10 hours on the shaking table;
6. the phosphate buffer with 0.1mol/L pH=7.4 washs 2 times;
7. add Tris and EDTA, making the content of Tris is 0.05mol/L, and the content that makes EDTA is 0.004mol/L, transfers pH=7.5, and making AC is 1.0mg/ml, 2-4 ℃ of preservation.
9. the preparation method of a kind of Chromogranin A chemical luminescence immune analysis reagent box according to claim 6, the step that its characteristic prepares said cleansing solution is:
Get: trishydroxymethylaminomethane 6.05g; NaCl8.55g; Tween-20 0.5ml; Add water to 1L; Transfer pH to 7.5 ± 0.1 with HCl.
10. the preparation method of a kind of Chromogranin A chemical luminescence immune analysis reagent box according to claim 6 is characterized in that the step of said preparation chemical luminous substrate A working fluid is:
(1) the Tris-HCl damping fluid of preparation 0.05mol/L pH=8.0;
(2) in the Tris-HCl damping fluid of the said step of 1000ml (1) preparation, add 3～5mmol luminol, mixing; The step of said preparation chemical luminous substrate B working fluid is:
(1) the Tris-HCl damping fluid of preparation 0.05mol/L pH=8.0;
(2) in the Tris-HCl damping fluid of the said step of 1000ml (1) preparation, add: 0.5～1.0mmol tetraphenylboron sodium; 1.42mmol the potassium ferricyanide; 0.38mmol cinnamic acid; 7.56mmol hydrogen peroxide, mixing.
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