CN101377513A - Chromogranin A chemiluminescence immune analysis quantitative determination reagent kit and preparing method thereof - Google Patents
Chromogranin A chemiluminescence immune analysis quantitative determination reagent kit and preparing method thereof Download PDFInfo
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- CN101377513A CN101377513A CN 200810104147 CN200810104147A CN101377513A CN 101377513 A CN101377513 A CN 101377513A CN 200810104147 CN200810104147 CN 200810104147 CN 200810104147 A CN200810104147 A CN 200810104147A CN 101377513 A CN101377513 A CN 101377513A
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Abstract
The invention relates to a chromogranin A chemiluminescent quantitative detection kit, which relates to the medical field of immunoassay. The invention provides a chromogranin A chemiluminescent immunoassay quantitative detection kit and a preparation method thereof. The kit is mainly composed of a chromogranin A antigen calibrator, a solid-phase vector which is coated by chromogranin A antigen, a chromogranin A specific antibody which is marked by enzyme, chemiluminescent substrate solution and concentrated washing solution. The kit adopts the double antibody sandwich one step method to detect the content of chromogranin A in human serum, can effectively improve the sensitivity and the stability, and has good application value in clinic.
Description
Technical field
The present invention relates generally to the immunoassay medical domain, and a kind of Chromogranin A chemiluminescence immune analysis quantitative determination reagent kit and preparation method thereof is provided.
Background technology
Chromogranin A (CgA) claims Chromogranin A, chromogranin A etc. again, is a kind of acidity, hydrophilic protein of being made up of 439 amino acid, is positioned at the chromaffin granule of neuroendocrine cell.It is a member in the neuropeptide family.The proteolysis of CgA has tissue specificity, and the difference of this protein fracture is relevant with its residing tissue.The CgA that tumour cell occurs in immunohistochemistry is relevant with the neuroendocrine primary tumor, and neuroendocrine tumor patient's change of serum C gA level increases.Change of serum C gA is used for neuroendocrine tumor mark preferably now, and therefore, the check of change of serum C gA can be used for the diagnosis and the treatment of neuroendocrine tumor.
Aspect pheochromocytoma and functional benign tumour, CgA detects than 24h urine 5-hydroxyindoleacetic acid (5-HIAA) has higher sensitivity, and therefore, many documents and expert advise that CgA is applied to a clinical line to be detected.The clinical meaning of CgA is:
(1) CgA can be used for distinguishing primary or the secondary hypertension that high blood sample that pheochromocytoma causes and non-pheochromocytoma cause.
(2) CgA is an extremely responsive and accurate mark of pancreas islet benign tumour and endocrine tumors, and biological property, design feature and the diseases range of its level and tumour are closely related.
(3) CgA can be used for diagnosing small-cell carcinoma of the lung, uses CgA and NSE joint-detection simultaneously, can improve the sensitivity of diagnosis.
(4) mensuration of change of serum C gA helps to discern the tumour of corticotropin (ACTH) and short cortex renis hormone releasing hormone (CRH) abnormal secretion, for example relevant with neuroendocrine benign tumour, thyroid gland medullary substance cancer and small-cell carcinoma of the lung.
(5) change of serum C gA level can be applicable to the diagnosis of prostate cancer, especially for prostate specific antigen (PSA) feminine gender or with being far apart the diagnosis of shifting case.CgA and PSA joint-detection can improve prostate cancer diagnosis and be worth.So change of serum C gA is the tumor markers preferably that often is applied to the NE differentiation prostate cancer now.
(6) according to the literature, the CgA level of Patients with Chronic Heart Failure increases, and the amplitude that CgA increases is directly relevant with the order of severity of heart failure.CgA is the independent indication factor of Patients with Chronic Heart Failure mortality ratio, if the CgA level of heart failure patient increases, then its death risk is corresponding increases.
Quantitative test for CgA, method commonly used at present is immunoradiometric assay (IRMA), radio immunoassay (RIA) and enzyme linked immunosorbent assay analysis method (ELISA), though immunoradiometric assay and radio immunoassay is highly sensitive, there are shortcomings such as complicated operation, measurement result instability, reagent holding time weak point, radioactive contamination in it; Enzyme linked immunosorbent assay analysis method then exists detection sensitivity not high enough, and sensing range is narrow, and influence factor is more, easily causes shortcomings such as false negative and false positive.Therefore said method all can not satisfy clinical requirement.
Summary of the invention
The present invention overcomes the deficiency of said method, chemiluminescence is learned effectively with the immunoassay of CgA combine, and has used simultaneously that luminous signal is strong, longer duration, the self-control chemical luminous substrate liquid of high s/n ratio more.Improve the accuracy of testing result, and can not produce radioactive contamination to external world.
The object of the present invention is to provide a kind of detection precision, sensitivity and stability higher, specificity and accuracy be Chromogranin A chemiluminescence immune analysis quantitative determination reagent kit preferably.
Chromogranin A chemiluminescence immune analysis quantitative determination reagent kit of the present invention is mainly by Chromogranin A antigen calibration object; The solid phase carrier of Chromogranin A antibody sandwich; The Chromogranin A specific antibody of enzyme labeling; The chemical luminous substrate liquid that enzyme acted on; Concentrated cleaning solution is formed.
In the mentioned reagent box, described solid phase carrier is microwell plate or wraps by pearl.
A further object of the present invention provides a kind of method for preparing the mentioned reagent box.
May further comprise the steps:
(a) with the calf serum be matrix, add Chromogranin A preparation Chromogranin A antigen calibration object.
(b) the Chromogranin A specific antibody with carbonate buffer solution and debita spissitudo is mixed and made into coating buffer, and it is carried on the solid phase carrier, 4 ℃ of placements are spent the night, and add the confining liquid for preparing after getting rid of coating buffer, room temperature is placed and was got rid of confining liquid, room temperature removal moisture drying 24 hours in 3 hours.
(c) with alkaline phosphatase or horseradish peroxidase-labeled Chromogranin A specific antibody.
(d) preparation 1,2-two oxidative ethane analog derivatives or luminol chemiluminescence substrate solution.
(e) preparation Tris-HCl or PBST concentrated cleaning solution.
(f) the Chromogranin A specific antibody of the above-mentioned Chromogranin A antigen of packing calibration object, enzyme labeling, chemical luminous substrate liquid and concentrated cleaning solution and be assembled into the finished product kit.
What kit of the present invention adopted is " double-antibody sandwich single stage method " reaction pattern, both effectively utilized the chemiluminescence principle, on the basis of enzyme-linked immuno assay, use the enzymatic luminous substrate again, it can not produce radioactive contamination to external world, and sensitivity and stability height, specificity and accuracy are good, can be clinical diagnosis more special, quick, reliable foundation is provided.
The present invention's " Chromogranin A chemiluminescence immune analysis quantitative determination reagent kit " can be used for detecting the CgA level among the neuroendocrine tumor patients serum, thereby carries out diagnosis and treatment to the neuroendocrine tumor patient.
Description of drawings
Fig. 1 is the calibration object linear graph in the prepared kit of embodiment 1
Fig. 2 exempts from the contrast that kit carries out clinical blood sample measured value for kit of the present invention with enzyme
Embodiment
Embodiment 1 using basic phosphatase systems produce Chromogranin A chemiluminescence immune analysis quantitative determination reagent kit of the present invention
One, the preparation of alkali phosphatase enzyme mark antibody
The CgA specific antibody is fully dialysed with the PBS damping fluid with classical glutaraldehyde cross-linking method and alkaline phosphatase coupling, adds isopyknic glycerine and preservation in low temperature refrigerator (20 ℃) in bond solution.Dilute with 20% cow's serum enzyme dilution when re-using.Draw through optimization Test, the best effort concentration range of enzyme labelled antibody is 1:5000～10000.
The prescription of employed 20% cow's serum enzyme dilution is:
NaH2PO4·2H2O 1.28mM 0.2g
Na2HPO4·12H2O 8.10mM 2.9g
NaCl 150mM 8.8g
NBCS 20% 200ml
Proclin300 0.1％ 1.0ml
Food Red 0.05 ‰ 1.0ml
Deionized water is settled to 1000mL
Two, the preparation of Chromogranin A antigen calibration object
With the calf serum is matrix, and it is formulated to add Chromogranin A, and its concentration is respectively 0,15,50,125,250,500ng/mL.
Three, the specific antibody bag is by the preparation of solid phase carrier.
(1) bag quilt
Adopting 0.05M pH value is that 9.6 the carbonate buffer solution and the Chromogranin A specific antibody of debita spissitudo purifying are mixed and made into coating buffer, and it is carried on the solid phase carrier;
Particularly, the prescription of described 0.05mol/L carbonate buffer solution is:
Natrium carbonicum calcinatum 1.59g
Sodium bicarbonate 2.94g
Deionized water 1000mL
Behind the dissolving mixing, adjust pH value to 9.6, add 5.0mg Chromogranin A antibody mixing, add then in each hole of microwell plate, every hole 110 μ L, 4 ℃ of placements are spent the night.
(2) sealing
The prescription of confining liquid is:
NaH
2PO
4·2H
2O 0.2g
Na
2HPO
4·12H
2O 2.9g
BSA 10g
Proclin300 1mL
Distilled water is settled to 1000mL
The mentioned reagent weighing is put into clean container well, add the distilled water constant volume, the dissolving mixing, adjusting the pH value is 7.0～7.6.
Get rid of coating buffer, the amount of confining liquid by 200 μ L/ holes added in the microwell plate, room temperature was placed 3 hours.Get rid of confining liquid, on thieving paper, pat dry.Vacuum sealing bag is carried out in the room temperature removal moisture drying immediately after 24 hours.Give over to standby.
Four, chemical luminous substrate
The compound method of the chemical luminous substrate liquid of alkaline phosphatase used in the present invention (ALP):
Tris 24g
NaCl 160g
KCl 4g
HCl 15mL
Distilled water 1000mL
Proclin?300 1mL
AMPPD 200mL
Five, the preparation prescription of 20 times of concentrated cleaning solutions
Tris 24g
NaCl 160g
KCl 4g
HCl 15mL
Deionized water 1000mL
Adjust pH value to 7.4.
Six, above-mentioned semi-manufacture of packing and form the finished product kit.
Embodiment 2 uses horseradish peroxidase systems produce Chromogranin A chemiluminescence immune analysis quantitative determination reagent kit of the present invention
One, the preparation of horseradish peroxidase-labeled antibody
Dissolving 4.4mg HRP adds 0.4mL sodium periodate (50mmol/L) stirring at room 20min in 1mL distilled water, through the 1mmol/L sodium-acetate buffer, the pH value is that 4.4 dialysis backs add 8mg Chromogranin A specific antibody, stirs 2h, uses 200mmol/L NaBH at last
4Reduce, after the dialysis of 0.02M PBS damping fluid, add equal-volume glycerine, preserve below-20 ℃.
Two, the preparation of chemical luminous substrate
1, chemical luminous substrate A liquid
Luminol 10mM 1.7716g
4-xenol 0.3mM 0.051g
4-iodobenzene boric acid 0.05mM 0.012g
Boric acid 11.4g
Borax 4.9g
The fixed molten 1000mL of distilled water
Adjust pH to 8.0～10.0
2, chemical luminous substrate B liquid
Urea peroxide 3.5mM 0.329g
Tween20 0.1％ 1ml
Na2HPO4·12H2O 51.58g
NaH2PO4·2H2O 8.74g
The fixed molten 1000mL of distilled water
Adjust pH to 7.0～7.6
A liquid mixes with B liquid equal proportion during use.
Three, the preparation prescription of 20 times of concentrated cleaning solutions
Na2HPO4·12H2O 58g
NaH2PO4·2H2O 2g
NaCl 160g
Tween-20 1mL
Proclin?300 1mL
Deionized water 1000mL
Adjust pH to 7.2～7.4
The compound method of other components is identical with the method for embodiment 1.
Embodiment 3～4 preparations Chromogranin A chemiluminescence immune analysis quantitative determination reagent kit of the present invention
As outside the carrier, all the other are all with identical with the method for embodiment 1 and 2 preparations quantitative determination reagent kit of the present invention divided by plastic bead.
The using method of embodiment 5 kits of the present invention
Before using this kit to experimentize, need to take out earlier solid phase carrier, calibration object/testing sample, labelled antibody solution, make them equilibrate to room temperature room temperature placement 15～30 minutes; Afterwards, be ready to suitable micro sample adding appliance and corresponding suction nozzle and check Chemiluminescence Apparatus and supplementary instrument, as wash plate machine etc., whether operate as normal.
Use this kit as follows according to the concrete operations step that the method for embodiment 1 experimentizes:
1) will test the microwell plate that needs is placed on the grillage;
2) add each concentration calibration object 0,15,50,125,250,500ng/mL and the every hole of testing sample in the reacting hole respectively and add 25 μ L, blank 1 hole is established in each test, and each hole adds enzymic-labelled antibody solution 100 μ L except that blank well then;
3) 30 seconds mixings of vibration on the micro oscillator;
4) with shrouding film shrouding, room temperature (20～27 ℃) incubation 60min;
5) discard solution in the hole with washing lotion, automatic washer or craft are washed plate 5 times, pat dry on thieving paper;
6) every hole adds luminous substrate liquid 100 μ L, fully shakes evenly room temperature (20～27 ℃) lucifuge reaction 10～30min with micro-oscillator.
7) on the chemiluminescence measuring instrument, measure the luminous intensity (RLU) in each hole in regular turn, 1 second/hole of Measuring Time;
8) read calibration object concentration value and its corresponding RLU value respectively, on the double logarithmic curve of setting up, set up typical curve (referring to accompanying drawing 1);
9) print test results report.
Embodiment 6 evaluations of kit of the present invention on methodology
The kit of preparation among the embodiment 1 is identified that kit method index of the present invention is as follows through a large amount of experiment showed, according to manufacturing conventional in this area and vertification regulation:
1) sensing range: 7.8～500ng/mL.
2) limit of identification: replicate determination 20 hole null value calibration object serum, calculate null value calibration object RLU mean value (X) and standard deviation (S), with X+2S is in the definite value substitution typical curve equation, obtains its corresponding concentration value, and it is 7.8ng/mL that pairing concentration value is limit of identification.
3) precision: the concentration of replicate determination 100 hole quality controlled serums, duplicate detection 3 times, each 20 holes of repeating, carrying out precision measures, precision (high, medium and low three quality control clearances) is 5.2% in the computational analysis,, precision between analysis (high, medium and low three quality control clearances) is 6.8%.
4) accuracy: the recovery experiment of a Senior Three concentration serum in hanging down respectively (concentration is 10ng/mL, 40ng/mL and 80ng/mL respectively), it on average is respectively 102%, 111% and 97%.
5) specificity: carry out cross reaction with its associated protein such as PSA, fPSA, CEA, the result shows that no cross reaction occurs.
6) stability: each reagent set placed 37 ℃ of baking ovens respectively 3 days, 7 days and 15 days, and experiment finds that each component still can keep stable.
Find that by above-mentioned technical indicator kit of the present invention is highly sensitive, high specificity, sensing range is wide, and good stability is applicable to clinical detection.Therefore the present invention be the clinical detection Chromogranin A, and auxiliary diagnosis and the observation of operation back provide a kind of more accurate, method easily and efficiently.
Embodiment 7 uses kit of the present invention is exempted from kit with enzyme and is carried out the contrast of blood sample measured value
Collect 111 parts of clinical tumor patients serum samples, patients with prostate cancer 40 examples wherein, pancreas benign tumour patient 16 examples, pheochromocytoma patient's 55 examples, 200 parts of normal human serum samples.Use the kit of the embodiment of the invention 1 and enzyme to exempt from diagnostic kit respectively and carry out blood examination, the statistical experiment result line correlation analysis of going forward side by side, these two kinds of method height correlations.
Kit of the present invention and enzyme are exempted from the clinical trial contrast of kit, and experimental result sees the following form 1:
Table 1 kit of the present invention and enzyme are exempted from the experimental comparison result of kit
Wherein the CgA mean value of 200 parts of normal person's blood samples is 52.87ng/mL, and normal reference value is less than 100ng/mL.More approaching by finding that relatively this law and enzyme are exempted from the kit testing result simultaneously, its related coefficient reaches 0.9468.But it is 77.48% to be higher than enzyme and to exempt from 73.87% of kit that kit of the present invention detects the recall rate of positive serum, wherein in detecting normal human serum, kit of the present invention does not detect the positive, and enzyme is exempted from kit to detect an example positive, shows that this kit obviously is better than enzyme linked immunological kit.
Claims (9)
1, Chromogranin A chemiluminescence immune analysis quantitative determination reagent kit is characterized in that, mainly by Chromogranin A antigen calibration object; The solid phase carrier of Chromogranin A antibody sandwich; The Chromogranin A specific antibody of enzyme labeling; Chemical luminous substrate liquid; Concentrated cleaning solution is formed.
2, kit as claimed in claim 1 is characterized in that, described calibration object is to be matrix with the calf serum, and adding Chromogranin A antigen is formulated; Described solid phase carrier is microwell plate or plastic bead; Described enzyme is alkaline phosphatase or horseradish peroxidase; Described chemical luminous substrate liquid is 1,2-two oxidative ethane analog derivative or luminols.
3, kit as claimed in claim 2, it is characterized in that, described chemical luminous substrate liquid luminol or different luminol comprise A liquid and B liquid, the prescription of described luminous substrate liquid A is: luminol 10mM, 4-xenol 0.3mM, 4-iodobenzene boric acid 0.05mM, boric acid 11.4g, borax 4.9g and 1000ml distilled water, pH8.0～10.0; The prescription of described luminous substrate liquid B is: urea peroxide 3.5mM, 1%Tween201ml, Na2HPO412H
2O 51.58g, NaH2PO42H
2O 8.74g and 1000ml distilled water, pH7.0～7.6.
4, kit as claimed in claim 1 is characterized in that, described concentrated cleaning solution is Tris-HCl cleansing solution or PBST cleansing solution, carries out 20 times dilution with deionized water earlier in use.
5, a kind of method for preparing the described kit of claim 1 is characterized in that may further comprise the steps: preparation Chromogranin A antigen calibration object; With Chromogranin A antibody sandwich solid phase carrier; With enzyme labeling Chromogranin A specific antibody; Preparation chemical luminous substrate liquid and concentrated cleaning solution; Chemical luminous substrate liquid and concentrated cleaning solution that Chromogranin A specific antibody, the enzyme of the above-mentioned Chromogranin A antigen of packing calibration object, enzyme labeling acted on; Be assembled into finished product.
6, method as claimed in claim 5 is characterized in that, described bag, is got rid of coating buffer confining liquid is carried on above-mentioned solid phase carrier for coating buffer is carried on the solid phase carrier by the step of solid phase carrier.
7, method as claimed in claim 5 is characterized in that, described enzyme is alkaline phosphatase or horseradish peroxidase.
8, method as claimed in claim 6 is characterized in that, described coating buffer is that 9.6 the carbonate buffer solution and the Chromogranin A antibody of debita spissitudo purifying are mixed and made into for 0.05M pH value.
9, method as claimed in claim 5 is characterized in that, described solid phase carrier is microwell plate or plastic bead; Described chemical luminous substrate liquid is 1,2-two oxidative ethane analog derivative or luminols; Described concentrated cleaning solution is Tris-HCl cleansing solution or PBST cleansing solution.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102226808A (en) * | 2011-03-28 | 2011-10-26 | 北京永瀚星港生物技术有限公司 | Trypsinogen-2 chemiluminescent immunoassay kit and preparation method thereof |
| 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 |
| CN106093416A (en) * | 2016-05-18 | 2016-11-09 | 北京北方生物技术研究所有限公司 | A kind of test kit of one-step method detection Procalcitonin. and preparation method thereof |
| CN106461673A (en) * | 2014-04-15 | 2017-02-22 | 塞尚公司 | Immunoassay and antibodies for the detection of chromogranin A |
| CN108780093A (en) * | 2016-03-09 | 2018-11-09 | 塞尚公司 | Chromogranin A as bladder carcinoma marker |
| CN109507432A (en) * | 2018-11-14 | 2019-03-22 | 嘉兴行健生物科技有限公司 | A kind of CGA detection reagent and detection reference interval and detection method |
| CN111500584A (en) * | 2020-04-23 | 2020-08-07 | 福州市长乐区宝爱冬医学技术有限公司 | Sequence and application of aptamer CGA02 for specifically recognizing chromogranin A |
| CN113702362A (en) * | 2021-08-27 | 2021-11-26 | 宁波熙宁检测技术有限公司 | Method for quantitatively detecting IFN-gamma concentration by using chemiluminescence method and detection kit thereof |
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2008
- 2008-04-16 CN CN 200810104147 patent/CN101377513A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102226808A (en) * | 2011-03-28 | 2011-10-26 | 北京永瀚星港生物技术有限公司 | Trypsinogen-2 chemiluminescent immunoassay kit and preparation method thereof |
| 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 |
| CN102520195B (en) * | 2011-12-30 | 2014-07-23 | 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 |
| CN106461673A (en) * | 2014-04-15 | 2017-02-22 | 塞尚公司 | Immunoassay and antibodies for the detection of chromogranin A |
| CN108780093A (en) * | 2016-03-09 | 2018-11-09 | 塞尚公司 | Chromogranin A as bladder carcinoma marker |
| CN106093416A (en) * | 2016-05-18 | 2016-11-09 | 北京北方生物技术研究所有限公司 | A kind of test kit of one-step method detection Procalcitonin. and preparation method thereof |
| CN106093416B (en) * | 2016-05-18 | 2018-10-12 | 北京北方生物技术研究所有限公司 | A kind of kit and preparation method thereof of one-step method detection Procalcitonin |
| CN109507432A (en) * | 2018-11-14 | 2019-03-22 | 嘉兴行健生物科技有限公司 | A kind of CGA detection reagent and detection reference interval and detection method |
| CN111500584A (en) * | 2020-04-23 | 2020-08-07 | 福州市长乐区宝爱冬医学技术有限公司 | Sequence and application of aptamer CGA02 for specifically recognizing chromogranin A |
| CN111500584B (en) * | 2020-04-23 | 2022-08-05 | 丽水君弘生物科技有限公司 | Sequence and application of aptamer CGA02 for specifically recognizing chromogranin A |
| CN113702362A (en) * | 2021-08-27 | 2021-11-26 | 宁波熙宁检测技术有限公司 | Method for quantitatively detecting IFN-gamma concentration by using chemiluminescence method and detection kit thereof |
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Owner name: BEIJING KEMEI BIOLOGICAL TECHNOLOGY CO., LTD. Free format text: FORMER OWNER: KEMEI DONGYA BIOLOGICAL TECHNOLOGY CO., LTD., BEIJING Effective date: 20111028 |
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Application publication date: 20090304 |