CN103048446A - Luteinizing hormone nano-magnetic particle chemiluminescence assay kit and preparation method thereof and assay method thereof - Google Patents
Luteinizing hormone nano-magnetic particle chemiluminescence assay kit and preparation method thereof and assay method thereof Download PDFInfo
- Publication number
- CN103048446A CN103048446A CN2012105713059A CN201210571305A CN103048446A CN 103048446 A CN103048446 A CN 103048446A CN 2012105713059 A CN2012105713059 A CN 2012105713059A CN 201210571305 A CN201210571305 A CN 201210571305A CN 103048446 A CN103048446 A CN 103048446A
- Authority
- CN
- China
- Prior art keywords
- antibody
- fluorescein
- luteinizing principle
- solution
- luteinizing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a luteinizing hormone nano-magnetic particle chemiluminescence assay kit and a preparation method thereof and an assay method thereof. The luteinizing hormone nano-magnetic particle chemiluminescence assay kit comprises a solution containing a fluorescein-labeled luteinizing hormone antibody, a suspension coated with magnetic particles of an anti-fluorescein antibody, and a solution containing an alkaline phosphatase-labeled luteinizing hormone antibody, wherein the alkaline phosphatase-labeled luteinizing hormone antibody is formed by connecting an alkaline phosphatase with a luteinizing hormone antibody through SMCC (4-(N-maleimidomethyl) cyclohexyl-1-succinimide carboxylate) and 2IT (2-imido sulfane hydrochloride). The luteinizing hormone nano-magnetic particle chemiluminescence assay kit can be used for quantitative determination of the luteinizing hormone at lower cost and higher accuracy and precision.
Description
Technical field
The invention belongs to biological technical field, be specifically related to chemical luminescence immune analysis reagent box of a kind of luteinizing principle that combines immune magnetic particle isolation technics and chemiluminescence immunoassay technology and preparation method thereof.The present invention also is particularly related to the preparation method of this kit.
Background technology
Luteinizing principle (Luteinizing Hormone, LH) is a kind of glycoprotein hormones that is produced by anterior pituitary basocyte secretion, and molecular weight is about 30,000 dalton, is comprised of the different subunits of two non-covalent combinations.Its alpha subunit has 92 amino acid residues, identical with LH, TSH, hCG.The amino acid sequence of β subunit and LH, TSH are very different, but very similar to hCG.
LH is subjected to the regulation and control of the gonadotropin-releasing hormone (GRH) (Gn-RH) of hypothalamus generation.The pituitary LH that studies show that to normal women is divided into two phases, namely presents intermittent pulsed secretion in the basal secretion phase in per approximately 2 hours.In menstrual cycle, LH and LH synergy cause that the cycle of ovary changes at female normal.2 summits that occur in mid-term menstrual cycle cause follicular rupture, discharge ripe ovum.Remaining ovarian follicle becomes functional corpus luteum, secretion progesterone and estradiol, thus these steroids apply the further secretion that positive and negative two kinds of feedback regulations affect LH to hypothalamus and hypophysis.To climacteric, hypo-ovaria, the secretion of estradiol reduce until final the end.Because removed the negative feedback that is applied on the hypothalamus so that in the circulation level of LH significantly increase.In males, LH promotes the testosterone that interstitial glands generates.And the sperm of coordinating to keep in the vas deferens with LH equally generates.The testicosteroid hormone is by regulating the LH level in the circulation to the negative feedback of hypothalamus and pituitary in response Gn-RH.The hypopituitarism that dyspituitarism causes can cause the decline of LH level and cause sterile.To after taking Gn-RH, measure the functional status that serum Lh content can effectively be judged hypophysis.Other has a kind of dynamic function test is to take estradiol to the amenorrhoea women, predicts that by measuring LH content the patient is to the reactivity of clomiphene treatment.LH and LH 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.At release peak and the ovary ovulation close relation of menstrual cycle LH, 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.
The immune analysis method that is used at present luteinizing principle (LH) mainly contains enzyme-linked immunosorbent assay, chemiluminescence immunoassay etc.The methodology limiting factors such as enzyme-linked immunosorbent assay exists sensitivity low, and narrow, the difficult realization of the range of linearity is full-automatic.Chemiluminescence immunoassay is a kind of immunoassay technology that grows up on the enzyme-linked immunosorbent assay basis, have highly sensitive, detect the advantages such as linear wide ranges, easy and simple to handle, automaticity height.At present chemiluminescence immunoassay technology has above-mentioned manyly be widely used a little because of it.
Yet, in the immune detection of reality, owing to impurity component contained in the testing sample is more, detection sensitivity and accuracy have been affected to a certain extent, so from the sample substrate of complexity, separate fast, be purified into the purpose determinand, it is one of difficult problem of facing of clinical examination worker.The magnetic particle immunoassay technology is to utilize the magnetic solid phase particle of synthesis of polymer material certain particle size size to make carrier, have the various immunologic active materials such as the antibody of specificity affinity or antigen on coated with methods such as physisorption, chemical couplings, have that velocity of separation is fast, efficient is high, the characteristics such as favorable repeatability, simple to operate, the biological character that do not affect separated cell or other biological material and function, adding orientable motion under the magnetic fields, so that some special composition is separated, concentrated or purifying.
The open CN101614742A of the open Chinese invention patent of Chinese invention patent discloses the magnetic microparticle chemiluminescence immune assay kit of luteinizing principle a kind of; this kit comprises luteinizing principle series calibration object; the magnetic particle solution that anti-fluorescein isothiocynate monoclonal antibody is coated; the luteinizing principle monoclonal antibody of marked by fluorescein isothiocyanate; the luteinizing principle monoclonal antibody of horseradish peroxidase-labeled; Chemoluminescent substrate, concentrated cleaning solution.Coated magnetic particle solution, the luteinizing principle antibody of marked by fluorescein isothiocyanate and the luteinizing principle monoclonal body antibody of horseradish peroxidase-labeled of anti-fluorescein isothiocynate monoclonal antibody consists of the immune response reagent that the immune response step is used in this kit.Although this kit can be realized detecting quickly and accurately, but wherein, the preparation process of the luteinizing principle monoclonal antibody of horseradish peroxidase-labeled is very loaded down with trivial details, technology stability is bad, and mark rate is low, limits its detection effect and particularly analyzes a precision and cause cost to increase.
Summary of the invention
Technical matters to be solved by this invention is to overcome the deficiencies in the prior art, a kind of chemical luminescence immune analysis reagent box of luteinizing principle is provided, it can prepare with lower cost, and can realize the accurate and high precision ground quantitative measurement of luteinizing principle.
The present invention also provides a kind of preparation method of chemical luminescence immune analysis reagent box of luteinizing principle simultaneously, the method process stabilizing, and cost is low, and the precision of gained kit is particularly analyzed a precision height.
For solving above technical matters, a kind of technical scheme that the present invention takes is:
The nano magnetic particulate chemistry luminescence assays kit of a kind of luteinizing principle (LH); it comprises the immune response reagent for the immune response step; this immune response reagent comprises the solution (being called for short the first reagent) of the luteinizing principle antibody that contains fluorescein (FITC) mark and is coated with the suspending liquid of the magnetic particle of anti-fluorescein antibody (being called for short the magnetic separation agent); particularly; described immune response reagent also comprises the solution (being called for short the second reagent) of the luteinizing principle antibody that contains alkaline phosphatase (ALP) mark, and the luteinizing principle antibody of this alkali phosphatase enzyme mark is connected and composed by 4-(N-maleimide ylmethyl) cyclohexane-1-carboxylic acid succinimide ester (SMCC) and 2-imino group sulfane hydrochloride (2IT) by alkaline phosphatase and luteinizing principle antibody.
Further, the described pH that contains the solution of fluorescein-labeled luteinizing principle antibody is 7 ~ 9, and the concentration of fluorescein-labeled luteinizing principle antibody wherein is 0.5 ~ 1 μ g/mL.The pH of the solution of the described luteinizing principle antibody that contains alkali phosphatase enzyme mark is 7 ~ 9, and wherein the concentration of the luteinizing principle antibody of alkali phosphatase enzyme mark is 0.5 ~ 1 μ g/mL.
According to the present invention, describedly contain fluorescein-labeled anti-luteinizing principle antibody and can easily prepare by ripe and stable technique.
According to the present invention, the described preparation that is coated with the magnetic particle of anti-fluorescein antibody also is comparatively to be easy to, and the existing ripe preparation technology of prior art can reference.For example can prepare by physisorption or the coated mode of chemical coupling of routine, be not particularly limited.As a kind of preferred embodiment of the present invention: this is coated with in the magnetic particle of anti-fluorescein antibody, phase chemistry coupling between anti-fluorescein antibody and the magnetic particle.In the described magnetic particle that is coated with anti-fluorescein antibody, phase chemistry coupling between anti-fluorescein antibody and the magnetic particle.
Those skilled in the art should know, and kit of the present invention can further include other and detects required reagent, for example substrate solution.But can buy separately or prepare such as other reagent such as substrate solutions, therefore, although can comprise these reagent in the kit, they be not essential for kit of the present invention.
The another technical scheme that the present invention takes is: a kind of preparation method of nano magnetic particulate chemistry luminescence assays kit of above-mentioned luteinizing principle; the step of suspending liquid that it comprises the solution for preparing respectively the described solution that contains fluorescein-labeled luteinizing principle antibody, the described luteinizing principle antibody that contains alkali phosphatase enzyme mark and is coated with the magnetic particle of anti-fluorescein antibody, wherein: the preparation process of the solution of the described luteinizing principle antibody that contains alkali phosphatase enzyme mark is as follows:
1. get the phosphate buffer of luteinizing principle antibody, add 2IT, at room temperature leave standstill reaction 15 ~ 25min, add glycine solution, room temperature leaves standstill 3 ~ 10min, by G-25 gel column desalination, collect the luteinizing principle antibody of activation, save backup under 2 ~ 8 ℃;
2. get the phosphate buffer of alkaline phosphatase, add 4-(N-maleimide ylmethyl) cyclohexane-1-carboxylic acid succinimide ester, room temperature leaves standstill reaction 25 ~ 35min, by G-25 gel column desalination, collect the alkaline phosphatase of activation, save backup under 2 ~ 8 ℃;
3. with step 1. gained activation luteinizing principle antibody and step 2. the alkaline phosphatase that activates of gained be that the ratio of 1:1 ~ 2 is mixed according to luteinizing principle antibody and the molecular proportion of alkaline phosphatase, under 2 ~ 8 ℃, leave standstill 12 ~ 24h, carry out purifying by Supperdex200 gel-purified post, the damping fluid that the solution of purifying is selected to have proper pH value is adjusted concentration and pH value, namely gets the described solution that contains the luteinizing principle antibody of alkali phosphatase enzyme mark.
Preferably, the purity of described luteinizing principle antibody, described alkaline phosphatase is all more than or equal to 95wt%, and the specific activity of described alkaline phosphatase surpasses 1000u/mg.
Preferably, step 2. in, the concentration of the phosphate buffer alkaline phosphatase of alkaline phosphatase is 5 ~ 10mg/mL.
Further, the preparation method of the described solution that contains fluorescein-labeled luteinizing principle antibody is as follows: the pH that preparation contains fluorescein is 9 ~ 10 damping fluid, then the molecular proportion according to fluorescein and anti-luteinizing principle antibody is the ratio of 20 ~ 200:1, be 9 ~ 10 damping fluid with the described pH that contains fluorescein with the pH of anti-luteinizing principle antibody be that 9 ~ 10 damping fluid mixes, behind the mixing, room temperature leaves standstill reaction, then reactant liquor is separated by the G-25 gel column, remove free fluorescein, obtain containing the solution of fluorescein-labeled anti-luteinizing principle antibody, then adjust concentration and pH with the damping fluid with proper pH value, and get final product.Wherein the damping fluid of proper pH value can be for for example containing the TRIS damping fluid of 0.5% bovine serum albumin(BSA), pH8.0.
Further, the preparation method of the suspending liquid of the described magnetic particle that is coated with anti-fluorescein antibody is as follows: make the magnetic particle that contains the carboxyl reactive group and anti-fluorescein antibody in the presence of the coupling agent carbodiimide, room temperature reaction 2 ~ 18 hours, reaction finishes, magnetic separates, remove supernatant, adjust pH and concentration with the damping fluid with proper pH value, and get final product, wherein said magnetic particle has superparamagnetism, its diameter is 0.5 ~ 2 μ m, on every gram magnetic particle with the content of carboxyl reactive group be not less than 0.4mmol; Described anti-fluorescein antibody is monoclonal antibody or polyclonal antibody, and purity is more than or equal to 90wt%, and dilution is tired greater than 1:100 ten thousand.Wherein the damping fluid of proper pH value can be for for example containing the TRIS damping fluid of 0.5% bovine serum albumin(BSA), pH8.0.
Preferably, the above-mentioned damping fluid with proper pH value is the TRIS damping fluid that contains 0.5% bovine serum albumin(BSA), pH8.0.
Fluorescein of the present invention can be known various fluoresceins, and commonly used have for example fluorescein isothiocynate, RB 200, a TRITC etc.
Step when kit of the present invention is applied to chemiluminescence immunoassay and detects is the same with traditional detection method, namely comprise the immune response step of carrying out successively, use magnetic to separate and step that washing facility washs immunoreactive reactant liquor and the reactant liquor after the washing in add substrate solution and utilize the step of chemiluminescence detector detection luminous intensity.
Preferably, described immune response step implementation is as follows: add for example 10 ~ 30 μ l sample to be tested stoste or dilutions in test sample tube, then add successively 30 ~ 100 μ l and contain the solution of fluorescein-labeled luteinizing principle antibody, the solution that 30 ~ 100 μ l contain the luteinizing principle antibody of alkali phosphatase enzyme mark, mixing, under 25 ~ 40 ℃, carry out the incubation first time, then add 30 ~ 100 μ l and be coated with the suspending liquid of the magnetic particle of anti-fluorescein antibody, mixing carries out the incubation second time under 25 ~ 40 ℃; Described substrate solution is alkaline phosphatase chemical luminous substrate solution.
Further, the time of incubation described first time can be 10 ~ 20min, is generally 15min; The time of incubation can be 3 ~ 15min for the second time, is generally 5min.
The step that adds substrate solution and utilize chemiluminescence detector to detect luminous intensity in the step that above-mentioned use magnetic separates and washing facility washs immunoreactive reactant liquor and the reactant liquor after the washing all can be implemented with reference to conventional method, and used equipment and device also are conventional.Wherein, alkaline phosphatase chemical luminous substrate solution also is known for those skilled in the art, commercially available acquisition.
Because the enforcement of above technical scheme, the present invention compared with prior art has following advantage:
1. the applicant finds, when taking SMCC and 2IT to carry out the coupling of alkaline phosphatase and luteinizing principle antibody, has with other method and compares higher coupling efficiency, when reducing preparation cost, is conducive to improve the detection effect.Therefore, three kinds of reagent in the kit of the present invention all can prepare by stable preparation technology, and production cost is low, and because preparation technology's stability, it is little that kit is analyzed difference between batch, and precision improves between the analysis of detection;
2. the preparation method of the solution of the luteinizing principle antibody that contains alkali phosphatase enzyme mark in the kit of the present invention, can be effectively with luteinizing principle antibody and alkaline phosphatase coupling, coupling efficiency is high, and it is low and guarantee the detection effect of kit further to reduce the cost of kit.
3, take kit of the present invention to detect, accuracy is good, and precision is high, and highly sensitive, sensing range is wide, the unordered pre-dilution of sample, simple to operate saving time.Compare with the method that adopts the import reagent box to detect, detection method of the present invention has significant advantage at cost.
Description of drawings
Fig. 1 is detection calibration product typical curves;
Fig. 2 is that matched curve is estimated in sensitivity;
Fig. 3 is serum sample testing result correlativity (wherein horizontal ordinate x is the kit sample measured value that embodiment 4 is prepared into, and concentration unit is mIU/mL, and ordinate y is Beckman company kit sample measured value, and concentration unit is mIU/mL).
Embodiment
For convenience of description, hereinafter, with the first reagent, the second reagent and magnetic separation agent represent respectively the solution that contains fluorescein-labeled luteinizing principle antibody among the present invention, contain alkaline phosphatase (ALP) mark luteinizing principle antibody solution and be coated with the suspending liquid of the magnetic particle of anti-fluorescein antibody.
The preparation of embodiment 1 first reagent
(1) material and instrument: with the anti-LH monoclonal antibody (purity surpasses 95wt%, and concentration is 2mg/mL, calls LH antibody in the following text) of phosphate buffer preservation; Fluorescein isothiocynate (FITC), the reagent such as sodium bicarbonate should reach chemical pure; The buying of G-25 gel-purified post is from GE company.
(2) preparation process:
1. use the FITC solution of the carbonate buffer solution preparation 0.5mg/mL of 0.1 ~ 0.2mol/L pH9.0 ~ 10.0;
2. add step according to LH antibody and FITC molecular proportion by the ratio of 1:20 in antibody-solutions and 1. joined FITC solution, mix, room temperature left standstill 12h hour, and reaction generates LH antibody-FITC connector;
3. will separate by the G-25 gel column through step reactant liquor 2., remove unreacted FITC, obtain containing the solution of LH antibody-FITC connector (being the LH antibody of FITC mark);
4. with step 3. the gained solution that contains LH antibody-FITC connector to be diluted to LH antibody-FITC connector concentration with the TRIS damping fluid of the 0.1mol/L that contains 0.5% bovine serum albumin(BSA) (BSA) pH8.0 be 0.5 ~ 1 μ g/mL, pH is 7 ~ 9, is the first reagent.
The preparation of embodiment 2 second reagent
(1) material and instrument: with the anti-LH monoclonal antibody (purity surpasses 95wt%, and concentration is 2mg/mL, calls the LH antibody-solutions in the following text) of phosphate buffer preservation; The alkaline phosphatase of preserving with phosphate buffer (ALP solution, ALP purity are about 99%, and specific activity is about 1500U/mg, and concentration is 10mg/mL); 5mg/mL SMCC solution, 10mg/mL2IT solution (SMCC and 2IT pressed powder are made into mentioned solution with DMF and use) (available from THERMO company); The chemical reagent such as TRIS should reach chemical pure; G-25 gel-purified post, AKTA-purifier100 protein purification instrument and Supperdex200 gel-purified post are GE company product.
(2) preparation process:
1. get 1mg LH antibody-solutions, add 2IT solution 2 ~ 4 μ l of 10mg/mL, room temperature leaves standstill 20min, adds the glycine solution 10 μ l of 0.1mol/L, and room temperature leaves standstill 5min.With G-25 gel column desalination, collect the antibody of activation, 2-8 ℃ saves backup;
2. get 1.5mg ALP solution, add SMCC solution 10 ~ 20 μ l of 5mg/mL, room temperature leaves standstill 30min, with G-25 gel column desalination, collects the ALP antibody of activation, and 2-8 ℃ saves backup;
3. the LH antibody with above-mentioned activation mixes with the ALP antibody of activation, leave standstill 12 ~ 24h under the 2-8 ℃ of condition, carry out purifying by Supperdex200 gel-purified post, obtain to contain the solution of antibody LH-ALP connector (the LH antibody of ALP mark), 2-8 ℃ saves backup;
4. use the TRIS damping fluid of the 0.1mol/L that contains 0.5% bovine serum albumin(BSA) (BSA) pH8.0 to be diluted to 0.5 ~ 1 μ g/mL the solution of antibody LH-ALP connector, pH7 ~ 9 are the second reagent.
The preparation of embodiment 3 magnetic separation agents
(1) material and instrument:
The suspending liquid of magnetic particle: magnetic particle content 5wt%, magnetic particle contain the active group of carboxyl (COOH), and every gram (g) magnetic particle (dry weight) carboxyl-content is not less than 0.4 mM (mmol), has superparamagnetism, and diameter is between 0.5-2 μ m;
Anti-FITC mAb: can be polyclonal antibody, also can be monoclonal antibody, and purity is more than the 90wt%, and dilution is tired above 1:100 ten thousand;
MES (MES), carbodiimide (EDC), TRIS and other reagent should reach chemical pure.
(2) preparation process:
1. get the suspending liquid of 100mg magnetic particle, magnetic divides the supernatant of leaving away, and uses 0.05mol/L, and 10mL is resuspended for pH4.5 ~ 5MES damping fluid;
2. the anti-FITC mAb that adds 2 ~ 4mg, room temperature suspendible 30 ~ 60min;
3. the EDC aqueous solution that adds the freshly prepared 10mg/mL of 0.5 ~ 1mL, room temperature suspendible 2 ~ 12h;
4. magnetic separates, and removes supernatant, uses the TRIS damping fluid of the 0.1mol/L that contains 0.5% bovine serum albumin(BSA) (BSA) pH8.0 to be resuspended to 1mg/mL, and pH8.0 is the magnetic separation agent.
The chemical luminescence immune analysis reagent box of embodiment 4 luteinizing principles
This kit comprises:
According to first reagent (concentration is 0.75 μ g/mL) of embodiment 1 method preparation, 50mL;
According to second reagent (concentration is 0.75 μ g/mL) of embodiment 2 methods preparation, 50mL;
Magnetic separation reagent 50mL according to the preparation of embodiment 3 methods.
The chemical luminescence immune analysis reagent box of embodiment 5 luteinizing principles
This kit comprises:
According to first reagent (concentration is 0.5 μ g/mL) of embodiment 1 method preparation, 5mL;
According to second reagent (concentration is 0.5 μ g/mL) of embodiment 2 methods preparation, 5mL;
Magnetic separation reagent 5mL according to the preparation of embodiment 3 methods.
Embodiment 6 takes the kit of embodiment 4 to carry out the quantitative detection of luteinizing principle
(1) detecting step:
1. immune response: in detector tube, add 20 μ l sample to be tested (serum) stostes, then add 50 μ l the first reagent, 50 μ l the second reagent, mixing, incubation 15min under 37 ± 1 ℃ of conditions; Add 50 μ l magnetic separation agents, mixing, incubation 5min under 37 ± 1 ℃ of conditions;
2. washing: make magnetic particle sedimentation in magnetic field, remove supernatant, add the cleaning fluid of 300 μ l, remove magnetic field, concussion makes the abundant suspendible of magnetic particle, and then magnetic separates, and removes supernatant.This step repeats 3 times;
3. add substrate solution and detect luminous intensity: in detector tube, add 100 μ l alkaline phosphatase chemical luminous substrate solution (Beijing Ah APCL-of this Bioisystech Co., Ltd I), concussion makes the abundant suspendible of magnetic particle, in 5 minutes, detect with luminous detector (MAGLIA60), detect the unit interval inner glow intensity.The result calculates the related description of consulting instrument.
(2) draw the calibration object typical curve
The calibration object typical curve is referring to Fig. 1.
(3) sensitivity evaluation
Detect " 0 " concentration samples, duplicate detection 20 times, calculate mean value (M) and the standard deviation (SD) of relative luminous intensity (RLU), and calculating M-2SD value, carry out 2 regression fits according to the concentration-RLU between zero-dose calibration object and the adjacent calibration object and draw linear function, the M+2SD value is brought in the above-mentioned equation, obtain corresponding concentration value, be lowest detectable limit.The sensitivity of this method is not more than 0.5mIU/mL.Wherein: A point luminous value is referring to table 1:
Table 1
The luminous average X=1719 of A point
SD=24.3
X+2SD=1767.7
B point luminous value is referring to table 2.
The luminous average X=56478 of B point
Table 2
| LH-STD-B(RLU) |
| 56470 |
| 56486 |
A, B point connect the some matched curve referring to Fig. 2.Sensitivity=0.002mIU/mL.
(4) precision evaluation
1. precision in analyzing
The kit of embodiment 4 is a collection of, measure respectively the serum of basic, normal, high three kinds of variable concentrations, 10 hole replicate determinations, the result is referring to table 3, and drawing the variation within batch coefficient is 2.95%～6.55%.
Precision test in table 3 is analyzed
| Measure serum-concentration (mIU/mL) | Measure number of times | CV (%) in analyzing |
| 12.20 | 10 | 6.55 |
| 75.25 | 10 | 3.43 |
| 120.36 | 10 | 2.95 |
2. precision between analyzing
The kit of embodiment 4 is got three batches, and every batch of kit is all measured the serum of basic, normal, high three kinds of variable concentrations, 10 hole replicate determinations.Every part of serum obtains 30 concentration measured values, and referring to table 4, the coefficient of variation between statistical study is 4.70%～6.95%.
Precision test between table 4 is analyzed
| Measure serum-concentration (mIU/mL) | Measure number of times | CV (%) in analyzing |
| 12.20 | 30 | 6.95 |
| 75.25 | 30 | 4.70 |
| 120.36 | 30 | 5.15 |
(5) accuracy estimating
Add different amount people LH standard items in 2 routine pooled serum samples, the serum that forms 3 concentration levels adds sample, and the additive volume is less than 10% of cumulative volume.Detect concentration of specimens, by following formula calculate recovery rate.This method serum matrix recovery is between 90-110%.Data are referring to table 5.
R: the recovery;
V: the volume that adds standard solution;
V
0: the volume of people source sample;
C: people source sample adds the detectable concentration behind the standard solution;
C
0: the detectable concentration of people source sample;
Cs: the concentration of standard solution.
Table 5 accuracy estimating-interpolation recovery experiment data
(6) kit Evaluation on specificity
To kit specificity check be choose with LH have similar structures follotropin (FSH),
Thyrotropic hormone (TSH), human chorionic gonadtropin (HCG) are mixed with the sample greater than physiological concentration, measure with this method.The results are shown in Table 6, this law and FSH, TSH, HCG no cross reaction.
The experiment of table 6 specificity
(7) correlativity evaluation
With the kit of embodiment 4 and the chemical luminescence reagent kit of Beckman company 100 parts of human serum samples are detected simultaneously.Its testing result is referring to accompanying drawing 3, take the serum Lh concentration of the survey of the inventive method as horizontal ordinate, does regretional analysis take the result of Beckman company kit measurement as ordinate, and dependent equation is: y=-0.07655+0.9988x, related coefficient is: 0.9831.Learn by statistics result and show, this method is good with external kit clinical sample measured value correlativity.
(8) Evaluation of Thermal Stability
Kit is carried out respectively 4 ℃ of 12 months and 37 ℃ of stability experiments of 7 days, the result show kit standard items luminous intensity variation, batch in and the indexs such as betweenrun precision, accuracy all within normal range, the kit term of validity can reach 12 months.
Embodiment 7 takes the kit of embodiment 5 to carry out the quantitative detection of luteinizing principle
(1) detecting step: with embodiment 6.
(2) sensitivity evaluation: the kit sensitivity among this embodiment is 0.002mIU/mL, reaches domestic and international similar reagent top standard, satisfies preferably clinical practice.
(3) precision evaluation: see precision all less than 10% with analysis in the kit analysis among this embodiment, difference between batch is little.
(4) evaluation of the accuracy: the kit serum among this embodiment adds the recovery between 90-110%, and the clinical practice measuring value accuracy is reliable.
Above-described embodiment only is explanation technical conceive of the present invention and characteristics; its purpose is to allow the personage who is familiar with technique can understand content of the present invention and according to this enforcement; can not limit protection scope of the present invention with this; all equivalences that Spirit Essence is done according to the present invention change or modify, and all should be encompassed within protection scope of the present invention.
Claims (10)
1. the nano magnetic particulate chemistry luminescence assays kit of a luteinizing principle; described kit comprises the immune response reagent for the immune response step; described immune response reagent comprises the solution that contains fluorescein-labeled luteinizing principle antibody and is coated with the suspending liquid of the magnetic particle of anti-fluorescein antibody; it is characterized in that: described immune response reagent also comprises the solution of the luteinizing principle antibody that contains alkali phosphatase enzyme mark, and the luteinizing principle antibody of this alkali phosphatase enzyme mark is connected and composed by 4-(N-maleimide ylmethyl) cyclohexane-1-carboxylic acid succinimide ester and 2-imino group sulfane hydrochloride by alkaline phosphatase and luteinizing principle antibody.
2. the nano magnetic particulate chemistry luminescence assays kit of luteinizing principle according to claim 1; it is characterized in that: the described pH that contains the solution of fluorescein-labeled luteinizing principle antibody is 7 ~ 9, and the concentration of fluorescein-labeled luteinizing principle antibody wherein is 0.5 ~ 1 μ g/mL.
3. the nano magnetic particulate chemistry luminescence assays kit of luteinizing principle according to claim 1 and 2 is characterized in that: in the described magnetic particle that is coated with anti-fluorescein antibody, and phase chemistry coupling between anti-fluorescein antibody and the magnetic particle.
4. the nano magnetic particulate chemistry luminescence assays kit of luteinizing principle according to claim 1 and 2; it is characterized in that: the pH of the solution of the described luteinizing principle antibody that contains alkali phosphatase enzyme mark is 7 ~ 9, and wherein the concentration of the luteinizing principle antibody of alkali phosphatase enzyme mark is 0.5 ~ 1 μ g/mL.
5. preparation method such as the nano magnetic particulate chemistry luminescence assays kit of the described luteinizing principle of each claim in the claim 1 ~ 4; the step that it comprises the suspending liquid of the solution for preparing respectively the described solution that contains fluorescein-labeled luteinizing principle antibody, the described luteinizing principle antibody that contains alkali phosphatase enzyme mark and the described magnetic particle that is coated with anti-fluorescein antibody is characterized in that: the preparation process of the solution of the described luteinizing principle antibody that contains alkali phosphatase enzyme mark is as follows:
1. get the phosphate buffer of luteinizing principle antibody, add 2-imino group sulfane hydrochloride, at room temperature leave standstill reaction 15 ~ 25min, add glycine solution, room temperature leaves standstill 3 ~ 10min, by G-25 gel column desalination, collect the luteinizing principle antibody of activation, save backup under 2 ~ 8 ℃;
2. get the phosphate buffer of alkaline phosphatase, add 4-(N-maleimide ylmethyl) cyclohexane-1-carboxylic acid succinimide ester, room temperature leaves standstill reaction 25 ~ 35min, by G-25 gel column desalination, collect the alkaline phosphatase of activation, save backup under 2 ~ 8 ℃;
3. with step 1. gained activation luteinizing principle antibody and step 2. the alkaline phosphatase that activates of gained be that the ratio of 1:1 ~ 2 is mixed according to luteinizing principle antibody and the molecular proportion of alkaline phosphatase, under 2 ~ 8 ℃, leave standstill 12 ~ 24h, carry out purifying by Supperdex200 gel-purified post, the damping fluid that the solution of purifying is selected to have proper pH value is adjusted concentration and pH value, namely gets the described solution that contains the luteinizing principle antibody of alkali phosphatase enzyme mark.
6. preparation method according to claim 5 is characterized in that: the purity of described luteinizing principle antibody, described alkaline phosphatase is all more than or equal to 95wt%, and the specific activity of described alkaline phosphatase surpasses 1000u/mg.
7. according to claim 5 or 6 described preparation methods, it is characterized in that: step 2. in, the concentration of the phosphate buffer alkaline phosphatase of alkaline phosphatase is 5 ~ 10mg/mL.
8. preparation method according to claim 5, it is characterized in that: the preparation method of the described solution that contains fluorescein-labeled luteinizing principle antibody is as follows: the pH that preparation contains fluorescein is 9 ~ 10 damping fluid, then the molecular proportion according to fluorescein and anti-luteinizing principle antibody is the ratio of 20 ~ 200:1, be 9 ~ 10 damping fluid with the described pH that contains fluorescein with the pH of anti-luteinizing principle antibody be that 9 ~ 10 damping fluid mixes, behind the mixing, room temperature leaves standstill reaction, then reactant liquor is separated by the G-25 gel column, remove free fluorescein, obtain containing the solution of fluorescein-labeled anti-luteinizing principle antibody, then adjust concentration and pH with the damping fluid with proper pH value, and get final product.
9. preparation method according to claim 5, it is characterized in that: the preparation method of the suspending liquid of the described magnetic particle that is coated with anti-fluorescein antibody is as follows: make the magnetic particle that contains the carboxyl reactive group and anti-fluorescein antibody in the presence of the coupling agent carbodiimide, room temperature reaction 2 ~ 18 hours, reaction finishes, magnetic separates, remove supernatant, adjust pH and concentration with the damping fluid with proper pH value, and get final product, wherein said magnetic particle has superparamagnetism, its diameter is 0.5 ~ 2 μ m, on every gram magnetic particle with the content of carboxyl reactive group be not less than 0.4mmol; Described anti-fluorescein antibody is monoclonal antibody or polyclonal antibody, and purity is more than or equal to 90wt%, and dilution is tired greater than 1:100 ten thousand.
10. according to claim 5 or 8 or 9 described preparation methods, it is characterized in that: described damping fluid with proper pH value is the TRIS damping fluid that contains 0.5% bovine serum albumin(BSA), pH 8.0.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210571305.9A CN103048446B (en) | 2012-12-25 | 2012-12-25 | Luteinizing hormone nano-magnetic particle chemiluminescence assay kit and preparation method thereof and assay method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210571305.9A CN103048446B (en) | 2012-12-25 | 2012-12-25 | Luteinizing hormone nano-magnetic particle chemiluminescence assay kit and preparation method thereof and assay method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103048446A true CN103048446A (en) | 2013-04-17 |
| CN103048446B CN103048446B (en) | 2015-02-04 |
Family
ID=48061160
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210571305.9A Active CN103048446B (en) | 2012-12-25 | 2012-12-25 | Luteinizing hormone nano-magnetic particle chemiluminescence assay kit and preparation method thereof and assay method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103048446B (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103293323A (en) * | 2013-06-14 | 2013-09-11 | 南京医科大学第二附属医院 | Luteinizing hormone nano-magnetic particle chemiluminescence quantitative immunoassay kit and preparation method thereof |
| CN103901188A (en) * | 2014-04-11 | 2014-07-02 | 苏州浩欧博生物医药有限公司 | Chemiluminescence quantitative detection kit for inhaled allergens, and preparation method and detection method thereof |
| CN103901215A (en) * | 2014-04-11 | 2014-07-02 | 苏州浩欧博生物医药有限公司 | Chemiluminescence quantitative detection kit for food allergens, and preparation method and detection method thereof |
| CN105116153A (en) * | 2015-08-07 | 2015-12-02 | 南通市伊士生物技术有限责任公司 | Semiquantitative detection system for luteinizing hormone and detection method thereof |
| CN105424682A (en) * | 2015-11-17 | 2016-03-23 | 苏州浩欧博生物医药有限公司 | Bevacizumab kit and drug-resistant antibody kit thereof |
| CN105445256A (en) * | 2014-09-02 | 2016-03-30 | 江苏泽成生物技术有限公司 | Luteinizing hormone (LH) quantitative determination kit, and making method and detection method thereof |
| CN109211810A (en) * | 2018-10-11 | 2019-01-15 | 郑州安图生物工程股份有限公司 | A kind of reagent and its rapid assay methods measuring the micro amino content in magnetic particle surface |
| CN109613276A (en) * | 2018-12-20 | 2019-04-12 | 北京贝尔生物工程股份有限公司 | A kind of anti-Miao Leguan hormone determination kit of people of highly sensitive, wide detection range |
| CN113933521A (en) * | 2021-09-26 | 2022-01-14 | 北京贝尔医疗设备有限公司 | Magnetic particle chemiluminescence detection kit and preparation method and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2668714A1 (en) * | 2006-11-14 | 2008-05-22 | Novartis Ag | Methods of treating, diagnosing or detecting cancer |
| CN101614742A (en) * | 2009-07-23 | 2009-12-30 | 清华大学 | The magnetic microparticle chemiluminescence immune assay determination kit of luteinizing principle |
| CN101937000A (en) * | 2010-08-05 | 2011-01-05 | 北京倍爱康生物技术有限公司 | Magnetic particle separation chemiluminescence immunoassay method of human cystatin C |
| WO2011159835A1 (en) * | 2010-06-16 | 2011-12-22 | University Of Pittsburgh-Of The Commonwealth System Of Higher Education | Antibodies to endoplasmin and their use |
| US20120141375A1 (en) * | 2002-06-14 | 2012-06-07 | Immunomedics, Inc. | Anti-Mucin Antibodies for Early Detection and Treatment of Pancreatic Cancer |
-
2012
- 2012-12-25 CN CN201210571305.9A patent/CN103048446B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120141375A1 (en) * | 2002-06-14 | 2012-06-07 | Immunomedics, Inc. | Anti-Mucin Antibodies for Early Detection and Treatment of Pancreatic Cancer |
| CA2668714A1 (en) * | 2006-11-14 | 2008-05-22 | Novartis Ag | Methods of treating, diagnosing or detecting cancer |
| CN101614742A (en) * | 2009-07-23 | 2009-12-30 | 清华大学 | The magnetic microparticle chemiluminescence immune assay determination kit of luteinizing principle |
| WO2011159835A1 (en) * | 2010-06-16 | 2011-12-22 | University Of Pittsburgh-Of The Commonwealth System Of Higher Education | Antibodies to endoplasmin and their use |
| CN101937000A (en) * | 2010-08-05 | 2011-01-05 | 北京倍爱康生物技术有限公司 | Magnetic particle separation chemiluminescence immunoassay method of human cystatin C |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103293323A (en) * | 2013-06-14 | 2013-09-11 | 南京医科大学第二附属医院 | Luteinizing hormone nano-magnetic particle chemiluminescence quantitative immunoassay kit and preparation method thereof |
| CN103293323B (en) * | 2013-06-14 | 2015-09-23 | 南京医科大学第二附属医院 | A kind of interstitialcellstimulating hormone (ICSH) nano magnetic particulate chemistry electrochemiluminescent immunoassay immue quantitative detection reagent box and preparation method thereof |
| CN103901188A (en) * | 2014-04-11 | 2014-07-02 | 苏州浩欧博生物医药有限公司 | Chemiluminescence quantitative detection kit for inhaled allergens, and preparation method and detection method thereof |
| CN103901215A (en) * | 2014-04-11 | 2014-07-02 | 苏州浩欧博生物医药有限公司 | Chemiluminescence quantitative detection kit for food allergens, and preparation method and detection method thereof |
| CN103901188B (en) * | 2014-04-11 | 2016-08-24 | 苏州浩欧博生物医药有限公司 | A kind of chemical luminescent analysis reagent kid sucking anaphylactogen and preparation method thereof and detection method |
| CN105445256A (en) * | 2014-09-02 | 2016-03-30 | 江苏泽成生物技术有限公司 | Luteinizing hormone (LH) quantitative determination kit, and making method and detection method thereof |
| CN105116153A (en) * | 2015-08-07 | 2015-12-02 | 南通市伊士生物技术有限责任公司 | Semiquantitative detection system for luteinizing hormone and detection method thereof |
| CN105116153B (en) * | 2015-08-07 | 2017-07-07 | 南通市伊士生物技术有限责任公司 | The half-quantitative detection system and its detection method of interstitialcellstimulating hormone (ICSH) |
| CN105424682A (en) * | 2015-11-17 | 2016-03-23 | 苏州浩欧博生物医药有限公司 | Bevacizumab kit and drug-resistant antibody kit thereof |
| CN109211810A (en) * | 2018-10-11 | 2019-01-15 | 郑州安图生物工程股份有限公司 | A kind of reagent and its rapid assay methods measuring the micro amino content in magnetic particle surface |
| CN109613276A (en) * | 2018-12-20 | 2019-04-12 | 北京贝尔生物工程股份有限公司 | A kind of anti-Miao Leguan hormone determination kit of people of highly sensitive, wide detection range |
| CN113933521A (en) * | 2021-09-26 | 2022-01-14 | 北京贝尔医疗设备有限公司 | Magnetic particle chemiluminescence detection kit and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103048446B (en) | 2015-02-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103048446B (en) | Luteinizing hormone nano-magnetic particle chemiluminescence assay kit and preparation method thereof and assay method thereof | |
| CN103063851B (en) | Free triiodothyronine nanometer magnetic particle chemiluminescence assay kit and preparation method thereof and detection method thereof | |
| CN103901203B (en) | Chemical luminescent analysis reagent kid of a kind of Procalcitonin and preparation method thereof and detection method | |
| CN102998467B (en) | β human chorionic gonadotrophin magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof | |
| Ozturk et al. | Differential production of human chorionic gonadotropin and free subunits in gestational trophoblastic disease | |
| CN103048452A (en) | Nanometer magnetic particle chemiluminiscence determination kit for antigen CA125 relating to tumor, as well as preparation method and determining method of same | |
| CN103293323B (en) | A kind of interstitialcellstimulating hormone (ICSH) nano magnetic particulate chemistry electrochemiluminescent immunoassay immue quantitative detection reagent box and preparation method thereof | |
| CN101545913A (en) | Chemoluminescence immunoassay measuring kit and preparation method thereof for triiodothyronine magnetic particles | |
| CN110862881A (en) | Special cleaning solution or diluent for full-automatic chemiluminescence determinator and preparation method thereof | |
| CN103063845A (en) | Nanometer magnetic particle chemiluminiscence kit, preparation method and detection method of hepatitis B virus surface-antibody | |
| CN108445230B (en) | Procalcitonin chemiluminescence detection reagent based on nano antibody and detection method | |
| CN101614742A (en) | The magnetic microparticle chemiluminescence immune assay determination kit of luteinizing principle | |
| CN103048477B (en) | Nanometer magnetic particle chemiluminescence detection kit for triiodothyronine as well as preparation method and detecting method of same | |
| CN103048473B (en) | Nano magnetic particle chemiluminiscence determining kit for promoting hormone generation by follicles and preparation and detection method thereof | |
| CN103063852B (en) | Free thyroxine nanometer magnetic particle chemiluminescence assay kit and preparation method thereof and detection method thereof | |
| CN103336115A (en) | Magnetic particle chemiluminescence immune assay kit for TPOAb and detecting method thereof | |
| CN103048453B (en) | Nanometer magnetic particle chemiluminescence detection kit for carbohydrate antigen CA19-9 as well as preparation method thereof and detecting method thereof | |
| CN103267866B (en) | A kind of FSH nano magnetic particulate chemistry electrochemiluminescent immunoassay immue quantitative detection reagent box and preparation method thereof | |
| CN103048475A (en) | Nano magnetic particle chemiluminiscence assay kit for free HCG (human chorionic gonadotropin) beta subunit, preparation method for Nano magnetic particle chemiluminiscence assay kit and detection method adopting Nano magnetic particle chemiluminiscence assay kit | |
| CN109459570A (en) | The colloid gold immune test paper preparation method of lead ion in a kind of quick detection blood of human body | |
| CN108593940A (en) | A kind of urine transferrins detection kit | |
| CN103048474A (en) | Kit for detecting nano magnetic particle chemiluminescence of hormothyrin and preparation method and detection method thereof | |
| CN109342719A (en) | A kind of preparation method of thyroid function multi-link detection reagent kit | |
| CN105758846A (en) | Chemiluminescence enzyme-linked immunosorbent assay reagent kit for detecting clenbuterol | |
| Xiao et al. | Development of a rapid and sensitive magnetic chemiluminescent enzyme immunoassay for detection of luteinizing hormone in human serum |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder | ||
| CP01 | Change in the name or title of a patent holder |
Address after: Xinghu Street Industrial Park of Suzhou city in Jiangsu province 215123 building C6 No. 218 Patentee after: Jiangsu Hao Bo biomedical Limited by Share Ltd Address before: Xinghu Street Industrial Park of Suzhou city in Jiangsu province 215123 building C6 No. 218 Patentee before: Suzhou Haooubo Biopharmaceutical Co., Ltd. |