CN103969438B - interleukin 6 detection kit - Google Patents
interleukin 6 detection kit Download PDFInfo
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- CN103969438B CN103969438B CN201410176133.4A CN201410176133A CN103969438B CN 103969438 B CN103969438 B CN 103969438B CN 201410176133 A CN201410176133 A CN 201410176133A CN 103969438 B CN103969438 B CN 103969438B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
The present invention relates to a kind of interleukin 6 detection kit.Kit comprises two strain specificity interleukin 6 monoclonal antibodies.The interleukin 6 of the monoclonal antibody and biotin labeled monoclonal antibody and tested sample of porous plate wrapping quilt forms the sandwich complex structure of " coated antibody-antigen-biotin labelled antibodies ".The Streptavidin that the biotin that antibody marks marks with HRP is combined, HRP and then produce signal with substrate reactions, in order to carry out interleukin 6 quantitative test.By kit of the present invention, to make it possible to rapidly, the interleukin 6 content quantitatively detected delicately in sample.
Description
Technical field
The invention belongs to molecular immunology field, be specifically related to a kind of kit detecting interleukin 6 (IL-6) protein content.
Background technology
Body there will be the state of a similar inflammatory reaction after being subject to dissimilar wound and stimulation, i.e. SIRS (SIRS).Impairment factor stimulates body inflammatory cell release inflammatory mediator in this course.Under normal circumstances, be of value to the post-traumatic recovery of body as defense mechanism, and can disappear voluntarily along with the recovery from illness of wound.But, if owing to infecting, again the reason such as damage make that this inflammatory reaction is excessive or the reaction time is long, body synthesis discharges too much inflammatory mediator, will cause chain reaction that is out of control and that amplify step by step.Body enters a kind of bad general body state, shows middle severe SIRS, and along with the state of an illness worsens further, severe multiple injuries secondary multiple organ dysfunction syndrome (MODS) will sequentially occur.Therefore, wound, SIRS, MODS are progressive processes in development, the intensity of inflammatory reaction and the development of patient trauma and lapse to closely related.
IL-6 is the precursor of calcitonin, is synthesized and secretion under physiological conditions by parafollicular cells of thyroid gland.Under normal circumstances, the existence of IL-6 in body, can not be detected, can be detected in the peripheral blood of critical patients, lung and hepatic tissue, and be considered to, to systemic infection, there is good diagnostic value and forewarning function.
IL-6 is produced by T lymphocyte, bone-marrow-derived lymphocyte and endothelial cell, and it can stimulate the propagation of T cell, B cell, and can promote that cytotoxic T cell breaks up, and improves the vigor of natural killer cell.Can detect in the early stage blood of bad person after wound that the exception of IL-6 raises.In SIRS process, first proinflammatory cytokine is activated, and then impels body to discharge IL-6, and the latter again positive feedback, in mononuclear phagocyte system and polymorphonuclear granulocyte, produces more various inflammatory mediator further.Therefore, IL-6 plays core regulating action in inflammatory reaction, is the important medium of inflammatory and immune response.
Summary of the invention
Embodiment there is provided a kind of ELISA kit detecting interleukin 6 in sample according to some, it comprises:
Be coated with the porous plate of the first interleukin 6 monoclonal antibody,
Reagent 1,
Reagent 2,
Reagent 3,
Sample dilution and
Substrate solution.
In some embodiments, be coated with the first interleukin 6 monoclonal antibody in the porous plate of the first interleukin 6 monoclonal antibody to be produced by the hybridoma of preserving number by CGMCCNo.8796 or 8797.
In some embodiments, biotin labeled interleukin 6 monoclonal antibody working fluid also made by reagent 1.It comprises sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, calf serum, biotin labeled second interleukin 6 monoclonal antibody and antiseptic.In some embodiments, the second interleukin 6 monoclonal antibody is produced by the hybridoma of preserving number by CGMCCNo.8797 or 8796.Second interleukin 6 monoclonal anti physical efficiency specific recognition in conjunction with the IL-6 in sample, the Streptavidin that the biotin that antibody marks marks with HRP is combined, HRP and then produce signal with substrate reactions.The signal produced is converted into concentration, for quantitative test.In some embodiments, reagent 1 comprises 5.8g/L sodium hydrogen phosphate, 0.593g/L sodium dihydrogen phosphate, 8.0g/L sodium chloride, 200ml/L calf serum, the biotin labeled second interleukin 6 monoclonal antibody of 0.5ml/LProclin-300 and 12.5mg/L.When the hybridoma that the first interleukin 6 monoclonal antibody is CGMCCNo.8796 by preserving number is produced, the hybridoma that the second interleukin 6 monoclonal antibody is CGMCCNo.8797 by preserving number is produced; Or when the hybridoma that the second interleukin 6 monoclonal antibody is CGMCCNo.8796 by preserving number is produced, the hybridoma that the first interleukin 6 monoclonal antibody is CGMCCNo.8797 by preserving number is produced.
In some embodiments, reagent 2 is used as the dilution of biotin labelled antibodies or is used as the dilution of horseradish peroxidase-labeled Streptavidin.In some embodiments, reagent 2 comprises sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, calf serum and antiseptic.In a particular embodiment, 5.8g sodium hydrogen phosphate, 0.593g sodium dihydrogen phosphate, 8.0g sodium chloride, 200ml calf serum and 0.5mlProclin-300 is comprised in the reagent 2 of 1000ml.
In some embodiments, reagent 3 is used as horseradish peroxidase-labeled Streptavidin working fluid.In some embodiments, reagent 3 comprises sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, calf serum, the Streptavidin of horseradish peroxidase-labeled and antiseptic.
In some embodiments, Sample dilution comprises sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, lowlenthal serum, polysorbas20 and antiseptic.In a particular embodiment, 1000ml Sample dilution is 10 ×, it comprises 58g/L sodium hydrogen phosphate, 5.94g/L sodium dihydrogen phosphate, 180g/L sodium chloride, 250ml/L lowlenthal serum, 10ml/L polysorbas20 and 5ml/L antiseptic.Sample dilution can be mixed with concentrated type, may not be.Technician is appreciated that other cycles of concentration is also included within the scope of the invention.
In some embodiments, substrate solution comprises: horseradish peroxidase substrate.In a particular embodiment, substrate solution comprises substrate solution A and substrate solution B.Substrate solution A is the citrate buffer solution containing urea peroxide.Substrate solution B is the citrate buffer solution containing TMB.
Should be appreciated that each reagent of the present invention can be mixed with concentrated type, may not be.In some embodiments, some preparation of reagents become concentrated type, and this can reduce transport, reservoir volume.Technician is appreciated that other cycles of concentration except instantiation is also included within the scope of the invention.
In some embodiments, sample from mammal, preferably from the mankind.Sample is selected from whole blood, blood plasma and serum.In a particular embodiment, sample is human serum.
In some embodiments, kit of the present invention can also comprise calibration object as required.Calibration object is used for the drafting of typical curve.Calibration object comprises the interleukin 6 of concentration known, and Interleukin-6 Concentration is within the scope of 0-1500pg/ml.In a particular embodiment, in calibration object Interleukin-6 Concentration be respectively 0,10,25,250,750,1500pg/ml.
In some embodiments, kit of the present invention can also comprise quality-control product as required.Quality-control product comprises the interleukin 6 of concentration known, and Interleukin-6 Concentration is within the scope of 200-1000pg/ml.In a particular embodiment, in quality-control product, Interleukin-6 Concentration is respectively 700pg/ml – 900pg/ml; 200pg/ml – 400pg/ml.
In some embodiments, calibration object or quality-control product are the serum of known IL-6 concentration; The human serum that serum can be any commercially available serum or collect from Clinical Institutions; It is through inactivation treatment, and anti-TP, HBsAg, anti-HCV and anti-HIV are feminine gender.In some embodiments, negative serum can also be used to dilute IL-6 sterling to prepare calibration object or quality-control product.
According to some embodiments, provide a kind of interleukin 6 monoclonal antibody, its hybridoma being CGMCCNo.8796 by preserving number is produced.
According to other embodiments, provide a kind of interleukin 6 monoclonal antibody, its hybridoma being CGMCCNo.8797 by preserving number is produced.
According to some embodiments, provide the purposes of interleukin 6 monoclonal antibody of the present invention in preparation ELISA diagnostic reagent.In a specific embodiment, the hybridoma that preserving number is CGMCCNo.8796 and 8797 is produced 2 strain interleukin 6 monoclonal antibodies jointly for the preparation of ELISA diagnostic reagent.
According to some embodiments, provide the hybridoma that preserving number is CGMCCNo.8796.
According to other embodiments, provide the hybridoma that preserving number is CGMCCNo.8797.
Embodiment
In order to make easy to understand of the present invention, set forth the present invention further below in conjunction with specific embodiment.The following provide the concrete material and source thereof that use in embodiment of the present invention.But should be understood that, these are only exemplary, be not intended to limit the present invention, all may be used for implementing the present invention with the type of following reagent and instrument, model, quality, character or the same or analogous material of function.
The preparation of embodiment 1.IL-6 recombinant protein
IL-6 coded sequence is obtained from ncbi database, build prokaryotic expression carrier pET32-IL6, abduction delivering IL-6 recombinant antigen, has immune response through western blot analyzing proteins, nickel affinity chromatography post purifying prepares IL-6 recombinant protein in a large number, and this albumen may be used for the preparation of antibody.
1.pET28-IL-6 expression vector establishment
IL-6 gene is bought in Wuhan Sanying Bio-Technology Co., Ltd., with reference to IL-6cDNA primers:
IL-6-F:cgggatccccagtacccccaggag (SEQIDNo.1) and
IL-6-R:ccgctcgagctacatttgccgaag(SEQIDNo.2)。
Pcr amplification IL-6 gene, PCR system:
PCR program:
94 DEG C 10min1 circulation
94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C 30s30 circulation
72 DEG C 10min1 circulation
PCR primer is connected with carrier T after using Ago-Gel to reclaim kit recovery, and 16 DEG C connect 1h and obtain IL-6-T, transformation of E. coli DH5 α.IL-6-T carrier is cut after qualification through enzyme, send company to check order.
Check order correct IL-6-T carrier and pET32a expression vector after BanmHI and XhoI enzyme cuts 3h,
The 16 DEG C of connections of T4DNA ligase are used to spend the night, transformation of E. coli DH5 α;
Connection product is added in 100 μ l bacillus coli DH 5 alpha competent cells, ice bath 20min, thermal shock 90s in 42 DEG C of water-baths, then 2-3min is on ice placed in rapidly, after adding the fresh LB nutrient culture media mixing of 900 μ l, 1h cultivated by 37 DEG C of shaking tables, and DH5 α is restore normal growth state.By the LB nutrient culture media of transformant solution coating containing ampicillin; 37 DEG C of constant temperature culture are spent the night.Qualification is cut through enzyme.By the recombinant plasmid transformed e. coli bl21 built.
Sequence is as follows through order-checking qualification:
ccagtacccccaggagaagattccaaagatgtagccgccccacacagacagccactcacctcttcagaacgaattgacaaacaaattcggtacatcctcgacggcatctcagccctgagaaaggagacatgtaacaagagtaacatgtgtgaaagcagcaaagaggcactggcagaaaacaacctgaaccttccaaagatggctgaaaaagatggatgcttccaatctggattcaatgaggagacttgcctggtgaaaatcatcactggtcttttggagtttgaggtatacctagagtacctccagaacagatttgagagtagtgaggaacaagccagagctgtgcagatgagtacaaaagtcctgatccagttcctgcagaaaaaggcaaagaatctagatgcaataaccacccctgacccaaccacaaatgccagcctgctgacgaagctgcaggcacagaaccagtggctgcaggacatgacaactcatctcattctgcgcagctttaaggagttcctgcagtccagcctgagggctcttcggcaaatgtag(SEQIDNo.3)。
The protein induced expression of 2.IL-6
The prokaryotic expression plasmid pET32-IL6 built transforms BL21 competent cell, and picking monoclonal bacterium colony 220rpm/min in the LB fluid nutrient medium (containing ampicillin) of 5ml, 37 DEG C of overnight incubation are cultivated.Incubated overnight bacterium is transferred in fresh LB fluid nutrient medium by 1:100,220rpm/min, and 37 DEG C of cultivations, treat bacterial concentration OD
600add IPTG when=0.6 to start to induce (final concentration is 0.25mM); 30 DEG C of 220rpm/min induce 4h to receive bacterium; Ultrasonication thalline, ultrasound condition: often process 3sec interval 7sec, ultrasonic 5min, output power 50%.9500rpm, 4 DEG C of centrifugal 20min, cleer and peaceful precipitation in separation.Sds polyacrylamide gel electrophoresis is identified.Result shows, and IL-6 albumen major part is solubility expression.
3.IL-6 protein purification
Abduction delivering 100ml bacterium liquid, 9500rpm, 4 DEG C of centrifugal 2min, collect thalline.Add sample-loading buffer 20ml (300mMNaCl, the 50mMNaH of precooling
2pO
4, 10mM imidazoles, 10mMTris alkali, pH8.0), resuspended thalline, ultrasonication thalline: often process 3sec interval 7sec, ultrasonic 20min, output power 50%.9500rpm, 4 DEG C of centrifugal 20min, cleer and peaceful precipitation in separation.
Before loading, supernatant be crossed the filter membrane of 0.22 μm.Irrigate Ni
2+-NTA affinity column, carries out slow wash-out with deionized water, avoids introducing bubble in post bed; Carry out pre-equilibration, loading with the sample-loading buffer of 10 times of column volumes, collect efflux; With dcq buffer liquid (300mMNaCl, the 50mMNaH of 20 times of bed volumes
2pO
4, 50mM imidazoles, 10mMTris alkali, pH8.0) and carry out rinsing, collect filtered solution.With elution buffer (300mMNaCl, the 50mMNaH of 5 times of bed volumes
2pO
4, 200mM imidazoles, 10mMTris alkali, pH8.0) and carry out wash-out, collect destination protein.Through BCA protein quantification kit, finally obtain 3mg, SDS-PAGE electrophoretic analysis, purity of protein is greater than 95%.
The immune response of 4.Western blotting qualification albumen
IL-6 antigen after purifying, through SDS-PAGE electrophoresis, takes out offset plate, is dipped in transfering buffering liquid by glue and balances 10min; According to size clip film and the filter paper 4 of glue, put into transfering buffering liquid and balance 10min.Pvdf membrane need be soaked saturated 3-5 second with pure methyl alcohol.Assembling transfer sandwich: spread respectively from down to up: 2 metafiltration paper-film-glue-2 metafiltration paper, every layer put well after, to rush bubble with test tube.Transfer, voltage is arranged: 24V, transfer time: 30min.Put film in 15ml Block buffer (1g/100mlBSA) 4 DEG C, spend the night.TBS/T washes 3 times (5min/T).Add the anti-IL-6 monoclonal antibody of Abcam, incubated at room 2h.TBS/T washes 3 times (5min/T).Add the sheep anti mouse that 1ml horseradish peroxidase (HRP) marks two resist, incubated at room 2h.TBS/T washes 3 times (5min/T).15mlTBS washes 2 times.Add 0.5ml chemoluminescence method liquid, be placed in gel imaging system and develop the color.Result show: anti-IL-6 monoclonal antibody can with IL-6 association reaction.
The preparation of embodiment 2. monoclonal antibody and qualification
1. immune Balb/c mouse
Choose 6 week age, female Balb/c mouse that body weight is about 20g, initial immunity, get the subcutaneous multi-point injection of above-mentioned IL-620-50 μ g Freund Freund's complete adjuvant respectively, 14th and within 28 days, carry out respectively second time and third time immunizing dose the same, the intraperitoneal injection of Freund Freund's incomplete adjuvant, merges first 3 days booster immunizations, and dosage 20-50 μ g is advisable, after 3 days, get spleen and merge.
2. the step of Fusion of Cells
Preparation feeder layer: get a non-immune Balb/c mouse, 6 weeks, draw neck to put to death, be immersed in 75% alcohol, 5min, cuts off skin by sterile scissors, expose peritonaeum, inject the nutrient solution (forbidding to puncture intestinal tube) of 6ml precooling with asepsis injector, repeatedly rinse, sucking-off washing fluid, washing fluid puts into 10ml centrifuge tube, 1200rpm/ is separated 6min, with the nutrient solution suspendible of 20% (v/v) hyclone (FCS), and adjustment cell number to 1 × 10
5/ ml, adds 96 orifice plates, and 100 μ l/ holes, put into 37 DEG C of CO
2incubator is cultivated.
Prepare immune spleen cell: get the Balb/c mouse that immunity is good, neck is drawn to put to death, asepticly get spleen, wash once with the incomplete nutrient solution of 10ml, spleen grinds, cross 200 order cell sieves, be transferred to by splenocyte in 10ml centrifuge tube, the centrifugal 10min of 800rpm, cell 10ml nutrient solution washes 2 times, cell count, gets 1 × 10
8splenic lymphocyte suspension is for subsequent use.
Preparation myeloma cell SP2/0: the growth myeloma cell that takes the logarithm is centrifugal, washes 2 times with serum-free medium, and counting, obtains 5 × 10
7cell is for subsequent use.
Fusion of Cells: myeloma cell and splenocyte are mixed in the ratio of 1:10, washes 1 time with the incomplete nutrient solution of serum-free in 50ml centrifuge tube, centrifugal, 1200rpm, 8min; Abandon supernatant, to exhaust residual liquid with suction pipe, in order to avoid affect polyglycol (PEG) concentration.Gently at the bottom of attack centrifuge tube, cell precipitation is slightly loosened.
Add 1ml45g/100mlPEG (molecular weight 4000) solution of 37 DEG C of pre-temperature, limit edged gentle agitation.37 DEG C of water-bath effect 90s.The incomplete nutrient solution adding 37 DEG C of pre-temperature, to stop PEG effect, adds 1ml, 2ml, 3ml, 4ml, 5ml and 6ml respectively every 2min.Centrifugal, 800rpm, 6min.Fill with clearly, select nutrient solution resuspended with containing 20% (v/v) calf serum HAT.By above-mentioned cell, be added in 96 orifice plates of existing feeder layer, every hole adds 100 μ l.Culture plate is put 37 DEG C, 5%CO
2cultivate in incubator.
3. the screening of hybridoma
After splenocyte and myeloma cell fusion 5 days, form the mixture of various kinds of cell, add HAT nutrient culture media 100 μ l, within the 10th day, change HT medium culture.When hybridoma is covered with 1/5 area at the bottom of hole, indirect elisa method can be adopted to detect culture supernatant, screening positive clone.Prepare IL-6 recombinant antigen coated elisa plate (5 μ g/ml) 100 μ l/ hole with embodiment 1,4 DEG C are spent the night, and by the liquid in ELISA Plate hole to the greatest extent, add PBST, repeated washing three times; Add confining liquid 200 μ l/ hole to close, be placed in 37 DEG C, 1 hour.Washing confining liquid, add 100 μ l cells and supernatant, positive control selects the immune serum of mouse, and negative control selects SP2/0 culture supernatant, and blank is cleansing solution, in 37 DEG C of standing 2h.Add HRP and mark sheep anti mouse two anti-(1:5000) 100 μ l/ hole, in 37 DEG C of standing 60min.Add PBST, repeated washing three times; Add substrate reactions liquid 100 μ l/ hole, put dark place for 37 DEG C and react 10 minutes.Add H
2sO
4(2mol/L) 50 μ l/ holes, cessation reaction.Microplate reader detects 450nm absorbance.Result successfully obtains the hybridoma cell strain of 2 strain secretion IL-6 monoclonal antibodies.
4. the cloning of hybridoma
Screen the positive colony that obtains, adopt limiting dilution assay to hybridoma cloning, clone first 1 day preparation feeder layer, the hybridoma that will the clone full nutrient culture media that cannots be used up dries up gently in culture hole, counting.Adjustment cell is 5 cell/ml.Get the Tissue Culture Plate of the feeder layer of preparation, every hole adds diluting cells 100 μ l.Hatch in 37 DEG C, 5%CO
2in incubator.Change liquid at the 7th day, within later every 3 days, change liquid 1 time.9 days visible cell Clone formation, ELISA method detects antibody titer.And by the cloning again of the strongest positive colony, until cell positive rate reaches 100%, can strain be determined; The cell expansion determining strain is cultivated.And send the preservation of preservation center.
5. the preservation of hybridoma
Hybridoma cell strain (systematic name IL-6 hybridoma cell strain) is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City) on March 5th, 2014, and deposit number is respectively CGMCCNo.8796, CGMCCNo.8797.
A large amount of productions of embodiment 3. monoclonal antibody
Possess 2 strain monoclonal antibody hybridoma cell strains: get 1 × 10
7cell concentration be injected in Balb/c mouse peritoneal, collect ascites after 10 days.By following steps monoclonal antibody purification:
1. the ascites of collecting gained is centrifugal with 2500rpm, gets supernatant.Add the saturated ammonium sulfate of isopyknic PBS (pH7.4) and 1/2 volume, 4 DEG C, standing 30min;
2. with 3000rpm, 4 DEG C, centrifugal 20min;
3. go precipitation, above reset and add isopyknic saturated ammonium sulfate, leave standstill 30min;
4. with 3000rpm, 4 DEG C, centrifugal 20min;
5. get precipitation, add 5ml physiological saline and 5ml saturated ammonium sulfate leaves standstill 30min;
6. with 3000rpm, 4 DEG C, centrifugal 20min;
7. precipitation adds the PB damping fluid (PH7.4) of 5ml physiological saline and 5ml0.02M;
8. add PB damping fluid balance ProteinG gel column (purchased from GE company) of 8 times of column volumes;
9. the mixed liquor in the 7th step is added in gel column;
10. use the PB buffer solution elution foreign protein of 10 times of column volumes;
11. collect with 0.2MpH2.8 glycine buffer antibody elution;
12. use regenerated liquid cleaning pillar;
13. with equilibrium liquid balance pillar.
The preparation of embodiment 4. IL-6-ELISA detection kit of the present invention committed step
1. preparation is coated with the porous plate of IL-6 monoclonal antibody:
A) bag quilt: adopt 0.05M, pH value to be that IL-6 monoclonal antibody (CGMCCNo.8796) prepared by the carbonate buffer solution of 9.6 and the embodiment 3 of debita spissitudo is mixed and wraps mixed liquid, and being loaded on microwell plate, 4 DEG C of bags are by 12h;
Carbonate buffer solution standard recipe:
| Natrium carbonicum calcinatum 1.600g |
| Sodium bicarbonate 2.940g |
| PH value 9.60-9.80 purified water is settled to 1000ml |
B) wash plate: dilute 20 times of concentrated washing lotion to 1 times concentration, use 1 times of concentration washing lotion to wash plate 2 times; 20 times of concentrated washing lotion standard recipes:
C) close: by confining liquid (sodium hydrogen phosphate 5.8g, sodium dihydrogen phosphate 0.593g, sodium chloride 8.0g, lowlenthal serum 100ml, Proclin-3000.5ml, purified water is settled to 1000ml) load on microwell plate, room temperature 2 hours, dry, dried overnight, obtain the porous plate being coated with IL-6 monoclonal antibody.As required, bag can be packed by plate.
2. the preparation (100 ×) of reagent 1:
Reagent 1 is used as biotin labeled interleukin 6 monoclonal antibody working fluid.
(1) markers step of biotin:
IL-6 monoclonal antibody (CGMCCNo.8797) 0.1mol/L sodium bicarbonate buffer liquid (pH8.0) is adjusted to 1mg/ml.With 0.1mol/L sodium bicarbonate buffer liquid (pH8.0), to protein enough hemodialysis.
Dissolve NHSB (N-hydroxy-succinamide biotin, purchased from Pierce) 1mg with 1mlDMSO, namely concentration is 1mg/ml.
150 μ lNHSB solution (namely containing NHSB150 μ g) are added to the monoclonal antibody solution after 1ml dialysis.
At room temperature Keep agitation 4 hours.
Add 6 μ l1mol/LNH
4cl (every 25 μ gNHSB add 1 μ l), at room temperature stirs 10 minutes.
At 4 DEG C, spend the night with PBS enough hemodialysis, to remove free NHSB.
Add 50% (v/v) glycerine, make biotin labeled interleukin 6 monoclonal antibody final concentration be 0.5mg/ml, put-20 DEG C of preservations.
(2) formula of the preparation (100 ×) of reagent 1:
5.8g/L sodium hydrogen phosphate, 0.593g/L sodium dihydrogen phosphate, 8.0g/L sodium chloride, 200ml/L calf serum, the biotin labeled second interleukin 6 monoclonal antibody of 0.5ml/LProclin-300,12.5mg/L is had, pH7.2-7.4 in 1000ml.
3. the preparation of reagent 2:
5.8g/L sodium hydrogen phosphate, 0.593g/L sodium dihydrogen phosphate, 8.0g/L sodium chloride, 200ml/L calf serum, 0.5ml/LProclin-300, pH7.2-7.4 is had in 1000ml reagent 2.
4. the preparation (100 ×) of reagent 3:
The Streptavidin (purchased from life) of 5.8g/L sodium hydrogen phosphate, 0.593g/L sodium dihydrogen phosphate, 8.0g/L sodium chloride, 200ml/L calf serum, 0.5ml/LProclin-300,12mg/L horseradish peroxidase-labeled is had in 1000ml.
5. the preparation (10 ×) of Sample dilution:
58g/L sodium hydrogen phosphate, 5.94g/L sodium dihydrogen phosphate, 180g/L sodium chloride, 250ml/L lowlenthal serum, 10ml/L polysorbas20 and 5ml/L antiseptic; PH value 6.20-6.60.
6. the preparation of substrate solution:
Horseradish peroxidase substrate: substrate solution A is the 10mmol/L citrate buffer solution containing 0.6 ‰ urea peroxides, and lucifuge stores; Substrate solution B is the 5mmol/L citrate buffer solution containing 0.4 ‰ TMB, and lucifuge stores.
7.IL-6 the preparation of calibration object:
With 1 × sample diluting liquid, high level positive serum (or IL-6 sterling) is diluted, makes the IL-6 concentration gradient of calibration object be 0,10,25,250,750,1500pg/ml.
As required, calibration object can be traced to the source to the reference material Interleukin-6HumanRecombinant (CatalogueNumber:CYT-213) of ProSpec company, purity >=97.0%.
8.IL-6 the preparation of quality-control product:
Quality-control product collects the people source sample (serum or blood plasma) from IL-6 clinical detection high level, and through inactivation treatment, its anti-TP, HBsAg, anti-HCV and anti-HIV are feminine gender after testing.Be diluted within the scope of normal concentration with 1 × Sample dilution.High level Quality Control: 700-900pg/ml; Low value Quality Control: 200-400pg/ml.2-8 DEG C saves backup.
Test case
The using method of test case 1. IL-6ELISA kit of the present invention
Sample requires: sample, without the need to special processing, adopts conventional medical technology to collect whole blood sample, draws serum or blood plasma for detecting after centrifuging.Whole blood sample please after room temperature places 2 hours or 4 DEG C are spent the night in 1000g centrifugal 20 minutes, getting supernatant can detect, or sample is put in-20 DEG C or-80 DEG C of preservations, but should avoid multigelation.Please do not use significant hemolysis, piarhemia or jaundice sample.
1. prepare before experiment
1.1. in 4 DEG C of refrigerators, take out kit, room temperature (18-25 DEG C) all should be equilibrated to.
1.2.20 use after times concentrated cleaning solution purified water or distilled water diluting 20 times.
1.3.10 use after times sample diluting liquid purified water or distilled water diluting 10 times.
2. test method
2.1. calibration object and sample to be tested is added: get the bag of sufficient amount by plate, be fixed on framework, calibration sample wells, sample to be tested hole and blank control wells are set respectively, record each hole site, except blank control wells, calibration object 100 μ l add dilution in calibration sample wells after, calibration object hole at zero point adds IL-6 sample diluting liquid 100 μ l, adds sample to be tested 100 μ l in sample to be tested hole; Add biotin labeling IL-6 antibody working fluid every hole 50 μ l, the two mixing adds room temperature reaction 120min simultaneously.
2.2. wash plate: discard liquid, thieving paper pats dry, cleansing solution is filled it up with in every hole, leaves standstill 5-10s, gets rid of cleansing solution, thieving paper pats dry, wash 5 times.
2.3. horseradish enzyme labeling Avidin: every hole adds the horseradish enzyme labeling Avidin working fluid 100 μ l after dilution, (blank control wells does not add) room temperature reaction 60min.
2.4. wash plate: discard liquid, thieving paper pats dry, cleansing solution is filled it up with in every hole, leaves standstill 5-10s, gets rid of cleansing solution, thieving paper pats dry, wash 5 times.
2.5. develop the color: every hole adds 50 μ l substrate solution A and 50 μ l substrate solution B, fully mixes, room temperature lucifuge reaction 15min.
2.6. stop: after colour developing, every hole adds 50 μ l stop buffers, fully mixes.
2.7. measure: after cessation reaction, under putting microplate reader 450nm wavelength (with blank well zeroing) or dual wavelength 450nm/630nm immediately, measure OD value.
2.8. calculate: according to the concentration of calibration object and the OD value of correspondence, use double-log linear fit mode (log (X)-log (Y)) result of calculation, result is multiplied by extension rate, is sample ultimate density.
The methodology index of the ELISA kit of test case 2. IL-6 of the present invention
1. accuracy: the recovery should between 95% ~ 105%;
2. blank detection limit: should not higher than 4.6pg/ml;
3. measuring system is linear: in 4.6pg/ml-1500pg/ml scope internal linear correlation coefficient r >=0.9900.
4. repeatability: variation within batch coefficient: CV
in batchshould not 5% be more than;
5. difference between batch: interassay coefficient of variation: CV
between batchshould not 10% be more than;
Table 1. accuracy, minimum detectability, system linear, repeated testing result
6. analyze specificity:
Detect IL-6 negative sample N1, positive sample P1 blood preparation respectively.
In N1, P1 sample, add the cross antigen of three concentration gradients respectively, detect cross antigen to the cross reaction situation of IL-6 antibody test, experimental result is in table 4:
IL-10(30pg/ml、20pg/ml、10pg/ml)、
Procalcitonin (400pg/ml, 200pg/ml, 100pg/ml),
TNF-α(25ng/ml、12.5ng/ml、6.5ng/ml)、
C reactive protein concentration (50ng/ml, 25ng/ml, 12.5ng/ml),
Calcitonin (30pg/ml, 15pg/ml, 7.5pg/ml).
Cross antigen usually has higher homology, sequence similarity with test substance or has the similar epi-position of structure.Can imagine, if when there are these cross antigens in sample, antibody may identify and in conjunction with these cross antigens, cause false positive.Therefore, the cross reactivity of diagnostic kit is an important index.This reliability for clinical effectiveness is extremely important.Interpretation of result: interleukin-10 is not more than 30pg/ml, calcitonin is not more than 30pg/ml, Procalcitonin is not more than 400pg/ml, c reactive protein is not more than 50ng/ml, TNF-α concentration when being not more than 25ng/ml, does not have a significant effect to IL-6 testing result.
7. stability: each component of kit 37 DEG C can place 6 days, preserves 3 months for 4 DEG C.
Table 2.37 DEG C shelf stability experimental result
Table 3.4 DEG C preservation condition stability inferior experimental result
R-H, R-M, R-L represent respectively: high level accuracy reference material, intermediate value accuracy reference material, low value accuracy reference material.
8. interfering material analysis:
8.1 haemoglobins:
Experimental result is in table 5.Conclusion: as minor hemolysis sample haemoglobin≤200mg/ml, on result substantially without impact, when significant hemolysis, testing result may cause false positive, therefore should not use significant hemolysis sample.
8.2 triglyceride:
Experimental result is in table 6.Conclusion: when triglyceride is in 1000mg/dl, IL-6 testing result is not had a significant impact.
8.3 cholerythrin:
Experimental result is in table 7.Interpretation of result: IL-6 testing result is had no significant effect when bilirubin concentration is not more than 20mg/dl, but sample mesobilirubin higher than 20mg/dl time, may false negative be caused.
8.4 liquaemins:
Experimental result is in table 8.Interpretation of result: IL-6 testing result is had no significant effect when concentration of heparin is not more than 40U/ml.
8.5EDTA.K2：
Experimental result is in table 9.Interpretation of result: IL-6 testing result is not had a significant effect when EDTA.K2 is not more than 600 μ g/ml.
8.6 biotins:
Experimental result is in table 10.Interpretation of result: IL-6 testing result is not had a significant effect when biotin concentration is not more than 20ng/ml.
The present invention adopts double antibody sandwich method to measure IL-6 level in sample, and the IL-6 of the antibody and biotin labelled antibodies and sample that namely ELISA Plate are wrapped quilt forms the sandwich complex structure of " coated antibody-antigen-biotin labelled antibodies ".The present invention adopts Avidin-Biotin-multienzyme complex to achieve the high sensitivity of detection effectively in addition.
Kit of the present invention can measure the content of IL-6 in serum on the one hand quickly and accurately, provides reliable clinical reference value for the early diagnosis of inflammation, early treatment; On the other hand owing to using the combination of two strain monoclonal antibody specifics, substantially increase the detection sensitivity of IL-6.
The present invention have easy to use, detect the advantages such as quick, sensitive, stable, easy and simple to handle.Be applicable to vast middle and small hospital use.
Claims (5)
1. detect an ELISA kit for interleukin 6 in sample, it comprises:
Be coated with the porous plate of the first interleukin 6 monoclonal antibody,
Reagent 1,
Reagent 2,
Reagent 3,
Sample dilution and
Substrate solution;
Wherein:
Reagent 1 comprises: sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, calf serum, biotin labeled second interleukin 6 monoclonal antibody and antiseptic;
Reagent 2 comprises: sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, calf serum and antiseptic;
Reagent 3 comprises: the Streptavidin of sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, calf serum, horseradish peroxidase-labeled and antiseptic;
Sample dilution comprises: sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, lowlenthal serum, polysorbas20 and antiseptic;
Substrate solution comprises: horseradish peroxidase substrate;
When the hybridoma that the first interleukin 6 monoclonal antibody is CGMCCNo.8796 by preserving number is produced, the hybridoma that the second interleukin 6 monoclonal antibody is CGMCCNo.8797 by preserving number is produced; Or
When the hybridoma that the second interleukin 6 monoclonal antibody is CGMCCNo.8796 by preserving number is produced, the hybridoma that the first interleukin 6 monoclonal antibody is CGMCCNo.8797 by preserving number is produced;
Sample from the mankind, and
Sample is selected from whole blood, blood plasma and serum.
2. the ELISA kit of interleukin 6 in detection sample according to claim 1, also comprises:
Calibration object, calibration object comprises the interleukin 6 of concentration known, and Interleukin-6 Concentration is within the scope of 0-1500pg/ml.
3. the ELISA kit of interleukin 6 in detection sample according to claim 1, also comprise quality-control product, quality-control product comprises the interleukin 6 of concentration known, and Interleukin-6 Concentration is within the scope of 200-1000pg/ml.
4. detect an ELISA kit for interleukin 6 in sample, it comprises:
Be coated with the porous plate of the first interleukin 6 monoclonal antibody,
Reagent 1,
Reagent 2,
Reagent 3,
Sample dilution and
Substrate solution;
Wherein:
Reagent 1 comprises: the biotin labeled second interleukin 6 monoclonal antibody of 5.8g/L sodium hydrogen phosphate, 0.593g/L sodium dihydrogen phosphate, 8.0g/L sodium chloride, 200ml/L calf serum, 0.5ml/LProclin-300 and 12.5mg/L;
Reagent 2 comprises: 5.8g/L sodium hydrogen phosphate, 0.593g/L sodium dihydrogen phosphate, 8.0g/L sodium chloride, 200ml/L calf serum and 0.5ml/LProclin-300;
Reagent 3 comprises: the Streptavidin of 5.8g/L sodium hydrogen phosphate, 0.593g/L sodium dihydrogen phosphate, 8.0g/L sodium chloride, 200ml/L calf serum, 0.5ml/LProclin-300 and 12mg/L horseradish peroxidase-labeled;
Sample dilution comprises: 58g/L sodium hydrogen phosphate, 5.94g/L sodium dihydrogen phosphate, 180g/L sodium chloride, 250ml/L lowlenthal serum, 10ml/L polysorbas20 and 5ml/L antiseptic;
Substrate solution is made up of substrate solution A and substrate solution B, and substrate solution A contains urea peroxide and citrate buffer solution, and substrate solution B contains tetramethyl benzidine and citrate buffer solution;
When the hybridoma that the first interleukin 6 monoclonal antibody is CGMCCNo.8796 by preserving number is produced, the hybridoma that the second interleukin 6 monoclonal antibody is CGMCCNo.8797 by preserving number is produced; Or
When the hybridoma that the second interleukin 6 monoclonal antibody is CGMCCNo.8796 by preserving number is produced, the hybridoma that the first interleukin 6 monoclonal antibody is CGMCCNo.8797 by preserving number is produced.
5. the purposes be combined in preparation ELISA diagnostic reagent of two strain interleukin 6 monoclonal antibodies below:
The interleukin 6 monoclonal antibody that the hybridoma being CGMCCNo.8796 by preserving number produces; And
The interleukin 6 monoclonal antibody that the hybridoma being CGMCCNo.8797 by preserving number produces.
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| CN103969438B (en) * | 2014-04-29 | 2016-03-30 | 北京普恩光德生物科技开发有限公司 | interleukin 6 detection kit |
| CN104330551B (en) * | 2014-11-17 | 2017-01-04 | 南方医科大学南方医院 | A kind of interleukin 6 quantitative determination reagent kit and preparation method thereof |
| EP3562845A2 (en) * | 2016-12-27 | 2019-11-06 | H. Hoffnabb-La Roche Ag | Novel biotin-specific monoclonal antibody and use thereof |
| CN107589250B (en) * | 2017-09-28 | 2020-08-25 | 国家食品安全风险评估中心 | TMB (Tetramethylbenzidine) two-component color developing liquid and kit with same |
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| CN108535494A (en) * | 2018-06-29 | 2018-09-14 | 湖南师范大学 | A kind of IL-6ELISA detection kits and its application method |
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| CN114217068A (en) * | 2021-10-26 | 2022-03-22 | 苏州新波生物技术有限公司 | Detection reagent strip for interleukin-6 and application thereof |
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