CN102507950A - Plasma HSP70 (Heat Shock Protein 70) antibody detection kit - Google Patents
Plasma HSP70 (Heat Shock Protein 70) antibody detection kit Download PDFInfo
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Abstract
The invention discloses a plasma HSP70 (Heat Shock Protein 70) antibody detection kit, belonging to the technical field of antibody detection. The plasma HSP70 antibody detection kit comprises an HSP70-cured enzyme-labeled plate, an HSP70 monoclonal antibody standard product, a horseradish peroxidase-labeled antibody, antibody diluents, a washing buffer solution, a substrate developing solution A, a substrate developing solution B and a stop solution. The plasma HSP70 antibody detection kit has high detection sensitivity, high accuracy and low cost and can detect the HSP70 antibodies in a tissue extracting solution, plasma and serum.
Description
Technical field
The invention belongs to the antibody test technical field, be specifically related to a kind of blood plasma HSP70 antibody assay kit.
Background technology
Heat-shock protein family (HSPs) is the albumen of one type of 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 received 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 induction type 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 like 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 in these processes, possibly play a significant role.The researcher finds that the autoantibody of Hsps in the generation of a lot of diseases, formation, prognosis process significantly changes, and prompting HSP70 antibody possibly have vital 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, causes that body produces HSP70 antibody.Recent discovers that in various rheumatic diseases and autoimmune disease, HSP70 can be discerned by body, produces autoantibody; Crowd's epidemiological study finds that HSP70 antibody and atherosclerotic progress and the order of severity are closely related.So set up fast, the method for HSP70 antibody in the reliable detection blood, important means will be provided for the monitoring of multiple disease.The method that detects HSP70 antibody now mainly contains Western Blotting, 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) that is the basis with 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 are with the further perfect ELISA method that developed of this 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 antibody kit mainly is the HSP70 antibody assay kit of the Stressgen company of the U.S. at present, and it has monopolized the market of HSP70 antibody assay kit basically; 50 of the about 5000 yuans of about working samples of price of 96 orifice plates in a detection of the kit box of the detection HSP70 antibody that present Stressgen company is commercially available, about 100 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 that a kind of detection sensitivity is high, accuracy by force, blood plasma HSP70 antibody assay kit efficiently.
A kind of blood plasma HSP70 antibody assay kit, this kit comprise antibody, antibody diluent, lavation buffer solution, substrate colour developing liquid A liquid, substrate display liquid B liquid and the stop buffer of the ELISA Plate that solidifies HSP70 albumen, HSP70 monoclonal antibody standard items, horseradish enzyme labeling;
Said HSP70 monoclonal antibody standard items are to process with following method:
(1) HIS-HSP70 albumen is recombinant expressed
Design upstream primer: 5 '-CGGAATTCATGGCCAAGAAAACAGCG-3 ', downstream primer: 5 '-CCCAAGCTTCTAATCCACCTCCTCGAT-3 ' earlier; Pcr amplification, double digestion are connected in the PET-32a carrier, transformed into escherichia coli, and the IPTG abduction delivering, purifying, SDS glue is identified purity, the dialysis postlyophilization is promptly processed the HIS-HSP70 recombinant protein, packing ,-70 ℃ are frozen;
(2) HSP70 monoclonal antibody standard items preparation
With the HIS-HSP70 recombinant protein of 2mg/ml as immunizing antigen; The female mouse of Balb/c of selecting 18-20g for use is as immune animal, and gets its splenocyte and SP2/0 Fusion of Cells, ELISA screening, subgroup identification, ascites preparation; Purifying HSP70 monoclonal antibody; Antibody purification is carried out protein quantification, and HSP70 monoclonal antibody standard items are processed in packing;
The ELISA Plate of said curing HSP70 albumen is to process with following method:
As envelope antigen, to contain the coating buffer coated elisa plate that concentration is the reorganization HIS-HSP70 recombinant protein of 1 μ g/ml, 4 ℃ are spent the night, and wash plate three times with the HIS-HSP70 recombinant protein, clap and do, 4 ℃ of preservations.
Consisting of of said antibody diluent: every 5ml contains NaCl 0.025g, KCl 0.00125g, KH
2PO
40.00125g, Na
2HPO
40.009g surplus is a water.
Consisting of of said lavation buffer solution: every 100ml contains NaCl 5g, KCl 0.125g, KH
2PO
40.125g, Na
2HPO
41.81g, Tween-20 20 μ l, surplus is a water.
Consisting of of said 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
212 μ l, surplus is a water.
Consisting of of said 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 said stop buffer: every 20ml contains H
2SO
41.15ml surplus is a water.
Beneficial effect of the present invention: detection sensitivity is high, and accuracy is strong, cost is low, can detect the HSP70 antibody in tissue extract, blood plasma and the serum simultaneously; Along with going deep into of research, blood plasma HSP70 antibody 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
Fig. 1 encapsulates the ELISA Plate preparation flow of HIS-HSP70 recombinant protein.
Fig. 2 HSP70 Monoclonal Antibody flow process.
Fig. 3 standard items HSP70 monoclonal antibody purifying protein electrophoretogram;
Among the figure, M is Protein Marker; 1 be the HSP70 monoclonal antibody sad-the ammonium sulfate purifying protein; 2 is Protein G purification column purifying HSP70 monoclonal antibody.
Fig. 4 HSP70 antibody assay kit detection sensitivity and sensing range curve.
Embodiment
Below in conjunction with accompanying drawing and specific embodiment the present invention is further specified.
The composition of embodiment 1HSP70 antibody assay kit
Blood plasma HSP70 antibody assay kit, this kit comprise antibody, antibody diluent, lavation buffer solution, substrate colour developing liquid A liquid, substrate display liquid B liquid and the stop buffer of the ELISA Plate that solidifies HSP70 albumen, HSP70 monoclonal antibody standard items, horseradish enzyme labeling; Wherein, horseradish enzymic-labelled antibody 0.5ml dilutes than 1: 1000 with front volume; Consisting of of antibody diluent: every 5ml contains NaCl 0.025g, KCl0.00125g, KH
2PO
40.00125g, Na
2HPO
40.009g surplus is a water; Consisting of of lavation buffer solution: every 100ml contains NaCl 5g, KCl 0.125g, KH
2PO
40.125g, Na
2HPO
41.81g, Tween-20 20 μ l, surplus is a water; Consisting of of 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
212 μ l, surplus is a water; Consisting of of 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 stop buffer: every 20ml contains H
2SO
41.15ml surplus is a water.
Preparation of embodiment 2HIS-HSP70 recombinant protein and purifying
The HIS-HSP70 recombinant protein is to process with following method:
1.) design the pcr amplification primer earlier:
The upper reaches: 5 ' CGGAATTCATGGCCAAGAAAACAGCG 3 '
Downstream: 5 ' CCCAAGCTTCTAATCCACCTCCTCGAT 3 '
Carry out pcr amplification then, amplification condition is:
95℃?4min；95℃?30s?56℃?60s?72℃?60s(30cycle)；72℃?10min。
Behind EcoRI and HinDIII double digestion, be connected into plasmid PET-32a.
2.) 37 ℃ of joltings of recombinant plasmid transformed e. coli bl21 add IPTG 0.5 μ l/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 GE company.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 appearance (1ml supernatant, a flow velocity 0.5ml/ml).
4.) eluent of collecting is got 100 μ l SDS glue and identify 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 ℃ subsequent use.
Reorganization HSP70 purity of protein and content detection adopt SDS glue to identify and the coomassie brilliant blue staining method respectively.Concrete grammar is:
The 300mmol/L miaow eluent of consulting is got 80 μ l during with purifying, adds 20,100 ℃ of sex change 5min of 5 * sample-loading buffer, and 10000g is centrifugal, gets appearance on the supernatant 10 μ l, 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 preparation that embodiment 3 solidifies the ELISA Plate of HSP70 albumen
The HIS-HSP70 albumen of gained is carried out ELISA Plate encapsulate, concrete steps are:
(1) get the HIS-HSP70 recombinant protein of purifying, be diluted to 1mg/ml with encapsulating damping fluid, 4 ℃ of preservations, subsequent use.
(2) with the aforesaid liquid coated elisa plate, every hole adds liquid 100 μ l, and 4 ℃ are spent the night.
(3) wash plate three times, clap and do; Every hole adds 150 μ l, 10% hyclone confining liquid, and 4 ℃ of sealings are spent the night.
(4) wash plate three times, clap and do, deposit for 4 ℃, subsequent use.
ELISA Plate to the resulting HIS-HSP70 of being coated with albumen is analyzed, and the HSP70 monoclonal antibody 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, measure the sensitivity of ELISA Plate with the ELISA method.The result: through protein purification, encapsulate after, obtain ELISA Plate, minimum degree of detecting (detection sensitivity) reaches 0.1-0.001ng.
When assembling kit of the present invention, one of ELISA Plate (96 holes encapsulate HIS-HSP70 albumen), 4 ℃ of preservations, unsuitable multigelation is valid for one year.
The preparation of embodiment 4HSP70 monoclonal antibody standard items
With the said 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 gets 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, and it is 1: 20000 that indirect ELISA is measured antibody titer.
The monoclonal antibody of gained is carried out sad-ammonium sulfate precipitation and two purifying of Protein G, and antibody purification adopts SDS glue to identify HSP70 monoclonal antibody purity; The Brad-Ford method that adopts is measured AC.The HSP70 monoclonal antibody is a positive control in the kit, is distributed into 100 μ g/ pipe, and-70 ℃ of preservations can use PBS or the dissolved in distilled water of different volumes, dilution to use during use.
Embodiment 5HSP70 antibody test
Adopt embodiment 1 described kit to carry out the HSP70 antibody test, concrete grammar is following:
1. HSP70 monoclonal antibody standard items positive control is diluted to 10 μ g/ml with the 1ml sample diluting liquid, further is diluted to 50,12.5,3.125,0.78,0.195ng/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 100 μ l, 37 ℃, 30 minutes respectively.
2. discard liquid, PBST washing three times is clapped and is done.
3. every hole adds enzyme labelled antibody 100 μ l, 37 ℃, 20 minutes.Lavation buffer solution is washed plate 3 times, and the every hole of 350 μ l//inferior is clapped and done.
4. every in regular turn hole adds each 90 μ l of substrate colour developing liquid AB, and 37 ℃ of lucifuges developed the color 15 minutes.
5. every in regular turn hole adds stop bath 90 μ l, cessation reaction.With ELIASA in 450nm wavelength readings (OD value).
Embodiment 6
The purpose of present embodiment is confirmed sensing range and the sensitivity that this kit detects.
Material: embodiment 1 said blood plasma HSP70 antibody assay kit.
Method: get positive control standard items and PBS and be made into 1 μ g/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
Embodiment 7
The purpose of present embodiment confirms 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
2SO
4, transfer 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 liquid in the 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, on coordinate axis, draws typical curve, 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 8
The purpose of present embodiment is to measure the stability of blood plasma HSP70 antibody assay kit, judges that through the degree of variation between kit inside and group confirm the stability of kit, concrete grammar is following:
1.HSP70 the inner coefficient of variation of ELISA kit is confirmed method: (25ng) each is measured 20 times separately on an ELISA Plate that encapsulates HSP70 albumen for 5ng, 12ng for three concentration known samples.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 confirmed method between the antibody assay kit reaction: 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: through two groups CV value, this kit has good stable property.
Embodiment 9 blood plasma HSP70 antibody assay kit detecting operation instructionss
Heat shock protein 70 (HSP70) antibody kit operation instructions
This kit only supplies research to use
Use
The ELISA standard measure is measured HSP70 antibody content in blood plasma and the serum.
Experimental principle
This kit is used HSP70 antibody horizontal in the enzyme-labeled immunity assay sample.HIS-HSP70 albumen with purifying encapsulates 96 hole ELISA Plates, processes insolubilized antibody, adds HSP70 monoclonal antibody, enzyme labelled antibody in the micropore of coating protein successively, through thorough washing back developing the color with substrate TMB.TMB changes into blueness under the catalysis of peroxidase, and under the effect of acid, changes into final yellow.The depth of color and the HSP70 AC in the sample are proportionate.Under the 450nm wavelength, measure absorbance (OD value), calculation sample concentration with ELIASA.
Kit is formed
ELISA Plate: (96 hole) (encapsulating HIS-HSP70 albumen).
Standard items (dried frozen aquatic products, positive control): 1 bottle, to face with preceding and be diluted to 1ml with sample diluting liquid, its concentration is 10000000pg/mL; After doing serial doubling dilution, 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.
Horseradish enzymic-labelled antibody: 1 * 500 μ l/ pipe.
Antibody diluent: 5ml/ bottle.
Lavation buffer solution: 100ml/ bottle.
Substrate colour developing liquid A:1 * 20ml/ bottle.
Substrate colour developing liquid B:1 * 20ml/ bottle.
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 the centrifugal 20min of 2000xg in room temperature, gets supernatant and can detect, or sample is put in-70 ℃ 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 the 2-8 ℃ of centrifugal 15min of 2000xg, or sample is put in-70 ℃ of preservations, but should avoids multigelation.
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 10 μ g/ml with the 1ml sample diluting liquid, further is diluted to 5012.5 3.125 0.78 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 100 μ l, 37 ℃, 30min respectively.
2. discard liquid, PBST washing three times is clapped and is done.
3. every hole adds enzyme labelled antibody 100 μ l, 37 ℃, 20min.PBST washing three times, the every hole of 350 μ l//inferior is clapped and is done.
4. every in regular turn hole adds each 90 μ l of substrate colour developing liquid AB, 37 ℃ of lucifuge colour developing 15min.
5. every in regular turn hole adds stop bath 90 μ l, cessation reaction.With ELIASA 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 2M H2SO4 at last.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 liquid in the ELISA Plate; Which floor thieving paper of place mat on experiment table, ELISA Plate are firmly clapped several times down; The lavation buffer solution of recommending at least in the 0.3ml 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 human serum (slurry) HSP70 antibody, and with other GAP-associated protein GAP no cross reaction.
Calculate
Concentration with reference material is horizontal ordinate, and the OD value is ordinate (a common coordinate), on coordinate axis, draws typical curve, 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 is prone to cause false positive.
2. an application of sample time preferably was controlled in 5 minutes, and is many like 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. like 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 in water-bath, heating 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 (6)
1. blood plasma HSP70 antibody assay kit; It is characterized in that this kit comprises antibody, antibody diluent, lavation buffer solution, substrate colour developing liquid A liquid, substrate display liquid B liquid and the stop buffer of the ELISA Plate that solidifies HSP70 albumen, HSP70 monoclonal antibody standard items, horseradish enzyme labeling;
Said HSP70 monoclonal antibody standard items are to process with following method:
(1) HIS-HSP70 albumen is recombinant expressed
Design upstream primer: 5 '-CGGAATTCATGGCCAAGAAAACAGCG-3 ', downstream primer: 5 '-CCCAAGCTTCTAATCCACCTCCTCGAT-3 ' earlier; Pcr amplification, double digestion are connected in the PET-32a carrier, transformed into escherichia coli, and the IPTG abduction delivering, purifying, SDS glue is identified purity, the dialysis postlyophilization is promptly processed the HIS-HSP70 recombinant protein, packing ,-70 ℃ are frozen;
(2) HSP70 monoclonal antibody standard items preparation
With the HIS-HSP70 recombinant protein of 2mg/ml as immunizing antigen; The female mouse of Balb/c of selecting 18-20g for use is as immune animal, and gets its splenocyte and SP2/0 Fusion of Cells, ELISA screening, subgroup identification, ascites preparation; Purifying HSP70 monoclonal antibody; Antibody purification is carried out protein quantification, and HSP70 monoclonal antibody standard items are processed in packing;
The ELISA Plate of said curing HSP70 albumen is to process with following method:
As envelope antigen, to contain the coating buffer coated elisa plate that concentration is the reorganization HIS-HSP70 recombinant protein of 1 μ g/ml, 4 ℃ are spent the night, and wash plate three times with the HIS-HSP70 recombinant protein, clap and do, 4 ℃ of preservations.
2. according to the said a kind of blood plasma HSP70 antibody assay kit of claim 1, it is characterized in that the consisting of of said antibody diluent: every 5ml contains NaCl 0.025g, KCl 0.00125g, KH
2PO
40.00125g, Na
2HPO
40.009g surplus is a water.
3. according to the said a kind of blood plasma HSP70 antibody assay kit of claim 1, it is characterized in that the consisting of of said lavation buffer solution: every 100ml contains NaCl 5g, KCl 0.125g, KH
2PO
40.125g, Na
2HPO
41.81g, Tween-20 20 μ l, surplus is a water.
4. according to the said a kind of blood plasma HSP70 antibody assay kit of claim 1, it is characterized in that the consisting of of said 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
212 μ l, surplus is a water.
5. according to the said a kind of blood plasma HSP70 antibody assay kit of claim 1, it is characterized in that the consisting of of said 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.
6. according to the said a kind of blood plasma HSP70 antibody assay kit of claim 1, it is characterized in that the consisting of of said stop buffer: every 20ml contains H
2SO
41.15ml surplus is a water.
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| CN102879584A (en) * | 2012-09-26 | 2013-01-16 | 中国人民解放军军事医学科学院基础医学研究所 | Human blood HSP70 antibody colloidal gold-labeled detection test strip and preparation method thereof |
| CN102890156A (en) * | 2012-09-26 | 2013-01-23 | 中国人民解放军军事医学科学院基础医学研究所 | Gold-labeled detection test strip of body fluid HSP70 gels and preparation method of gold-labeled detection test strip |
| CN104597249A (en) * | 2015-01-15 | 2015-05-06 | 贵州大学 | Mo Hsp70 protein based indirect ELISA (enzyme-linked immunosorbent assay) kit and using method thereof |
| CN104730241A (en) * | 2015-03-02 | 2015-06-24 | 深圳市第二人民医院 | Kit used for detecting enzymatic activity of cardiovascnlar and cerebrovascular diseases |
| CN109613255A (en) * | 2018-11-21 | 2019-04-12 | 北京利德曼生化股份有限公司 | For detecting the magnetic microparticle separating chemiluminescence immunoassay of heat shock protein 70 (HSP70) |
| CN113063937A (en) * | 2021-03-16 | 2021-07-02 | 首都医科大学附属北京佑安医院 | Evaluation marker for chronic acute liver failure and application thereof |
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| 陈胜: "用酶联免疫吸附法测定血浆HSP70抗体滴度", 《中华劳动卫生职业病杂志》 * |
| 陶娟: "系统性红斑狼疮患者抗 HSP70和HSP60 抗体的检测", 《中国麻风皮肤病杂志》 * |
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| CN102879584A (en) * | 2012-09-26 | 2013-01-16 | 中国人民解放军军事医学科学院基础医学研究所 | Human blood HSP70 antibody colloidal gold-labeled detection test strip and preparation method thereof |
| CN102890156A (en) * | 2012-09-26 | 2013-01-23 | 中国人民解放军军事医学科学院基础医学研究所 | Gold-labeled detection test strip of body fluid HSP70 gels and preparation method of gold-labeled detection test strip |
| CN104597249A (en) * | 2015-01-15 | 2015-05-06 | 贵州大学 | Mo Hsp70 protein based indirect ELISA (enzyme-linked immunosorbent assay) kit and using method thereof |
| CN104730241A (en) * | 2015-03-02 | 2015-06-24 | 深圳市第二人民医院 | Kit used for detecting enzymatic activity of cardiovascnlar and cerebrovascular diseases |
| CN109613255A (en) * | 2018-11-21 | 2019-04-12 | 北京利德曼生化股份有限公司 | For detecting the magnetic microparticle separating chemiluminescence immunoassay of heat shock protein 70 (HSP70) |
| CN113063937A (en) * | 2021-03-16 | 2021-07-02 | 首都医科大学附属北京佑安医院 | Evaluation marker for chronic acute liver failure and application thereof |
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