CN103344774A - Chemiluminescence immune assay kit and method for magnetic particle of human estradiol (E2) - Google Patents
Chemiluminescence immune assay kit and method for magnetic particle of human estradiol (E2) Download PDFInfo
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- CN103344774A CN103344774A CN2013103015502A CN201310301550A CN103344774A CN 103344774 A CN103344774 A CN 103344774A CN 2013103015502 A CN2013103015502 A CN 2013103015502A CN 201310301550 A CN201310301550 A CN 201310301550A CN 103344774 A CN103344774 A CN 103344774A
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Abstract
The invention relates to a magnetic separation enzymatic chemiluminescence immune assay method for human estradiol (E2), belonging to the technical field of immune assay. The method comprises the following steps of: enabling E2 in a sample to be detected, a calibrated sample or a quality control sample and an alkaline phosphatase (AP) labeled E2 derivative to compete to be combined with a fluorescein isothiocyanate (FITC) labeled E2 monoclonal antibody; then, adding the magnetic particle, connecting an antigen-antibody complex to the magnetic particle through the special combination of an anti-FITC antibody and FITC, and directly settling the complex in an external magnetic field to separate the complex from other uncombined substances without centrifuging, wherein the magnetic particle is connected with the anti-FITC antibody, and the complex is formed through immunoreaction. The combination of chemiluminescence and the magnetic particle is realized by using a kit provided by the invention; an approximately homogeneous reaction system is provided; compared with the prior art, the kit provided by the invention has various advantages of higher sensitivity, wide linearity range, high speed and the like; in addition, the cost of a product is greatly reduced; and the kit is wide in application prospect on the aspects of clinical examination and the like.
Description
Technical field
The invention belongs to the immune detection analysis technical field, relate to the magnetic microparticle chemiluminescence immune assay kit and the method for testing thereof that detect estradiol E2 content in the serum.
Background technology
Estradiol (E2) be in the body mainly by a kind of natural estrogen of ovary maturity folliculus secretion, can promote the growth of growing, impel secondary sex characters of female sex organ, to the influence of hypothalamic pituitary axis.Estradiol concentration inspection in the serum is one of hypothalamus-hypophysis-gonadal axis functional parameter, to diagnosing some endocrine and gynecological disease certain value and significance is arranged.The change of estradiol content can show different pathologies.The change of estradiol content can show different pathologies.Estradiol increases visible women's sex premature, gemellary pregnancy and 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, discover estradiol content level and some tumour in the human body, as breast cancer, the cancer of the uterus, liver cancer etc. significant correlativity is arranged.Therefore, invent a kind of highly sensitive, easy and simple to handle, the 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.
The chemiluminescence immunoassay detection technique is eighties of last century eighties, the emerging technology that grows up continue Enzyme-multiplied immune technique and after putting immune technology, have high sensitivity, high specific with respect to both chemiluminescence immunoassay technology of back, easy and simple to handle, quick, the mark bond is stable, therefore characteristics such as simultaneously "dead" isotope damage and pollution are extensively promoted the use of in the clinical detection analysis in recent years.
Summary of the invention
The technical issues that need to address of the present invention are to provide a kind of estradiol (E2) quantitative determination reagent kit and detection method thereof, adopt this kit to carry out E2 and detect and to have higher sensitivity and specificity, operate easylier, and detection time is shorter.
The invention provides and detect the E2 chemical luminescence immune analysis reagent box, it is characterized in that this kit comprises: magnetic particle reagent; The E2 calibration object; The E2 quality-control product; The anti-reagent of E2; The E2 derivant; Cleaning concentrate; Luminous substrate solution; The sample dilution.
Described magnetic particle reagent is for connecting the magnetic particle solution of goat-anti FITC antibody;
Described calibration object and quality-control product are the BSA damping fluid that contains a certain amount of E2 antigen;
Described anti-reagent is the E2 monoclonal anti body fluid of fluorescein isothiocynate (FITC) mark;
Described E2 derivant is the E2 derivant of alkaline phosphatase (AP) mark;
Described cleaning concentrate is the damping fluid that contains Tris, NaCl and surfactant etc.;
Described luminous substrate solution is the damping fluid that contains Lumigen APS-5 luminescent solution;
The sample-pretreating method of detection E2 provided by the present invention may further comprise the steps:
1, sample pre-treatments
To clinical serum, centrifugal 5 minutes of 3000rpm gets upper strata liquid and can carry out assay determination.Must not deposit above 48 hours for testing sample 2-8 ℃, if do not detect in 48 hours, preserve below Ying Yu-20 ℃, but should be above 30 days.
2, prepare before the experiment
Need before the experiment all reagent are placed to room temperature; Be used for covering the plastic foil of magnetic separator when being ready to disposable flat based tubes and magnetic separator and incubation; Regulating the water bath temperature is 37 ℃; Prepare chemical luminescence detector, and read over the instrument operation instructions.
3, reagent is prepared
Experiment is preceding puts abundant mixing on the mixing instrument with each reagent in the kit; Should be even suspension behind the magnetic particle reagent mixing, do not have obvious aggegation.
4, utilize the chemical luminescence immune analysis reagent box test sample of above-mentioned detection E2.
Detection method of the present invention is as follows:
(1) application of sample and immune response: in each flat based tubes, add 30 μ l E2 calibration objects, quality-control product or sample to be tested; The anti-reagent of 60 μ l, behind the mixing, 37 ℃ of incubations 20 minutes; 60 μ l enzyme labeling E2 derivants, behind the mixing, 37 ℃ of incubations 20 minutes; Add 60 μ l magnetic particle reagent, behind the mixing, 37 ℃ of incubations 5 minutes;
(2) washing: flat based tubes was left standstill 2 minutes at magnetic separator, and camber line is toppled over supernatant then, test tube and magnetic separator together is upside down on the thieving paper pat dry; Add 300 μ l cleaning fluids in every pipe, behind the mixing, flat based tubes was left standstill 2 minutes at magnetic separator, camber line is toppled over supernatant then, test tube and magnetic separator together is upside down on the thieving paper pat dry; Repeat twice.
(3) add luminous substrate solution: each test tube adds 200 μ l luminous substrate.
(4) read luminous value: the luminous value of measuring every pipe with Chemiluminescence Apparatus.
(5) as running into high value HOOK sample, for fear of high value HOOK effect occurring, the suggestion clinician selects suitable extension rate that sample is diluted according to all the other indexs.
The present invention has the following advantages:
1, uses magnetic bead to be solid phase, make immune response more near liquid phase, react more abundant and rapid, and make the immune complex of combination be more prone to separate, reduced non-specific adsorption.
2, the anti-reagent that uses is the monoclonal antibody potpourri, makes immunoreactive affinity higher, and the production differences between batches of monoclonal antibody are relatively little, easier assurance product batch between stable.
3, use chemical luminous substrate solution, the sensitivity of detection is improved, and the range of linearity is wideer.
4, the foundation of this method can provide the immunologic detection method of a kind of convenience, high sensitivity, accuracy and stability for the exploitation of other kits.
Main innovation part of the present invention is:
1, kit of the present invention combines chemiluminescence with immune magnetic particle, and a kind of reaction system near homogeneous phase is provided, and compared with prior art, kit of the present invention has higher detection sensitivity and specificity, and has reached preferable performance parameter.
2, the magnetic particle reagent in the kit, anti-reagent, calibration object, quality-control product, cleaning concentrate, luminous substrate solution and sample dilution are the optimization formulas under this reaction system, and giving the use effect phase of kit and detecting performance provides powerful guarantee.
The magnetic microparticle chemiluminescence immune assay kit of detection of the present invention E2 mainly adopts competition law quantitatively to detect the content of E2 in the sample such as human serum; Pre-treatment requirement to sample is low, and the pre-treatment process is simple, and energy is quick, high flux detects gross sample; Adopted high special monoclonal antibody and super paramagnetic, high dispersive, magnetic particle that specific surface area is big, main agents provides with the form of working fluid, and the method for inspection is convenient and easy, has specificity height, highly sensitive, characteristics such as degree of accuracy is high, accuracy height.Magnetic microparticle chemiluminescence immune assay kit of the present invention, simple in structure, easy to use, low price, carrying convenience are compared with enzyme linked immunological kit on the market etc.; the range of linearity is wide; effectively avoid hook effect, do not need the sample dilution, be applicable to the quantitative of batch samples screening.
Description of drawings
Fig. 1 is E2 chemiluminescence immunoassay examination criteria curve of the present invention, and wherein, horizontal ordinate is the concentration of E2, and ordinate is relative luminous intensity (RLU).
Embodiment
Embodiment 1
One, magnetic bead buffer solution formulation operations rules: 1L is example with preparation:
1, according to amount of preparation, select suitable vessel, add the pure water of 0.7kg 700ml;
2, take by weighing Tris 6.02g, NaN
30.99g in container, mixing, stirring at room 2 hours;
3, detect pH value of solution, should be about 10;
4, modulation pH value is 8.0;
5, measure Tween-20 2.76ml; Take by weighing neomycinsulphate 0.99g; Tetracycline 0.03g; BSA 4.97g stirred adjust pH to 8.0 ± 0.05 1 hour in container;
6, be settled to 1L, adjust pH to 8.0 ± 0.05;
7, filter with the 0.22um filter, be stored in 4 ℃.
Two, the preparation process of magnetic particle reagent
Be that 0.1 micron tri-iron tetroxide microballoon activates with glutaraldehyde with diameter, the room temperature mixing is after 4 hours, with 0.01mol/L PBS pH7.4 buffer solution for cleaning three times, and suspends with this solution, and concentration is 50-100mg/ml; Then, add goat-anti FITC antibody 100 μ g in every milliliter of suspension, in 37 ℃ of mixing incubations 3-8 hour; Sealed 40 minutes in 37 ℃ with isopyknic 0.01mol/L PBS 5%BSA pH7.4 damping fluid; At last, use 0.5%BSA 0.02mol/L Tris-HCl pH8.0 buffer solution for cleaning three times, and prepare certain density working fluid with above-mentioned magnetic bead buffer solution.
Embodiment 2
One, the coupling of alkaline phosphatase AP and E2 derivant
Use the SMCC activator to activate, generate maleimide-E2 derivant; Maleimide-E2 derivant contain can with the maleimide base group of-SH radical reaction, add and contain-alkaline phosphatase of SH, E2 derivant and alkaline phosphatase can be linked together.
Two, the coupling of fluorescein isothiocynate FITC and anti-E2 monoclonal antibody
The conjugate of FITC molecule and E2 monoclonal antibody is to carry out mark with common alkalies labelling method antagonist.When FITC reacted with antibody protein in alkaline solution, the amino sulphur carbon amine bond with fluorescein of the r of lysine was closed on the protein, forms the FITC-protein conjugate, i.e. fluorescence antibody or fluorescence bond.Generally multipotency is in conjunction with 15-20, and an IgG molecule can be in conjunction with the FITC of 2-8 molecule, and its reaction equation is as follows:
FITC-N=C=S+-NH
2-protein → FITC-NS-C-NH
2-protein
Embodiment 3
The preparation of calibration object/quality-control product:
1, peak: maximum concentration point is X, and impact point concentration is A, B, and C, D, E, F, during G preparation V volume solution, the volume that needs to add raw material is respectively: table 1
| Concentration | Add calibration object dilution volume | Add the X volume |
| A | V-A*V/X | A*V/X |
| B | V-B*V/X | B*V/X |
| C | V-C*V/X | C*V/X |
| D | V-D*V/X | D*V/X |
| E | V-E*V/X | E*V/X |
| F | V-F*V/X | F*V/X |
| G | V-G*V/X | G*V/X |
2, estradiol (E2) is measured kit E2 calibration object raw material (concentration is 50ng/ml) and is mixed with sample dilution (specifically fill a prescription and see embodiment 4) that concentration point is 0,60,120,300,750,1600,3200pg/ml; Quality-control product concentration is respectively 120,750U/ml.
3, fully the dissolving after, post label in 2-8 ℃ of preservation, the term of validity is 12 months.
Embodiment 4
One, cleaning concentrate formulation operations rules: 1L is example with preparation:
1, according to amount of preparation, select suitable containers, add water outlet 700ml, take by weighing Tris 12.11g; NaCl 312.43g; Tween-20 27.74g; Bronidox 1g; Triton X-100 1g;
2, placed 18 hours, transfer pH to 8.6 ± 0.05; Add pure water to 1L, filter.
Carry out 15 times of dilutions when 3, using.
Two, luminous substrate solution formulation operations rules: 1L is example with preparation:
1, according to amount of preparation, select suitable containers, take by weighing Tris 36.36g; Na
2SO
310mg; SDS 1.0g; Lucigenin 3.27mg; Tween-20 0.31ml transfers pH to 9.35; Add pure water to 1L, filter.
2, test luminous value
Embodiment 5
People's estradiol E2 magnetic microparticle chemiluminescence immune detection:
Operation steps:
(1) application of sample and immune response: in each flat based tubes, add 30 μ l E2 calibration objects, quality-control product or sample to be tested; The anti-reagent of 60 μ l, behind the mixing, 37 ℃ of incubations 20 minutes; 60 μ l enzyme labeling E2 derivants, behind the mixing, 37 ℃ of incubations 20 minutes; Add 60 μ l magnetic particle reagent, behind the mixing, 37 ℃ of incubations 5 minutes.
(2) washing: flat based tubes was left standstill 2 minutes at magnetic separator, and camber line is toppled over supernatant then, test tube and magnetic separator together is upside down on the thieving paper pat dry; Add 300 μ l cleaning fluids in every pipe, behind the mixing, flat based tubes was left standstill 2 minutes at magnetic separator, camber line is toppled over supernatant then, test tube and magnetic separator together is upside down on the thieving paper pat dry; Repeat twice.
(3) add luminous substrate solution: each test tube adds 200 μ l luminous substrate.
(4) read luminous value: the luminous value of measuring every pipe with Chemiluminescence Apparatus.
Testing result:
Detection curve is seen Fig. 1.
Zero standard is carried out 20 repeated tests on schedule, get the mean value that zero standard measures on schedule and add 2 times standard deviation, be its sensitivity.The sensitivity of this method is≤1U/ml.
Clinical testing:
One, detects data
Sample picks up from the normal health check-up of 1000 examples, blood donor.The equal non-communicable disease of sample physical examination result does not have blood transfusion and major operation history in half a year, the women is not in the gestational period and lactation.Measured value is carried out statistical analysis, draw normal serum term of reference: the male sex＜43.42pg/ml, be in the follicular phase women for 8.9-35.27pg/ml, be in the women 106.51-164.38pg/ml onset of ovulation, luteal phase, the women was 35.27-119.38pg/ml, postmenopausal women＜48.50pg/ml compares with the kit that external renowned company produces, and the testing result of kit of the present invention is more accurate.
Notebook data is only for reference, and different regions, Different Individual and employing distinct methods detect, and measured E2 level also can be different, advises the range of normal value of each laboratory foundation oneself.The E2 value that can not only draw with this method is made diagnosis, only as the intermediate data reference role, should comprise patient's concrete condition and treatment situation in conjunction with clinical other analyses result.E2 concentration value and this kit measurement result in the sample that obtains by additive method do not have direct comparability.The sample that exceeds the kit measurement scope, system can't provide definite numerical value.Measure its definite result as desire, measure again after the suggestion dilution.The testing result of this kit only supplies clinical reference, can not be separately as the foundation of making a definite diagnosis or get rid of case, and for reaching diagnostic purpose, this testing result will be used in combination with clinical examination, medical history and other inspection.This product can be used for the mensuration of serum sample, and the reliability that is used for other body fluid samples E2 concentration determination is fully confirmed.
Two, kit performance index of the present invention
Sensitivity for analysis is defined as: to the mensuration of 20 zero calibration objects, get its mean deviation of 2 times, its corresponding concentration on typical curve is sensitivity for analysis;
Sensitivity for analysis :≤1U/ml;
Precision: variation within batch CV%≤10.0%; Batch variation CV%≤15.0%;
Linear coefficient: r 〉=0.9900;
The range of linearity: 8.0-3000pg/ml;
Specificity: measure the cross reacting material of high concentration, the result is as follows:
| Potential cross reaction thing | Testing result | Interference |
| Oestrone (Estrone) 10000pg/mL | 8.10pg/ml | ≤0.01% |
| Estriol (E3) 10000pg/ml | 7.35pg/ml | ≤0.01% |
Claims (4)
1. magnetic microparticle chemiluminescence immunity detection reagent that people's estradiol is E2, it is characterized in that: the E2 derivant competition of the E2 in sample to be tested, calibration object or the quality-control product and enzyme labeling is in conjunction with the E2 monoclonal antibody of fluorescein isothiocynate (FITC) mark, adding subsequently is connected with the magnetic particle of anti-fluorescein antibody, is combined with the specificity of fluorescein by anti-fluorescein antibody antigen antibody complex is connected on the magnetic particle; The enzyme that uses in the described enzyme labeling E2 derivant is alkaline phosphatase AP; The coupling method of described enzyme marking reagent is with N-succinamide 3-(2-pyridine dimercapto) the E2 derivant of propionic acid [SPDP] activation mixes with the ratio of 1:0.5-2 with the alkaline phosphatase of Traunt ' s reagent activation, and uses the gel chromatography column separating purification; Employed substrate solution is the damping fluid that contains Lumigen APS-5 luminescent solution.
2. E2 magnetic microparticle chemiluminescence immune assay kit as claimed in claim 1 is characterized in that this kit comprises: magnetic particle reagent; The E2 calibration object; The E2 quality-control product; The anti-reagent of E2; The E2 derivant; Cleaning concentrate; Luminous substrate solution; The sample dilution;
Described magnetic particle reagent is for connecting the magnetic particle solution of goat-anti FITC antibody;
Described calibration object and quality-control product are the BSA damping fluid that contains a certain amount of E2 antigen;
Described anti-reagent is the E2 monoclonal anti body fluid of fluorescein isothiocynate (FITC) mark;
Described E2 derivant is the E2 derivant of alkaline phosphatase (AP) mark;
Described cleaning concentrate is the damping fluid that contains Tris, NaCl and surfactant etc.;
Described luminous substrate solution is the damping fluid that contains Lumigen APS-5 luminescent solution;
Described sample dilution is the solution that contains BSA.
3. as each described kit among the claim 1-2, it is characterized in that the using method of described kit is as follows:
(1) application of sample and immune response: in each flat based tubes, add 30 μ l E2 calibration objects, quality-control product or sample to be tested; The anti-reagent of 60 μ l, behind the mixing, 37 ℃ of incubations 20 minutes; 60 μ l enzyme labeling E2 derivants, behind the mixing, 37 ℃ of incubations 20 minutes; Add 60 μ l magnetic particle reagent, behind the mixing, 37 ℃ of incubations 5 minutes;
(2) washing: flat based tubes was left standstill 2 minutes at magnetic separator, and camber line is toppled over supernatant then, test tube and magnetic separator together is upside down on the thieving paper pat dry; Add 300 μ l cleaning fluids in every pipe, behind the mixing, flat based tubes was left standstill 2 minutes at magnetic separator, camber line is toppled over supernatant then, test tube and magnetic separator together is upside down on the thieving paper pat dry; Repeat twice;
(3) add luminous substrate solution: each test tube adds 200 μ l luminous substrate;
(4) read luminous value: the luminous value of measuring every pipe with Chemiluminescence Apparatus.
4. kit as claimed in claim 2 is characterized in that, when described sample to be tested is high value HOOK sample, uses the sample dilution that sample is diluted as required.
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| CN103823064A (en) * | 2014-03-03 | 2014-05-28 | 中华人民共和国张家港出入境检验检疫局 | Vomitoxin quantitative determination kit and use method thereof |
| CN106526165A (en) * | 2016-11-08 | 2017-03-22 | 北京久峰润达生物技术有限公司 | Quantitative measurement kit for glypican and detection method thereof |
| CN107941790A (en) * | 2017-11-28 | 2018-04-20 | 泰州泽成生物技术有限公司 | Magnetism particulate immuno chemistry luminescence method measures the kit and its detection method of feritin |
| CN108303554A (en) * | 2018-02-08 | 2018-07-20 | 江苏麦得科生物科技有限公司 | A kind of magnetic microparticle chemiluminescence immune assay kit of anti-gyneduct hormone |
| CN108445236A (en) * | 2018-02-07 | 2018-08-24 | 深圳市新产业生物医学工程股份有限公司 | Steroid derivatives and its preservation liquid and its application |
| CN108982835A (en) * | 2018-05-31 | 2018-12-11 | 湖南远璟生物技术有限公司 | A kind of estradiol magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof |
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| CN103823064B (en) * | 2014-03-03 | 2016-05-25 | 中华人民共和国张家港出入境检验检疫局 | A kind of vomitoxin immue quantitative detection reagent box and using method thereof |
| CN106526165A (en) * | 2016-11-08 | 2017-03-22 | 北京久峰润达生物技术有限公司 | Quantitative measurement kit for glypican and detection method thereof |
| CN107941790A (en) * | 2017-11-28 | 2018-04-20 | 泰州泽成生物技术有限公司 | Magnetism particulate immuno chemistry luminescence method measures the kit and its detection method of feritin |
| CN108445236A (en) * | 2018-02-07 | 2018-08-24 | 深圳市新产业生物医学工程股份有限公司 | Steroid derivatives and its preservation liquid and its application |
| CN108445236B (en) * | 2018-02-07 | 2021-04-20 | 深圳市新产业生物医学工程股份有限公司 | Steroid derivative, preservation solution thereof and application thereof |
| CN108303554A (en) * | 2018-02-08 | 2018-07-20 | 江苏麦得科生物科技有限公司 | A kind of magnetic microparticle chemiluminescence immune assay kit of anti-gyneduct hormone |
| CN108982835A (en) * | 2018-05-31 | 2018-12-11 | 湖南远璟生物技术有限公司 | A kind of estradiol magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof |
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