CN101639478A - Kit for detecting estradiol by utilizing magnetic particle chemiluminescence immunoassay - Google Patents
Kit for detecting estradiol by utilizing magnetic particle chemiluminescence immunoassay Download PDFInfo
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- CN101639478A CN101639478A CN200910091848A CN200910091848A CN101639478A CN 101639478 A CN101639478 A CN 101639478A CN 200910091848 A CN200910091848 A CN 200910091848A CN 200910091848 A CN200910091848 A CN 200910091848A CN 101639478 A CN101639478 A CN 101639478A
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Abstract
The invention discloses a kit for detecting the estradiol (E2) by utilizing magnetic particle chemiluminescence immunoassay. The kit comprises estradiol series calibrator, magnetic particle solution enveloped by the second antibody, estradiol marked by horse radish peroxidase, estradiol antibody, chemiluminescence substrate liquid and concentrated washing liquid. The invention further discloses apreparation method of the kit. The kit of the invention can quantitively detect the content of estradiol in the serum and blood plasma samples of human body, and can simultaneously detect a large number of samples, thus having advantages of simpleness, convenience, rapidness, stability, high sensitivity and the like, and providing a extremely valuable detection method for clinical diagnosis and scientific research.
Description
Technical field
The invention belongs to the immunoassay field, be specifically related to a kind of kit that utilizes magnetic microparticle chemiluminescence immunological technique detection estradiol and preparation method thereof.Kit of the present invention combines immune magnetic particle isolation technics and chemiluminescence immunoassay technology.
Background technology
Estradiol (E
2) be the strongest a kind of of biologically active in the female sex hormone, mainly synthetic by ovary, minority is by acth secretion, and its physiological effect mainly impels the growth of growing, impel secondary sex characters of female sex organ, to the influence of hypothalamic pituitary axis.Estradiol concentration is checked one of hypothalamus-hypophysis-gonadal axis functional parameter in the serum, to diagnosing some endocrine and gynecological disease certain value and significance is arranged.The change of estradiol content can show different pathologies.Estradiol increases visible women's sex premature, gemellary pregnancy or multifetation, ovary sex cord mesenchymal tumor, carcinoma of endometrium, breast cancer, gynaecomastia, males with coronary disease, male sex's systemic loupus erythematosus (SLE), cirrhosis; Estradiol reduces and to show as women's sexual dyspenesis, Turner syndrome (Turner ' s Syndrome), amenorrhoea, female acyesis, Stein-Leventhal syndrome, sheehan's syndrome, completeness or partial hydatidiform mole, ectopic pregnancy.In addition, the human body estradiol is to promoting growing, keep fertility and metabolism being had material impact of female reproductive system.In recent years, people discover estradiol content level and some tumour in the human body, as breast cancer, the cancer of the uterus, liver cancer etc. significant correlativity are arranged, and countries such as America and Europe forbid in succession or strictness bans use of.Therefore, invent a kind of highly sensitive, easy and simple to handle, analytical approach that expense is lower and suitable and domestic reagent, for popularizing of domestic clinical diagnosis, and the research of family planning all is highly significant.
At present, immune analysis method can provide the wide region of analyte to detect and very low detectability, and expense is cheap, for the detection of analyte provides quick, easy, a high-sensitivity analysis method.Therefore, immunoassay has been widely used in the research of aspects such as clinical diagnosis and biological chemistry as a kind of important analytical approach.The method that estradiol commonly used detects has radio immunoassay (radioimmunoassay, RIA), enzyme immunoassay (EIA) (enzyme linked immunosorbentassay, ELISA) and chemiluminescence immunoassay (chemiluminescence immunoassay, CLIA) etc.Yet radioelement is to the widespread use of effects limit RIA such as the pollution of environment and half life period be short and IRMA; Enzyme immuning adsorpting analysis method is low because of sensitivity, sensing range is narrow etc. can't satisfy clinical requirement.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.This method has advantages such as specificity height, highly sensitive, quick, easy and "dead" pollution, has been widely used in fields such as medical science, biological and chemical.
In the immune detection of reality,, detection sensitivity and accuracy have been influenced to a certain extent owing to impurity component contained in the testing sample is more.Immunity magnetic particle technology is to utilize the magnetic solid phase particle of the synthetic certain particle size size of macromolecular material to make carrier, with method bags such as physisorption, chemical coupling by on have the specificity affinity various immunologic active materials (antigen or antibody), making its sensitization is immune magnetic particle, has that velocity of separation is fast, efficient is high, favorable repeatability; Simple to operate; Do not influence characteristics such as the biological character of separated cell or other biomaterial and function, adding directed movement under the action of a magnetic field, make some special composition be separated, concentrate or purifying.Adopt immune magnetic particle technology to combine and detect determinand, can improve the sensitivity and the accuracy of detection greatly with chemiluminescence immunoassay technology.The novel part of this technology is: adopt small magnetic particle as solid phase carrier, can increase bag by surface area, thereby increased effective package amount of antibody, increased contact area and the substrate light-emitting area of antigen, antibody, improved the sensitivity and the sensing range of reaction.
Kit of the present invention is that chemiluminescence immune assay is combined with immune magnetic particle isolation technics, lack highly sensitive, detection time, need not expensive instrument, conservation greatly, and easy and simple to handle, applicability is wide, both can be applicable to the open luminous measuring instrument of semi-automatic chemistry, also can be used for full automatic measuring system, helped lend some impetus to application and the development of chemiluminescence immunoassay technology in clinical examination, detection.
Summary of the invention
The object of the present invention is to provide a kind of kit that utilizes the magnetic microparticle chemiluminescence immunological technique to detect estradiol.
The present invention also aims to provide a kind of preparation method of mentioned reagent box.
A kind of kit that utilizes the magnetic microparticle chemiluminescence immunological technique to detect estradiol, described kit comprise estradiol series calibration object, the magnetic particle solution of two anti-bag quilts, estradiol, estradiol antibody, chemical luminous substrate liquid and the concentrated cleaning solution of horseradish peroxidase mark.
Described two anti-are polyclonal antibody, the polyclonal antibody of preferred goat-anti rabbit.
Described estradiol antibody is the anti-estradiol polyclonal antibody of rabbit.
Described chemical luminous substrate liquid is A liquid and B liquid, wherein:
Chemical luminous substrate A liquid is that the pH value is 8.0～10.0 the 0.1～0.2M boric acid-borate buffer solution that contains 8～10mM luminol, 0.1～0.3mM 4-xenol and 0.05～0.1mM 4-iodobenzene boric acid.
Chemical luminous substrate B liquid is that the pH value is 7.0～7.6 the 0.1～0.2M phosphate buffer that 3.5～5mM urea peroxide and concentration of volume percent are 0.1～0.5% Tween20 that contains.
The preferred pH value of described chemical luminous substrate A liquid is 8.5 the 0.2M boric acid-borate buffer solution that contains 10mM luminol, 0.3mM 4-xenol and 0.05mM 4-iodobenzene boric acid.
The preferred pH value of described chemical luminous substrate B liquid be 7.4 contain the 3.5mM urea peroxide and concentration of volume percent is the 0.2M phosphate buffer of 0.1%Tween20.
Described concentrated cleaning solution is that pH is that 7.2～7.4 the mass percent concentration that contains is that 10～20% NaCl, concentration of volume percent are 0.1～0.25M phosphate buffer of the antiseptic Proclin 300 of 0.05～0.1% polysorbas20 and 0.05～0.1%.
Described concentrated cleaning solution is 25 times of concentrated cleaning solutions, and preferably this cleansing solution is that pH is that 7.4 the mass percent concentration that contains is that 20% NaCl, concentration of volume percent are the 0.25M phosphate buffer of the antiseptic Proclin 300 of 0.1% polysorbas20 and 0.1%.
The preparation method of mentioned reagent box comprises following operation steps:
1) preparation of estradiol series calibration object: spend the hormone human serum the pure product of estradiol are diluted to calibration object, the concentration range of its calibration object is 0-1000pg/mL;
2) preparation of the magnetic particle solution of goat-anti rabbit polyclonal antibody bag quilt: the magnetic particle for preparing goat-anti rabbit polyclonal antibody bag quilt by the glutaraldehyde two-step approach, and to be dissolved in the pH value be 7.4, contain concentration of volume percent and be 0.1～0.3% Tween-20 and mass percent concentration and be in 0.01～0.05M phosphate lavation buffer solution of 0.05～0.1% sodium azide antiseptic, preparation concentration is the working fluid of 5～10mg/mL, the concrete operations step is as follows: with particle diameter is that the magnetic particle of 2～3 μ m activates with glutaraldehyde, stirring at room, behind the mixing 2 hours, add magnetic field (magnetic field intensity 2000 Gausses), leave standstill 20～25min, pour out supernatant, with the pH value is that 7.4 0.01mol/L phosphate buffer cleans 3～5 times, and suspend with this damping fluid, concentration is 50～100mg/mL; Goat-anti rabbit polyclonal antibody 60～100 μ g that add purifying in every milliliter of suspending liquid, after 4 ℃ stirring is spent the night down, add magnetic field, leave standstill 10～15min, pour out supernatant, sealed 3～4 hours in room temperature with the phosphate buffer (pH is 7.2) that contains 0.2%～1.0% bovine serum albumin(BSA), 0.5%～1.0% casein, 0.02mol/L; Be 7.4 and to be dissolved in the pH value be 7.4, contain percent by volume is that 0.1～0.3% Tween-20 and mass percent are that the phosphate lavation buffer solution of 0.01～0.05M of 0.05～0.1% sodium azide antiseptic cleans 3～5 times with the pH value at last, and be the working fluid of 5～10mg/mL with the magnetic particle concentration that this solution is prepared goat-anti rabbit polyclonal antibody bag quilt; Magnetic particle solution is 4 ℃ of preservations.
3) estradiol Polyclonal Antibody Preparation:
A) preparation of estradiol antigen: use the mixed acid anhydride method to prepare the estradiol active ester, under the condition of phosphate buffer, prepare estradiol-BSA compound with estradiol active ester and BSA again;
B) estradiol Polyclonal Antibody Preparation: choose new zealand rabbit, the estradiol-BSA compound for preparing with step a) carries out immunity, and initial dose is 2.0mg/, the dose of back reinforcement all around is 1.0mg/, after the injection, two months booster shots doses in every interval only are 1.0mg/, inject continuously 3 times again for the second time, after three weeks, heart blood sampling, separation of serum, purifying and the estradiol polyclonal antibody, use antibody diluent that it is diluted, dilution ratio is 1: 1000～1: 3000;
4) preparation of horseradish peroxidase mark estradiol: adopt the glutaraldehyde method of improvement to prepare horseradish peroxidase mark estradiol, the enzyme labeling thing that obtains is diluted to 1: 1000 with it～1: 3000 working fluid by volume with enzyme labeling thing dilution.
5) preparation of chemical luminous substrate liquid:
A) chemical luminous substrate liquid A:
In 0.1～0.2M boric acid-borate buffer solution, add the following composition of final concentration: 8～10mM luminol, 0.1～0.3mM 4-xenol and 0.05～0.1mM 4-iodobenzene boric acid, regulate pH value to 8.0～10.0;
B) chemical luminous substrate liquid B:
Adding final concentration in 0.1～0.2M phosphate buffer is 3.5～5mM urea peroxide, and adding Tween20 then, to make its concentration of volume percent in damping fluid be 0.1～0.5%, regulates pH value to 7.0～7.6.
6) preparation of 25 times of concentrated cleaning solutions:
In 0.1～0.25M phosphate buffer, add NaCl, polysorbas20 and antiseptic Proclin 300, wherein, the mass percent concentration of NaCl is 10～20%, the concentration of volume percent of polysorbas20 is 0.05～0.1%, the concentration of volume percent of antiseptic Proclin 300 is 0.05～0.1%, regulates pH of buffer to 7.2～7.4 then.
7) packing step 1)～6) in Zhi Bei the reagent and the kit of packing into.
Described carrier magnetic particle is 2～3 μ m particle diameters, tri-iron tetroxide kernel, the surperficial polymkeric substance that has amino reactive group that wraps up.
Described antibody diluent is to contain the 0.01M PBS that mass percent concentration is 1%BSA and 0.5% gelatin hydrolysate.
Kit of the present invention can also comprise reaction tube, some reagent that enzyme labeling thing dilution etc. makes things convenient for kit to use, and the material of reaction tube is polystyrene, tygon, polypropylene or glass.
The purity of the pure product of described estradiol is greater than 90%.
Kit of the present invention adopts the reaction pattern of " competition law ", has effectively utilized chemiluminescence binding immunoassay magnetic particle know-why, E in detection by quantitative human serum, the plasma sample
2Content, can detect a large amount of samples simultaneously, have advantages such as easy, quick, sensitive, stable, high sensitivity, detection highly sensitive in similar kit.Kit of the present invention is simple in structure, easy to use, low price, carrying convenience is compared with the Enzymoimmune reagent kit on the market, linear performance is all fine, and clinical coincidence rate also improves greatly, and this kit is suitable for industrialization, has better market prospect, be particularly suitable for vast middle and small hospital and promote the use of, for clinical diagnosis and research work provide a kind of very valuable detection means.
Description of drawings
Fig. 1 is the calibration object linear graph in the prepared kit of embodiment 1, i.e. logit-log typical curve;
Fig. 2 is that kit of the present invention is with the correlation curve of external RIA kit to clinical blood sample measured value comparison.
Embodiment
The reagent source that uses in following examples is as follows:
The pure product of estradiol are available from U.S. Fitzgerald company, purity 99%;
The goat-anti rabbit polyclonal antibody of purifying is available from gondola Adaltis company;
Magnetic particle is available from Italian Adaltis company, and magnetic particle surface parcel has the polymkeric substance of amino reactive group;
Horseradish peroxidase is available from U.S. Sigma company;
Normal PHS is from hospital blood bank.
The preparation of the part reagent that uses in following examples:
Confining liquid: add bovine serum albumin(BSA) and casein in the 0.02mol/L phosphate buffer, make the mass percent concentration of the two be respectively 1.0% and 0.5%, pH to 7.2 is regulated in the dissolving back.
The preparation of enzyme labeling thing dilution: add following component and make its mass percent concentration in damping fluid be in the 0.02M phosphate buffer: 0.076% potassium chloride, 1%BSA and 0.1%Proclin300,0.05 ‰ Food Red is regulated pH value to 7.5.
The preparation of antibody diluent: adding mass percent concentration in 0.01M PBS is 1%BSA and 0.5% gelatin hydrolysate.
The preparation I of embodiment 1 kit of the present invention
1), the preparation of estradiol series calibration object
A) go the preparation of hormone human serum:
Normal PHS's equal portions of 60mL are packed in four volumetric flasks; add the 8.0g activated charcoal then respectively; inversion mixes with vortex mixer; behind the vibration 8h; with the centrifugal 20min of 6000rpm; with supernatant liquid filtering, add concentration of volume percent in the filtrate and be 0.5 ‰ antiseptic Proclin-300, freezing preservation.
B) preparation of estradiol calibration object:
Use steps A) preparation go the hormone human serum with E
2Pure product are diluted to calibration object, packing E
2Pure product concentration is respectively 0,15,50,150,500, totally 6 bottles of the calibration objects of 1000pg/mL.
2), goat-anti rabbit polyclonal antibody bag is by the preparation of magnetic particle solution
The magnetic particle solution for preparing goat-anti rabbit polyclonal antibody bag quilt by the glutaraldehyde two-step approach, the concrete operations step is as follows: with particle diameter is that the magnetic particle of 2.8 μ m activates with glutaraldehyde, stirring at room, behind the mixing 2 hours, add the magnetic field that magnetic field intensity is 2000 Gausses, leave standstill 25min, pour out supernatant, with the pH value is that 7.4 0.01mol/L phosphate buffer cleans three times, and suspends with this damping fluid, and concentration is 100mg/mL; The goat-anti rabbit polyclonal antibody 100 μ g that add purifying in every milliliter of suspending liquid, 4 ℃ stir down spend the night after, add magnetic field, magnetic field intensity is 2000 Gausses, leaves standstill 10min, pours out supernatant, with confining liquid in room temperature sealing 3 hours; At last with the pH value be 7.4, to contain concentration of volume percent be that 0.3% Tween-20 and mass percent concentration are that the 0.05M phosphate buffer of 0.1% sodium azide antiseptic cleans 4 times, and being mixed with the working fluid of 5mg/mL with this damping fluid, the magnetic particle solution of this antibody sandwich is 4 ℃ of preservations.
3), E
2Polyclonal Antibody Preparation
A) preparation of estradiol antigen: use the mixed acid anhydride method to prepare the estradiol active ester, under the condition of phosphate buffer, prepare estradiol-BSA compound with estradiol active ester and BSA again;
B) estradiol Polyclonal Antibody Preparation: it is (female to choose two new zealand rabbits, male each one, body weight 1.5～2.0kg), carry out immunity with the estradiol for preparing in the step a)-BSA compound, initial dose is 2.0mg/, the dose of back reinforcement all around is 1.0mg/, for the second time after the injection, two months booster shots doses in every interval only are 1.0mg/, inject continuously 3 times, after three weeks again, the heart blood sampling, centrifuging serum, adopt saturated ammonium sulfate (saturated ammonium sulfate, SAS) the method purifying and the estradiol polyclonal antibody, use antibody diluent that it is diluted, dilution ratio is 1: 2000.
4), the preparation of horseradish peroxidase (HRP) mark estradiol
Adopt the glutaraldehyde method of improveing with horseradish peroxidase mark estradiol, concrete labeling process is as follows:
Take by weighing 5mg HRP is dissolved among the PH 6.80.1mol/L PBS that 0.1ml contains 1.25% glutaraldehyde, put room temperature 18 hours, remove unnecessary glutaraldehyde through sephadex G-25 chromatography, add physiological saline to 0.5ml, the 1mol/L carbonate buffer solution that adds pure product of 5mg estradiol and 0.05ml PH 9.6 then, mixing the back placed 24 hours in 4 ℃ of refrigerators, the lysine solution that adds 0.05ml 0.2mol/L, room temperature was put 2 hours, with PH 7.2,0.15mol/L PBS dialysed overnight, the centrifugal precipitation of removing, supernatant is enzyme labeling thing (can add equal-volume glycerine, behind the mixing, airtight freezing preservation), make working fluid with the above-mentioned enzyme labeling thing of enzyme labeling thing diluted, dilution ratio is 1: 1000;
5), chemical luminous substrate liquid
A) preparation of A liquid
Luminol 1.7716g (10mM)
4-xenol 0.051g (0.3mM)
4-iodobenzene boric acid 0.012g (0.05mM)
Boric acid 11.4g
Borax 4.9g
Distilled water is settled to 1000mL
PH is adjusted to 8.5
B), the preparation of B liquid:
Urea peroxide 0.329g (3.5mM)
Tween20 1mL (percent by volume 0.1%)
Na
2HPO
4·12H
2O 51.58g
NaH
2PO
4·2H
2O 8.74g
Distilled water is settled to 1000mL
PH is adjusted to 7.4
A liquid mixes with B liquid equal proportion during use.
6), 25 times of concentrated cleaning solutions
Na
2HPO
4·12H
2O 72.5g
NaH
2PO
4·2H
2O 2.5g
NaCl 200g
Tween-20 1mL
Proclin 3001mL
Distilled water is settled to 1000mL
Adjust pH to 7.4
Use 25 times of distilled water dilutings during use.
7), the kit of packing into after the packing of above-mentioned steps products obtained therefrom is kit of the present invention.With the kit that detects 50 samples is example, comprising:
A) calibration object is totally 6 bottles, the 3mL/ bottle;
B) the magnetic particle solution of goat-anti rabbit polyclonal antibody bag quilt is 1 bottle, altogether 6mL;
C) horseradish peroxidase mark estradiol solution is 1 bottle, 6mL;
D) E
21 bottle of polyclonal antibody solution, 6mL;
F) chemical luminous substrate A liquid is 1 bottle, 1 bottle of 12mL, B liquid, 12mL; According to use amount A liquid and B liquid equal-volume are mixed before use;
G) concentrated cleaning solution is 1 bottle, and 5mL dilutes 25 times with deionized water during use;
H) reaction tube of polystyrene material is 1 bag, and 50, every pipe 10mm diameter * 50mm length.
The preparation II of embodiment 2 kits of the present invention
1), the preparation of estradiol series calibration object, same example I
2), goat-anti rabbit polyclonal antibody bag is by the preparation of magnetic particle solution
The magnetic particle solution for preparing goat-anti rabbit polyclonal antibody bag quilt by the glutaraldehyde two-step approach, the concrete operations step is as follows: with particle diameter is that the magnetic particle of 2.0 μ m activates with glutaraldehyde, stirring at room, behind the mixing 2 hours, add the magnetic field that magnetic field intensity is 2000 Gausses, leave standstill 25min, pour out supernatant, with the pH value is that 7.4 0.01mol/L phosphate buffer cleans three times, and suspends with this damping fluid, and concentration is 50mg/mL; The goat-anti rabbit polyclonal antibody 80 μ g that add purifying in every milliliter of suspending liquid, 4 ℃ stir down spend the night after, add magnetic field, wherein, magnetic field intensity is 2000 Gausses, leaves standstill 15min, pours out supernatant, with confining liquid with magnetic particle in room temperature sealing 4 hours; At last with the pH value be 7.4, to contain concentration of volume percent be that 0.2% Tween-20 and mass percent concentration are that the phosphate lavation buffer solution of the 0.01M of 0.1% sodium azide antiseptic cleans 4 times, and the magnetic particle of antibody sandwich is mixed with the working fluid of 7.5mg/mL with this damping fluid.Magnetic particle solution is 4 ℃ of preservations.
3), E
2Polyclonal Antibody Preparation, except that the antibody dilution that will make and dilution ratio are 1: 3000, other same example I.
4), the same example I of preparation of horseradish peroxidase mark estradiol (enzyme labeling thing), with the dilution of enzyme labeling thing, dilution ratio is 1: 2000 with enzyme labeling thing dilution;
5), chemical luminous substrate liquid
A) preparation of A liquid:
Luminol 1.4173g (8mM)
4-xenol 0.017g (0.1mM)
4-iodobenzene boric acid 0.024g (0.1mM)
Boric acid 11.4g
Borax 4.9g
Distilled water is settled to 1000mL
PH is adjusted to 9.0
B), the preparation of B liquid:
Urea peroxide 0.470g (5mM)
Tween20 2mL (volume ratio number percent 0.2%)
Na
2HPO
4·12H
2O 51.58g
NaH
2PO
4·2H
2O 8.74g
Distilled water is settled to 1000mL
PH is adjusted to 7.6
A liquid mixes with B liquid equal proportion during use.
6), 25 times of concentrated cleaning solutions
Na
2HPO
4·12H
2O 36.25g
NaH
2PO
4·2H
2O 1.25g
NaCl 100g
Tween-20 0.5mL
Proclin?300 0.5mL
Distilled water is settled to 1000mL
Adjust pH to 7.4
Use 25 times of distilled water dilutings during use.
7), the kit of packing into after the packing of above-mentioned steps products obtained therefrom is kit of the present invention.With the kit that detects 50 samples is example, comprising:
A) calibration object is totally 6 bottles, the 3mL/ bottle;
B) the magnetic particle solution of goat-anti rabbit polyclonal antibody bag quilt is 1 bottle, altogether 6mL;
C) horseradish peroxidase mark estradiol solution is 1 bottle, 6mL;
D) E
21 bottle of polyclonal antibody solution, 6mL;
F) chemical luminous substrate A liquid is 1 bottle, 1 bottle of 12mL, B liquid, 12mL; According to use amount A liquid and B liquid equal-volume are mixed before use;
G) concentrated cleaning solution is 1 bottle, and 5mL dilutes 25 times with deionized water during use;
H) reaction tube of polythene material is 1 bag, and 50, every pipe 10mm diameter * 50mm length.
The preparation III of embodiment 3 kits of the present invention
1), the preparation of estradiol series calibration object, same example I
2), goat-anti rabbit polyclonal antibody bag is by the preparation of magnetic particle solution
The magnetic particle solution for preparing goat-anti rabbit polyclonal antibody bag quilt by the glutaraldehyde two-step approach, the concrete operations step is as follows: with particle diameter is that the magnetic particle of 3.0m activates with glutaraldehyde, stirring at room, behind the mixing 2 hours, add the magnetic field that magnetic field intensity is 2000 Gausses, leave standstill 20min, pour out supernatant, with the pH value is that 7.4 0.01mol/L phosphate buffer cleans three times, and suspends with this damping fluid, and concentration is 75mg/mL; The goat-anti rabbit polyclonal antibody 60 μ g that add purifying in every milliliter of suspending liquid, 4 ℃ stir down spend the night after, add magnetic field, wherein, magnetic field intensity is 2000 Gausses, leaves standstill 10min, pours out supernatant, with confining liquid with magnetic particle in room temperature sealing 3 hours; At last with the pH value be 7.4, to contain concentration of volume percent be that 0.1% Tween-20 and mass percent concentration are that the phosphate lavation buffer solution of the 0.05M of 0.05% sodium azide antiseptic cleans 4 times, and is mixed with the working fluid of 10mg/mL with this solution.Magnetic particle solution is 4 ℃ of preservations.
3), E
2Polyclonal Antibody Preparation, except that the antibody dilution that will make and dilution ratio are 1: 1000, other same example I.
4), the same example I of preparation of horseradish peroxidase mark estradiol enzyme labeling thing, with the dilution of enzyme labeling thing, dilution ratio is 1: 3000 with enzyme labeling thing dilution;
5), chemical luminous substrate liquid
A) preparation of A liquid:
Luminol 1.5944g (9mM)
4-xenol 0.034g (0.2mM)
4-iodobenzene boric acid 0.012g (0.05mM)
Boric acid 11.4g
Borax 4.9g
Distilled water is settled to 1000mL
PH is adjusted to 10
B), the preparation of B liquid:
Urea peroxide 0.376g (4mM)
Tween20 5mL (percent by volume is 0.5%)
Na
2HPO
4·12H
2O 51.58g
NaH
2PO
4·2H
2O 8.74g
Distilled water is settled to 1000mL
PH is adjusted to 7.0
A liquid mixes with B liquid equal proportion during use.
6), 25 times of concentrated cleaning solutions
Na
2HPO
4·12H
2O 29g
NaH
2PO
4·2H
2O 1g
NaCl 150g
Tween-20 0.75mL
Proclin?300 0.75mL
Distilled water is settled to 1000mL
Adjust pH to 7.2
Use 25 times of distilled water dilutings during use.
7), the kit of packing into after the packing of above-mentioned steps products obtained therefrom is kit of the present invention.With the kit that detects 50 samples is example, comprising:
A) calibration object is totally 6 bottles, the 3mL/ bottle;
B) the magnetic particle solution of goat-anti rabbit polyclonal antibody bag quilt is 1 bottle, altogether 6mL;
C) horseradish peroxidase mark estradiol solution is 1 bottle, 6mL;
D) E
21 bottle of polyclonal antibody solution, 6mL;
F) chemical luminous substrate A liquid is 1 bottle, 1 bottle of 12mL, B liquid, 12mL; According to use amount A liquid and B liquid equal-volume are mixed before use;
G) concentrated cleaning solution is 1 bottle, and 5mL dilutes 25 times with deionized water during use;
H) reaction tube of glass material is 1 bag, and 50, every pipe 10mm diameter * 50mm length.
The using method of embodiment 4 kits of the present invention
1), sample pre-treatments to be checked
Vein extracts people's empty stomach blood sample in morning, and the centrifugal 5min of 3000rpm gets upper serum analysis.
2), detection method
Use the kit of the present invention of embodiment 1 preparation the human serum sample to be analyzed according to following steps:
A) cleansing solution preparation: 25 times of concentrated cleaning solution adding distil water dilutions that kit is provided become 1 times for 25 times;
B) with magnetic particle solution, horseradish peroxidase mark estradiol and the E of estradiol series calibration object, goat-anti rabbit polyclonal antibody bag quilt
2Polyclonal antibody takes out from 4 ℃ of refrigerators, places 15min, and balance is to room temperature;
C) be fixed on the grillage after will testing the round bottom polystyrene test tube numbering that needs;
D) will add each 50 μ L of testing sample and each concentration calibration object in the above-mentioned test tube successively, blank 1 pipe is established in each test, and each pipe adds horseradish peroxidase mark estradiol, E except that the blank pipe then
2Each 100 μ L of magnetic particle solution of polyclonal antibody and many anti-bag quilts, 37 ℃ of reaction 50min;
E) test tube rack is placed separate 5min on the magnetic separator, discard solution in the pipe then,, wash 5 times, on thieving paper, pat dry with the good cleansing solution of above-mentioned dilution;
F) chemical luminous substrate liquid A and B were mixed by 1: 1 after, every pipe adds chemical luminous substrate liquid 200 μ L, abundant mixing, 37 ℃ of incubation 10min place in the magnetic separator, 10min is placed in the dark place;
G) on the chemiluminescence measuring instrument, measure the luminous intensity (RLU) of each pipe in regular turn, 1 second/pipe of Measuring Time;
H) set up typical curve: respectively calibration object concentration and corresponding RLU are taken the logarithm, on log-log plot, set up typical curve, Log value with calibration object concentration is a horizontal ordinate, the Logit value of RLU is drawn typical curve for ordinate, see accompanying drawing 1, curvilinear equation is: logit Y=-2.3376log X+4.983, r=0.9993.
I) on typical curve, find the concentration of this serum estradiol with test serum RLU value.
The methodology calibrating of embodiment 5 kits of the present invention
According to vertification regulation conventional in this area the kit of preparation among the embodiment 1 is examined and determine, respectively precision, accuracy, sensitivity, specificity and the stability of kit are examined and determine.
1), kit sensitivity experiment
Sensitivity be on a given level of significance can with other dosage of zero-dose phase region, be meant the minimum detectable activity (be designated as zero standard on schedule on average deduct 2SD) of analytical approach.Press 10 S0 standard points of kit measurement of using method embodiment 1 preparation of embodiment 4, calculate the mean value and the standard deviation of its luminous intensity, bring linear equation into, calculate corresponding concentration value, and repeating this operation 5 times, the minimum detectability that obtains this kit is 2.51pg/mL.And with the comparison of other method, see Table 1.
Table 1 kit of the present invention and additive method sensitivity contrast and experiment
As can be seen from Table 1, the method detection sensitivity of this kit employing is apparently higher than additive method.
2), kit precision is measured
The kit of preparation among the embodiment 1 is got three batches respectively carry out the precision experiment, measure basic, normal, high three kinds of serum by the using method of embodiment 4 respectively without concentration, 10 pipe replicate determinations repeat the batch variation (table 2) that draws the variation within batch of each analysis and repeatedly analyze 3 times.In its batch and interassay coefficient of variation all less than 15%.
Table 2 method precision is investigated
3), kit accuracy determination
Accuracy is meant the degree of measured value near true value, represents with the recovery usually.Select three parts of human serums for use, under optimum experimental condition, measure concentration, in serum, add concentration then respectively and be 0,30,80 and the E of 200pg/mL
2Calibration object, use embodiment 1 kit embodiment 4 using method to each concentration do 3 parallel, calculate recovery rate.The result is as shown in table 3, shows E
2Recovery of standard addition between 93.3%～106%.
Table 3 sample recovery rate
4) kit specificity experiment
Choose common estradiol analogue, be mixed with the clinical sample of concentration, measure by the using method of embodiment 4 with the kit of embodiment 1 much larger than physiological concentration.
The result shows, intersecting all less than 2.0% of some common steroid hormones and estradiol antibody illustrates that the selected antibody specificity of this method is higher, can satisfy the requirement of estradiol in the accurate mensuration human serum.
Some common estradiol analogues of table 4 and the cross reacting rate (n=5) between the antibody
5), kit stability experiment
Each reagent in the kit of embodiment 1 is experimentized behind placement 3d, 7d, the 10d down at 4 ℃ and 37 ℃.Experimental result shows that all within normal range, different condition all less than significantly influence, then illustrates the stable fine of this product to experimental result to the kit index of embodiment 1.
As follows through experiment showed, that kit method of the present invention is learned index:
Sensing range: 0～1000pg/mL;
Sensitivity: minimum detects and is limited to 2.51pg/mL;
Precision: less than 15% (n=3);
Accuracy: average recovery rate is between 93.3%～106%;
Specificity: with E
1, E
3, other analogs such as T, DHT cross reacting rate less than 2.0%;
Stability: each reagent set splits 4 ℃ and 37 ℃, and behind investigation 3d, 7d, the 10d, each component is still stable.
Embodiment 6 kits of the present invention are with the comparison of RIA kit to clinical blood serum sample measured value
With the kit (using method is seen embodiment 4) of embodiment 1 preparation with available from the RIA kit of French Cisbio International company 105 parts of clinical blood serum samples are detected respectively.Its testing result is seen accompanying drawing 2, with the blood serum sample E of kit measurement of the present invention
2The result is a horizontal ordinate, is that ordinate is done regretional analysis with the result of RIA kit measurement, and dependent equation is: Y=1.0782X-3.2188, correlation coefficient r=0.9892.Learn result by statistics and show, the inventive method is good with the clinical blood sample measured value of RIA kit correlativity.
Claims (10)
1, a kind of kit that utilizes the magnetic microparticle chemiluminescence immunological technique to detect estradiol; it is characterized in that described kit comprises estradiol series calibration object, the magnetic particle solution of two anti-bag quilts, estradiol, estradiol antibody, chemical luminous substrate liquid and the concentrated cleaning solution of horseradish peroxidase mark.
2, kit according to claim 1 is characterized in that, described two anti-are polyclonal antibody.
3, kit according to claim 1 and 2 is characterized in that, described two anti-are the polyclonal antibody of goat-anti rabbit.
4, kit according to claim 1 is characterized in that, described estradiol antibody is the anti-estradiol polyclonal antibody of rabbit.
5, kit according to claim 1 is characterized in that, described chemical luminous substrate liquid is A liquid and B liquid, wherein:
Chemical luminous substrate A liquid is that the pH value is 8.0～10.0 the 0.1～0.2M boric acid-borate buffer solution that contains 8～10mM luminol, 0.1～0.3mM 4-xenol and 0.05～0.1mM 4-iodobenzene boric acid;
Chemical luminous substrate B liquid is that the pH value is 7.0～7.6 the 0.1～0.2M phosphate buffer that 3.5～5mM urea peroxide and concentration of volume percent are 0.1～0.5% Tween20 that contains.
6, kit according to claim 5 is characterized in that, described chemical luminous substrate A liquid is that the pH value is 8.5 the 0.2M boric acid-borate buffer solution that contains 10mM luminol, 0.3mM 4-xenol and 0.05mM 4-iodobenzene boric acid;
Described chemical luminous substrate B liquid be the pH value be 7.4 contain the 3.5mM urea peroxide and concentration of volume percent is the 0.2M phosphate buffer of 0.1%Tween20.
7, kit according to claim 1, it is characterized in that described concentrated cleaning solution is that pH is that 7.2～7.4 the mass percent concentration that contains is that 10～20% NaCl, concentration of volume percent are 0.1～0.25M phosphate buffer of the antiseptic Proclin 300 of 0.05～0.1% polysorbas20 and 0.05～0.1%.
8, kit according to claim 7, it is characterized in that, described concentrated cleaning solution is 25 times of concentrated cleaning solutions, and this cleansing solution is that pH is that 7.4 the mass percent concentration that contains is that 20% NaCl, concentration of volume percent are the 0.25M phosphate buffer of the antiseptic Proclin 300 of 0.1% polysorbas20 and 0.1%.
9, the preparation method of the described kit of claim 1 is characterized in that, comprises following operation steps:
1) preparation of estradiol series calibration object: spend the hormone human serum the pure product of estradiol are diluted to calibration object, the concentration range of its calibration object is 0～1000pg/mL;
2) preparation of the magnetic particle solution of goat-anti rabbit polyclonal antibody bag quilt: the magnetic particle for preparing goat-anti rabbit polyclonal antibody bag quilt by the glutaraldehyde two-step approach, and to be dissolved in the pH value be 7.4, contain concentration of volume percent is that 0.1～0.3% Tween-20 and mass percent concentration are that preparation concentration is the working fluid of 5～10mg/mL in 0.01～0.05M phosphate lavation buffer solution of 0.05～0.1% sodium azide antiseptic;
3) estradiol Polyclonal Antibody Preparation:
A) preparation of estradiol antigen: use the mixed acid anhydride method to prepare the estradiol active ester, under the condition of phosphate buffer, prepare estradiol-BSA compound with estradiol active ester and BSA again;
B) estradiol Polyclonal Antibody Preparation: choose new zealand rabbit, the estradiol-BSA compound for preparing with step a) carries out immunity, and initial dose is 2.0mg/, the dose of back reinforcement all around is 1.0mg/, after the injection, two months booster shots doses in every interval only are 1.0mg/, inject continuously 3 times again for the second time, after three weeks, heart blood sampling, separation of serum, purifying and the estradiol polyclonal antibody, use antibody diluent that it is diluted, dilution ratio is 1: 1000～1: 3000;
4) preparation of horseradish peroxidase mark estradiol: adopt the glutaraldehyde method of improvement to prepare horseradish peroxidase mark estradiol, the enzyme labeling thing that obtains is diluted to 1: 1000 with it～1: 3000 working fluid by volume with enzyme labeling thing dilution;
5) preparation of chemical luminous substrate liquid:
A) chemical luminous substrate liquid A:
In 0.1～0.2M boric acid-borate buffer solution, add the following composition of final concentration: 8～10mM luminol, 0.1～0.3mM4-xenol and 0.05～0.1mM 4-iodobenzene boric acid, regulate pH value to 8.0～10.0;
B) chemical luminous substrate liquid B:
Adding final concentration in 0.1～0.2M phosphate buffer is 3.5～5mM urea peroxide, and adding Tween20 then, to make its concentration of volume percent in damping fluid be 0.1～0.5%, regulates pH value to 7.0～7.6;
6) preparation of 25 times of concentrated cleaning solutions:
In 0.1～0.25M phosphate buffer, add NaCl, polysorbas20 and antiseptic Proclin 300, wherein, the mass percent concentration of NaCl is 10～20%, the concentration of volume percent of polysorbas20 is 0.05～0.1%, the concentration of volume percent of antiseptic Proclin 300 is 0.05～0.1%, regulates pH of buffer to 7.2～7.4 then;
7) packing step 1)～6) in Zhi Bei the reagent and the kit of packing into.
10, preparation method according to claim 9 is characterized in that, described carrier magnetic particle is 2～3 μ m particle diameters, tri-iron tetroxide kernel, the surperficial polymkeric substance that has amino reactive group that wraps up.
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