CN103293323A - Luteinizing hormone nano-magnetic particle chemiluminescence quantitative immunoassay kit and preparation method thereof - Google Patents
Luteinizing hormone nano-magnetic particle chemiluminescence quantitative immunoassay kit and preparation method thereof Download PDFInfo
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- 102000009151 Luteinizing Hormone Human genes 0.000 title claims abstract description 123
- 108010073521 Luteinizing Hormone Proteins 0.000 title claims abstract description 123
- 238000002360 preparation method Methods 0.000 title claims abstract description 52
- 239000006249 magnetic particle Substances 0.000 title claims abstract description 27
- 238000003018 immunoassay Methods 0.000 title abstract description 5
- 229940040129 luteinizing hormone Drugs 0.000 title abstract 6
- 238000003908 quality control method Methods 0.000 claims abstract description 32
- 238000006243 chemical reaction Methods 0.000 claims abstract description 26
- 238000005406 washing Methods 0.000 claims abstract description 25
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 13
- 108090000790 Enzymes Proteins 0.000 claims abstract description 12
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- 238000001514 detection method Methods 0.000 claims abstract description 12
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Abstract
The invention discloses a luteinizing hormone nano-magnetic particle chemiluminescence quantitative immunoassay kit. The kit comprises a luteinizing hormone calibration product, a nano-magnetic particle suspension coupled with streptavidin, a biotin-marked luteinizing hormone antibody, a luteinizing hormone antibody enzyme binding substance, a luteinizing hormone quality control product, a chemiluminescence solution A and a chemiluminescence solution B, a 20-times concentrated washing solution and a reaction tube, wherein the used enzyme is horseradish peroxidase, the purity RZ of the horseradish peroxidase is not less than 3.0, and the activity is not less than 250U/mL. In addition, the invention further discloses a preparation method of the kit. Compared with the existing kit, the kit disclosed by the invention has the advantages of high sensitivity, a wide range of measurable concentrations, long effective period of a reagent, high degree of automation in detection and the like, and is simple to operate.
Description
Technical field
The present invention relates to the immunoassay medical domain, concrete, the invention provides a kind of interstitialcellstimulating hormone (ICSH) (LH) nanometer magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof.
Background technology
(Luteotropic hormone LH) is secreted by the adenohypophysis basophil interstitialcellstimulating hormone (ICSH).In the male sex, can stimulate interstitial glands secretion male sex hormone, at women's moderate stimulation ovarian secretion female estrogen.Interstitialcellstimulating hormone (ICSH) (LH) causes ovulation and corpus luteum to form in the menstrual cycle that ovary is kept in collaborative follicular stimulating hormone (FSH) acting in conjunction of women.The generation of LH is subjected to the control of hypothalamus gonadotropin releasing hormone, regulated and control by the positive-negative feedback of ovary.LH and FSH joint-detection are mainly differentiated primary (ovary) or Secondary cases (hypophysis) amenorrhoea in the women; Low for discriminating primary or Secondary cases testicular function the male sex; Simultaneously can differentiate the true property of preadolescence children or false precocity.
Release peak and ovary ovulation at menstrual cycle LH have substantial connection, and the LH peak indicates ovary ovulation in 24-36 hour once appearance, therefore can monitor the serum Lh peak value in the menstrual cycle, becomes pregnant the time to determine the best.This testing result is represented with milli-international unit/milliliter (MIU/ML).
The method that detects interstitialcellstimulating hormone (ICSH) (LH) is differentiated fluoroimmunoassay (number of patent application: 200810043552.5) if having time at present, adopt long-acting fluorescent marker such as rare earth metal (Eu, Tb, Sm, Dy) mark, differentiate non-specific fluorescence by central bay, but the method need be utilized rare earth metal, the cost height, the mark difficulty reaches, and is not easy to obtain.
The most popular method that detects LH in addition is chemiluminescence immunoassay; the magnetic microparticle chemiluminescence immune assay method; radio immunoassay than before, enzyme-linked immunosorbent assay; in detection sensitivity, sensing range, detection time and automation mechanized operation raising has greatly been arranged; and do not pollute the person's that obtains the clinical position favor.
Prior art generally adopts the fluorescein system, and (number of patent application: 200910089583.9,201210571305.9), this system reagent box term of validity is short, and instability is still accurate inadequately on the measured value;
Other has technology, and (number of patent application: be direct len antibody on magnetic particle 201110257444.X), the operation easier height is not easy to obtain.
Summary of the invention
The problem to be solved in the present invention provides quantitative detection kit of chemiluminescence immunoassay of interstitialcellstimulating hormone (ICSH) and preparation method thereof, avoid the reagent term of validity of radioimmunoassay short, had shortcomings such as radioactive contamination, complex operation, and it is low to have solved sensitivity, measured value is accurate inadequately, sensing range is narrow, the cost height, the defective that the shelf-life is short.
For solving the problems of the technologies described above, the technical solution used in the present invention is: interstitialcellstimulating hormone (ICSH) nanometer magnetic microparticle chemiluminescence immune quantitative detection reagent box comprises: the interstitialcellstimulating hormone (ICSH) calibration object; Coupling has the nanometer magnetic particle suspending liquid of Streptavidin; Biotin labeled interstitialcellstimulating hormone (ICSH) antibody; Interstitialcellstimulating hormone (ICSH) abzyme bond, used enzyme are horseradish peroxidase, horseradish peroxidase purity RZ 〉=3.0, activity 〉=250U/mL; Interstitialcellstimulating hormone (ICSH) quality-control product, quality-control product comprise the low value quality-control product of concentration 5mIU/mL and the high value quality-control product of 120mIU/mL; Chemical luminescence for liquid A liquid and B liquid, A liquid are 5mmol/L, the Tris-HCl damping fluid of pH8.6, and contain final concentration 0.7g/L luminol and final concentration 0.08g/L p-iodophenol in this damping fluid; B liquid is the 0.675g/L urea peroxide; 20 times of concentrated washing lotions; Reaction tube.
Further, described nanometer magnetic particle is the tri-iron tetroxide that the surface parcel has amino or carboxyl reactive group, particle diameter 10-50nm.
Further, the material of described reaction tube is transparent polystyrene, tygon, polypropylene or glass.
The preparation method of kit may further comprise the steps:
(1) preparation of interstitialcellstimulating hormone (ICSH) calibration object:
Interstitialcellstimulating hormone (ICSH) antigen is mixed with the dense liquid storage of calibration object with lowlenthal serum, calibrates with national calibration object, dense liquid storage is diluted to working concentration with lowlenthal serum, be respectively 0,2,10,25,100,250mIU/mL, be calibration object concentration;
(2) preparation of interstitialcellstimulating hormone (ICSH) quality-control product:
With lowlenthal serum above-mentioned dense liquid storage is diluted to 5mIU/mL and 120mIU/mL respectively, and calibrates with national calibration object; As the low value quality-control product, 120mIU/mL is as high value quality-control product with 5mIU/mL;
(3) preparation of nanometer magnetic particle-Streptavidin suspending liquid:
A, the preparation of ferriferrous oxide nano magnetic particle
Adopt the precipitation method to prepare the ferriferrous oxide nano magnetic particle, concrete preparation method is as follows: 1) with FeCl
36H
2O and FeCl
24H
2O joins in the distilled water with mol ratio 2:1, the vigorous stirring dissolving; 2) under nitrogen environment, add 0.5M ammoniacal liquor in above-mentioned iron salt solutions, transfer pH9-10,65 ℃ of temperature of reaction, reaction time 45min; 3) after reaction finishes, be washed with distilled water to neutrality, abandon supernatant, in 60 ℃ of oven dry, namely get the ferriferrous oxide nano magnetic particle of 10-50nm;
The coupling of B, nanometer magnetic bead surface carboxyl
Adopt dispersion copolymerization method to carry out coupling, concrete preparation method is as follows: ultrasonic being dispersed in 10% polyglycol (PEG8000) solution of nanometer magnetic particle of getting above-mentioned preparation, get magnetic fluid solution, 1:10 adds absolute ethyl alcohol by volume in the magnetic fluid solution, stir 30min after, move into and have stirrer, condenser pipe, in the three-necked bottle of nitrogen inlet, add crosslinking chemical N, N '-methylene-bisacrylamide; Under protection of nitrogen gas, be warming up to 60 ± 1 ℃, constant temperature stirs 30min, add benzoyl peroxide afterwards successively, consumption is magnetic fluid consumption 3%, and stirring rate is about 500rpm, volume of styrene is with magnetic fluid solution, and the acrylic acid volume is 1/4 of magnetic fluid solution, keeps stream of nitrogen gas, all the other conditions remain unchanged, reaction 8-10h, and products therefrom leaves standstill, use the distilled water cyclic washing, regulate pH=1 with hydrochloric acid again, soak 24h, leave standstill; Use the distilled water cyclic washing again, remove the Fe that does not coat
3O
4Magnetic is put into 50 ℃ of following dry 24h of vacuum drying chamber to the product that precipitates, and obtains the nanometer magnetic particle that the surface is associated with carboxyl;
The preparation of C, nanometer magnetic particle-Streptavidin suspending liquid, preparation 1L, method is as follows:
Get 100mL0.1M2-morpholino b acid (MES) damping fluid, adding 10mg surface is associated with the nanometer magnetic particle of carboxyl, stirring at room 40min, add the 3.5mg Streptavidin afterwards, add 8mg/mL carbodiimide (EDC) solution then, behind the 2-8 ℃ of reaction 1h, with 0.01M PBS damping fluid washing 3 times, get final product to 1L with 0.01M PBS is fixed molten at last;
(4) preparation of biotin labeled interstitialcellstimulating hormone (ICSH) antibody
Get 0.5mg interstitialcellstimulating hormone (ICSH) antibody, with borate buffer solution dialysis 1～3h under 2～8 ℃; Antibody after the dialysis is added the 25ug biotin, add dimethyl sulfoxide (DMSO) simultaneously, making dimethyl sulfoxide (DMSO) final mass concentration is 5-10%, slowly vibration, lucifuge reaction 3h; In above-mentioned solution, add 250uL1M ammonium chloride solution, reacting at normal temperature without light 30-60min; With 0.01M PBS solution 2～8 ℃ of down dialysis 2 days, during change liquid 3-5 time;
(5) preparation of interstitialcellstimulating hormone (ICSH) abzyme bond
After adopting the improvement sodium periodate oxidation that interstitialcellstimulating hormone (ICSH) antibody and horseradish peroxidase are carried out coupling, with the enzyme dilution it is diluted to working concentration 1:3000-5000, and adds 15% enzyme stabilizers, be stored in 2～8 ℃;
Improvement sodium periodate oxidizing process step comprises:
The A:HRP activation
1) configuration 10mg/mL HRP solution;
2) configuration 12.8mg/mL sodium periodate NaIO
4Solution;
3) with above-mentioned 1) and 2) obtain solution 1:1 mixing by volume, 4 ℃ of lucifuges reaction 30min;
4) configuration concentration is the glycol water of 20uL/mL, with above-mentioned solution 3) mix with equal volume, reacting at normal temperature without light 20min, activation is namely finished, and puts-20 ℃ and preserves (holding time is no more than 3 months).
B, interstitialcellstimulating hormone (ICSH) labeling of monoclonal antibody
1) raw material to be marked is packed in the bag filter, with the carbonate buffer solution of 0.05M pH9.6, dialysis 30min;
2) the mark raw material is mixed by mass ratio 1:2 with the HRP of activation, during 4 ℃ of dialysis 24h(, change liquid 2-3 time with the 0.05M carbonate buffer solution afterwards);
3) configuration concentration is the NaBH of 2mg/mL
4Aqueous solution adds the NaBH that 80uL prepares by 1mgHRP
4The ratio of aqueous solution is mixed, and in 4 ℃ of lucifuge reaction 2h;
4) with above-mentioned steps 3) marking fluid finished in 4 ℃ of dialysis 24h, adds equal-volume glycerine ,-20 ℃ of preservations with 0.01M PBS.
Comprise 10mL/L2M NaOH in the enzyme dilution, 15g/L NaCl, 10g/LBSA, 5g/L Dextran T-2000(is available from Sigma company), 1.05g/L Triton X-100(is available from Sigma company), the 2.5mL/L gentamicin sulphate, (famille rose is powder solid to the 1mL/L famille rose, being mixed with concentration 40mg/mL uses later on), 2g/L Tween-20(is available from Sigma company), 1mL/L ProClin300(is available from Sigma company);
The preparation of (6) 20 times of concentrated washing lotions
20 times of concentrated washing lotions comprise the 58g/L sodium hydrogen phosphate, 5.92g/L sodium dihydrogen phosphate, 180g/L NaCl, 10mL/LTween-20 and 2%Proclin300;
(7) preparation of chemical luminescence for liquid A liquid and B liquid
A liquid is the 0.7g/L luminol, and 0.08g/L p-iodophenol, damping fluid are the 5mmol/L Tris-HCl of pH8.6, keep in Dark Place; B liquid is the 0.675g/L urea peroxide, prepares with process water; A liquid and B liquid 5min before use mix;
(8) assembling: mentioned reagent is assembled into box, is stored in 2～8 ℃;
(9) kit that adopts the preparation of this method is carried out physical examination, measured value and the stability of the linearity of accuracy, dose-response curve, precision, specificity, sensitivity, quality-control product are measured.
Principle of the present invention is, adopt the LH in double antibody sandwich method mensuration serum or the blood plasma, in Avidin-nanometer magnetic particle suspending liquid, add biotin-LH antibody conjugates, compatible reaction by Avidin and biotin, form magnetic particle-Avidin-biotin-interstitialcellstimulating hormone (ICSH) antibody complex, add sample and enzyme, by antigen-antibody reaction, form magnetic particle-Avidin-biotin-interstitialcellstimulating hormone (ICSH) antibody-interstitialcellstimulating hormone (ICSH)-interstitialcellstimulating hormone (ICSH) antibody-HRP compound, with magnetic field compound is adsorbed on the test tube bottom, wash free composition, add the substrate working fluid, under the oxygenant effect, HRP catalysis luminol generates the aminophthalic acid ion that is in excited state, when it returns to ground state, discharge the photon of 425nm, measured the luminous value RLU of each well in the 5th minute.The RLU of sample and sample interstitialcellstimulating hormone (ICSH) concentration are proportionate.Interstitialcellstimulating hormone (ICSH) concentration in the sample is according to the Log(X that is set up by calibration object interstitialcellstimulating hormone (ICSH) concentration and corresponding RLU)-Log(Y) mathematical model is carried out quantitatively, thus detect the interstitialcellstimulating hormone (ICSH) content in human serum, the blood plasma.
The interstitialcellstimulating hormone (ICSH) nanometer magnetic microparticle chemiluminescence immunological quantitative determining kit of this patent invention has the following advantages:
(1) highly sensitive, the sensitivity for analysis of this kit is not higher than 0.2mIU/mL.
(2) specificity is good, and this product cross reaction do not occur to human chorionic gonadotrophin (200000mIU/mL), thyrotropic hormone (500 μ IU/mL), follicle-stimulating hormone (FSH) (500mIU/mL).
(3) accuracy is good, and imprecision is not higher than 5% in batch, and imprecision is not higher than 10% between batch.
(4) cost is low, compares with like product on the market, and this kit is functional, and cost is low, has clinical value.
(5) have good stability, this product can be deposited more than 7 days at 37 ℃, can deposit 1 year at 2～8 ℃.
Description of drawings
Fig. 1 is the measurement result comparison diagram that kit measurement interstitialcellstimulating hormone (ICSH) of the present invention and Luo Shi measure interstitialcellstimulating hormone (ICSH), the interstitialcellstimulating hormone (ICSH) value that records for this kit of ordinate wherein, horizontal ordinate is Luo Shi kit measurement interstitialcellstimulating hormone (ICSH) value, two kinds of method related coefficients (r)=0.9601, straight-line equation y=1.1034x-0.669.
Embodiment
Embodiment 1: preparation interstitialcellstimulating hormone (ICSH) nanometer magnetic microparticle chemiluminescence immunological quantitative determining kit
(1) preparation of interstitialcellstimulating hormone (ICSH) calibration object:
Interstitialcellstimulating hormone (ICSH) antigen (production of Fitzgerald company) is mixed with the dense liquid storage of calibration object with lowlenthal serum (available from Zhengzhou Yi Kang bioengineering company limited), with national calibration object (lot number: 150531-0211, specification: 530mIU/ props up) calibrate, dense liquid storage is diluted to working concentration with lowlenthal serum, be respectively 0,2,10,25,100,250mIU/mL is calibration object concentration;
(2) preparation of interstitialcellstimulating hormone (ICSH) quality-control product:
With lowlenthal serum above-mentioned dense liquid storage is diluted to 5mIU/mL and 120mIU/mL respectively, and calibrates with national calibration object; As the low value quality-control product, 120mIU/mL is as high value quality-control product with 5mIU/mL;
(3) preparation of nanometer magnetic particle-Streptavidin suspending liquid:
A, the preparation of ferriferrous oxide nano magnetic particle
Adopt the precipitation method to prepare the ferriferrous oxide nano magnetic particle, concrete preparation method is as follows: 1) with FeCl
36H
2O and FeCl
24H
2O joins in the distilled water with mol ratio 2:1, the vigorous stirring dissolving; 2) under nitrogen environment, add 0.5M ammoniacal liquor in above-mentioned iron salt solutions, transfer pH9-10,65 ℃ of temperature of reaction, reaction time 45min; 3) after reaction finishes, be washed with distilled water to neutrality, abandon supernatant, in 60 ℃ of oven dry, namely get the ferriferrous oxide nano magnetic particle of 10-50nm;
The coupling of B, nanometer magnetic bead surface carboxyl
Adopt dispersion copolymerization method to carry out coupling, concrete preparation method is as follows: get in the ultrasonic 10%PEG8000 of the being dispersed in solution of nanometer magnetic particle of above-mentioned preparation, get magnetic fluid solution, 1:10 adds absolute ethyl alcohol by volume in the magnetic fluid solution, stir 30min after, move into and have stirrer, condenser pipe, in the three-necked bottle of nitrogen inlet, add crosslinking chemical N, N '-methylene-bisacrylamide; Under protection of nitrogen gas, be warming up to 60 ± 1 ℃, constant temperature stirs 30min, add benzoyl peroxide afterwards successively, consumption is magnetic fluid consumption 3%, and stirring rate is about 500rpm, volume of styrene is with magnetic fluid solution, and the acrylic acid volume is 1/4 of magnetic fluid solution, keeps stream of nitrogen gas, all the other conditions remain unchanged, reaction 8-10h, and products therefrom leaves standstill, use the distilled water cyclic washing, regulate p H=1 with hydrochloric acid again, soak 24h, leave standstill; Use the distilled water cyclic washing again, remove the Fe that does not coat
3O
4Magnetic is put into 50 ℃ of following dry 24h of vacuum drying chamber to the product that precipitates, and obtains the nanometer magnetic particle that the surface is associated with carboxyl;
The preparation of C, nanometer magnetic particle-Streptavidin suspending liquid, preparation 1L, method is as follows:
Get 100mL0.1M MES damping fluid, adding 10mg surface is associated with the nanometer magnetic particle of carboxyl, stirring at room 40min, add the 3.5mg Streptavidin afterwards, add 8mg/mLEDC solution then, behind the 2-8 ℃ of reaction 1h, with 0.01M PBS damping fluid washing 3 times, get final product to 1L with 0.01M PBS is fixed molten at last;
(4) preparation of biotin labeled interstitialcellstimulating hormone (ICSH) antibody
Get 0.5mg interstitialcellstimulating hormone (ICSH) antibody, with borate buffer solution dialysis 1～3h under 2～8 ℃; Antibody after the dialysis is added the 25ug biotin, add dimethyl sulfoxide (DMSO) simultaneously, making dimethyl sulfoxide (DMSO) final mass concentration is 5-10%, slowly vibration, lucifuge reaction 3h; In above-mentioned solution, add 250uL1M ammonium chloride solution, reacting at normal temperature without light 30-60min; With 0.01M PBS solution 2～8 ℃ of down dialysis 2 days, during change liquid 3-5 time;
(5) preparation of interstitialcellstimulating hormone (ICSH) abzyme bond
After adopting the improvement sodium periodate oxidation that interstitialcellstimulating hormone (ICSH) antibody and horseradish peroxidase are carried out coupling, (its composition comprises 5g/LMES with the enzyme dilution, 10mL/L2M NaOH, 15g/L NaCl, 10g/LBSA, 5g/L glucosan T-2000(Dextran T-2000) (available from Sigma company), 1.05g/L Triton X-100 (Triton X-100) (available from Sigma company), 2.5mL/L gentamicin sulphate, (famille rose is powder solid to the 1mL/L famille rose, being mixed with concentration 40mg/mL uses later on), 2g/L Tween-20(is available from Sigma company), 1mL/L ProClin300(is available from Sigma company)) it is diluted to working concentration 1:3000-5000, and add 15% enzyme stabilizers, be stored in 2～8 ℃;
The preparation of (6) 20 times of concentrated washing lotions
20 times of concentrated washing lotions comprise the 58g/L sodium hydrogen phosphate, 5.92g/L sodium dihydrogen phosphate, 180g/L NaCl, 10mL/L Tween-20 and 2%Proclin300;
(7) preparation of chemical luminescence for liquid A liquid and B liquid
A liquid is the 0.7g/L luminol, and 0.08g/L p-iodophenol, damping fluid are the 5mmol/L Tris-HCl of pH8.6, keep in Dark Place; B liquid is the 0.675g/L urea peroxide, prepares with process water; A liquid and B liquid 5min before use mix;
(8) assembling: mentioned reagent is assembled into box, is stored in 2～8 ℃;
(9) kit that adopts the preparation of this method is carried out physical examination, measured value and the stability of the linearity of accuracy, dose-response curve, precision, specificity, sensitivity, quality-control product are measured.
Embodiment 2: the inspection of kit of the present invention
(1) physical examination: liquid component should be clarified, and does not have precipitation or floccus; Other components should not have packages in damaged condition.
(2) accuracy: kit calibration object and national standard product series are carried out assay determination simultaneously, with the match of double-log mathematical model, require two not obvious parallel deviates of dose-response curve (t check, | t|＜2.447); Be reference substance with interstitialcellstimulating hormone (ICSH) national standard product, with the match of double-log mathematical model, the mean value of the measured value of kit calibration object and sign value ratio should be in 0.90～1.10 scope.
(3) linearity of dose-response curve: with two reading mathematical model matches, dose-response curve correlation coefficient r absolute value in the 0.2-250mIU/mL concentration range is not less than 0.9900.
(4) sensitivity for analysis: the kit sensitivity for analysis is not higher than 0.2mIU/mL.
(5) precision: the high value of 10 hole replicate determinations and low value quality-control product, the mean concentration of calculating measurement result (
) and standard deviation (SD), imprecision in batch
Use 3 batches of products to carry out 3 tests, the mean concentration of calculating measurement result (
) and standard deviation (SD), imprecision between batch
The result should meet batch interior imprecision (CV%) should not be higher than 5%; Imprecision (CV%) should not be higher than 10% between batch.
(6) measured value of quality-control product: the quality-control product of the high value in replicate determination 10 holes and low value, with Log (X)-Log (Y) mathematical model match, the quality-control product measured value should be in allowed band, and low value quality-control product measured value is at 4-6mIU/mL, and high value quality-control product measured value is at 96-144mIU/mL.
(7) specificity:
Cross reaction meets following table and requires:
(8.) stability: placed 7 days for 37 ℃, measured value should meet above-mentioned every requirement.
Embodiment 3: the using method of kit of the present invention
(1) kit to be checked was descended balance 30 minutes in room temperature (18～25 ℃).
(2) preparation washing lotion: will concentrate washing lotion by 1:20 dilution (the 1mL washing lotion adds 19mL distilled water) with distilled water.If concentrate washing lotion crystallization is arranged, can place room temperature or 37 ℃ with concentrating washing lotion, dilute again after the dissolving to be crystallized.
(3) preparation luminescent solution: use and got an amount of luminescent solution A in preceding 5 minutes and mix with luminescent solution B equal-volume.
(4) reaction tube is numbered, in test tube, add 25-50uL calibration object or serum specimen successively, 50uL magnetic-particle-Streptavidin suspending liquid, 50uL biotin-interstitialcellstimulating hormone (ICSH) antibody conjugates, 50-100uL interstitialcellstimulating hormone (ICSH) abzyme bond, 37 ℃ of following oscillating reactions 30min, test tube rack placed separate 5min on the magnetic separator, pour out supernatant then, add the 500uL washing lotion, fully behind the mixing, on magnetic separator, separate, pour out washing lotion, repeat 3 times, in each pipe, add chemical luminous substrate liquid 100uL, fully mixing is secretly put 5min, measures the luminous value (RLU) of each pipe at the tubular type Chemiluminescence Apparatus, Log value with calibration object concentration is horizontal ordinate, Log with luminous value is ordinate, and the drawing standard curve can calculate the concentration of interstitialcellstimulating hormone (ICSH) according to the luminous value of serum specimen.
Embodiment 4: the evaluation of methodology result of this kit
Sensing range: scope is 0.2～250mIU/mL, measures after should diluting earlier greater than the sample of 250mIU/mL for concentration again.
Sensitivity: 0.2mIU/mL.
Precision: less than 5%.
Accuracy: the mean value of the recovery is in 0.90～1.10 scope.
Specificity: with the cross reaction coefficient of thyrotropic hormone (TSH), human chorionic gonadotrophin (hCG), follicle-stimulating hormone (FSH) (FSH) less than 1%.
The quality-control product measured value: all in allowed band, low value quality-control product measured value is at 4-6mIU/mL for the measured value of low value quality-control product (QcL) and high value quality-control product (QcH), and high value quality-control product measured value is at 96-144mIU/mL.
Stability: each reagent component in the kit is placed 7d down in 37 ℃, have good stability.
Embodiment 5: the clinical comparison experiment of this kit
The kit of this patent invention has carried out clinical examination, total sample number 120 examples of this clinical testing, earlier with after the test of interstitialcellstimulating hormone (ICSH) Luo Shi detection kit, measure with the kit (chemiluminescence) of this patent invention again, the result shows, straight-line equation is y=1.1034x-0.669, coefficient R=0.9601.As seen kit and hospital's measured value of this method preparation have consistance preferably.With the SPSS13.0 statistical analysis software related coefficient is carried out t check (inspection level α=0.05), P＜0.001, the related intimate degree of the interstitialcellstimulating hormone (ICSH) value of two kinds of method mensuration is conspicuousnesses, as seen the interstitialcellstimulating hormone (ICSH) value of two kinds of method mensuration is closely related, the diagnosis capability that kit is described is stronger, can promote clinical practice.
In order to determine the clinical reference value of this kit, adopt this kit to detect to 1033 parts of normal human serums, plasma samples, the result shows that the reference value (term of reference) of this kit is the male sex: 1～11mIU/mL; Women: follicular phase: 1～17mIU/mL, the onset of ovulation: 24～98mIU/mL, luteal phase: 0.5～19mIU/mL, climacteric: 14～54mIU/mL.
Claims (4)
1. interstitialcellstimulating hormone (ICSH) nanometer magnetic microparticle chemiluminescence immune quantitative detection reagent box is characterized in that described kit comprises:
1) interstitialcellstimulating hormone (ICSH) calibration object, concentration are 0,2,10,25,100,250mIU/mL;
2) coupling has the nanometer magnetic particle suspending liquid of Streptavidin;
3) biotin labeled interstitialcellstimulating hormone (ICSH) antibody;
4) interstitialcellstimulating hormone (ICSH) abzyme bond, used enzyme are horseradish peroxidase, horseradish peroxidase purity RZ 〉=3.0, activity 〉=250U/mL;
5) interstitialcellstimulating hormone (ICSH) quality-control product; Quality-control product comprises the low value quality-control product of concentration 5mIU/mL and the high value quality-control product of 120mIU/mL;
6) chemical luminescence for liquid A liquid and B liquid; A liquid is 5mmol/L, the Tris-HCl damping fluid of pH8.6, and contain final concentration 0.7g/L luminol and final concentration 0.08g/L p-iodophenol in this damping fluid; B liquid is the 0.675g/L urea peroxide;
7) 20 times of concentrated washing lotions;
8) reaction tube.
2. interstitialcellstimulating hormone (ICSH) nanometer magnetic microparticle chemiluminescence immune quantitative detection reagent box according to claim 1 is characterized in that, described nanometer magnetic particle is the tri-iron tetroxide that the surface parcel has amino or carboxyl reactive group, particle diameter 10-50nm.
3. interstitialcellstimulating hormone (ICSH) nanometer magnetic microparticle chemiluminescence immune quantitative detection reagent box according to claim 1 is characterized in that the material of described reaction tube is transparent polystyrene, tygon, polypropylene or glass.
4. a method for preparing the described kit of the arbitrary claim of described claim 1-3 is characterized in that, may further comprise the steps:
(1) preparation of interstitialcellstimulating hormone (ICSH) calibration object:
Interstitialcellstimulating hormone (ICSH) antigen is mixed with the dense liquid storage of calibration object with lowlenthal serum, calibrates with national calibration object, dense liquid storage is diluted to working concentration with lowlenthal serum, be respectively 0,2,10,25,100,250mIU/mL;
(2) preparation of interstitialcellstimulating hormone (ICSH) quality-control product:
With lowlenthal serum above-mentioned dense liquid storage is diluted to 5mIU/mL and 120mIU/mL respectively, and calibrates with national calibration object; As the low value quality-control product, 120mIU/mL is as high value quality-control product with 5mIU/mL;
(3) preparation of nanometer magnetic particle-Streptavidin suspending liquid:
A, the preparation of ferriferrous oxide nano magnetic particle
Adopt the precipitation method to prepare the ferriferrous oxide nano magnetic particle, concrete preparation method is as follows: 1) with FeCl
36H
2O and FeCl
24H
2O joins in the distilled water with mol ratio 2:1, the vigorous stirring dissolving; 2) under nitrogen environment, add 0.5M ammoniacal liquor in above-mentioned iron salt solutions, transfer pH9-10,65 ℃ of temperature of reaction, reaction time 45min; 3) after reaction finishes, be washed with distilled water to neutrality, abandon supernatant, in 60 ℃ of oven dry, namely get the ferriferrous oxide nano magnetic particle of 10-50nm;
The coupling of B, nanometer magnetic bead surface carboxyl
Adopt dispersion copolymerization method to carry out coupling, concrete preparation method is as follows: get in the ultrasonic 10%PEG8000 of the being dispersed in solution of nanometer magnetic particle of above-mentioned preparation, get magnetic fluid solution, 1:10 adds absolute ethyl alcohol by volume in the magnetic fluid solution, stir 30min after, move into and have stirrer, condenser pipe, in the three-necked bottle of nitrogen inlet, add crosslinking chemical N, N '-methylene-bisacrylamide; Under protection of nitrogen gas, be warming up to 60 ± 1 ℃, constant temperature stirs 30min, add benzoyl peroxide afterwards successively, consumption is magnetic fluid consumption 3%, and stirring rate is about 500rpm, volume of styrene is with magnetic fluid solution, and the acrylic acid volume is 1/4 of magnetic fluid solution, keeps stream of nitrogen gas, all the other conditions remain unchanged, reaction 8-10h, and products therefrom leaves standstill, use the distilled water cyclic washing, regulate p H=1 with hydrochloric acid again, soak 24h, leave standstill; Use the distilled water cyclic washing again, remove the Fe that does not coat
3O
4Magnetic is put into 50 ℃ of following dry 24h of vacuum drying chamber to the product that precipitates, and obtains the nanometer magnetic particle that the surface is associated with carboxyl;
The preparation of C, nanometer magnetic particle-Streptavidin suspending liquid, preparation 1L, method is as follows:
Get 100mL0.1M MES damping fluid, adding 10mg surface is associated with the nanometer magnetic particle of carboxyl, stirring at room 40min, add the 3.5mg Streptavidin afterwards, add 8mg/mLEDC solution then, behind the 2-8 ℃ of reaction 1h, with 0.01M PBS damping fluid washing 3 times, get final product to 1L with 0.01M PBS is fixed molten at last;
(4) preparation of biotin labeled interstitialcellstimulating hormone (ICSH) antibody
Get 0.5mg interstitialcellstimulating hormone (ICSH) antibody, with borate buffer solution dialysis 1～3h under 2～8 ℃; Antibody after the dialysis is added the 25ug biotin, add dimethyl sulfoxide (DMSO) simultaneously, making dimethyl sulfoxide (DMSO) final mass concentration is 5-10%, slowly vibration, lucifuge reaction 3h; In above-mentioned solution, add 250uL1M ammonium chloride solution, reacting at normal temperature without light 30-60min; With 0.01M PBS solution 2～8 ℃ of down dialysis 2 days, during change liquid 3-5 time;
(5) preparation of interstitialcellstimulating hormone (ICSH) abzyme bond
After adopting the improvement sodium periodate oxidation that interstitialcellstimulating hormone (ICSH) antibody and horseradish peroxidase are carried out coupling, with the enzyme dilution it is diluted to working concentration 1:3000-5000, and adds 15% enzyme stabilizers, be stored in 2～8 ℃;
The preparation of (6) 20 times of concentrated washing lotions
20 times of concentrated washing lotions comprise the 58g/L sodium hydrogen phosphate, 5.92g/L sodium dihydrogen phosphate, 180g/L NaCl, 10mL/L Tween-20 and 2%Proclin300;
(7) preparation of chemical luminescence for liquid A liquid and B liquid
A liquid is the 0.7g/L luminol, and 0.08g/L p-iodophenol, damping fluid are the 5mmol/L Tris-HCl of pH8.6, keep in Dark Place; B liquid is the 0.675g/L urea peroxide, prepares with process water; A liquid and B liquid 5min before use mix;
(8) assembling: mentioned reagent is assembled into box, is stored in 2～8 ℃;
(9) kit that adopts the preparation of this method is carried out physical examination, measured value and the stability of the linearity of accuracy, dose-response curve, precision, specificity, sensitivity, quality-control product are measured.
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