CN107991492A - A kind of detection method of the immue quantitative detection reagent box of insulin-like growth factor binding protein-3 and its non-diagnostic purpose - Google Patents
A kind of detection method of the immue quantitative detection reagent box of insulin-like growth factor binding protein-3 and its non-diagnostic purpose Download PDFInfo
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Abstract
The present invention relates to detection technique field, the immue quantitative detection reagent box of more particularly to a kind of insulin-like growth factor binding protein 3 and its detection method of non-diagnostic purpose.The immue quantitative detection reagent box includes：3 monoclonal antibodies of IGFBP, 3 recombinant antigen standard items of IGFBP, biotinylated 3 polyclonal antibodies of IGFBP, the Streptavidin of enzyme mark, chromogenic substrate, terminate liquid.3 immue quantitative detection reagent box high sensitivities of IGFBP of the present invention, high specificity, reproducible, stability is good.3 immue quantitative detection reagent boxes of IGFBP of the present invention are using IGFBP 3 in serum in double site enzyme-linked immunosorbent assay analytic approach detection human body, can be used as the detection kit for being used for clinical diagnosis dwarfism and GHD.
Description
Technical field
The present invention relates to detection technique field, more particularly to a kind of insulin-like growth factor binding protein-3 quantifies
The detection method of detection kit and its non-diagnostic purpose.
Background technology
Dwarfism refers to that the height of children is less than same gender, same to age, 2 standard deviations of agnate children's average height,
The annual speed of growth is less than 5 centimetres of persons.Dwarfism is a kind of very serious disease of puberty children, with heredity, environment, interior point
It is related many factors such as to secrete, wherein internal system is an important factor for causing dwarfism.Growth hormone (Growth
Hormone, GH), insulin-like growth factor-i (insulin-like growth factors-1, IGF-1) and insulin
Like growth factor associated proteins -3 (IGF-binding proteins-3, IGFBP-3) have collectively constituted GH-IGF-1 axis, its
Important regulating and controlling effect is played in the growth and development process of children.IGF-1 secretes after being stimulated by GH, and GH is sent out by IGF-1
Wave the growth function of promoting human body respectively to organize.And IGFBP-3 is main carriers of the IGF-1 in blood circulation, IGF-1 can be extended and existed
It is half-life period in blood circulation, also the closest with the relation of GH.Since the secretion of IGF-1 and IGFBP-3 can direct reactant
The level of interior GH, and the change without circadian rhythm, therefore study report and think that IGFBP-3 and IGF-1 can be used as dwarfism
With the diagnosis index of growth hormone deficiency (growth hormone deficienty, GHD).
The detection kit listing of existing relevant IGFBP-3, the kit include 96 orifice plates, standard items, detection at present
Solution A, detection solution B, tmb substrate and dense cleaning solution, experiment flow include：
1. the preparation of standard items, reagent and sample before experiment；
2. (standard items and sample) 100 μ L are loaded, when 37 DEG C of incubations 1 are small；
3. suction is abandoned, add 100 μ L of detection solution A, when 37 DEG C of incubations 1 are small；
4. board-washing 3 times；
5. adding 100 μ L of detection solution B, 37 DEG C are incubated 30 minutes；
6. board-washing 5 times；
7. adding 90 μ L of tmb substrate, 37 DEG C are incubated 10-20 minutes；
8. add 50 μ L of terminate liquid, immediately 450nm readings.
But using the kit detection IGFBP-3 concentration when there are sensitivity it is poor the problem of.Therefore how to divide completely
From with purifying IGFBP-3, be current urgent problem so as to improve the sensitivity of IGFBP-3 immue quantitative detection reagent boxes.
The content of the invention
In view of this, the present invention provides a kind of insulin-like growth factor binding protein-3 immue quantitative detection reagent box and
The detection method of its non-diagnostic purpose.The IGFBP-3 immue quantitative detection reagent boxes high sensitivity, high specificity, stability are good.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
The present invention provides a kind of immue quantitative detection reagent box of insulin-like growth factor binding protein-3, the quantitative detection
Kit includes：IGFBP-3 monoclonal antibodies, IGFBP-3 recombinant antigen standard items, biotinylated IGFBP-3 Anti-TNF-αs
Body, the Streptavidin of enzyme mark, chromogenic substrate, terminate liquid.
Preferably, the concentration of IGFBP-3 monoclonal antibodies is 0.1~10 μ g/mL.
Preferably, the concentration of IGFBP-3 monoclonal antibodies is 0.5~4.0 μ g/mL.
It is highly preferred that the concentration of IGFBP-3 monoclonal antibodies is 1.0 μ g/mL.
Preferably, the concentration of IGFBP-3 recombinant antigen standard items is 50~100ng/mL.
Preferably, the concentration of IGFBP-3 recombinant antigens standard items is 50~60ng/mL.
It is highly preferred that the concentration of IGFBP-3 recombinant antigen standard items is 50ng/mL.
Preferably, the concentration of biotinylated IGFBP-3 polyclonal antibodies is 0.1~1.0 μ g/mL.
Preferably, the concentration of biotinylated IGFBP-3 polyclonal antibodies is 0.25~1.0 μ g/mL.
It is highly preferred that the concentration of biotinylated IGFBP-3 polyclonal antibodies is 0.25 μ g/mL.
Preferably, biotinylated IGFBP-3 polyclonal antibodies are the biotinylated IGFBP-3 Anti-TNF-αs in rabbit source
Body.
Preferably, the Streptavidin of enzyme mark is the Streptavidin of HRP (horseradish peroxidase) marks.
Preferably, chromogenic substrate is tetramethyl benzidine.
Present invention also offers a kind of side of the detection insulin-like growth factor binding protein-3 concentration of non-diagnostic purpose
Method, includes the following steps：
Step 1：IGFBP-3 monoclonal antibodies are coated in surface of solid phase carriers, are closed；
Step 2：A kind of, biotinylated IGFBP-3 in IGFBP-3 recombinant antigens standard items or sample is polyclonal
Antibody is incubated jointly with IGFBP-3 monoclonal antibodies；
Step 3：The Streptavidin for adding enzyme mark is incubated；
Step 4：Add chromogenic substrate to develop the color, detect OD values after color development stopping under the wavelength of 450nm；
Step 5：Standard curve is drawn according to the OD values of the IGFBP-3 recombinant antigen standard items of gradient concentration, then basis
The OD values and standard curve of sample, obtain the concentration of IGFBP-3 in sample.
Preferably, coated temperature is 4 DEG C in step 1, when the time is 8~12 small.
Preferably, the temperature being incubated in step 2 is 37 DEG C, when the time is 1~2 small.
Preferably, the temperature being incubated in step 2 is 37 DEG C, when the time is 1 small.
Preferably, the temperature being incubated in step 3 is 37 DEG C, when the time is 1~2 small.
Preferably, the temperature being incubated in step 3 is 37 DEG C, when the time is 1 small.
Preferably, the extension rate of the Streptavidin of enzyme mark is 500~1500.
Preferably, the extension rate of the Streptavidin of enzyme mark is 500.
In embodiment provided by the invention, the gradient concentration of IGFBP-3 recombinant antigen standard items is：Initial concentration is
50ng/mL, by 2 times of gradient dilutions, 8 gradients.
The present invention provides the immue quantitative detection reagent box of a kind of insulin-like growth factor binding protein-3 and its non-diagnostic
The detection method of purpose.The immue quantitative detection reagent box includes：IGFBP-3 monoclonal antibodies, IGFBP-3 recombinant antigen standard items,
Biotinylated IGFBP-3 polyclonal antibodies, the Streptavidin of enzyme mark, chromogenic substrate, terminate liquid.What the present invention had has
Beneficial effect is：
1st, IGFBP-3 immue quantitative detection reagent boxes high sensitivity of the present invention, high specificity, reproducible, stability is good, detection
Limit low.
2nd, IGFBP-3 immue quantitative detection reagent boxes of the present invention are to utilize double site enzyme-linked immunosorbent assay analytic approach detection people
IGFBP-3 in internal serum, can be as the detection kit for clinical diagnosis dwarfism and GHD.
Brief description of the drawings
Fig. 1 shows bacterium colony PCR identification pMC1-IGFBP3-mFc recon clones, 1-6：PMC1-IGFBP3-mFc recons
Clone；M1：5Kb DNA marker (5kb, 3kb, 2kb, 1kb, 750bp, 500bp, 250bp, 100bp)；M2：2Kb DNA
Marker (2kb, 1kb, 750bp, 500bp, 250bp, 100bp)；
Fig. 2 shows that IGFBP3-mFc recombinant antigens just purify SDS-PAGE electrophoresis, M：Marker；1：Cell expresses supernatant；
2：Cell expression supernatant after concentration；3：Penetrate liquid；4：Destination protein eluent；
Fig. 3 shows IGFBP3-mFc recombinant antigen reduced form SDS-PAGE electrophoresis, M：Marker；BSA：Various concentrations
BSA；IGFBP3-mFc：Various concentrations；IGFBP3-mFc；
Fig. 4 shows that antigen-antibody ELISA is combined；
Fig. 5 shows that IGFBP-3 monoclonal antibodies purify collection of illustrative plates；Peak 1：Foreign protein peak；Peak 2：The 0.1M glycine solutions elution egg of pH3.3
White peak；Peak 3：Foreign protein peak；
Fig. 6 shows the non-reduced SDS-PAGE electrophoresis of IGFBP-3 monoclonal antibodies after purification, M：Marker 170kD；1-3 is three batches
IGFBP3 monoclonal antibodies after purification；
Fig. 7 shows IGFBP-3 monoclonal antibody purity detecting results；
Fig. 8 shows IGFBP-3PCR product electrophoretograms, M：Marker；1：PCR product；
Fig. 9 shows that bacterium solution PCR identifies electrophoretogram, M Maker；1st, 2,3 be bacterium colony PCR product；
Figure 10 shows that plasmid double digestion identifies electrophoretogram, M Maker；1st, 2,3 be small pumping plasmid double digestion product；
Figure 11 shows pET-22b-IGFBP3 induced expression electrophoretograms, M：Maker；1：The pET-22b-IGFBP3 not induced；
2、3、4、5：PET-22b-IGFBP3 after induction；
Figure 12 shows IGFBP3-ELISA quantitative measurement standard curves；
Figure 13 shows that the method for the present invention detects children serum IGFBP-3 content distribution figures.
Embodiment
The invention discloses the immue quantitative detection reagent box of a kind of insulin-like growth factor binding protein-3 and its non-diagnostic
The detection method of purpose, those skilled in the art can use for reference present disclosure, be suitably modified technological parameter realization.Especially need to refer to
Go out, all similar substitutions and modifications are apparent to those skilled in the art, they are considered as including
In the present invention.The method of the present invention and application are described by preferred embodiment, and related personnel can substantially not take off
Method described herein and application are modified or suitably change and combine from present invention, spirit and scope, is come real
Now and using the technology of the present invention.
Term is explained：
Insulin-like growth factor-i (insulin-like growth factors-1, IGF-1) is a kind of promotion cell
Growth, the factor with insulin-like metabolic effect.IGF-1 is by the multifactor adjusting such as nutritional status, hormone, heredity, in baby
The growth of youngster and persistently carry out being of great significance on anabolic action in adult body.
Insulin-like growth factor binding protein-3 (IGF-binding proteins-3, IGFBP-3) is energy and pancreas islet
The regulatory protein that plain like growth factor (IGFs) combines, adjusts the binding ability of IGFs and its acceptor (IGFR), influences under IGFR
Signal strength in signal transduction pathway is swum, regulates and controls growth and the propagation of target cell.
The immue quantitative detection reagent box of insulin-like growth factor binding protein-3 provided by the invention and its non-diagnostic purpose
Detection method in agents useful for same, instrument can be bought by market.
With reference to embodiment, the present invention is further explained：
The preparation of 1 IGFBP-3 monoclonal antibodies of embodiment
(1) structure of IGFBP-3 immunizing antigens, expression and purifying
A) IGFBP-3 immunizing antigens expression vector establishment
Synthesis IGFBP3 genes simultaneously be inserted into cloning vector pUC57-simple, synthetic primer, using following primer from
IGFBP3 genes are expanded in pUC57simple-IGFBP3.
KF08YF1F1:TTGTAAAACGACGGCCAGTGAATTGGAG
KF08YF1R1:GTGGGGCCTCGTGGTCCCTGGAAATAC
Labelled antigen, TEV- mIgG2aFc bases are expanded using following primer from pUC57simple-KF001AG2aFc
Cause.
KF08YF1F2:GGACCACGAGGCCCCACAAT
KF08YF1R2:ATGCTTCCGGCTCGTATGTTGTGT
Two above fragment is connected and composed SP-IGFBP3-TEV-mIgG2aFc genetic fragments by over-lap PCR.Utilize Xho
I/BamH I enzymes digestion fragments are simultaneously inserted into pMC1 carriers.
B) identification of pMC1-IGFBP3-mFc recons
Qualification result is shown in Fig. 1.
C) purifying of IGFBP-3 recombinant antigens
The pMC1-IGFBP3-mFc recombinant plasmids of structure are transiently transfected into HEK293F suspension cells, collect cell conditioned medium,
Purified with GE companies protein A prepacked columns (HiTrap protein AHP), obtain purity>90% destination protein
IGFBP3-mFc, the results are shown in Figure 2：
By purification process, final sample IGFBP3-mFc recombinant antigens are obtained, 12%SDS-PAGE testing results, such as scheme
Shown in 3.
(2) structure stablizes the hybridoma of expression IGFBP-3 monoclonal antibodies
A) animal immune
The female Balb/c mouse of 56 week old are taken, are immunized with IGFBP3-mFc recombinant antigens, subcutaneous multiple spot is immunized,
Immunizing dose is 50ug/；Mouse rat-tail takes blood to carry out serum titer detection after 10 days immune for the second time, 3 before cell fusion
My god, direct intrasplenic injection booster immunization is carried out, dosage is 50ug/.After 4 fundamental immunities, rat-tail takes Balb/c mouse
Its antibody titer is surveyed in blood examination, the results are shown in Table 1：
1 rat-tail of table takes blood examination to survey antibody titer result
As can be known from the results of Table 1, the serum antibody titer of 5 immunized mices has all reached 10-4, illustrate that immune effect is preferable.
B) cell fusion
According to table 1 as a result, take immune mouse spleen cell and Syngenic mice myeloma cell SP2/0, PEG is used as fusion
Agent, is merged in 5: 1 ratio, fusion rate 100%.The culture medium containing 1% HAT and HT is respectively adopted afterwards to carry out
Selectivity culture.
As a result it is 17.5% with indirect ELISA primary dcreening operation positive rate；Take the higher positive hole cell of indirect ELISA screening OD values
2 limiting dilutions are carried out, establish the hybridoma cell strain of the anti-IGFBP-3 monoclonal antibodies of stably excreting, the results are shown in Table 2 and figure
4。
2 antigen-antibody ELISA of table is combined
Antibody (the H008- of the 7 strain of hybridoma strains secretion obtained it can be seen from the result of table 2 and Fig. 5
11E7.8.5、H008-17H6.8.7、H009-11B12.3.6、H009-13E2.9.5、 H009-3G6.8.7、H008-
11E11.3.9.13, H009-8D7.5.3) with the EC50 numerical value of antigen binding all than the positive control antibodies (PCAb) of commercialization
It is small, illustrate that the affinity of the antibody of these hybridoma cell strains secretion is all very strong or even better than the positive control antibodies of commercialization
Very much, the preparation for illustrating anti-IGFBP-3 monoclonal antibodies is successful.
C) purifying of IGFBP3 monoclonal antibodies
Large-scale culture IGFBP3 hybridomas, collect cell conditioned medium, centrifugal filtration processing.It is pure using protein A
Change column to be purified.0.02M phosphate buffers are equilibrium liquid, and the 0.1M glycine solutions that PH is 3.3 are eluent, 20% ethanol
To preserve liquid.As a result as illustrated in Figures 5 and 6.
D) IGFBP3 monoclonal antibodies purity detecting
Purity detecting the results are shown in Table 3, Fig. 7.
3 IGFBP-3 monoclonal antibody purity detecting results of table
By protein A column purifications, the IGFBP-3 monoclonal antibody purity of acquisition reaches more than 98%.
The acquisition of 2 IGFBP-3 recombinant antigens of embodiment
Using modern genetic engineering technique construction IGFBP-3 expression vectors, recombination expression, protein of interest, obtain high
Purity recombinant antigen.
(1) structure of IGFBP-3 recombinant antigens expression vector
A) IGFBP-3 recombinant antigens complete sequence
TATACCATGGGCGCGAGCTCGGCGGGCTTGGGTCCCGTGGTGCGCTGCGAGCCGTG
CGACGCGCGTGCACTGGCCCAGTGCGCGCCTCCGCCCGCCGTGTGCGCGGAGCTGG
TGCGCGAGCCGGGCTGCGGCTGCTGCCTGACGTGCGCACTGAGCGAGGGCCAGCCG
TGCGGCATCTACACCGAGCGCTGTGGCTCCGGCCTTCGCTGCCAGCCGTCGCCCGAC
GAGGCGCGACCGCTGCAGGCGCTGCTGGACGGCCGCGGGCTCTGCGTCAACGCTAG
TGCCGTCAGCCGCCTGCGCGCCTACCTGCTGCCAGCGCCGCCAGCTCCAGGAAATGC
TAGTGAGTCGGAGGAAGACCGCAGCGCCGGCAGTGTGGAGAGCCCGTCCGTCTCCA
GCACGCACCGGGTGTCTGATCCCAAGTTCCACCCCCTCCATTCAAAGATAATCATCA
TCAAGAAAGGGCATGCTAAAGACAGCCAGCGCTACAAAGTTGACTACGAGTCTCAG
AGCACAGATACCCAGAACTTCTCCTCCGAGTCCAAGCGGGAGACAGAATATGGTCC
CTGCCGTAGAGAAATGGAAGACACACTGAATCACCTGAAGTTCCTCAATGTGCTGA
GTCCCAGGGGTGTACACATTCCCAACTGTGACAAGAAGGGATTTTATAAGAAAAAG
CAGTGTCGCCCTTCCAAAGGCAGGAAGCGGGGCTTCTGCTGGTGTGTGGATAAGTAT
GGGCAGCCTCTCCCAGGCTACACCACCAAGGGGAAGGAGGACGTGCACTGCTACAG
CATGCAGAGCAAGCTCGAGCGC
B) IGFBP-3 recombinant antigens primer synthesizes
IGFBP3 sense primers：5'TATACCATGGGCGCGAGCTCGGCGGGCTTG 3' NcoI
IGFBP3 anti-sense primers：5'GCGCTCGAGCTTGCTCTGCATGCTGTAGCAGTGC 3' XhoI
C) IGFBP-3 recombinant antigens PCR amplification
Amplified band is shown in Fig. 8.
D) structure of IGFBP-3 recons pET-22b-IGFBP3
With NcoI and XhoI double digestions PCR product and expression vector pET-22b, connected using DNA ligase, build pET-
22b-IGFBP3 expression vectors.Recon transformed competence colibacillus Escherichia coli, bacterium solution PCR identifications are identified with digestion.As a result such as Fig. 9
Shown in 10.
Bacterium solution PCR and the successful bacterial strain of double digestion, after conservation, take 1ml bacterium solutions to be sequenced.Sequencing result and IGFBP-
Identification is compared in 3 complete sequences.All IGFBP-3 recons are shown by above method bacterium solution PCR, double digestion and sequencing result
PET-22b-IGFBP3 is built successfully.
(2) expression of IGFBP-3 recombinant antigens
Analyzed after recon pET-22b-IGFBP3 induced expressions through 12%SDS-PAGE, as a result such as Figure 11.Due to
IGFBP-3 recombinant antigens carry His labels, therefore nickel ion column can be used to be purified.
3 IGFBP-3 kits of embodiment and detection method
Kit forms：IGFBP-3 monoclonal antibodies (self-control, its Homemade method are shown in embodiment 1), IGFBP-3 restructuring are anti-
Former (self-control, its Homemade method are shown in embodiment 2), biotinylated polyclonal rabbit-anti IGFBP-3, Streptavidin HRP, tetramethyl
Base benzidine (TMB), terminate liquid.
Detection method：Using Elisa double-antibody methods, IGFBP-3 monoclonal antibodies are primary antibody, and IGFBP-3 recombinant antigens are detection
Sample, the polyclonal rabbit-anti IGFBP-3 combinations IGFBP-3 recombinant antigens of biotin, shake in 96 hole elisa Plates and mix, 37 DEG C
It is incubated.Streptavidin HRP (horseradish peroxidase) is secondary antibody, and 37 DEG C are incubated.The chromogenic substrate of single component is added after board-washing
Tetramethyl benzidine (TMB), 37 DEG C of incubations, forms color.It is eventually adding terminate liquid, color development stopping reaction.TMB is in HRP enzymes
The lower conversion au bleu of catalysis, and final yellow is changed under the action of an acid.IGFBP-3 in the depth and sample of color
It is proportionate.Absorbance (OD values) is measured under 450nm wavelength with microplate reader, IGFBP-3 in sample is calculated by standard curve
Concentration.
The optium concentration of antibody and enzyme marking reagent is determined using Checkerboard titration method：1. IGFBP-3 antibody is coated with concentration：0.5、
1.0、2.0、4.0μg/mL；2. the polyclonal rabbit-anti IGFBP-3 concentration of biotin：0.25、 0.5、1.0μg/mL；3. strepto- is affine
Plain HRP：1:500、1:1000、1:1500.
By the optimization of above parameter, when the concentration of IGFBP-3 monoclonal antibodies is 1.0 μ g/mL, biotin multi-clone rabbit is anti-
IGFBP3 concentration is 0.25 μ g/mL, when Streptavidin HRP extension rates are 500, the detection starting of IGFBP-3 recombinant antigens
Concentration is 50ng/mL, and by 2 times of gradient dilutions, 8 gradients, the related coefficient of obtained Elisa mark songs (Figure 12) is 95.78%.
After testing, the detection range of IGFBP-3 samples is 0.39ng/mL-50ng/mL, and lowest detection is limited to 0.39ng/
mL。
The batch detection verification of 4 clinical samples of embodiment
1st, serum is handled：
Extract children blood 2mL using heparin tube, blood be placed at room temperature 1 it is small when or so, after solidification, put at 4 DEG C overnight,
Serum is separated out, 4000rpm is centrifuged, 10 minutes, collects serum, be stored in -20 DEG C, it is to be detected.
2nd, Enzyme-linked Immunosorbent Assay (enzyme-linked immunosorbent assay, ELISA) detects：
(1) wrapper sheet：IGFBP-3 antibody is diluted with coating buffer, is coated with 96 hole elisa Plates, 100 μ L/ holes, 4 DEG C of refrigerator overnights；
(2) detain dry coating buffer, 1 × PBST board-washings 1 time, per hole 300 μ L, 3min after detain it is dry；
(3) 1%BSA confining liquids are added, per hole 300mL, are incubated 1h, 37 DEG C；
(4) dry confining liquid is detained, 1 × PBST board-washings 1 time, per 300 μ L of hole, 3min/ times.
(5) by certain gradient dilution IGFBP-3 standard items, detection sample, per 100 μ L of hole, 3 multiple holes；
(6) Biotin rabbit-anti IGFBP-3 polyclonal antibodies are diluted by a certain percentage, are separately added into ELISA Plate, per 100 μ of hole
L, 3 multiple holes, mix with standard items/sample liquid, 37 DEG C of incubation 1h；
(7) detain and do how anti-sample liquid, 1 × PBST board-washings 3 times, per 300 μ L of hole, 3min/ times.
(8) add and dilute (500-1000 times) Streptavidin HRP enzyme marks with 0.1%BSA, per 100 μ L of hole, be incubated 1h,
37℃；
(9) dry enzyme standard liquid is detained, 1 × PBST board-washings 5 times, per 300 μ L of hole, 3min/ times.
(10) TMB nitrite ions are added, per 100 μ L of hole, 37 DEG C are incubated 15-30min, lucifuge.
(11) it is not required to detain dry nitrite ion, is directly added into terminate liquid, every 100 μ L, OD is detected under the wavelength of microplate reader 450nm
Value.
(12) 3 multiple holes/sample, are averaged.IGFBP-3 standard items by setting gradient concentration make standard curve,
Calculate the concentration of IGFBP-3 albumen in sample.
3rd, result of study and analysis：
The present embodiment acquires the serum sample of 200 less than 12 years old healthy childrens altogether, and sex ratio and ratio of age are not
Require.Document (《[Xu Shanshan, Gu Xuefan, Pan Hui, Zhu Huijuan, Gong Fengying, Li Yufeng, Xing Yan plum children and youth serum pancreases
The normal reference value of island element growth factor-1 and insulin gene associated proteins -3 research [J] clinical pediatric magazines, 2009,27
(12):1105-1110.》) report 6-18 Sui serum I GFBP-3 content of children, the IGFBP-3 of less than 12 years old healthy children contains
It is the testing result of the IGFBP-3 immue quantitative detection reagent boxes of the present invention, two groups of contrasts that scope, which is measured, in 2 μ g/mL-12 μ g/mL, Figure 13
There was no significant difference (P>0.05).Therefore, IGFBP-3 detection kits testing result of the invention and document report reference value phase
Symbol, can obtain basically identical result when detecting.
5 precision contrast test of embodiment
The present embodiment is detected the IGFBP-3 of the present invention by being detected to 200 children serum sample IGFBP-3
Kit and the IGFBP-3 detection kits of DSL companies of U.S. production carry out precision contrast.The IGFBP-3 detections of control
Kit and the IGFBP-3 detection kits of the present invention are same class products, its listing and extensive use at home, performance
Stablize, obtain the accreditation of most of hospital doctors, be suitable as the contrast agents of IGFBP-3 detection kits of the present invention.
Collection, processing and the detection method of clinical samples are same as above, and the clinical test results of gained are as shown in table 4 below：
4 200 pattern detection results of table
| Group | Sample number | Sensitivity | Specificity | Positive coincidence rate | Negative match-rate | P |
| Test group | 200 | 128/128=100% | 50/72=69.4% | 128/150=85.3% | 50/50=100% | >0.05 |
| Control group | 200 | 131/131=100% | 48/69=69.6% | 131/150=87.3% | 48/50=96% | >0.05 |
Statistical result prompting in table 4, IGFBP-3 detections and both control group results no significant difference (P>
0.05)。
Therefore the present embodiment is by being detected 200 children serum sample IGFBP-3, by the IGFBP-3 of the present invention
Detection kit is compared with the IGFBP-3 detection kit contrast agents that DSL companies of the U.S. produce, the results show：This hair
Bright good relationship, can obtain basically identical as a result, being fully able to meet that clinical analysis is used in clinical analysis.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
- A kind of 1. immue quantitative detection reagent box of insulin-like growth factor binding protein-3, it is characterised in that the quantitative detection Kit includes：IGFBP-3 monoclonal antibodies, IGFBP-3 recombinant antigen standard items, biotinylated IGFBP-3 Anti-TNF-αs Body, the Streptavidin of enzyme mark, chromogenic substrate, terminate liquid.
- 2. immue quantitative detection reagent box according to claim 1, it is characterised in that the IGFBP-3 monoclonal antibodies it is dense Spend for 0.1~10 μ g/mL.
- 3. immue quantitative detection reagent box according to claim 1, it is characterised in that the IGFBP-3 recombinant antigens standard items Concentration be 50~100ng/mL.
- 4. immue quantitative detection reagent box according to claim 1, it is characterised in that biotinylated more grams of the IGFBP-3 The concentration of grand antibody is 0.1~1.0 μ g/mL.
- 5. immue quantitative detection reagent box according to claim 1, it is characterised in that the Streptavidin of enzyme mark is The Streptavidin of HRP marks.
- 6. immue quantitative detection reagent box according to any one of claim 1 to 5, it is characterised in that the chromogenic substrate is Tetramethyl benzidine.
- A kind of 7. method of the detection insulin-like growth factor binding protein-3 concentration of non-diagnostic purpose, it is characterised in that bag Include following steps：Step 1：IGFBP-3 monoclonal antibodies are coated in surface of solid phase carriers, are closed；Step 2：By a kind of, biotinylated IGFBP-3 polyclonal antibodies in IGFBP-3 recombinant antigens standard items or sample with The IGFBP-3 monoclonal antibodies are incubated jointly；Step 3：The Streptavidin for adding enzyme mark is incubated；Step 4：Add chromogenic substrate to develop the color, detect OD values after color development stopping under the wavelength of 450nm；Step 5：Standard curve is drawn according to the OD values of the IGFBP-3 recombinant antigen standard items of gradient concentration, then according to sample OD values and standard curve, obtain the concentration of IGFBP-3 in sample.
- 8. the method according to the description of claim 7 is characterized in that coated temperature described in step 1 is 4 DEG C, the time for 8~ 12 it is small when.
- 9. the method according to the description of claim 7 is characterized in that the temperature being incubated described in step 2 be 37 DEG C, the time 1 ~2 it is small when；The temperature being incubated described in step 3 is 37 DEG C, when the time is 1~2 small.
- 10. the method according to any one of claim 7 to 9, it is characterised in that the Streptavidin of the enzyme mark Extension rate is 500~1500.
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| CN111398598A (en) * | 2020-03-16 | 2020-07-10 | 迪瑞医疗科技股份有限公司 | Chemiluminescence detection kit for insulin-like growth factor binding protein-3 and preparation method thereof |
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