CN103048446B - 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
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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 a kind ofly combine chemical luminescence immune analysis reagent box of the luteinizing principle of immune magnetic particle isolation technics and chemiluminescence immunoassay technology and preparation method thereof.The present invention is also in particular to the preparation method of this kit.
Background technology
Luteinizing principle (Luteinizing Hormone, LH) secretes by anterior pituitary basocyte a kind of glycoprotein hormones produced, and molecular weight is about 30,000 dalton, is made up of the different subunits of two Non-covalent binding.Its alpha subunit has 92 amino acid residues, identical with LH, TSH, hCG.Amino acid sequence and LH, TSH of β subunit are very different, but very similar to hCG.
The regulation and control of the gonadotropin-releasing hormone (GRH) (Gn-RH) that LH produces by hypothalamus.The research of normal women is shown that pituitary LH is divided into two phases, namely within about every 2 hours while basal secretion phase, presents intermittent pulses secretion.In the female normal menstrual cycle, LH and LH synergy causes the mechanical periodicity of ovary.2 cause follicular rupture at the summit occurred mid-term menstrual cycle, discharge ripe ovum.Remaining ovarian follicle becomes functional corpus luteum, secretion progesterone and estradiol, and these steroids apply positive and negative two kinds of feedback regulations to hypothalamus and hypophysis thus affect the further secretion of LH.To climacteric, hypo-ovaria, the secretion of estradiol reduces until finally terminate.Owing to relieving the negative feedback be applied on hypothalamus, the level of LH in circulation is significantly increased.In males, LH promotes the testosterone that interstitial glands generates.And coordinate equally to maintain the Sperm specific enzyme in vas deferens together with LH.Testicosteroid hormone is by regulating the LH level in circulation to the negative feedback of hypothalamus and pituitary in response Gn-RH.The hypopituitarism that dyspituitarism causes can cause LH level decline and cause sterile.To after taking Gn-RH, measure the functional status that serum Lh content effectively can judge hypophysis.Separately having a kind of dynamic function to test is take estradiol to amenorrhoea women, predicts the reactivity of patient to clomiphene by measuring LH content.LH and LH joint-detection, mainly differentiates primary (ovarian) or Secondary cases (nanosom) amenorrhoea in women; The male sex for differentiate primary or Secondary cases testicular function low; The true property of prepubertal children or false precocity can be differentiated simultaneously.Ovulate on the release peak of menstrual cycle LH and ovary close relation, LH peak, once appearance, indicates ovary ovulation in 24-36 hour, therefore can monitor serum Lh peak value in the menstrual cycle, to determine best Time to pregnancy.
Immune analysis method at present for luteinizing principle (LH) mainly contains enzyme-linked immunosorbent assay, chemiluminescence immunoassay etc.It is low to there is sensitivity in enzyme-linked immunosorbent assay, and the range of linearity is narrow, not easily realize the methodology limiting factors such as full-automatic.Chemiluminescence immunoassay is a kind of immunoassay technology grown up on enzyme-linked immunosorbent assay basis, have highly sensitive, detect linear wide ranges, easy and simple to handle, the advantages such as automaticity is high.Current chemiluminescence immunoassay technology has above-mentioned to be manyly widely used a little because of it.
But, in the immune detection of reality, because impurity component contained in testing sample is more, have impact on detection sensitivity and accuracy to a certain extent, so from the sample substrate of complexity quick separating, be purified into object determinand, be one of difficult problem of facing of clinical examination worker.Magnetic particle immunoassay technology utilizes the Magnetic solid phases particulate of synthesis of polymer material certain particle size size to make carrier, with the method such as physisorption, chemical coupling bag, above be there is the various immunologic active materials such as the antibody of specificity affinity or antigen, have that velocity of separation is fast, efficiency is high, favorable repeatability, simple to operate, the feature such as biological character and function that do not affect separated cell or other biological material, orientable motion under additional magnetic fields, makes some special composition be separated, concentrates or purifying.
The magnetic microparticle chemiluminescence immune assay kit that the open CN101614742A of the open Chinese invention patent of Chinese invention patent has disclosed luteinizing principle a kind of; this kit comprises luteinizing principle series of calibration product; the magnetic particle solution of anti-fluorescein isothiocynate monoclonal antibody bag quilt; the luteinizing principle monoclonal antibody of marked by fluorescein isothiocyanate; the luteinizing principle monoclonal antibody of horseradish peroxidase-labeled; Chemoluminescent substrate, concentrated cleaning solution.In this kit, the luteinizing principle monoclonal antibodies of the anti-magnetic particle solution of fluorescein isothiocynate monoclonal antibody bag quilt, the luteinizing principle antibody of marked by fluorescein isothiocyanate and horseradish peroxidase-labeled forms the immune response reagent of immune response step.Although this kit can realize 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 results and particularly analyze a precision and cause cost to increase.
Summary of the invention
Technical matters to be solved by this invention overcomes 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 luteinizing principle accurately and the quantitative measurement of high precision ground.
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 particularly to analyze a precision high.
For solving above technical matters, a kind of technical scheme that the present invention takes is:
A kind of nano magnetic particulate chemistry luminescent assay kit of luteinizing principle (LH), it comprises the immune response reagent for immune response step, this immune response reagent comprises the solution (being called for short the first reagent) of the luteinizing principle antibody marked containing fluorescein (FITC) and is coated with the suspending liquid (be called for short Magneto separate reagent) of magnetic particle of anti-fluorescein antibody, particularly, described immune response reagent also comprises the solution (being called for short the second reagent) of the luteinizing principle antibody marked containing alkaline phosphatase (ALP), the luteinizing principle antibody of this alkali phosphatase enzyme mark is connected and composed by 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester (SMCC) and 2-iminothiolane hydrochloride (2IT) by alkaline phosphatase and luteinizing principle antibody.
Further, the pH of the described solution containing fluorescein-labeled luteinizing principle antibody is 7 ~ 9, and the concentration of fluorescein-labeled luteinizing principle antibody is wherein 0.5 ~ 1 μ g/mL.The pH of the solution of the described luteinizing principle antibody containing 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, described easily can be prepared by ripe and stable technique containing fluorescein-labeled anti-luteinizing principle antibody.
According to the present invention, described in be coated with the magnetic particle of anti-fluorescein antibody preparation be also comparatively be easy to, and prior art have ripe preparation technology can reference.Such as can be prepared by mode by conventional physisorption or chemical coupling bag, 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 magnetic particle.Describedly be coated with in the magnetic particle of anti-fluorescein antibody, phase chemistry coupling between anti-fluorescein antibody and magnetic particle.
Those skilled in the art should know, kit of the present invention can further include other detect needed for reagent, such as substrate solution.But such as other reagent such as substrate solution can be bought separately or prepare, therefore, although can comprise these reagent in kit, they are for not essential 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 luminescent assay kit of above-mentioned luteinizing principle; it comprise the described solution containing fluorescein-labeled luteinizing principle antibody of preparation respectively, the described luteinizing principle antibody containing alkali phosphatase enzyme mark solution and be coated with the step of suspending liquid of magnetic particle of anti-fluorescein antibody, wherein: the preparation process of the solution of the described luteinizing principle antibody containing 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 at 2 ~ 8 DEG C;
2. the phosphate buffer of alkaline phosphatase is got, add 4-(N-maleimidomethyl) 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 at 2 ~ 8 DEG C;
3. the step luteinizing principle antibody that 1. gained activates is mixed according to the ratio that luteinizing principle antibody and the molecular proportion of alkaline phosphatase are 1:1 ~ 2 with the step alkaline phosphatase that 2. gained activates, 12 ~ 24h is left standstill at 2 ~ 8 DEG C, purifying is carried out by Supperdex200 gel-purified post, the solution of purifying selects damping fluid adjustment concentration and the pH value with proper pH value, obtains the solution of the described luteinizing principle antibody containing alkali phosphatase enzyme mark.
Preferably, described luteinizing principle antibody, the purity of described alkaline phosphatase are all more than or equal to 95wt%, and the specific activity of described alkaline phosphatase is more than 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 containing fluorescein-labeled luteinizing principle antibody is as follows: the pH of preparation containing fluorescein is the damping fluid of 9 ~ 10, then be the ratio of 20 ~ 200:1 according to the molecular proportion of fluorescein and anti-luteinizing principle antibody, by the described pH containing fluorescein be 9 ~ 10 damping fluid and the pH of anti-luteinizing principle antibody be 9 ~ 10 damping fluid mixes, after mixing, room temperature leaves standstill reaction, then reactant liquor is separated by G-25 gel column, the fluorescein that removing is free, obtain the solution containing fluorescein-labeled anti-luteinizing principle antibody, then with damping fluid adjustment concentration and the pH with proper pH value, obtain.Wherein the damping fluid of proper pH value can be the TRIS damping fluid such as containing 0.5% bovine serum albumin(BSA), pH8.0.
Further, the described preparation method being coated with the suspending liquid of the magnetic particle of anti-fluorescein antibody is as follows: make magnetic particle containing carboxyl-reactive group and anti-fluorescein antibody under the existence of coupling agent carbodiimide, room temperature reaction 2 ~ 18 hours, reaction terminates, Magneto separate, remove supernatant, by damping fluid adjustment pH and the concentration with proper pH value, obtain, wherein said magnetic particle has superparamagnetism, its diameter is 0.5 ~ 2 μm, on every gram of 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 and is greater than 1:100 ten thousand.Wherein the damping fluid of proper pH value can be the TRIS damping fluid such as containing 0.5% bovine serum albumin(BSA), pH8.0.
Preferably, the above-mentioned damping fluid with proper pH value is the TRIS damping fluid containing 0.5% bovine serum albumin(BSA), pH8.0.
Fluorescein of the present invention can be known various fluoresceins, and conventional has such as fluorescein isothiocynate, RB 200, TRITC etc.
The step that kit of the present invention is applied to when chemiluminescence immunoassay detects is the same with traditional detection method, namely comprises the immune response step of carrying out successively, the step using Magneto separate and washing facility to wash immunoreactive reactant liquor and in the reactant liquor after washing, adds substrate solution and utilize the step of chemiluminescence detector detection luminous intensity.
Preferably, described immune response step is specifically implemented as follows: in test sample tube, add such as 10 ~ 30 μ l sample to be tested stoste or dilutions, then the solution that 30 ~ 100 μ l contain the solution of fluorescein-labeled luteinizing principle antibody, 30 ~ 100 μ l contain the luteinizing principle antibody of alkali phosphatase enzyme mark is added successively, mixing, first time incubation is carried out at 25 ~ 40 DEG C, then the suspending liquid that 30 ~ 100 μ l are coated with the magnetic particle of anti-fluorescein antibody is added, mixing, carries out second time incubation at 25 ~ 40 DEG C; 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 second time incubation can be 3 ~ 15min, is generally 5min.
The step that above-mentioned use Magneto separate and washing facility wash immunoreactive reactant liquor and add substrate solution and the step utilizing chemiluminescence detector to detect luminous intensity all can refer to conventional method implements in the reactant liquor after washing, equipment used and device are also conventional.Wherein, alkaline phosphatase chemical luminous substrate solution is also known for those skilled in the art, commercially available acquisition.
Due to the enforcement of above technical scheme, the present invention compared with prior art tool has the following advantages:
1. applicant finds, when taking SMCC and 2IT to carry out the coupling of alkaline phosphatase and luteinizing principle antibody, has and compares higher coupling efficiency with other method, while reduction preparation cost, is conducive to improving Detection results.Therefore, three kinds of reagent in kit of the present invention all can be prepared by stable preparation technology, and production cost is low, and due to the stability of preparation technology, kit assay difference between batch is little, and between the analysis of detection, precision improves;
2. the preparation method of the solution of the luteinizing principle antibody containing alkali phosphatase enzyme mark in kit of the present invention, can effectively by luteinizing principle antibody and alkaline phosphatase coupling, coupling efficiency is high, reduces the low Detection results with guaranteeing kit of cost of kit further.
3, take kit of the present invention to detect, accuracy is good, and precision is high, highly sensitive, and sensing range is wide, the unordered pre-dilution of sample, simple to operately saves time.Compared with the method adopting import reagent box to carry out detecting, detection method of the present invention has significant advantage on cost.
Accompanying drawing explanation
Fig. 1 is detection calibration product typical curves;
Fig. 2 is sensitivity evaluation matched curve;
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, represent the solution containing fluorescein-labeled luteinizing principle antibody in the present invention, the solution of luteinizing principle antibody marked containing alkaline phosphatase (ALP) with the first reagent, the second reagent and Magneto separate reagent respectively and be coated with the suspending liquid of magnetic particle of anti-fluorescein antibody.
The preparation of embodiment 1 first reagent
(1) material and instrument: the anti-LH monoclonal antibody (more than 95wt%, concentration is 2mg/mL to purity, calls LH antibody in the following text) of preserving with phosphate buffer; 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. the FITC solution of the carbonate buffer solution preparation 0.5mg/mL of 0.1 ~ 0.2mol/L pH9.0 ~ 10.0 is used;
2. the ratio being 1:20 according to LH antibody and FITC molecular proportion adds step and is 1. joined FITC solution in antibody-solutions, and mix, room temperature leaves standstill 12h hour, and reaction generates LH antibody-FITC connector;
3. be separated through step reactant liquor 2. by G-25 gel column, remove unreacted FITC, obtain the solution containing LH antibody-FITC connector (i.e. the LH antibody of FITC mark);
4. the step solution that 3. gained contains LH antibody-FITC connector being diluted to LH antibody-FITC connector concentration with the TRIS damping fluid of the 0.1mol/L containing 0.5% bovine serum albumin(BSA) (BSA) pH8.0 is 0.5 ~ 1 μ g/mL, and pH is 7 ~ 9, is the first reagent.
The preparation of embodiment 2 second reagent
(1) material and instrument: the anti-LH monoclonal antibody (more than 95wt%, concentration is 2mg/mL to purity, calls LH antibody-solutions in the following text) of preserving with phosphate buffer; With the alkaline phosphatase (ALP solution, ALP purity is about 99%, and specific activity is about 1500U/mg, and concentration is 10mg/mL) that phosphate buffer is preserved; 5mg/mL SMCC solution, 10mg/mL2IT solution (SMCC and 2IT pressed powder is made into above-mentioned solution with DMF and uses) (purchased 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 Products.
(2) preparation process:
1. get 1mg LH antibody-solutions, add 2IT solution 2 ~ 4 μ l of 10mg/mL, room temperature leaves standstill 20min, and add the glycine solution 10 μ l of 0.1mol/L, room temperature leaves standstill 5min.With G-25 gel column desalination, collect the antibody of activation, 2-8 DEG C saves backup;
2. get 1.5mg ALP solution, add SMCC solution 10 ~ 20 μ l of 5mg/mL, room temperature leaves standstill 30min, and with G-25 gel column desalination, collect the ALP antibody of activation, 2-8 DEG C saves backup;
3. the LH antibody of above-mentioned activation is mixed with the ALP antibody of activation, 12 ~ 24h is left standstill under 2-8 DEG C of condition, carry out purifying by Supperdex200 gel-purified post, obtain the solution containing antibody LH-ALP connector (the LH antibody of ALP mark), 2-8 DEG C saves backup;
4. the solution of the antibody LH-ALP connector TRIS damping fluid of the 0.1mol/L containing 0.5% bovine serum albumin(BSA) (BSA) pH8.0 is diluted to 0.5 ~ 1 μ g/mL, pH7 ~ 9, is the second reagent.
The preparation of embodiment 3 Magneto separate reagent
(1) material and instrument:
The suspending liquid of magnetic particle: magnetic particle content 5wt%, magnetic particle is containing carboxyl (COOH) active group, 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 μm;
Anti-FITC antibody: can be polyclonal antibody also can be monoclonal antibody, and purity is more than 90wt%, and dilution is tired more than 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, Magneto separate removes supernatant, and with 0.05mol/L, pH4.5 ~ 5MES damping fluid, 10mL is resuspended;
2. the anti-FITC antibody of 2 ~ 4mg is added, room temperature suspendible 30 ~ 60min;
3. the EDC aqueous solution of the freshly prepared 10mg/mL of 0.5 ~ 1mL is added, room temperature suspendible 2 ~ 12h;
4. Magneto separate, removes supernatant, is resuspended to 1mg/mL, pH8.0, is Magneto separate reagent with the TRIS damping fluid of the 0.1mol/L containing 0.5% bovine serum albumin(BSA) (BSA) pH8.0.
The chemical luminescence immune analysis reagent box of embodiment 4 luteinizing principle
This kit comprises:
The first reagent (concentration is 0.75 μ g/mL) prepared according to embodiment 1 method, 50mL;
The second reagent (concentration is 0.75 μ g/mL) prepared according to embodiment 2 method, 50mL;
The magnetic separation reagent 50mL prepared according to embodiment 3 method.
The chemical luminescence immune analysis reagent box of embodiment 5 luteinizing principle
This kit comprises:
The first reagent (concentration is 0.5 μ g/mL) prepared according to embodiment 1 method, 5mL;
The second reagent (concentration is 0.5 μ g/mL) prepared according to embodiment 2 method, 5mL;
The magnetic separation reagent 5mL prepared according to embodiment 3 method.
Embodiment 6 takes the kit of embodiment 4 to carry out the quantitative detection of luteinizing principle
(1) detecting step:
1. immune response: add 20 μ l sample to be tested (serum) stostes in detector tube, then adds 50 μ l first reagent, 50 μ l second reagent, mixing, incubation 15min under 37 ± 1 DEG C of conditions; Add 50 μ l Magneto separate reagent, mixing, incubation 5min under 37 ± 1 DEG C of conditions;
2. wash: 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, then Magneto separate, 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 substrates solution (Beijing Apis Biotechnology Co., Ltd. APCL-I), concussion makes the abundant suspendible of magnetic particle, detected with luminometer (MAGLIA60) in 5 minutes, detect unit interval inner glow intensity.Result calculates the related description of consulting instrument.
(2) calibration object typical curve is drawn
Calibration object typical curve is see 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 calculate M-2SD value, carry out 2 regression fits according to the concentration-RLU between zero-dose calibration object and adjacent calibration object and draw linear function, M+2SD value is brought in above-mentioned equation, obtains 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 see table 1:
Table 1
The luminous average X=1719 of A point
SD=24.3
X+2SD=1767.7
B point luminous value is see table 2.
The luminous average X=56478 of B point
Table 2
| LH-STD-B(RLU) |
| 56470 |
| 56486 |
A, B point connects some matched curve see Fig. 2.Sensitivity=0.002mIU/mL.
(4) precision evaluation
1. precision in analyzing
By a collection of for the kit of embodiment 4, measure the serum of basic, normal, high three kinds of variable concentrations respectively, 10 hole replicate determinations, result, see table 3, show that variation within batch coefficient is 2.95% ~ 6.55%.
The test of interior precision analyzed by table 3
| 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, often criticizes the serum that kit all measures basic, normal, high three kinds of variable concentrations, 10 hole replicate determinations.Every part of serum obtains 30 concentration measured values, and see table 4, the coefficient of variation between statistical study is 4.70% ~ 6.95%.
Precision test between table 4 analysis
| 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
In 2 routine pooled serum samples, add different amount people LH standard items, the serum forming 3 concentration levels adds sample, and additive volume is less than 10% of cumulative volume.Detect concentration of specimens, by the following formulae discovery recovery.This method serum matrix recovery is between 90-110%.Data are see table 5.
R: the recovery;
V: the volume adding standard solution;
V
0: the volume of people source sample;
C: people source sample adds the detectable concentration after 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 specific assay be choose have similar structures with LH follotropin (FSH),
Thyrotropic hormone (TSH), human chorionic gonadtropin (HCG), be mixed with the sample being greater than physiological concentration, measure with this method.The results are shown in Table 6, this law and FSH, TSH, HCG no cross reaction.
Table 6 specificity experiments
(7) relativity 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 see accompanying drawing 3, and the serum Lh concentration of survey is in the process of the present invention horizontal ordinate, and with the result of Beckman company kit measurement for ordinate does regretional analysis, dependent equation is: y=-0.07655+0.9988x, and related coefficient is: 0.9831.Show through statistical procedures result, this method is good with external kit clinical sample measured value correlativity.
(8) Evaluation of Thermal Stability
4 DEG C of 12 months and 37 DEG C of stability experiments of 7 days are carried out respectively to kit, result show kit standard product luminous intensity change, batch in and the index 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 in this embodiment is 0.002mIU/mL, reaches domestic and international similar reagent top standard, meets clinical practice preferably.
(3) precision evaluation: see that precision is all less than 10% with analysis in the kit assay in this embodiment, difference between batch is little.
(4) evaluation of the accuracy: the kit serum TIANZHU XINGNAO Capsul in this embodiment is between 90-110%, and clinical practice measuring value accuracy is reliable.
Above-described embodiment is only for illustrating technical conceive of the present invention and feature; its object is to person skilled in the art can be understood content of the present invention and implement according to this; can not limit the scope of the invention with this; all equivalences done according to Spirit Essence of the present invention change or modify, and all should be encompassed within protection scope of the present invention.
Claims (8)
1. the chemical luminescence immune analysis reagent box of a luteinizing principle, described kit comprises the immune response reagent for immune response step, described immune response reagent comprises solution containing fluorescein-labeled luteinizing principle antibody and is coated with the suspending liquid of magnetic particle of anti-fluorescein antibody, it is characterized in that: described immune response reagent also comprises the solution of the luteinizing principle antibody containing alkali phosphatase enzyme mark, the luteinizing principle antibody of this alkali phosphatase enzyme mark is connected and composed by 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester and 2-iminothiolane hydrochloride by alkaline phosphatase and luteinizing principle antibody, the pH of the solution of the described luteinizing principle antibody containing alkali phosphatase enzyme mark is 7 ~ 9, wherein the concentration of the luteinizing principle antibody of alkali phosphatase enzyme mark is 0.5 ~ 1 μ g/ml,
The described preparation method being coated with the suspending liquid of the magnetic particle of anti-fluorescein antibody is as follows: make magnetic particle containing carboxyl-reactive group and anti-fluorescein antibody under the existence of coupling agent carbodiimide, room temperature reaction 2 ~ 18 hours, reaction terminates, Magneto separate, remove supernatant, by damping fluid adjustment pH and the concentration with proper pH value, obtain, wherein said magnetic particle has superparamagnetism, its diameter is 0.5 ~ 2 μm, on every gram of 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 and is greater than 1:100 ten thousand.
2. the chemical luminescence immune analysis reagent box of luteinizing principle according to claim 1, it is characterized in that: the pH of the described solution containing fluorescein-labeled luteinizing principle antibody is 7 ~ 9, and the concentration of fluorescein-labeled luteinizing principle antibody is wherein 0.5 ~ 1 μ g/ml.
3. the chemical luminescence immune analysis reagent box of luteinizing principle according to claim 1 and 2, is characterized in that: described in be coated with in the magnetic particle of anti-fluorescein antibody, phase chemistry coupling between anti-fluorescein antibody and magnetic particle.
4. the preparation method of the chemical luminescence immune analysis reagent box of the luteinizing principle any one of claims 1 to 3 as described in claim, it comprise the described solution containing fluorescein-labeled luteinizing principle antibody of preparation respectively, the described luteinizing principle antibody containing alkali phosphatase enzyme mark solution and described in be coated with the step of the suspending liquid of the magnetic particle of anti-fluorescein antibody, it is characterized in that: the preparation process of the solution of the described luteinizing principle antibody containing alkali phosphatase enzyme mark is as follows:
1. the phosphate buffer of luteinizing principle antibody is got, add 2-iminothiolane 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 at 2 ~ 8 DEG C;
2. the phosphate buffer of alkaline phosphatase is got, add 4-(N-maleimidomethyl) 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 at 2 ~ 8 DEG C;
3. the step luteinizing principle antibody that 1. gained activates is mixed according to the ratio that luteinizing principle antibody and the molecular proportion of alkaline phosphatase are 1:1 ~ 2 with the step alkaline phosphatase that 2. gained activates, 12 ~ 24h is left standstill at 2 ~ 8 DEG C, purifying is carried out by Supperdex200 gel-purified post, the solution of purifying selects damping fluid adjustment concentration and the pH value with proper pH value, obtains the solution of the described luteinizing principle antibody containing alkali phosphatase enzyme mark;
The described preparation method being coated with the suspending liquid of the magnetic particle of anti-fluorescein antibody is as follows: make magnetic particle containing carboxyl-reactive group and anti-fluorescein antibody under the existence of coupling agent carbodiimide, room temperature reaction 2 ~ 18 hours, reaction terminates, Magneto separate, remove supernatant, by damping fluid adjustment pH and the concentration with proper pH value, obtain, wherein said magnetic particle has superparamagnetism, its diameter is 0.5 ~ 2 μm, on every gram of 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 and is greater than 1:100 ten thousand.
5. preparation method according to claim 4, is characterized in that: described luteinizing principle antibody, the purity of described alkaline phosphatase are all more than or equal to 95wt%, and the specific activity of described alkaline phosphatase is more than 1000u/mg.
6. the preparation method according to claim 4 or 5, is characterized in that: step 2. in, the concentration of the phosphate buffer alkaline phosphatase of alkaline phosphatase is 5 ~ 10mg/ml.
7. preparation method according to claim 4, it is characterized in that: the preparation method of the described solution containing fluorescein-labeled luteinizing principle antibody is as follows: the pH of preparation containing fluorescein is the damping fluid of 9 ~ 10, then be the ratio of 20 ~ 200:1 according to the molecular proportion of fluorescein and anti-luteinizing principle antibody, by the described pH containing fluorescein be 9 ~ 10 damping fluid and the pH of anti-luteinizing principle antibody be 9 ~ 10 damping fluid mixes, after mixing, room temperature leaves standstill reaction, then reactant liquor is separated by G-25 gel column, the fluorescein that removing is free, obtain the solution containing fluorescein-labeled anti-luteinizing principle antibody, then with damping fluid adjustment concentration and the pH with proper pH value, obtain.
8. the preparation method according to claim 4 or 7, is characterized in that: the described damping fluid with proper pH value is the TRIS damping fluid containing 0.5% bovine serum albumin(BSA), pH8.0.
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| 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 |
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| 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 |
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| 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 |
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| 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 |
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