CN101105498A - Human, rat, mouse HSP70 double antibody sandwich method detection reagent kit - Google Patents
Human, rat, mouse HSP70 double antibody sandwich method detection reagent kit Download PDFInfo
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- CN101105498A CN101105498A CNA2007100586249A CN200710058624A CN101105498A CN 101105498 A CN101105498 A CN 101105498A CN A2007100586249 A CNA2007100586249 A CN A2007100586249A CN 200710058624 A CN200710058624 A CN 200710058624A CN 101105498 A CN101105498 A CN 101105498A
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Abstract
The invention discloses a test kit of HSP70 double antibody sandwich method used in the sample drawn from human, big mice and small mice, and comprises a kit body, an enzyme-labeled plate of solidified HSP70 monoclonal antibody provided in the kit body and substances provided in the kit body; the substances provided in the kit body comprises a HIS-HSP70 protein standard reagent, a HSP70 polyclonal antibody labeled by biotin, a sample dilution solution, a cleaning buffer solution, an antibody solution, a horseradish enzyme-labeled avidin, a horseradish enzyme-labeled avidin dilution solution, a substrate visualization solution A solution, a substrate visualization solution B solution and a stop solution. The invention has the advantages of high detection sensitivity, big accuracy and low cost; the invention can detect the HSP70 protein in a cell lysis solution, a tissue extracting solution, plasma and serum drawn from human, big mice and small mice at the same time.
Description
Technical field
The present invention relates to a kind of biological technology products, relate in particular to detection kit of a kind of double antibodies sandwich method fast detecting HSP70 albumen and preparation method thereof.
Background technology
Heat-shock protein family (HSPs) is the albumen of a class high conservative, HSP70 is the important member in the heat-shock protein family, and it is divided into the composing type of induction type.When body was subjected to that multiple undesirable element stimulates in the external environment, the HSP70 protein expression of induction type raise.The reparation of HSP70 albumen pair cell damage, to survive and keep normal cell function be essential.Cause performance molecular chaperones effect in the cellular stress damage in environmental stimulus, pathogenic bacterial infection, disease, stop protein aggregation, promote the folding again of injury protein.Most mammal, only HSP70 expresses under stressed condition, and just can detect behind significant cell and the physical stress, but people and primate, the HSP70 of induction type has a reference value, the infection that under some specific conditions, causes as overheated, chemical noxious material exposure, anoxic, ischemic, infection, autoimmune disease, apoptosis, organ transplant, bacterium and virus, in the angiocardiopathies such as atherosclerotic, the HSP70 of biosome expresses significantly and raises; In the normal aging process, spermatogenesis, menstruation, in the normal physiological processes such as athleticism, the HSP70 of biosome expresses also and significantly raises, and points out it may play a significant role in these processes.The HSP70 that it is generally acknowledged induction type is intracellular albumen, but discover the HSP70 and the HSP70 antibody that under normal person's peripheral circulation and some diseases state, can detect solubility, intracellular HSP70 can be discharged into the extracellular, and extracellular HSP70 becomes the focus of scientific research.Recent research mainly is to inquire into HSP70 as the cytoprotection medicine, the responsive biological warning index of and cellular stress unbalance as interior environment.The method that detects HSP70 now mainly contains SABC method, Western Bloting, Elisa method.The whole bag of tricks corroborates each other, and has simultaneously the influence of detection sensitivity, professional, installations and facilities condition and cost again.At present, the enzyme linked immunological absorption determining adsorption (Elisa) based on the serological reaction principle is the method that is widely accepted and promotes.
1969, Avrameas was applied to the enzyme len antibody to learn and learns a skill clearly, and Engvall in 1971 and Perlmann further improve this method and developed the ELISA method.Voller in 1974 etc. are applied to elisa technique the antibody test of humans and animals.Afterwards, the method further develops, and is widely used in the body fluid Protein Detection of humans and animals.Compare with other detection methods, the ELISA detection method has that cost is low, checkout facility equipment simply, characteristics rapidly and efficiently, and in the middle of updating, direct ELISA, indirect ELISA, monoclonal antibody body ELISA have been arranged, microwave ELISA, double-antibodies sandwich ELISA.Commercially available detection HSP70 kit mainly is that the double antibodies sandwich method detects at present, mainly is the HSP70 detection kit of the Stressgen company of the U.S., and it has monopolized the market of HSP70 detection kit basically; 50 of the about 10000 yuans of about working samples of price of 96 orifice plates in a detection of the kit box of the detection HSP70 that present Stressgen company is commercially available, about 200 yuans of each sample detection expense.Sales company's major part of China's this respect is agency or import packing, also have idol that the report of development is voluntarily arranged, but its detection sensitivity is low, and sensing range is narrow, is far from reaching the degree of production testing.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, provide a kind of detection sensitivity height, accuracy by force, people, rat, mouse HSP 70 double antibody sandwich method detection reagent kit efficiently.
Technical scheme of the present invention is summarized as follows:
A kind of people, rat, mouse HSP 70 double antibody sandwich method detection reagent kit, comprise box body, be located at the ELISA Plate of the curing HSP70 monoclonal antibody in the box body and be located at the interior material of box body that the material in the box body comprises that HIS-HSP70 protein standard substance, biotin labeled HSP70 polyclonal antibody, sample diluting liquid, lavation buffer solution, antibody diluent, horseradish enzyme labeling Avidin, horseradish enzyme labeling Avidin dilution, substrate colour developing liquid A liquid, substrate show liquid B liquid and stop buffer;
Described HIS-HSP70 protein standard substance is to make with following method:
Design upstream primer earlier: 5 ' CGGAATTCATGGCCAAGAAAACAGCG 3 ', downstream primer: 5 ' CCCAAGCTTCTAATCCACCTCCTCGAT 3 ', pcr amplification, double digestion are connected in the PET-32a carrier, transformed into escherichia coli, the IPTG abduction delivering, purifying, SDS glue is identified purity, the dialysis postlyophilization, promptly make the HIS-HSP70 protein standard substance, packing ,-70 ℃ are frozen;
Described biotin labeled HSP70 polyclonal antibody is to make with following method:
(1) preparation HSP70 polyclonal antibody: selecting the Healthy female rabbit for use is immune animal, with the described HIS-HSP70 albumen of 2mg/ml as immunizing antigen, 4 ℃ of preservations; Carry out the inoculation program, the 0.5ml immunizing antigen is added the 0.5ml Freund's complete adjuvant, fully mix, at the subcutaneous multi-point injection in rabbit back, every some 0.2ml, lower limb groin 0.2ml; After one month, the 0.5ml immunizing antigen is added the 0.5ml Freund's complete adjuvant, fully mix, at the subcutaneous multi-point injection in rabbit back, every some 0.2ml, lower limb groin 0.2ml; After another month, with the injection of 0.5ml immunizing antigen ear vein; After 7-10 days, arteria carotis is taken a blood sample entirely again, separation of serum, and adding mass percentage concentration is the thimerosal aqueous solution of 0.8-1.2%, preserves down, gets HSP70 polyclonal antibody serum for-70 ℃; Adopt ammonium sulfate precipitation method purifying HSP70 polyclonal antibody serum;
(2) the HSP70 polyclonal antibody is carried out biotin labeling:
1) preparation biotin N-maloyl imines ester: get biotin 1g and be suspended in 10-18ml N, dinethylformamide, add 0.4-0.8gN-maloyl imines and 0.6-1.0g dicyclohexylcarbodiimide, place in the closed container room temperature lower magnetic force beating action 8-12 hour, filter, filtrate adds 8-12mL ether washing through the rotation evaporate to dryness, continues to add the 160-220ml isopropyl alcohol and make its recrystallization, obtain the white powder crystal ,-30 ℃ of packing are frozen;
2) biotin and HSP70 polyclonal antibody lotus root connection:
1. get described biotin N-maloyl imines ester powder, be prepared into 1-2mg/ml solution with dimethyl sulfoxide, standby;
2. with the HSP70 polyclonal antibody of purifying NaHCO with 0.1mol/L pH9.0
3Be made into 2-4mg/ml solution;
3. be 1 by volume with the solution that 2. step prepares 1. with step: 4-8 mixes, and 20-30 ℃ is stirred 3.5-5.5h;
4. make above-mentioned mixing material with 0.05mol/L under 4 ℃ of conditions, pH7.2 PBS dialysis 8-12 hour adds equal-volume glycerine, and-20 ℃ of packing are deposited;
The ELISA Plate of described curing HSP70 monoclonal antibody is to make with following method:
With the described HIS-HSP70 albumen of 2mg/ml as immunizing antigen, the female mouse of Balb/c of selecting 18-20g for use is as immune animal, and get its splenocyte and SP2/0 Fusion of Cells, ELISA screening, subgroup identification, ascites preparation, monoclonal antibody purification, with the monoclonal antibody coated elisa plate of purifying, 4 ℃ of preservations.
Consisting of of described sample diluting liquid: every 100ml contains NaCl 0.5g, KCl 0.0125g, KH
2PO
40.0125g, Na
2HPO
40.181g surplus is a water.
Every 100ml that consists of of described lavation buffer solution contains NaCl 5g, KCl 0.125g, KH
2PO
40.125g, Na
2HPO
41.81g, Tween-20 20ul, surplus is a water.
Consisting of of described antibody diluent: every 5ml contains calf serum 500ul, mass percentage concentration is 1% thimerosal aqueous solution 100ul, the potassium ferricyanide 50ul of 100mM, Tween-20 2.5ul, the gentamicin 25ul of 10mg/ml, surplus is the PBS of the 0.01M of pH7.4.
Consisting of of described horseradish enzyme labeling Avidin dilution: every 5ml contains NaCl 0.025g, KCl 0.00125g, KH
2PO
40.00125g, Na
2HPO
40.009g surplus is a water.
Consisting of of described substrate colour developing liquid A liquid: every 20ml contains sodium acetate 0.544g, citric acid 0.064g, and concentration expressed in percentage by volume is 30% H
2O
212ul, surplus is a water.
Consisting of of described substrate colour developing liquid B liquid: every 20ml contains EDTA-Na 0.008g, citric acid 0.038g, and glycerine 2ml, tetramethyl benzidine 0.008g, surplus is a water, keeps in Dark Place.
Consisting of of described stop buffer: every 20ml contains dense H
2SO
41.15ml surplus is a water.
Advantage of the present invention is: the detection sensitivity height, accuracy is strong, cost is low, can detect the HSP70 albumen in people, rat, mouse cell lysate, tissue extract, blood plasma and the serum simultaneously.
Along with going deep into of research, HSP70 may be as the early warning molecular marked compound of disease and the drug targets of prevention from suffering from the diseases, this kit will more applications in clinical detection, kit will have bigger application prospect.
Description of drawings
The biotin labeled polyclonal antibody preparation flow of Fig. 1;
The ELISA Plate preparation flow of Fig. 2 Sheet clonal antibody;
Fig. 3 standard items positive control HSP70 protein purification figure;
Fig. 4 HSP70 detection kit detection sensitivity and sensing range curve.
Embodiment
The present invention is further illustrated below in conjunction with specific embodiment.
A kind of people, rat, mouse HSP 70 double antibody sandwich method detection reagent kit, comprise box body, be located at the ELISA Plate of the curing HSP70 monoclonal antibody in the box body and be located at the interior material of box body that the material in the box body comprises that HIS-HSP70 protein standard substance, biotin labeled HSP70 polyclonal antibody, sample diluting liquid, lavation buffer solution, antibody diluent, horseradish enzyme labeling Avidin, horseradish enzyme labeling Avidin dilution, substrate colour developing liquid A liquid, substrate show liquid B liquid and stop buffer.
The HIS-HSP70 protein standard substance is to make with following method:
1.) design the pcr amplification primer earlier:
Upstream: 5 ' CGGAATTCATGGCCAAGAAAACAGCG 3 '
Downstream: 5 ' CCCAAGCTTCTAATCCACCTCCTCGAT 3 '
Pcr amplification, amplification condition is:
95℃4min；95℃30s 56℃ 60s 72℃60s(30cycle)；72℃10min
Behind EcoR I and HinDIII double digestion, be connected into plasmid PET-32a
2.) 37 ℃ of joltings of recombinant plasmid transformed e. coli bl21 add IPTG 1.0ul/ml during OD value 0.5, induce 4h, ultrasonication, centrifugal collection supernatant.
3.) supernatant of collecting is carried out purifying by the HISTrap purification kit operation instructions of Pharmacia Corp.Through groping best purification condition be: the 1ml purification column,------post cleansing solution washing 8ml---the 20mmol/L miaow eluent wash-out 8ml that consults---300mmol/L miaow consult eluent wash-out---collects the 2-4ml eluent to column equilibration liquid balance 6ml to go up sample (1ml supernatant, flow velocity 0.5ml/ml).
4.) eluent of collecting is got 100ul SDS glue and identified purity, in all the other bag filters of packing into, dialysed overnight in the PBS liquid is changed liquid therebetween three times.
5.) liquid subpackage in the bag filter is gone in the freeze drying pipe, total protein concentration is 1mg in every pipe, and freeze drying is powdered, put into-70 ℃ standby.
Reorganization HSP70 purity of protein of the present invention and content detection adopt SDS glue to identify and the coomassie brilliant blue staining method respectively.Concrete grammar is:
1.SDS glue is identified reorganization HSP70 purity of protein
The 300mmol/L miaow eluent of consulting is got 80ul during with purifying, adds 20,100 ℃ of sex change 5min of 5 * sample-loading buffer, and 10000g is centrifugal, gets supernatant 10ul and goes up sample, 10%SDS glue, 80v, 10min, 180v, 45min; Examine and dye decolouring, the glue map analysis.The result: reorganization HSP70 purity of protein 〉=95%, as shown in Figure 3, the albumen of HSP70 shown in the arrow.
Fig. 3 is standard items positive control HSP70 protein purification figure, and wherein swimming lane 1 is normal bacterium, and 2 for inducing bacterium, and 3-7 is the HSP70 of branch's purifying, the albumen of HSP70 shown in the arrow.
2. the coomassie brilliant blue staining method of determination of protein concentration employing is a technology as well known to those skilled in the art.
The HIS-HSP70 protein standard substance is a positive control in the kit, is distributed into 100ug/ pipe, and-20 ℃ of preservations can be used with 1ml PBS or dissolved in distilled water, dilution during use.
Biotin labeling target HSP70 protein polyclone antibody is to make with following method:
(1) preparation HSP70 protein polyclone antibody
Select for use the above Healthy female rabbit of 2kg as immune animal, the HIS-HSP70 albumen for preparing with the embodiment 2 of 2mg/ml is immunizing antigen ,-4 ℃ of preservations; Carry out the inoculation program then, the 0.5ml immunizing antigen is added the 0.5ml Freund's complete adjuvant, fully mix, at the subcutaneous multi-point injection in rabbit back, every some 0.2ml, lower limb groin 0.2ml;
After one month, the 0.5ml immunizing antigen is added the 0.5ml Freund's complete adjuvant, fully mix, at the subcutaneous multi-point injection in rabbit back, every some 0.2ml, lower limb groin 0.2ml;
After one month, with the injection of 0.5ml immunizing antigen ear vein;
After 7-10 days, arteria carotis is taken a blood sample entirely, separation of serum, and adding mass percentage concentration is 1% thimerosal aqueous solution (also can select 0.8% or 1.2% thimerosal aqueous solution for use, become new embodiment) ,-70 ℃ of preservations obtain HSP70 polyclonal antibody serum 35ml;
The HSP70 polyclonal antibody serum of being gathered is carried out purifying: employing be ammonium sulfate precipitation method, concrete method is:
1.) get serum 10ml, put on the magnetic stirring apparatus and stir, and adding 30ml sodium acetate under the room temperature (60mmol/L, pH4.0), with 0.1mmol/LNaOH adjust pH to 4.5 (also can be 4.2 or 4.8);
2.) slowly drip sadly, continue to stir 30min to final concentration 100ul/ml;
3.) the centrifugal 30min of 10000g abandons precipitation, and filter paper filtering adds 10 * PBS of 1/10 volume, adjust pH to 7.4;
4.) slowly add saturated ammonium sulfate to 45% saturation degree of precooling, 4 ℃ are spent the night behind the mixing;
5.) the centrifugal 30min of 3000g uses 0.01mol/L PBS (pH7.4) resuspended, and dialyses with bag filter;
6.) antibody is quantitative: Coomassie brilliant blue quantivative approach as described above, and the antibody concentration of acquisition is 26mg/ml ,-70 ℃ of preservations,
The HSP70 polyclonal antibody of being gathered is carried out check and analysis, is 1: 10 1: 20 1 with the pure liquid doubling dilution of HSP70 polyclonal antibody: 40......1: 256000 with indirect elisa method mensuration antibody titer.The result: tiring of the polyclonal antibody behind the purifying is 1: 128000, obtains the polyclonal antibody 5ml of purifying;
(2) the HSP70 polyclonal antibody behind the purifying is carried out biotin labeling
1.) biotin N-maloyl imines ester (biotinyl-N-hydroxy-succinnimide, preparation BNHS):
Get biotin 1g and be suspended in 12ml (also can select 10ml or 8ml for use), N, dinethylformamide (DMF), add people 0.6g (also can select 0.4g or 0.8g for use) N-maloyl imines and 0.8g (also can select 0.6g or 1.0g for use) dicyclohexylcarbodiimide, place in the closed container, room temperature lower magnetic force beating action 10 hours, (also can select for use 8 hours or 12 hours), filter, filtrate is through the rotation evaporate to dryness, add 10ml (also can select 8ml or 12ml for use) ether washing, continuing adds 200ml (also can select 160ml or 220ml for use) isopropyl alcohol and makes its recrystallization, obtains the white powder crystal.-30 ℃ of packing are frozen;
2.) biotin and antibody lotus root connection:
(1) get the BNHS powder, be prepared into 1mg/ml (also can select 2mg/ml for use) solution with dimethyl sulfoxide (DMSO), standby;
(2) with the HSP70 polyclonal antibody of purifying NaHCO with 0.1mol/L pH9.0
3Be made into 2mg/ml (also can select 3mg/ml, 4mg/ml for use) solution;
(3) be that 1: 8 (also can select for use 1: 4 or 1: 6) mixes with step (1) by volume with the solution that step (2) prepares, stirring at room 4h (also can select 20 ℃ and stir 5.5h or 30 ℃ of stirring 3.5h);
Make above-mentioned mixing material with 0.05mol/L under (4) 4 ℃ of conditions, pH7.2 PBS dialysed overnight adds equal-volume glycerine, and-20 ℃ of packing are deposited.
The HSP70 polyclonal antibody of mark biotin is diluted to 1mg/ml, standby.The HSP70 albumen of purifying is diluted to 1mg/ml, 0.1mg/ml, 0.01mg/ml, 0.001mg/ml, 0.0001mg/ml, 0.00001mg/ml, 0.000001mg/ml measures the sensitivity of biotin labeled HSP70 polyclonal antibody with indirect ELISA method.The result: behind purifying, biotin labeling, obtain antibody 3ml, minimum degree of detecting (detection sensitivity) reaches 0.01-0.001ng.
During kit of the present invention, biotin labeled HSP70 polyclonal antibody solution is added 0.1% Sodium azide in assembling, aseptic subpackaged after, sealing, cold storage (70 ℃), standby.Can be kept at 4 ℃ in use, should not move repeatedly and melt the term of validity 1 year.Each kit 50ul dilutes with PBS or distilled water during use at 1: 2000.
Solidify the preparation of the ELISA Plate of HSP70 monoclonal antibody:
With the described HIS-HSP70 albumen of 2mg/ml as immunizing antigen, the female mouse of Balb/c of selecting 18-20g for use is as immune animal, and get its splenocyte and SP2/0 Fusion of Cells, ELISA screening, then to the monoclonal antibody subgroup identification, the ascites preparation, monoclonal antibody purification is analyzed the monoclonal antibody of gained, filter out the wide spectrum cell strain of monoclonal antibody that can discern people, rat, the natural HSP70 albumen of mouse, it is 1: 20000 that indirect ELISA is measured antibody titer.
Monoclonal antibody to gained is carried out the ELISA Plate antibody sandwich, and concrete steps are:
(1) get and screen and the monoclonal antibody concentrate of purifying, be cushioned liquid and be diluted to 1mg/ml with wrapping, 4 ℃ of preservations, standby.
(2) with the aforesaid liquid coated elisa plate, every hole adds liquid 100ul, and 4 ℃ are spent the night.
(3) wash plate three times, pat dry; Every hole adds 150ul 10% hyclone confining liquid, and 4 ℃ of sealings are spent the night.
(4) wash plate three times, pat dry, deposit for 4 ℃, standby.
The resulting ELISA Plate that is coated with monoclonal antibody is analyzed, the HSP70 albumen of purifying is diluted to 1mg/ml, 0.1mg/ml, 0.01mg/ml, 0.001mg/ml, 0.0001mg/ml, 0.00001mg/ml 0.000001mg/ml measures the sensitivity of ELISA Plate with indirect ELISA method.The result: behind antibody purification, bag quilt, obtain ELISA Plate, minimum degree of detecting (detection sensitivity) reaches 0.1-0.01ng.
When assembling kit of the present invention, one of ELISA Plate (96 holes, bag is by the HSP70 monoclonal antibody), 4 ℃ of preservations, unsuitable multigelation is valid for one year.
Other main materials and medicine in the kit, 4 ℃ of preservations.
(1) sample diluting liquid: every 100ml contains NaCl 0.5g, KCl 0.0125g, KH
2PO
40.0125g, Na
2HPO
40.181g surplus is a water;
(2) lavation buffer solution: every 100ml contains NaCl 5g, KCl 0.125g, KH
2PO
40.125g, Na
2HPO
41.81g, Tween-20 20ul, surplus is a water;
(3) antibody diluent: every 5ml contains calf serum 500ul, the 1% thimerosal 100ul that mass percent is, and the potassium ferricyanide 50ul of 100mM, Tween-20 2.5ul, the gentamicin 25ul of 10mg/ml, surplus is the PBS of the 0.01M of pH7.4;
(4) horseradish enzyme labeling Avidin: be HRP-avidin, packing, 4 ℃ of preservations, standby.Each kit, 5ul,
(5) horseradish enzyme labeling Avidin dilution: every 5ml contains NaCl 0.025g, KCl 0.00125g, KH
2PO
40.00125g, Na
2HPO
40.009g surplus is a water;
(6) substrate colour developing liquid A liquid: every 20ml contains sodium acetate 0.544g, citric acid 0.064g, and concentration expressed in percentage by volume is 30% H
2O
212ul, surplus is a water;
(7) substrate colour developing liquid B liquid: every 20ml contains EDTA-Na 0.008g, citric acid 0.038g, and glycerine 2ml, tetramethyl benzidine 0.008g, surplus is a water, keeps in Dark Place;
(8) stop buffer: every 20ml contains H
2SO
41.15ml surplus is a water,
Kit of the present invention can be according to the actual needs that detects unit, and each kit is assembled into the antibody and the medicine and reagent amount that can detect large sample.
People of the present invention, rat, mouse HSP70 detection kit is on existing ELISA method, adopt the double antibodies sandwich method to detect HSP70, and can detect the people simultaneously, rat, the HSP70 of mouse, that detects is highly sensitive, detection sensitivity can reach 195pg/ml, sensing range is 390pg～50000pg/ml, than the high 5-10 of detection kit of StressGen company doubly, reagent cost is cheap, be easy to realize mass production, the testing cost of each sample is about about 40 yuans, well below external like product, has very great help for the function of studying HSP70.
The present invention carries out HSP70 double antibodies sandwich method and detects, and concrete grammar is as follows:
1. the standard items positive control is diluted to 10ug/ml with the 1ml sample diluting liquid, further is diluted to 50 12.5 3.1250.78 0.195ng/ml.
2. application of sample: establish blank well, 5 gauge orifices, testing sample hole respectively.Except that blank well, surplus hole adds standard solution or testing sample 100ul, 37 ℃, 120 minutes respectively.
2. discard liquid, pat dry, need not wash.
3. every hole adds polyclonal antibody solution (biotin labeling) 100ul, 37 ℃, 60 minutes.Lavation buffer solution is washed plate 3 times, the every hole of 350ul//time, pat dry.
4. every hole adds HRP-avidin 100ul, and 37 ℃, 60 minutes, wash plate 5 times, pat dry.
5. every in regular turn hole adds each 90ul of substrate colour developing liquid AB, and 37 ℃ of lucifuges developed the color 30 minutes.
Every in regular turn hole adds stop bath 90ul, cessation reaction.With microplate reader in 450nm wavelength readings (OD value).
The purpose of present embodiment is determined sensing range and the sensitivity that this kit detects.
Material: this detection kit.
Method: get positive control standard items and PBS and be made into 1ug/ml, doubling dilution becomes 10 pipes, is undertaken by the method among the embodiment 6, and two multiple holes are measured.
The result: as shown in Figure 4, this kit detects the sensitivity 195pg/ml of HSP70, and sensing range is 390pg～50000pg/ml
| Concentration ng | 100 | 50 | 25 | 12.5 | 6.25 | 3.125 | 1.56 | 0.78 | 0.39 | 0.195 | 0.0975 | Blank |
| The OD value | 2.664 | 2.768 | 1.592 | 0.926 | 0.609 | 0.401 | 0.284 | 0.198 | 0.162 | 0.128 | 0.045 | 0.032 |
Embodiment 8
The purpose of present embodiment determines that yin and yang attribute is differentiated, accuracy compares.
Each experiment stays a hole as the blank hole of returning to zero, and this hole does not add any reagent, just adds substrate solution and 2N H at last
2SO4 transfers the OD value to zero with this hole earlier during measurement; The ratio of testing sample OD value and negative control OD value was more than or equal to 2.1 o'clock, and testing sample is positive, otherwise negative.For preventing sample evaporation, during experiment reaction plate is put in the closed enclosure that is covered with wet cloth.
Plate washing method: manual plate washing method: inhale and remove (untouchable wooden partition) or get rid of the interior liquid of ELISA Plate; Which floor thieving paper of place mat on experiment table, ELISA Plate are firmly clapped several times down; In the lavation buffer solution 0.35ml filling orifice of recommending, soaked 1-2 minute, as required, repeat this process for several times.Automatically wash plate:, should after skilled the use, use again in the formal experimentation if automatic washer is arranged.
Calculate: the concentration with reference material is horizontal ordinate, and the OD value is an ordinate, draws typical curve on coordinate axis, and OD value is per sample found corresponding concentration by typical curve; Multiply by extension rate again; Or the linear regression equation that calculates typical curve with the concentration and the OD value of reference material, the OD value substitution equation with sample calculates sample concentration, multiply by extension rate again, is the actual concentrations of sample.
Embodiment 9
The purpose of present embodiment is to measure the stability of HSP70 EIISA detection kit, judges that by the degree of variation between kit inside and group determine the stability of kit, concrete grammar is as follows:
1.HSP70 the inner coefficient of variation of ELISA kit is determined method: (25ng) each measures 20 times separately three concentration known samples on a HSP70 immune response plate for 5ng, 12ng.Coefficient of variation computing method:
CV=criticizes internal standard difference S/ mean value * 100%
Result: the value CV=8.2% of the coefficient of variation＜10%.
2.HSP70 the coefficient of variation is determined method between the reaction of ELISA kit: the sample of three concentration known is respectively measured separately 20 times by four researchists.Coefficient of variation CV=between the kit reaction criticizes a standard deviation S/ and criticizes a mean value * 100%CV=7.6%＜10%.
The result: by two groups CV value, this kit has good stable.
The present embodiment purpose is to determine that this kit detects the HSP70 species specificity.
The described HSP70 double antibodies sandwich of present embodiment method detection kit, comprise box body, be located at ELISA Plate (monoclonal antibody bag quilt) and the interior material of box body in the box body, material in the box body comprises the HIS-HSP70 protein standard substance, biotin labeled HSP70 polyclonal antibody, sample diluting liquid, lavation buffer solution, antibody diluent, horseradish enzyme labeling Avidin, horseradish enzyme labeling Avidin dilution, substrate colour developing liquid A liquid, substrate shows liquid B liquid and stop buffer, described polyclonal antibody and monoclonal antibody are recombinated HSP70 as immunizing antigen by 2mg/ml respectively, and immune rabbit and mouse are prepared from.
Embodiment 11
In the Monoclonal Antibody process in this kit, we have filtered out the cell strain of monoclonal antibody of a strain wide spectrum, indirect elisa method determines that it can discern people, rat, the natural HSP70 albumen of mouse, and we further study this kit specific detection people, rat, mouse HSP70 albumen.Concrete grammar is:
Material: HSP70 EIISA kit; Human serum (1: 10), blood plasma (1: 10), cell extract (stoste), tissue extract (stoste)
Method:
1. the standard items positive control is diluted to 10ug/ml with the 1ml sample diluting liquid, further is diluted to 50 12.5 3.1250.78 0.195ng/ml.
2. application of sample: establish blank well, 5 gauge orifices, testing sample hole respectively.Except that blank well, surplus hole adds standard solution or testing sample 100ul, 37 ℃, 120 minutes respectively.
2. discard liquid, pat dry, need not wash.
3. every hole adds polyclonal antibody solution (biotin labeling) 100ul, 37 ℃, 60 minutes.Lavation buffer solution is washed plate 3 times, the every hole of 350ul//time, pat dry.
4. every hole adds HRP-avidin 100ul, and 37 ℃, 60 minutes, wash plate 5 times, pat dry.
5. every in regular turn hole adds each 90ul of substrate colour developing liquid AB, and 37 ℃ of lucifuges developed the color 30 minutes.
Every in regular turn hole adds stop bath 90ul, cessation reaction.With microplate reader in 450nm wavelength readings (OD value).Instrument: microplate reader, wash plate machine etc.
The result:
| Sample (people) | Serum | Blood plasma | The liver cancer tissue extract | The umbilical vein cell extract |
| Final concentration (ng/ml) | 86 | 280 | 130 | 0.9 |
This kit can detect natural HSP70 albumen in the different body fluid of people.
Embodiment 12
Method is with the method among the embodiment 11, and different is that material is the serum (1: 10) of rat, blood plasma (1: 10), cell extract (stoste), tissue extract (stoste)
Instrument: microplate reader, wash plate machine etc.
The result:
| Sample (rat) | Serum | Blood plasma | The hepatic tissue extract | The smooth muscle cell extract |
| Concentration (ng/ml) | 36 | 372 | 94 | 1.2 |
This kit can detect natural HSP70 albumen in the different body fluid of rat.
Embodiment 13
Method is with the method among the embodiment 11, and different is that material is the serum (1: 10) of mouse, blood plasma (1: 10), cell extract (stoste), tissue extract (stoste)
Instrument: microplate reader, wash plate machine etc.
The result:
| Sample (mouse) | Serum | Blood plasma | The hepatic tissue extract | The SP2/0 cell extract |
| Concentration (ng/ml) | 23 | 156 | 45 | 0.201 |
This kit can detect natural HSP70 albumen in the different body fluid of mouse.
This ELISA kit can be recombinated in specific detection; Other kind comprises that the Hsp70 of monkey, hamster, pig, ox, sheep and dog does not detect.
Embodiment 14
HSP70 ELISA kit detecting operation instructions
Heat shock protein 70 (HSP70) ELISA
The kit operation instructions
This kit only uses for research
Use
Double antibodies sandwich ELISA standard measure is measured HSP70 content in people, rat, mouse cell lysate, tissue extract, blood plasma and the serum.
Experimental principle
This kit is used HSP70 level in the double-antibody sandwich enzyme-labeled immunity assay sample.By microwell plate, make insolubilized antibody with the antibody purified bag, add the Avidin of HSP70 antigen, the biotinylated mouse HSP70 of Chinese People's Anti-Japanese Military and Political College antibody, HRP mark in the anti-micropore of Sheet successively, through thorough washing back with substrate TMB colour developing.TMB changes into blueness under the catalysis of peroxidase, and changes into final yellow under the effect of acid.The depth of color and the HSP70 in the sample are proportionate.Under the 450nm wavelength, measure absorbance (0D value), calculation sample concentration with microplate reader.
Kit is formed
1. ELISA Plate: (96 hole) (a Sheet clonal antibody).
2. standard items (dried frozen aquatic products, positive control): 1 bottle, face with preceding and be diluted to 1ml with sample diluting liquid, its concentration is 10000000pg/mL, do serial doubling dilution after, be diluted to 50000pg/mL respectively, 25000pg/mL, 12500pg/mL, 6250pg/mL, 3125pg/mL, 1562.5pg/mL, 781.25pg/mL, 390pg/mL, 195pg/mL, sample diluting liquid face in preceding 15 minutes and prepare directly as normal concentration 0pg/mL.
3. biotin labeling polyclonal antibody: 1 * 50ul/ manages.
4. antibody diluent: 5ml/ bottle.
5. sample diluting liquid: 100ml/ bottle.
6. enzyme mark Avidin: 1 * 0.5ml/ manages.
7. enzyme is marked the Avidin dilution: 1 * 5ml/ bottle.
8. lavation buffer solution: 1 * 100ml/ bottle.
9. liquid A:1 * 20ml/ bottle develops the color.
10. liquid B:1 * 20ml/ bottle develops the color.
11. stop buffer: 1 * 20ml/ bottle.
The collection of sample and preservation
1. serum: sample is please placed 2 hours or the 4 ℃ backs of spending the night in 1000 * g centrifugal 20 minutes in room temperature, gets supernatant and can detect, or sample is put in-20 ℃ of preservations, but should avoid multigelation.
2. blood plasma: available EDTA or heparin be as anti-coagulants, after the collection of specimens in 30 minutes in centrifugal 15 minutes of 2-8 ℃ of 1000 * g, or sample is put in-70 ℃ of preservations, but should avoids multigelation.
3. tissue extract: get flesh tissue or liquid nitrogen cryopreservation tissue and add extract homogenate, centrifugal 1000g 10min extracts albumen, measures immediately, or sample is put in-70 ℃ of preservations, but should avoid multigelation.
4. cell extract: 1 * PBS gives a baby a bath on the third day after its birth time, adds lysis liquid and scrapes and get 4 ℃ of centrifugal 10000g 10min.Get supernatant and measure (concentration that suitably increases cell).
Annotate: sample hemolysis can influence last testing result, so the haemolysis sample should not carry out this detection.Test sample is looked concrete condition and is carried out suitable dilution or increase concentration determination.Will not precipitate suction when getting supernatant, influence testing result.
Operation steps
1. the standard items positive control is diluted to 10ug/ml with the 1ml sample diluting liquid, further is diluted to 50 12.5 3.1250.78 0.195ng/ml.
2. application of sample: establish blank well, 5 gauge orifices, testing sample hole respectively.Except that blank well, surplus hole adds standard solution or testing sample 100ul, 37 ℃, 120 minutes respectively.
2. discard liquid, pat dry, need not wash.
3. every hole adds polyclonal antibody solution (biotin labeling) 100ul, 37 ℃, 60 minutes.Lavation buffer solution is washed plate 3 times, the every hole of 350ul//time, pat dry.
4. every hole adds HRP-avidin 100ul, and 37 ℃, 60 minutes, wash plate 5 times, pat dry.
5. every in regular turn hole adds each 90ul of substrate colour developing liquid AB, and 37 ℃ of lucifuges developed the color 30 minutes.
Every in regular turn hole adds stop bath 90ul, cessation reaction.With microplate reader in 450nm wavelength readings (OD value).
Annotate:
1. each experiment stays a hole as the blank hole of returning to zero, and this hole does not add any reagent, just adds substrate solution and 2NH at last
2SO4.Transfer the OD value to zero with this hole earlier during measurement.
2. for preventing sample evaporation, during test reaction plate is put in the closed enclosure that is covered with wet cloth.
Plate washing method
Manual plate washing method: inhale and remove (untouchable wooden partition) or get rid of the interior liquid of ELISA Plate; Which floor thieving paper of place mat on experiment table, ELISA Plate are firmly clapped several times down; The lavation buffer solution recommended at least in the 0.4ml filling orifice, was soaked 1-2 minute, as required, repeat this process several.
Automatically wash plate:, should after skilled the use, use again in the formal experimentation if automatic washer is arranged.
Specificity
This kit can detect simultaneously the reorganization or natural rat HSP70, also can detect people, the natural HSP70 of mouse, and with other associated protein no cross reaction.
Calculate
Concentration with reference material is horizontal ordinate, and the OD value is ordinate (a common coordinate), draws typical curve on coordinate axis, and OD value is per sample found corresponding concentration by typical curve; Multiply by extension rate again; Or the linear regression equation that calculates typical curve with the concentration and the OD value of reference material, the OD value substitution equation with sample calculates sample concentration, multiply by extension rate again, is the actual concentrations of sample.
Points for attention
1. washing process is extremely important, and inadequate washing easily causes false positive.
2. an application of sample time preferably was controlled in 5 minutes, and is many as sample quantity, recommends to use volley of rifle fire application of sample.
3. do typical curve when please measuring at every turn, preferably do multiple hole.
4. as test substance too high levels in the sample, measure again after please diluting earlier, please multiply by extension rate during calculating at last.
5. when preparation standard items, detection solution working fluid,, can not obscure please with corresponding diluent preparing.
6. substrate please keep in Dark Place.
Sensing range:
390pg-50000pg/ml
Explanation
1. kit is preserved :-20 ℃ (long period is time spent not); 2-8 ℃ (when frequently using).
2. the term of validity: 6 months
3. dense cleansing solution has and salts out, the hydrotropy of can heating in water-bath during dilution.
4. may contain a little water sample material in the enzyme linked plate holes of just having opened, this is a normal phenomenon, can not have any impact to experimental result.
Claims (8)
1. a people, rat, mouse HSP 70 double antibody sandwich method detection reagent kit, comprise box body, be located at the ELISA Plate of the curing HSP70 monoclonal antibody in the box body and be located at the interior material of box body that the material in the box body comprises that HIS-HSP70 protein standard substance, biotin labeled HSP70 polyclonal antibody, sample diluting liquid, lavation buffer solution, antibody diluent, horseradish enzyme labeling Avidin, horseradish enzyme labeling Avidin dilution, substrate colour developing liquid A liquid, substrate show liquid B liquid and stop buffer;
Described HIS-HSP70 protein standard substance is to make with following method:
Design upstream primer earlier: 5 ' CGGAATTCATGGCCAAGAAAACAGCG 3 ', downstream primer: 5 ' CCCAAGCTTCTAATCCACCTCCTCGAT 3 ', pcr amplification, double digestion are connected in the PET-32a carrier, transformed into escherichia coli, the IPTG abduction delivering, purifying, SDS glue is identified purity, the dialysis postlyophilization, promptly make the HIS-HSP70 protein standard substance, packing ,-70 ℃ are frozen;
Described biotin labeled HSP70 polyclonal antibody is to make with following method:
(1) preparation HSP70 polyclonal antibody: selecting the Healthy female rabbit for use is immune animal, with the described HIS-HSP70 albumen of 2mg/ml as immunizing antigen, 4 ℃ of preservations; Carry out the inoculation program, the 0.5ml immunizing antigen is added the 0.5ml Freund's complete adjuvant, fully mix, at the subcutaneous multi-point injection in rabbit back, every some 0.2ml, lower limb groin 0.2ml; After one month, the 0.5ml immunizing antigen is added the 0.5ml Freund's complete adjuvant, fully mix, at the subcutaneous multi-point injection in rabbit back, every some 0.2ml, lower limb groin 0.2ml; After another month, with the injection of 0.5ml immunizing antigen ear vein; After 7-10 days, arteria carotis is taken a blood sample entirely again, separation of serum, and adding mass percentage concentration is the thimerosal aqueous solution of 0.8-1.2%, preserves down, gets HSP70 polyclonal antibody serum for-70 ℃; Adopt ammonium sulfate precipitation method purifying HSP70 polyclonal antibody serum;
(2) the HSP70 polyclonal antibody is carried out biotin labeling:
1) preparation biotin N-maloyl imines ester: get biotin 1g and be suspended in 10-18ml N, dinethylformamide, add 0.4-0.8gN-maloyl imines and 0.6-1.0g dicyclohexylcarbodiimide, place in the closed container room temperature lower magnetic force beating action 8-12 hour, filter, filtrate adds 8-12mL ether washing through the rotation evaporate to dryness, continues to add the 160-220ml isopropyl alcohol and make its recrystallization, obtain the white powder crystal ,-30 ℃ of packing are frozen;
2) biotin and HSP70 polyclonal antibody lotus root connection:
1. get described biotin N-maloyl imines ester powder, be prepared into 1-2mg/ml solution with dimethyl sulfoxide, standby;
2. with the HSP70 polyclonal antibody of purifying NaHCO with 0.1mol/L pH9.00
3Be made into 2-4mg/ml solution;
3. be 1 by volume with the solution that 2. step prepares 1. with step: 4-8 mixes, and 20-30 ℃ is stirred 3.5-5.5h;
4. make above-mentioned mixing material with 0.05mol/L under 4 ℃ of conditions, pH7.2 PBS dialysis 8-12 hour adds equal-volume glycerine, and-20 ℃ of packing are deposited;
The ELISA Plate of described curing HSP70 monoclonal antibody is to make with following method:
With the described HIS-HSP70 albumen of 2mg/ml as immunizing antigen, the female mouse of Balb/c of selecting 18-20g for use is as immune animal, and get its splenocyte and SP2/0 Fusion of Cells, ELISA screening, subgroup identification, ascites preparation, monoclonal antibody purification, with the monoclonal antibody coated elisa plate of purifying, 4 ℃ of preservations.
2. a kind of people according to claim 1, rat, mouse HSP 70 double antibody sandwich method detection reagent kit is characterized in that consisting of of described sample diluting liquid: every 100ml contains NaCl0.5g, KCl0.0125g, KH
2PO
40.0125g, Na
2HPO
40.181g surplus is a water.
3. a kind of people according to claim 1, rat, mouse HSP 70 double antibody sandwich method detection reagent kit is characterized in that the every 100ml of consisting of of described lavation buffer solution contains NaCl 5g, KCl 0.125g, KH
2PO
40.125g, Na
2HPO
41.81g, Tween-20 20ul, surplus is a water.
4. a kind of people according to claim 1, rat, mouse HSP 70 double antibody sandwich method detection reagent kit, it is characterized in that consisting of of described antibody diluent: every 5ml contains calf serum 500ul, mass percentage concentration is 1% thimerosal aqueous solution 100ul, the potassium ferricyanide 50ul of 100mM, Tween-20 2.5ul, the gentamicin 25ul of 10mg/ml, surplus is the PBS of the 0.01M of pH7.4.
5. a kind of people according to claim 1, rat, mouse HSP 70 double antibody sandwich method detection reagent kit is characterized in that consisting of of described horseradish enzyme labeling Avidin dilution: every 5ml contains NaCl 0.025g, KCl 0.00125g, KH
2PO
40.00125g, Na
2HPO
40.009g surplus is a water.
6. a kind of people according to claim 1, rat, mouse HSP 70 double antibody sandwich method detection reagent kit, it is characterized in that consisting of of described substrate colour developing liquid A liquid: every 20ml contains sodium acetate 0.544g, citric acid 0.064g, concentration expressed in percentage by volume is 30% H
2O
212ul, surplus is a water.
7. a kind of people according to claim 1, rat, mouse HSP 70 double antibody sandwich method detection reagent kit, it is characterized in that consisting of of described substrate colour developing liquid B liquid: every 20ml contains EDTA-Na 0.008g, citric acid 0.038g, glycerine 2ml, tetramethyl benzidine 0.008g, surplus is a water, keeps in Dark Place.
8. a kind of people according to claim 1, rat, mouse HSP 70 double antibody sandwich method detection reagent kit is characterized in that consisting of of described stop buffer: every 20ml contains dense H
2SO
41.15ml surplus is a water.
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