CN101368972A - Chemical luminescence immune assay determination reagent kit for hydrocortisone and preparation method thereof - Google Patents
Chemical luminescence immune assay determination reagent kit for hydrocortisone and preparation method thereof Download PDFInfo
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- CN101368972A CN101368972A CN 200810103268 CN200810103268A CN101368972A CN 101368972 A CN101368972 A CN 101368972A CN 200810103268 CN200810103268 CN 200810103268 CN 200810103268 A CN200810103268 A CN 200810103268A CN 101368972 A CN101368972 A CN 101368972A
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Abstract
The invention provides a cortisol chemiluminescence immunoassay test kit and a preparing method thereof, which is applied to quantitative analysis of the cortisol in blood serum. The test kit effectively adopts the biotin-avidin system to ensure the stability of the immunoreaction system, and also utilizes the chemiluminescence technique to ensure detecting sensitivity.
Description
Technical field
The invention belongs to the clinical blood technical field of immunoassay, particularly, the invention provides a kind of chemical luminescence immune assay determination reagent kit for hydrocortisone and preparation method thereof.This kit is highly sensitive, and high specificity is easy and simple to handle, and is reliable and stable.
Background technology
(Cortisol Cor) is the glucocorticoid of adrenal cortex zona fasciculata secretion to cortisol, to sugar in the body, protein, fatty metabolism and keep a series of vital movements such as body water, electrolyte balance and play important regulating action.Cortisol also is main irritability hormone simultaneously, under wound, infection, motion, situation such as excited, tangible rising is arranged all, the acth secretion cortisol hyperfunction of a variety of causes or low, equal obvious normal physiological functions of interference body, produce characteristic clinical, therefore measuring cortisol has clinical widely and scientific research value.
Chemiluminescence immunoassay technology is that combine of growing up of the immune analysis method of high-sensitive chemiluminescence analysis method and high specificity is had the new immuno analytical method of the high selectivity of the high sensitivity of chemiluminescence analysis and immunoassay.(biotin-avidin system BAS) has multistage signal amplification, and does not increase non-specific interference biotin-avidin system, and has highly sensitive, characteristics such as specificity good, stability is high, applicability is strong and experimental cost is low.
Utilize physisorption on solid phase, can be existed the monoclonal antibody consumption big merely the monoclonal antibody bag, shortcomings such as loss of activity is big, and process optimization is comparatively loaded down with trivial details, and with the streptavidin bag by on solid phase carrier, with the capture antibody biotinylation, above problem can be readily solved.The present invention will catch the monoclonal antibody biotinylation, and with alkaline phosphatase (ALP) mark cortisol antigen to be measured, biotinylation monoclonal antibody and enzyme-labelled antigen are diluted to working solution with dilution with certain proportion.During detecting operation blood serum sample, biotinylation monoclonal anti liquid solution and enzyme-labelled antigen solution are added the solid phase that is coated with streptavidin successively, through after the immune response, the immune complex of the streptavidin-biotinylated antibody that on solid phase, forms-determined antigen or streptavidin-biotinylated antibody-enzyme-labelled antigen, add chemical luminous substrate then, utilize Chemiluminescence Apparatus to detect relative luminous intensity, just can quantitative test serum in the content of cortisol.
The present invention is by the chemiluminescence light signal of the highly sensitive characteristics of detection, rather than the color signal of the chromogenic reaction of traditional enzyme-linked immuno assay, and its sensitivity is greatly enhanced, and can reach 10
-18Mol/mL.And the easy operation of having inherited traditional enzyme-linked immune analytic method can realize fast detection the in enormous quantities, and applicability is wider, and is easier in clinical diagnosis and research work application.
Summary of the invention
The preparation method who the purpose of this invention is to provide a kind of chemical luminescence immune assay determination reagent kit for hydrocortisone and this kit.
Chemical luminescence immune assay determination reagent kit for hydrocortisone according to the present invention comprises: the cortisol calibration object; The solid phase of streptavidin bag quilt; Biotinylation cortisol monoclonal antibody; The cortisol antigen of alkaline phosphatase mark; Chemical luminous substrate; And cleansing solution.
Cortisol series calibration object is matrix with the calf serum, and the pure product of adding cortisol are formulated.
Streptavidin is dissolved in carbonic acid buffer, is coated on then on the solid phase carrying agent, and solid phase carrier can be for being coated with microwell plate, plastic bead, plastic tube.
Biotinylation cortisol monoclonal antibody is by the cortisol monoclonal antibody and the biotin coupling formation of purifying, working concentration 0.02 μ g/mL.
The used marker enzyme of labelled antigen is an alkaline phosphatase, and alkali phosphatase enzyme mark adopts the glutaraldehyde cross-linking method of improvement, and gained enzyme-labelled antigen working concentration is 1:1000-4000.
According to chemical luminescence immune assay determination reagent kit for hydrocortisone preparation method of the present invention, may further comprise the steps: preparation cortisol calibration object; With the streptavidin bag by solid phase carrier, cortisol monoclonal antibody biotinylation; With the anti-cortisol antigen of alkali phosphatase enzyme mark; Concentrated cleaning solution; Chemical luminous substrate; The packing of above-mentioned semi-manufacture component and be assembled into finished product.
The preparation of 1 calibration object
The pure product of cortisol add the calibration object matrix solution, prepare an intermediate concentration calibration object strong solution, adopt the method for pointwise dilution then, are mixed with 0,10,40,100,200, the serial calibration object of 500ng/mL, and calibration object is proofreaied and correct with national standard.Described matrix solution is 100% calf serum deactivation, centrifugal, filtration, adds 0.1% Proclin-300.Cortisol calibration object after the concentration calibration is used the 3mL Clear glass bottles and jars, and the packing of 0.5ml/ bottle is labelled, freezing or 2～8 ℃ of preservations.
The preparation of 2 solid phase carriers
1) bag quilt
Adopting 0.05mol/L, pH value is that 7.2 the PBS and the cortisol monoclonal antibody of debita spissitudo are mixed and made into coating buffer, and it is carried on the solid phase carrier, and solid phase carrier is microwell plate, plastic bead or plastic tube.
2) sealing
The preparation confining liquid comprises 0.2g NaH based on the described confining liquid of 1000mL
2PO
42H
2O, 2.9gNaH
2PO
412H
2O, 100ml calf serum and 1mLProclin-300, the pH value of described confining liquid is 7.0～7.4, then the gained confining liquid is carried on the solid phase carrier after the above-mentioned washing.
3) packing, preservation
Remove the lock solution on insolubilized antibody surface, be placed on the drying room air dry, use aluminium foil bag, put into drying agent, sealing, labelled, 2～8 ℃ of preservations.
The preparation of 3 enzymic-labelled antibodies
The cortisol antigen of enzyme labeling adopts the glutaraldehyde cross-linking method mark alkaline phosphatase (ALP) of improvement, with dense enzymic-labelled antibody, to working concentration, described enzyme becomes the work enzyme solutions with the enzyme diluted with the enzyme diluted, and the enzymic-labelled antibody dilution ratio is 1:1000-1:4000.With the packing on demand of 15mL white plastic bottle, labelled, 2～8 ℃ of preservations.
The preparation of 4 biotinylated antibodies
Pure monoclonal antibody of cortex and biotin form biotinylated antibody by coupling reaction, with dense biotinylated antibody, arrive working concentration, the about 0.02 μ g/mL of described antibody working solution concentration with the enzyme diluted.With the packing on demand of 15mL white plastic bottle, labelled, 2～8 ℃ of preservations
5 cleansing solutions and luminous substrate packing
Cleansing solution and chemical luminous substrate, packing as required, labelled, 2～8 ℃ of preservations.
The method according to this invention, the finished product of described finished product assembling comprises: cortisol calibration object one cover, insolubilized antibody one bag, one bottle of biotinylated antibody solution, one bottle of enzyme-labelled antigen working solution, one bottle of cleansing solution, one bottle of chemical luminous substrate, one of kit operation instructions and other annex assemble.
The present invention has set up the detection method of human serum cortisol, can detection by quantitative go out the cortisol content in the patients serum, and according to how many auxiliary judgment treatment of diseases effect and change of illness state thereof of cortisol content.Every index of this chemical luminescence immune assay determination reagent kit for hydrocortisone all reaches the analytic approach level of similar import reagent box, and is highly sensitive, specificity good, and is easy and simple to handle, reliable and stable.
Description of drawings
Fig. 1 is calibration object linear graph (the accompanying drawing concentration unit: ng/mL) in the prepared kit of embodiment 1.
Embodiment
One, the preparation of cortisol calibration object
With the calf serum is matrix, and the dense raw material of people's seminal plasma of purifying is formed with the national standard product serial dilution that is as the criterion.Calf serum needs 56 ℃ of deactivations 30 minutes before using, and adds 0.1%Proclin-300.0,10,40,100,200, totally 6 bottles of the calibration objects of 500ng/mL the packing concentration range is:.
Two, the preparation of enzyme mark cortisol antigen
(1) Gai Liang glutaraldehyde cross-linking method mark alkaline phosphatase (ALP)
1) get ALP 1.0mg and cortisol antigen 2.0mg, be dissolved in respectively in the physiological saline, mix, final volume is controlled at 0.50ml;
2) glutaraldehyde of adding 0.50ml 1%, behind the room temperature magnetic agitation 15min, lucifuge reaction 4h;
3) the monoethanolamine 0.10ml of adding 1.0mol/L, room temperature incubation 2h;
4) add the magnetic agitation dialyzed overnight with PBS under 4 ℃ of conditions, change twice of dislysate;
5) product of will dialysing moves into reagent bottle and adds equal-volume glycerine, and mixing adds 1% BSA, and airtight freezing preservation is standby.
(2) enzyme-labelled antigen dilution
Contain in the 1000mL enzyme dilution: sodium dihydrogen phosphate 0.2g, sodium hydrogen phosphate 2.9g, sodium chloride 8.8g, BSA 5g, Proclin-300 1ml.PH adjusts to 7.0-7.2.
(3) enzymic-labelled antibody working solution concentration is selected
Evidence, adopting the square formation method to select the working concentration scope of enzyme-labelled antigen is 1:1000～4000.
(4) enzymic-labelled antibody working solution preparation
With described (2) dilution,, dense enzyme-labelled antigen is diluted to working solution according to (3) described dilution ratio.
Three, with the streptavidin bag by solid phase carrier
(1) bag quilt
Adopting 0.05mol/L pH value is that 7.2 the PBS and the streptavidin of debita spissitudo are mixed and made into coating buffer, and it is carried on the solid phase carrier;
Particularly, described method for coating can comprise:
The 0.05mol/L pH value that can adopt 1000mL is the sodium dihydrogen phosphate that 7.2 PBS comprises 2.2g, 12.9 the deionized water solution of the dibastic sodium phosphate of gram and 9 gram sodium chloride, be mixed and made into coating buffer with the streptavidin of debita spissitudo, add then in each hole of microwell plate, every hole 110 μ L, 4 ℃ are spent the night, and it is carried on the solid phase carrier;
(2) sealing
With load weighted NaH
2PO
42H
2O 0.2g, Na
2HPO
412H
2O 2.9g, calf serum 100mL puts into clean container, and add distilled water and be settled to 1000ml, the dissolving mixing, adjusting the pH value is 7.0-7.2.
Every hole adds confining liquid 300 μ L respectively, and room temperature was placed 3 hours.Get rid of confining liquid, vacuum sealing bag is carried out in room temperature removal moisture drying 24 hours.2～8 ℃ of preservations of labeling postposition.
Four, biotinylation cortisol Monoclonal Antibody
(1) biotinylation cortisol Monoclonal Antibody
1) biotin (BNHS) is dissolved in N, dinethylformamide (DMF) is made into 1mg/mL;
2) with 0.1mol/L pH value be 9.0 NaHCO
3The cortisol monoclonal antibody dilution of purifying is 2mg/mL;
3) be that 1:7 or weight ratio 1:5 mix by BNHS with the antibody volumetric ratio, react 2～4h under the stirring at room;
4) bag filter of packing into is 7.2 PBS4 ℃ of dialyzed overnight with 0.05mol/L pH value, and bond adds equal-volume glycerine, preserves standby below-20 ℃.
(2) biotinylation cortisol monoclonal antibody working solution preparation
With the dense biotinylated antibody of (1) gained with diluted to 0.2 μ g/mL, standby.
Five, chemical luminous substrate liquid
The compound method of chemical luminous substrate liquid used in the present invention:
Adding Tris and dense HCl are made into the Tris-HCl damping fluid of 0.1mol/L pH8.5 in distilled water.In this damping fluid of 1000mL, add NaCl 160g, KCl 4g, AMPPD 200ml adds 0.1% Proclin-300, mixes, and directly uses after the packing.
Six, 20 times of concentrated cleaning solutions
With load weighted Na
2HPO
412H
2O 58g, NaH
2PO
42H
2O 2g, NaCl 160g puts in the clean volumetric flask, is settled to 1000mL with deionized water, adds Tween-20 and Proclin 300 each 1mL adjustment pH to 7.2～7.4, and packing is standby.
Seven, semi-manufacture and finished product are formed
Above-mentioned steps gained semi-manufacture and essential annexes such as product description become the finished product kit through the packing assembling.Calibration object in the kit of the present invention is a liquid; Label also is the working solution that has been diluted to working concentration, and insolubilized antibody all can directly use for to wrap by good in advance.
Embodiment 2～3 preparations chemical luminescence immune assay determination reagent kit for hydrocortisone of the present invention
Divided by respectively with plastic bead, plastic tube as outside the carrier, all the other all prepare chemical luminescence immune assay determination reagent kit for hydrocortisone with the method identical with embodiment 1.
The using method of embodiment 4 kits of the present invention
Before using this kit to experimentize, need to take out earlier insolubilized antibody, calibration object/test sample, label 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 lath (wrapped by good streptavidin, 96/48 hole is removable to be the 12/6*8*1 hole) that needs is placed on the grillage;
2) add testing sample and each concentration calibration object in the reacting hole respectively, the every hole of calibration object adds each 25 μ l of 0,1.0,4.0,10.0,20.0,50.0 μ g/dL, blank 1 hole is established in each test, and each hole adds biotinylated antibody and enzyme-labelled antigen solution 50 μ l except that blank well then;
3) 30 seconds mixings of vibration on the micro oscillator;
4) room temperature (18-26 ℃) incubation is 45 minutes;
5) discard solution in the hole, wash plate 5 times, on thieving paper, pat dry with automatic washer or craft;
6) every hole adds luminous substrate 50 μ l, vibration mixing, room temperature (22～28 ℃) reaction 30～90 minutes;
7) survey relative light unit (RLU), Measuring Time 0.1-1 second/hole with Chemiluminescence Apparatus;
8) respectively calibration object concentration and corresponding RLU are taken the logarithm, on the double logarithmic curve of setting up, set up typical curve (seeing accompanying drawing 1), on typical curve, find the concentration of this serum cortisol, calculating testing result with each test serum RLU value;
9) print test results report.
The methodology of embodiment 5 kits of the present invention is identified
The kit of preparation among the embodiment 1 is identified that experiment showed, kit of the present invention when being used for Cor concentration level mensuration through a large amount of, this methodology index is as follows according to manufacturing conventional in this area and vertification regulation:
Embodiment 6 kits of the present invention are with the clinical blood sample measured value comparison of external kit
Reagent of the present invention and external authoritative reagent reach 99.9% to 873 parts of total coincidence rates of clinical blood serum sample testing result, and data are as follows:
From the data analysis of listed two kinds of reagent testing results, have 2 parts of feminine genders and external authoritative reagent testing result not to be inconsistent in the reagent testing result feminine gender of the present invention, this difference may since the immune raw material that reagent adopts or detection architecture sensitivity difference cause.
Kit specimen in use amount of the present invention is few, and as long as one-time detection is 25 μ l, vitro detection to tested object without any toxic and side effect.The chemiluminescence enzyme immunoassay method that the present invention simultaneously adopts does not produce radioactive contamination, and each index also is better than the ELISA adsorption analysis method (ELISA) of present widespread use.Be consistent with external authoritative diagnostic reagent analysis result.The present invention is highly sensitive, high specificity, and sensing range is wide, and is simple to operate, "dead" pollution, the kit cost is low, and clinical applicability is strong, more is applicable to China clinical detection laboratory.
Claims (8)
1. a chemical luminescence immune assay determination reagent kit for hydrocortisone is characterized in that, described kit comprises: the cortisol calibration object; The streptavidin bag is by solid phase carrier; Biotinylation cortisol monoclonal antibody; The cortisol antigen of alkaline phosphatase mark; Chemical luminous substrate; And cleansing solution.
2. kit as claimed in claim 1 is characterized in that, the solid phase carrier of described streptavidin bag quilt is microwell plate, plastic bead or plastic tube.
3. kit as claimed in claim 1 is characterized in that, the corresponding chemical luminous substrate of described alkaline phosphatase is 1,2-two oxidative ethane analog derivatives.
4. kit as claimed in claim 1, it is characterized in that, described chemical luminous substrate 1,2-two oxidative ethane analog derivatives are (diamantane)-1,2-two oxidative ethanes, 3-(2 '-the spiral diamantane)-4-methoxyl-4-(3 " the phosphorus acyloxy) phenyl-1,2-two oxidative ethanes, CSPD or CDP-Star.
5. kit as claimed in claim 1, it is characterized in that, described chemical luminous substrate is a solution, it comprises 24g Tris, 160g NaCl, 4g KCl, 15mL HCl, 200mL 3-(2 '-spiral diamantane)-4-methoxyl-4-(3 " phosphorus acyloxy) phenyl-1,2-two oxidative ethanes (AMPPD), 1mL Proclin 300,1000mL distilled water.
6. a preparation method who prepares the described kit of claim 1 is characterized in that may further comprise the steps: the preparation of cortisol calibration object; On solid phase carrier, wrap by streptavidin cortisol monoclonal antibody biotinylation; Marker enzyme on the cortisol antigen; The preparation of concentrated cleaning solution; Above-mentioned cortisol calibration object, streptavidin bag are by the packing of cortisol antigen, chemical luminous substrate and the cleansing solution of solid phase, biotinylated antibody, enzyme labeling; And be assembled into finished product.
7. method as claimed in claim 6, it is characterized in that, described streptavidin bag be may further comprise the steps by the preparation of solid phase carrier: it is that the streptavidin of 7.2 phosphate buffer (PBS) and debita spissitudo is mixed and made into coating buffer that (1) bag is used 0.05mol/L pH value, and it is carried on the solid phase carrier; (2) sealing preparation confining liquid comprises 0.2g NaH based on the described confining liquid of 1000mL
2PO
42H
2O, 2.9gNaH
2PO
412H
2O, 100mL calf serum and 1mLProclin-300, the pH value of described confining liquid is 7.0～7.4, then the gained confining liquid is carried on the solid phase carrier after the above-mentioned washing.
8. method as claimed in claim 7 is characterized in that, the solid phase carrier of described streptavidin bag quilt is microwell plate, plastic bead or plastic tube.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810103268 CN101368972A (en) | 2008-04-02 | 2008-04-02 | Chemical luminescence immune assay determination reagent kit for hydrocortisone and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810103268 CN101368972A (en) | 2008-04-02 | 2008-04-02 | Chemical luminescence immune assay determination reagent kit for hydrocortisone and preparation method thereof |
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| CN101368972A true CN101368972A (en) | 2009-02-18 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103353535A (en) * | 2013-08-02 | 2013-10-16 | 南通大学 | Pretreatment method for detecting cortisol content in hair |
| CN104479021A (en) * | 2014-10-13 | 2015-04-01 | 东南大学 | A nanometer antibody aiming at cortisol and a coding sequence thereof |
| CN105158041A (en) * | 2015-09-09 | 2015-12-16 | 中国农业大学 | Fur cortisol hormone extraction method |
| CN111175493A (en) * | 2020-02-27 | 2020-05-19 | 江苏泽成生物技术有限公司 | Magnetic particle chemiluminescence method human cortisol determination kit and use method thereof |
-
2008
- 2008-04-02 CN CN 200810103268 patent/CN101368972A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103353535A (en) * | 2013-08-02 | 2013-10-16 | 南通大学 | Pretreatment method for detecting cortisol content in hair |
| CN103353535B (en) * | 2013-08-02 | 2015-07-22 | 南通大学 | Pretreatment method for detecting cortisol content in hair |
| CN104479021A (en) * | 2014-10-13 | 2015-04-01 | 东南大学 | A nanometer antibody aiming at cortisol and a coding sequence thereof |
| CN105158041A (en) * | 2015-09-09 | 2015-12-16 | 中国农业大学 | Fur cortisol hormone extraction method |
| CN111175493A (en) * | 2020-02-27 | 2020-05-19 | 江苏泽成生物技术有限公司 | Magnetic particle chemiluminescence method human cortisol determination kit and use method thereof |
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Application publication date: 20090218 |