CN101672851B - Preparation of Human Tissue Kallikrein ELISA Kit - Google Patents
Preparation of Human Tissue Kallikrein ELISA Kit Download PDFInfo
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- CN101672851B CN101672851B CN 200910063379 CN200910063379A CN101672851B CN 101672851 B CN101672851 B CN 101672851B CN 200910063379 CN200910063379 CN 200910063379 CN 200910063379 A CN200910063379 A CN 200910063379A CN 101672851 B CN101672851 B CN 101672851B
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- 101000605522 Homo sapiens Kallikrein-1 Proteins 0.000 title claims abstract description 41
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- 238000000034 method Methods 0.000 claims abstract description 17
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- 101710176219 Kallikrein-1 Proteins 0.000 claims abstract description 4
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Abstract
The preparation of the ELISA kit for human tissue kallikrein realizes the technical scheme as follows: an ELISA kit for measuring the content of human tissue kallikrein, which is characterized in that: a) the method adopted by the kit is a double-antibody sandwich method; b) a biotin-avidin amplification system is added; c) the standard substance is human tissue kallikrein which is expressed and purified by pronucleus; d) the coating antibody is a polyclonal antibody of anti-human tissue kallikrein, and the detection antibody is a biotinylated monoclonal antibody thereof; e) the sensitivity is 0.3ng/ml-500 ng/ml. The kit has the characteristics of high sensitivity, strong specificity and good repeatability. The fusion protein expressed by the expression is used as a standard product, and the correlation coefficient of a standard curve is as high as 0.99. A large batch of human plasma is used as a sample to be detected, wherein the difference between groups is less than 10 percent.
Description
Technical field
The present invention relates to a kind of human tissue kallikrein fusion, is a kind of ELISA kit of human tissue kallikrein content detection.
Background technology
Human tissue kallikrein (HK1) is a class serine protease, brings into play a series of biological actions, called after KLK1 thereby can promote low molecule kininogen to discharge vasoactive peptide.In the model of hypertension, angiocardiopathy and the kidney trouble of humans and animals, all can see the reduction of HK content.We find in genetically modified animal model that HK1 has and reduce blood pressure, and improve insulin resistance, improve the effect of renal function.The level of HK1 may have important predictive value in the process of the genesis of people's angiocardiopathy and kidney trouble.Therefore, the efficient detection kit of setting up the HK1 assay all has important meaning in clinical and scientific research.
In view of prediction and the therapeutic value of HK1 in angiocardiopathy and kidney trouble, have the assay ELISA kit of KLK1 abroad.Its method that adopts is Inhibition ELISA, and used antibody is a kind of polyclonal antibody.Therefore, its method is complicated, calculates loaded down with trivial detailsly, and be easy to produce non-specific colour developing and cause the larger and expensive China's actual conditions that do not meet of resultant error, and domestic kit without this type of.These problems inventor for above-mentioned existence will adopt up-to-date double-antibody sandwich elisa technology, thereby and added biotin-avidin system and simplified operating process, improved specificity and sensitivity.We utilize the coated elisa plate of the polyclonal antibody of HK1, can fusion and the HK1 that capture HK1 as much as possible, and (biotin-HTK) can be identified the one section specific epi-position of the HK1 on the elisa plate to biotinylated monoclonal antibody specifically.Therefore, the ELISA kit of human tissue kallikrein that we produce has highly sensitive, high specificity, and characteristics easy and simple to handle be the more important thing is more economic and practical.
Summary of the invention
The ELISA kit that the purpose of this invention is to provide a kind of human tissue kallikrein assay.
The realization technical solution of the present invention is as follows:
A kind of ELISA kit of human tissue kallikrein assay is characterized in that:
A) method of this kit employing is double antibody sandwich method;
B) added the biotin-avidin amplification system;
C) standard items are the human tissue kallikrein of prokaryotic expression and purifying;
D) its coated antibody is the polyclonal antibody of anti-human tissue kallikrein, and detecting antibody is its biotinylated monoclonal antibody;
E) sensitivity is 0.3ng/ml-500ng/ml.
The method of the ELISA kit of human tissue kallikrein assay is characterized in that comprising the steps:
A) the coated elisa plate of the coated antibody of 1: 6400 times of dilution, and place 4 ℃ to hatch 24 hours;
B) the 1%BSA sealing is 1 hour;
C) doubling dilution standard items, and add respectively the sample of standard items and 1: 10 times of dilution, in 37 ℃ of reactions 60 minutes;
D) add the detection antibody of 1: 800 times of dilution and hatched 40 minutes in 37 ℃;
E) add the Avidin 25 minutes of the horseradish peroxidase-labeled of 1: 2000 times of dilution.
F) TMB colour developing and reading.
The preparation method of the coated reaction plate of specific human tissue kallikrein fusion polyclonal antibody is prepared from through the following steps:
1) acquisition of human tissue kallikrein fusion:
A. people's tissue and peptide discharge the prokaryotic expression of enzyme fusion proteins (HK-4T1): take pcDNA-HK as template, with Auele Specific Primer pcr amplification KLK1 genes of interest, genes of interest is cloned in the PEGX-4T1 prokaryotic expression carrier, transforms DH5 α competence bacterium.Extract the clone according to amicillin resistance, extract the plasmid DNA of transformant, identify, obtain the fragment consistent with the purpose fragment, be judged to positive findings.The recombinant plasmid HK/4T1 of genes of interest is transformed bacillus coli DH 5 alpha, and after IPTG induced, mycoprotein showed that through SDS-PAGE protein electrophoresis and coomassie brilliant blue staining a dense newly-increased protein band that dyes is arranged about the 55KD place greatly.Identify through Westernbloting, can with human tissue kallikrein monoclonal antibody generation strong positive reaction, the HK-4T1 fusion has stronger immunogenicity.
The great expression of b.HK-4T1 fusion, purifying: through inducing in a large number, express, inclusion body purification obtains a large amount of HK-4T1 fusions.
2) preparation of anti-HK-4T1 fusion polyclonal antibody: the fusion of purifying and the fully emulsified rear immunizing rabbit of Freund's complete adjuvant are obtained polyclonal antibody, through affinitive layer purification as coated antibody.
3) with the coated damping fluid of 0.05M PH9.6 carbonate coated antibody is diluted to suitable concentration after, in the reacting hole of each ELISA Plate, add 0.1ml, 4 ℃ are spent the night.Discard solution in the hole next day, washes 3 times with PBST, each 3 minutes, then pats dry; Obtain human tissue kallikrein fusion polyclonal antibody coated elisa plate.
4) every hole adds 200ul1%BSA-PBS, and 4 ℃ are spent the night.Discard solution in the hole next day, wash 3 times with lavation buffer solution, and each 3 minutes, pat dry and seal, namely obtain the coated reaction plate of human tissue kallikrein fusion polyclonal antibody.
The ELISA kit of this human tissue kallikrein content detection is characterized in that: described kit by above-mentioned specific anti-human tissue and peptide discharge that the coated plate of enzyme polyclonal antibody, sample diluent, cleansing solution, standard items, biotinylation are two anti-, enzyme mark Avidin, nitrite ion, stop buffer consist of.Specifically be divided into: (1) cleansing solution: the phosphate buffer (PBST) that contains 0.1%Tween-20; (2) sample diluent: the phosphate buffer (PBS) that contains 1%BSA; (3) standard items: above-mentioned human tissue kallikrein fusion HK-4T1; (4) biotinylation two is anti-: biotin labeled people's tissue and peptide discharge the enzyme monoclonal antibody; (5) enzyme mark Avidin: horseradish peroxidase-labeled Streptavidin (Streptavidin/HRP); (6) nitrite ion: TMB (tetramethyl benzidine) uses liquid; (7) stop buffer: 2M H2SO4 solution.
Kit of the present invention has highly sensitive, high specificity, the characteristics of good reproducibility.The fusion that utilizes above-mentioned expression is standard items the most, and the related coefficient of its typical curve is up to 0.99.Adopt human plasma in enormous quantities as sample to be tested, wherein difference and group difference all are lower than 10% in the group.
Description of drawings
Fig. 1 is the SDS-PAGE of human tissue kallikrein fusion HK-4T1.
Fig. 2 is the SDS-PAGE of anti-HK-4T1 recombinant protein polyclonal antibody.
Fig. 3 is the Western bloting of human tissue kallikrein fusion.
Fig. 4 is the Western bloting of anti-HK-4T1 recombinant protein polyclonal antibody.
Fig. 5 is the typical curve of this ELISA kit.
Embodiment
Following instance is used for illustrating the present invention, but is not used for limiting the scope of the invention.
The preparation of ELISA kit of human tissue kallikrein
1. reagent
(1) coated damping fluid (PH9.60.05M carbonate buffer solution):
Na2CO3 1.59 grams
NaHCO3 2.93 grams
Adding distil water is to 1000ml;
(2) lavation buffer solution (PH7.4PBS): 0.15M
KH2PO4 0.2 gram
Na2HPO412H2O 2.9 grams
NaCl 8.0 grams
KCl 0.2 gram
Tween-20 0.1% 0.5ml adding distil water is to 1000ml;
(3) sample diluent:
Bovine serum albumin(BSA) (BSA) 0.1 gram adds lavation buffer solution to 100ml;
(4) standard items:
The human tissue kallikrein fusion, self-produced during for this experiment;
(5) biotinylation two is anti-:
Biotin labeled monoclonal antibody of anti-human tissue kallikrein, for this laboratory self-produced;
(6 enzyme mark Avidins:
The HRP labelled streptavidin, biological company limited produces for Wuhan doctor's moral;
(7) stop buffer (2M H2SO4):
Distilled water 178.3ml dropwise adds the concentrated sulphuric acid (98%) 21.7ml.
(8) substrate buffer solution (PH5.0 phosphoric acid Jujube citric acid):
0.2M Na2 HPO4 (28.4 gram/L) 25.7ml
0.1M citric acid (19.2 gram/L) 24.3ml
Adding distil water 50ml.
(6) TMB (tetramethyl benzidine) uses liquid:
TMB (10mg/5ml absolute ethyl alcohol) 0.5ml
Substrate buffer solution (PH5.5) 10ml
0.75％H2O2 32μl
2. key instrument
(1) microplate reader: ELX800Universal Microplate reader
(2) ELISA reaction plate: Corning incorporated costar R 96Well EIA/RIA plate
3. method
The selection of the anti-optimum concentration of enzyme mark Avidin
(1) with 100ng/ml biotinylation two anti-being coated with, washing.
(2) enzyme mark Avidin is done to add respectively in the coated hole insulation, washing after a series of dilutions with dilution.
(3) add the substrate colour developing.After the acid adding cessation reaction, reading absorbance (A), get the A value at 1.0 enzyme mark Avidin dilutability, is 1: 2000 as the suitableeest titre of enzyme mark Avidin.
The selection of biotinylation two anti-optium concentrations
(1) discharges enzyme fusion proteins with 100ng/ml people's tissue and peptide and be coated with washing.
(2) do to add respectively in the coated hole insulation, washing after a series of dilutions with dilution with biotinylation two is anti-.
(3) add the enzyme mark Avidin that the suitableeest titre is diluted, insulation, washing.
(4) add the substrate colour developing.After the acid adding cessation reaction, reading absorbance (A), get the A value at 1.0 biotinylation two anti-dilutabilitys, is 1: 500 as biotinylation two the suitableeest anti-titres.
The chessboard titrimetry is selected the suitableeest titre of coated antibody
(1) with coating buffer coated antibody is done to be coated with after a series of dilutions washing.
(2) will enter tissue and peptide and discharge the dilution of enzyme fusion proteins proportioning as standard items, human plasma sample doubling dilution is as positive control, and the sample dilution is as negative control, and application of sample is incubated, washing.
(3) biotinylation two of the suitableeest titre dilution of adding is anti-, insulation, washing.
(4) add the enzyme mark Avidin that the suitableeest titre is diluted, insulation, washing.
(5) add the substrate colour developing.After the acid adding cessation reaction, read absorbance (A).
(6) selecting the A value of positive control is between 0.8-1.0, and the A value of negative control is less than the suitableeest coated concentration of dilutability conduct of 0.1 coated antibody, and its dilution titer is 1: 3200; The plasma sample optimum dilution degree is 1: 10; The sensing range of typical curve is 7-1000ng/ml.
The formation of kit
Described kit has that anti-human tissue and peptide discharge that the coated enzyme reaction plate of enzyme fusion proteins, sample diluent, biotinylation are two anti-, enzyme mark Avidin, nitrite ion, stop buffer consist of, and concrete component is:
(1) cleansing solution: the phosphate buffer (PBST) that contains 0.1%Tween-20;
(2) sample diluent: the phosphate buffer (PBS) that contains 1%BSA;
(3) standard items: above-mentioned human tissue kallikrein fusion HK-4T1;
(4) biotinylation two is anti-: biotin labeled people's tissue and peptide discharge the enzyme monoclonal antibody;
(5) enzyme mark Avidin: horseradish peroxidase-labeled Streptavidin (Streptavidin/HRP);
(6) nitrite ion: TMB-H2O2 system;
(7) stop buffer: 2M H2SO4 solution.
Utilize the mentioned reagent box to adopt the ELISA method to detect the content that people's tissue and peptide in the blood plasma discharge enzyme:
(1) blood plasma to be measured adds the blood plasma to be measured of dilution in 1: 10 to coated good enzyme reaction plate, and hatched 1 hour for 37 ℃ in the 100ul/ hole, washes plate 3 times;
(2) add biotinylation two and resist, the biotinylation two that adds dilution in 1: 500 is anti-, and hatched 40 minutes for 37 ℃ in the 100ul/ hole, washes plate 3 times;
(3) enzyme-added mark Avidin adds the enzyme mark Avidin that dilutes at 1: 2000, and hatched 25 minutes for 37 ℃ in the 100ul/ hole, washes plate 3 times;
(4) colour developing is washed and is added the TMB-H2O2 substrate 100ul of system, room temperature reaction 10 minutes behind the plate;
(5) every hole adds 2MH2SO450ul, cessation reaction;
(6) result judges, blank zeroing is read 450nm wavelength place absorbance, i.e. the OD450 value;
(7) result of calculation calculates the content that the tissue of people in the blood plasma and peptide discharge enzyme according to typical curve.
Embodiment 2
Comparison with external kit
The range of sensitivity of external kit is 0.4ng/ml-25ng/ml; The sensitivity of this kit is 0.3ng/ml-500ng/ml, and its sensing range is higher than external kit.The method that external kit adopts is the indirect competitive ELISA method, and its calculating is loaded down with trivial details, and the method that this kit adopts is double antibody sandwich method, calculates more intuitively easy.
Embodiment 3
Utilize this kit to detect the content of HK1 in 124 routine normal persons' the blood plasma, do three multiple holes for every group, repeat once after 6 months at the interval again.Difference is 1.7% in its group, and group difference is 8%.The plasma content of normal person's HK1 is 252 ± 93ng/ml.
Claims (1)
1. the ELISA kit of a human tissue kallikrein assay is characterized in that:
A) method of this kit employing is double antibody sandwich method;
B) added the biotin-avidin amplification system;
C) standard items are the human tissue kallikrein of prokaryotic expression and purifying;
D) its coated antibody is the polyclonal antibody of anti-human tissue kallikrein, and detecting antibody is its biotinylated monoclonal antibody;
E) the coated elisa plate of the coated antibody of 1: 6400 times of dilution, and place 4 ℃ to hatch 24 hours;
F) the 1%BSA sealing is 1 hour;
G) sensitivity is 0.3ng/ml-500ng/ml.
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| CN104049082B (en) * | 2014-05-30 | 2016-03-16 | 华中科技大学同济医学院附属同济医院 | Human tissue kallikrein activity detection kit and application thereof |
| CN105985437B (en) * | 2015-01-30 | 2019-04-05 | 江苏众红生物工程创药研究院有限公司 | 1 antibody of anti-human tissue kallikrein and its application |
| CN105985436B (en) * | 2015-01-30 | 2019-04-12 | 江苏众红生物工程创药研究院有限公司 | Anti-human kininogenase antibody and its application |
| CN105987998B (en) * | 2015-01-30 | 2017-12-29 | 江苏众红生物工程创药研究院有限公司 | Human tissue kallikrein 1ELISA immue quantitative detection reagent boxes |
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|---|---|---|---|---|
| CN101210926A (en) * | 2006-12-27 | 2008-07-02 | 中国科学院大连化学物理研究所 | Reagent kit for detecting human serum tissue kallikrein and its preparation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101210926A (en) * | 2006-12-27 | 2008-07-02 | 中国科学院大连化学物理研究所 | Reagent kit for detecting human serum tissue kallikrein and its preparation method |
Non-Patent Citations (2)
| Title |
|---|
| ELISA法测定人血液中组织激肽释放酶含量的研究;刘蔚等;《中国医科大学学报》;19951231;第24卷(第5期);448-450 * |
| 刘蔚等.ELISA法测定人血液中组织激肽释放酶含量的研究.《中国医科大学学报》.1995,第24卷(第5期),448-450. |
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