WO2015165357A1 - 白介素6检测试剂盒 - Google Patents

白介素6检测试剂盒 Download PDF

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WO2015165357A1
WO2015165357A1 PCT/CN2015/077288 CN2015077288W WO2015165357A1 WO 2015165357 A1 WO2015165357 A1 WO 2015165357A1 CN 2015077288 W CN2015077288 W CN 2015077288W WO 2015165357 A1 WO2015165357 A1 WO 2015165357A1
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interleukin
monoclonal antibody
reagent
accession number
produced
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French (fr)
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于晖
李雨心
王旭
陈勤慧
李鑫
刘琦
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北京普恩光德生物科技开发有限公司
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Publication of WO2015165357A1 publication Critical patent/WO2015165357A1/zh

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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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • 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
    • C07K16/244—Interleukins [IL]
    • C07K16/248—IL-6
    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/5412—IL-6

Definitions

  • the invention belongs to the field of molecular immunology, and particularly relates to a kit for detecting the content of interleukin 6 (IL-6) protein.
  • IL-6 interleukin 6
  • the body will develop an inflammatory-like state after being subjected to different types of trauma and irritation, namely systemic inflammatory response syndrome (SIRS).
  • SIRS systemic inflammatory response syndrome
  • the damaging factors stimulate the inflammatory cells of the body to release inflammatory mediators.
  • the body synthesizes and releases too much inflammatory mediator, which causes a chain reaction that is out of control and scaled up.
  • the body enters a poor systemic state and exhibits moderate to severe SIRS. As the condition worsens, severe multiple injuries secondary to multiple organ dysfunction syndrome (MODS) occur sequentially. Therefore, trauma, SIRS, and MODS are a gradual process in development, and the intensity of inflammatory response is closely related to the development and outcome of patient trauma.
  • IL-6 is a precursor of calcitonin, which is synthesized and secreted by thyroid C cells under physiological conditions. Under normal circumstances, the presence of IL-6 can not be detected in the body, can be detected in the peripheral blood, lung and liver tissues of critically ill patients, and is considered to have a good diagnostic value and early warning effect on systemic infection.
  • IL-6 is produced by T lymphocytes, B lymphocytes and endothelial cells. It stimulates the proliferation of T cells and B cells, promotes the differentiation of cytotoxic T cells, and enhances the viability of natural killer cells. Abnormal elevation of IL-6 can be detected in the early blood of patients with post-traumatic defects. During the SIRS process, pro-inflammatory cytokines are first activated, which then prompts the body to release IL-6, which in turn is fed back to the mononuclear macrophage system and polymorphonuclear granulocytes, further producing more various inflammatory mediators. Therefore, IL-6 plays a central regulatory role in the inflammatory response and is an important mediator of inflammatory immune responses.
  • an ELISA test for detecting interleukin 6 in a sample is provided a kit containing:
  • the first interleukin 6 monoclonal antibody in a multiwell plate coated with a first interleukin 6 monoclonal antibody is produced by hybridoma cells of accession number CGMCC No. 8796 or 8797.
  • Reagent 1 is also used as a biotinylated interleukin 6 monoclonal antibody working solution. It comprises disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, calf serum, a biotinylated second interleukin 6 monoclonal antibody and a preservative.
  • the second interleukin 6 monoclonal antibody is produced by hybridoma cells with accession number CGMCC No. 8797 or 8796.
  • the second interleukin 6 monoclonal antibody specifically recognizes and binds IL-6 in the sample, and the labeled biotin on the antibody binds to HRP-labeled streptavidin, which in turn reacts with the substrate to generate a signal. The resulting signal is converted to concentration for quantitative analysis.
  • Reagent 1 comprises 5.8 g/L disodium hydrogen phosphate, 0.593 g/L sodium dihydrogen phosphate, 8.0 g/L sodium chloride, 200 ml/L calf serum, 0.5 ml/L Proclin-300, And 12.5 mg/L biotinylated second interleukin 6 monoclonal antibody.
  • the second interleukin 6 monoclonal antibody is produced by a hybridoma cell having the accession number CGMCC No. 8797; or when the second When the interleukin 6 monoclonal antibody is produced by a hybridoma cell having the accession number CGMCC No. 8796, the first interleukin 6 monoclonal antibody is produced by a hybridoma cell having the accession number CGMCC No. 8797.
  • Reagent 2 is used as a dilution of a biotinylated antibody or as a dilution of horseradish peroxidase labeled streptavidin.
  • Reagent 2 comprises disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, calf serum, and a preservative.
  • 1000 ml of Reagent 2 comprises 5.8 g disodium hydrogen phosphate, 0.593 g sodium dihydrogen phosphate, 8.0 g sodium chloride, 200 ml calf serum, and 0.5 ml Proclin-300.
  • Reagent 3 is used as horseradish peroxidase-labeled streptavidin Working fluid.
  • Reagent 3 comprises disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, calf serum, horseradish peroxidase-labeled streptavidin, and a preservative.
  • the sample diluent comprises disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, goat serum, Tween 20, and a preservative.
  • the 1000 ml sample diluent is 10X, which comprises 58 g/L disodium hydrogen phosphate, 5.94 g/L sodium dihydrogen phosphate, 180 g/L sodium chloride, 250 ml/L goat serum, 10 ml/L. Tween 20 and 5ml/L preservative.
  • the sample diluent can be formulated as a concentrated type or not. The skilled person will appreciate that other concentration factors are also included within the scope of the invention.
  • the substrate fluid comprises: a horseradish peroxidase substrate.
  • the substrate fluid comprises substrate A and substrate B.
  • Substrate A is a citrate buffer containing urea peroxide.
  • Substrate B is a citrate buffer containing TMB.
  • the reagents of the present invention may be formulated in a concentrated form or may not be. In some embodiments, some of the agents are formulated in a concentrated form, which can reduce shipping and storage volumes. The skilled person will appreciate that other concentration factors other than the specific examples are also included in the scope of the present invention.
  • the sample is from a mammal, preferably from a human.
  • the sample is selected from the group consisting of whole blood, plasma, and serum.
  • the sample is human serum.
  • the kit of the invention may also include a calibrator as desired.
  • Calibrators are used for the drawing of standard curves.
  • the calibrator contains a known concentration of interleukin 6 with a concentration of interleukin 6 in the range of 0-1500 pg/ml. In a specific embodiment, the concentration of interleukin 6 in the calibrator is 0, 10, 25, 250, 750, 1500 pg/ml, respectively.
  • kits of the invention may also include controls as needed.
  • the control contains a known concentration of interleukin 6 with a concentration of interleukin 6 in the range of 200-1000 pg/ml.
  • the interleukin 6 concentration in the control is from 700 pg/ml to 900 pg/ml; 200 pg/ml to 400 pg/ml, respectively.
  • the calibrator or control is a serum of known IL-6 concentration; the serum can be any commercially available serum, or human serum collected from a clinical facility; it is inactivated, and anti-TP, HBsAg, anti-HCV and anti-HIV were negative.
  • negative serum IL-6 can also be diluted with negative serum to prepare a calibrator or control.
  • an interleukin 6 monoclonal antibody is produced, which is produced by a hybridoma cell having the accession number CGMCC No. 8796.
  • an interleukin 6 monoclonal antibody is produced which is produced by a hybridoma cell having the accession number CGMCC No. 8797.
  • an interleukin 6 monoclonal antibody of the invention in the preparation of an ELISA diagnostic reagent.
  • the hybridoma cells of accession numbers CGMCC No. 8796 and 8797 produce two interleukin 6 monoclonal antibodies for use in the preparation of ELISA diagnostic reagents.
  • a hybridoma cell with accession number CGMCC No. 8796 is provided.
  • hybridoma cells with accession number CGMCC No. 8797 are provided.
  • the IL-6 coding sequence was obtained from the NCBI database, and the prokaryotic expression vector pET32-IL6 was constructed to induce the expression of IL-6 recombinant antigen.
  • the protein was immunoreactive by Western blot analysis, and the IL-6 recombinant protein was prepared by nickel affinity chromatography. This protein can be used for the preparation of antibodies.
  • the IL-6 gene was purchased from Wuhan Sanying Biotechnology Co., Ltd., and the primers were designed with reference to the IL-6 cDNA sequence:
  • IL-6-F cgggatccccagtacccccaggag (SEQ ID No. 1)
  • IL-6-R ccgctcgagctacatttgccgaag (SEQ ID No. 2).
  • the PCR product was recovered using an agarose gel recovery kit and ligated to the T vector, and ligated at 16 ° C for 1 h to obtain IL-6-T, which was transformed into Escherichia coli DH5 ⁇ .
  • the IL-6-T vector was identified by enzyme digestion and sent to the company for sequencing.
  • the cells were digested with BanmH I and Xho I for 3 h.
  • T4 DNA ligase was ligated overnight at 16 ° C to transform E. coli DH5 ⁇ ;
  • the ligation product was added to 100 ⁇ l of E. coli DH5 ⁇ competent cells, ice-bathed for 20 min, heat-shocked in a 42 ° C water bath for 90 s, then rapidly placed on ice for 2-3 min, added to 900 ⁇ l of fresh LB medium and mixed, 37 ° C
  • the shaker was cultured for 1 h to restore DH5 ⁇ to normal growth state.
  • the transformed cell solution was plated with LB medium containing kanamycin; cultured at 37 ° C under constant temperature overnight. Identification by enzyme digestion.
  • the constructed recombinant plasmid was transformed into E. coli BL21.
  • the constructed prokaryotic expression plasmid pET32-IL6 was transformed into BL21 competent cells, and the monoclonal colonies were picked up in 5 ml of LB liquid medium (containing ampicillin) at 220 rpm/min, and cultured at 37 ° C overnight. The overnight culture was transferred to fresh LB liquid medium at 1:100, cultured at 220 rpm/min, 37 ° C.
  • IPTG was added to start induction (final concentration was 0.25 mM); 30 ° C 220 rpm /min induced 4h collection; ultrasonically disrupted cells, ultrasonic conditions: 7sec per treatment for 3sec, ultrasound for 5min, output power 50%.
  • the supernatant and the precipitate were separated by centrifugation at 9500 rpm for 20 min at 4 °C. Identification by SDS polyacrylamide gel electrophoresis. The results showed that most of the IL-6 protein was soluble.
  • the cells were induced to express 100 ml of bacteria, centrifuged at 9500 rpm for 2 min at 4 ° C, and the cells were collected.
  • Add 20 ml of pre-cooled loading buffer 300 mM NaCl, 50 mM NaH 2 PO 4 , 10 mM imidazole, 10 mM Tris base, pH 8.0
  • resuspend the cells and ultrasonically disrupt the cells: 7 sec apart for 3 sec, ultrasonic for 20 min, output
  • the power is 50%.
  • the supernatant and the precipitate were separated by centrifugation at 9500 rpm for 20 min at 4 °C.
  • Ni 2+ -NTA affinity chromatography column was prepared, slowly eluted with deionized water to avoid introducing bubbles into the column bed; pre-equilibration with 10 column volume of loading buffer, loading, collecting effluent Rinsing was carried out with 20 bed volumes of wash buffer (300 mM NaCl, 50 mM NaH 2 PO 4 , 50 mM imidazole, 10 mM Tris base, pH 8.0) and the filtrate was collected.
  • the protein of interest was collected by elution with 5 column volumes of elution buffer (300 mM NaCl, 50 mM NaH 2 PO 4 , 200 mM imidazole, 10 mM Tris base, pH 8.0). Quantified by BCA protein quantification kit, finally obtained 3mg, SDS-PAGE electrophoresis analysis, protein purity greater than 95%.
  • elution buffer 300 mM NaCl, 50 mM NaH 2 PO 4 , 200 mM imidazole, 10 mM Tris base, pH 8.0.
  • the purified IL-6 antigen was electrophoresed by SDS-PAGE, and the rubber plate was taken out, and the gel was immersed in the transfer buffer for 10 min; the membrane and the filter paper were cut according to the size of the gel, and the transfer was slowed down. Balance in the rinse for 10 min.
  • the PVDF membrane should be saturated with pure methanol for 3-5 seconds. Assembling transfer sandwiches: separately from bottom to top: 2 layers of filter paper-film-glue-2 layer filter paper. After each layer is placed, use the test tube to remove air bubbles. Transfer, voltage setting: 24V, transfer time: 30min.
  • the membrane was placed in 15 ml blocking buffer (1 g / 100 ml BSA) at 4 ° C overnight.
  • TBS/T was washed 3 times (5 min/T).
  • Abcam anti-IL-6 monoclonal antibody was added and incubated for 2 h at room temperature.
  • TBS/T was washed 3 times (5 min/T).
  • 1 ml of horseradish peroxidase (HRP)-labeled goat anti-mouse secondary antibody was added and incubated for 2 h at room temperature.
  • TBS/T was washed 3 times (5 min/T). Wash 2 times with 15ml TBS.
  • 0.5 ml of chemiluminescence solution was added and developed in a gel imaging system. The results showed that the anti-IL-6 monoclonal antibody can bind to IL-6.
  • mice Female Balb/c mice weighing 6 weeks and weighing about 20g were selected for initial immunization.
  • the above-mentioned IL-620-50 ⁇ g plus Freund's complete adjuvant was injected subcutaneously, and the second and third were performed on the 14th and 28th day respectively.
  • the sub-immunization dose was the same as above, and the incomplete peri-injection of Jiafu's incomplete adjuvant was used to strengthen the immunization 3 days before the fusion.
  • the dose was 20-50 ⁇ g, and after 3 days, the spleen fusion was performed.
  • Preparation of feeder cell layer Take an unimmunized Balb/c mouse for 6 weeks, sacrifice it by neck, soak it in 75% alcohol, 5 min, cut the skin with sterile scissors, expose the peritoneum, and inject 6 ml with a sterile syringe.
  • Pre-cooled culture solution (do not pierce the intestine), rinse repeatedly, aspirate the rinse solution, place the rinse solution in a 10ml centrifuge tube, 1200rpm/separate for 6min, mix with 20% (v/v) fetal bovine serum (FCS)
  • FCS fetal bovine serum
  • Preparation of immune spleen cells Take well-immunized Balb/c mice, sacrifice the neck, take the spleen aseptically, wash once with 10 ml of incomplete culture solution, spleen and crush, pass through 200 mesh cell sieve, transfer the spleen cells to 10 ml centrifugation. In the tube, centrifuge at 800 rpm for 10 min, the cells were washed twice with 10 ml of the culture solution, the cells were counted, and 1 ⁇ 10 8 spleen lymphocyte suspension was taken for use.
  • Cell fusion the myeloma cells and the spleen cells were mixed together in a ratio of 1:10, washed once with a serum-free incomplete culture solution in a 50 ml centrifuge tube, centrifuged, 1200 rpm, 8 min; Discard the supernatant and use a pipette to remove residual liquid to avoid affecting the polyethylene glycol (PEG) concentration. Gently hit the bottom of the tube to loosen the cell pellet slightly.
  • PEG polyethylene glycol
  • a solution of 1 ml of 45 g/100 ml of PEG (molecular weight 4000) pre-warmed at 37 ° C was added and shaken gently while adding. 37 ° C water bath for 90s. An incomplete medium pre-warmed at 37 ° C was added to terminate the action of PEG, and 1 ml, 2 ml, 3 ml, 4 ml, 5 ml and 6 ml were added every 2 min. Centrifugation, 800 rpm, 6 min. The supernatant was filled and resuspended in a culture medium containing 20% (v/v) calf serum HAT. The above cells were added to a 96-well plate of an existing feeder cell layer, and 100 ⁇ l was added to each well. The plate was incubated at 37 ° C in a 5% CO 2 incubator.
  • IL-6 recombinant antigen-coated enzyme plate 5 ⁇ g/ml 100 ⁇ l/well in Example 1 and incubate at 4°C overnight, pour the liquid in the well of the microtiter plate, add PBST, repeat the washing three times; add blocking solution 200 ⁇ l The wells were blocked and placed at 37 ° C for 1 hour.
  • the blocking solution was washed, 100 ⁇ l of the cell culture supernatant was added, the positive control was selected from the immune serum of the mouse, the negative control was selected as the SP2/0 culture supernatant, and the blank was washed, and allowed to stand at 37 ° C for 2 h.
  • HRP-labeled goat anti-mouse secondary antibody (1:5000) 100 ⁇ l/well was added and allowed to stand at 37 ° C for 60 min.
  • PBST was added, and the washing was repeated three times; 100 ⁇ l/well of the substrate reaction solution was added, and the reaction was allowed to stand in the dark at 37 ° C for 10 minutes.
  • the reaction was terminated by adding H 2 SO 4 (2 mol/L) at 50 ⁇ l/well.
  • the plate reader measures the absorbance at 450 nm. As a result, two hybridoma cell lines secreting IL-6 monoclonal antibodies were successfully obtained.
  • the positive clones were screened, and the hybridomas were cloned by limiting dilution method.
  • the feeder cells were prepared 1 day before the cloning, and the hybridoma cells to be cloned were gently dried from the culture wells with incomplete medium and counted. Adjust the cells to 5 cells/ml. A cell culture plate of the prepared feeder cell layer was taken, and 100 ⁇ l of the diluted cells were added to each well. Incubate in a 37 ° C, 5% CO 2 incubator. The liquid was changed on the 7th day, and the liquid was changed once every 3 days. Cell clone formation was observed in 9 days, and antibody titer was detected by ELISA. The strongest positive clone was cloned again until the cell positive rate reached 100%, and the fixed strain was established; And send it to the depository for preservation.
  • Hybridoma cell line (classified name IL-6 hybridoma cell line) on March 5, 2014 Nissho is located in the General Microbiology Center of China Microbial Culture Collection Management Committee (No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing). The preservation numbers are CGMCC No.8796 and CGMCC No.8797 respectively.
  • a) coating a 0.05 M, pH 9.6 carbonate buffer was mixed with an appropriate concentration of the IL-6 monoclonal antibody prepared in Example 3 (CGMCC No. 8796) to prepare a coating mixture, and It is loaded on a microplate and coated at 4 ° C for 12 h;
  • Blocking The blocking solution (5.8 g of disodium hydrogen phosphate, 0.593 g of sodium dihydrogen phosphate, 8.0 g of sodium chloride, 100 ml of goat serum, 0.5 ml of Proclin-300, and purified water to 1000 ml) was loaded on a microplate. After 2 hours at room temperature, dried and dried overnight, a porous plate coated with IL-6 mAb was obtained. The coated panels can be packaged as needed.
  • Reagent 1 was used as a biotinylated interleukin 6 monoclonal antibody working solution.
  • the IL-6 monoclonal antibody (CGMCC No. 8797) was adjusted to 1 mg/ml with 0.1 mol/L sodium bicarbonate buffer (pH 8.0). The protein was dialyzed extensively with 0.1 mol/L sodium bicarbonate buffer (pH 8.0).
  • NHBS N-hydroxysuccinimide biotin, purchased from Pierce
  • the dialysis was repeated overnight at 4 ° C with PBS to remove free NHSB.
  • 1000ml contains 5.8g/L disodium hydrogen phosphate, 0.593g/L sodium dihydrogen phosphate, 8.0g/L sodium chloride, 200ml/L calf serum, 0.5ml/L Proclin-300, 12.5mg/L biotin.
  • 1000ml reagent 2 contains 5.8g/L disodium hydrogen phosphate, 0.593g/L sodium dihydrogen phosphate, 8.0g/L sodium chloride, 200ml/L calf serum, 0.5ml/L Proclin-300, pH 7.2-7.4 .
  • 1000ml contains 5.8g/L disodium hydrogen phosphate, 0.593g/L sodium dihydrogen phosphate, 8.0g/L sodium chloride, 200ml/L calf serum, 0.5ml/L Proclin-300, 12mg/L horseradish. Oxidase-labeled streptavidin (purchased from life).
  • Horseradish peroxidase substrate is a 10 mmol/L citrate buffer containing 0.6 ⁇ carbamide peroxide, stored in the dark; substrate B is 5 mmol/L citrate buffer containing 0.4 ⁇ TMB. , protected from light storage.
  • High-value positive serum (or pure IL-6) was diluted with 1 ⁇ sample dilution to give a calibrator IL-6 concentration gradient of 0, 10, 25, 250, 750, 1500 pg/ml.
  • the calibrator is traceable to ProSpec's reference material Interleukin-6 Human Recombinant (Catalogue Number: CYT-213) as needed, with a purity of ⁇ 97.0%.
  • control samples were collected from human samples (serum or plasma) with high values of IL-6 clinical tests. After inactivation, the anti-TP, HBsAg, anti-HCV and anti-HIV were negative. Dilute to a specified concentration range with a 1 ⁇ sample dilution. High value quality control: 700-900pg/ml; low value quality control: 200-400pg/ml. Store at 2-8 °C for later use.
  • Test Example 1 Method of Using IL-6 ELISA Kit of the Present Invention
  • Sample requirements Specimens are not subject to special treatment. Whole blood samples are collected using conventional medical techniques. After centrifugation, serum or plasma is taken for testing. Whole blood samples should be placed at room temperature for 2 hours or 4 °C overnight, centrifuged at 1000g for 20 minutes, the supernatant can be detected, or the specimens can be stored at -20 ° C or -80 ° C, but repeated freezing and thawing should be avoided. Please do not use severe hemolysis, lipemia or jaundice specimens.
  • the 20-fold concentrated washing solution was diluted 20 times with purified water or distilled water and used.
  • sample dilution is diluted 10 times with purified water or distilled water.
  • Washing the plate Discard the liquid, pat dry on the absorbent paper, fill each well with the washing solution, let stand for 5-10s, remove the washing solution, pat dry on the absorbent paper, and wash 5 times.
  • Horseradish enzyme labeled avidin 100 ⁇ l of the diluted horseradish-labeled avidin working solution was added to each well, and the blank control well was not added at room temperature for 60 min.
  • Washing the plate Discard the liquid, pat dry on the absorbent paper, fill each well with the washing solution, let stand for 5-10s, remove the washing solution, pat dry on the absorbent paper, and wash 5 times.
  • the OD value was measured at a wavelength of 450 nm (zeroed with a blank well) or at a dual wavelength of 450 nm / 630 nm.
  • Test Example 2 Methodological indicators of the ELISA kit for IL-6 of the present invention
  • the recovery rate should be between 95% and 105%;
  • Blank detection limit should not be higher than 4.6pg/ml
  • the blood samples of the IL-1 negative sample N1 and the positive sample P1 were separately detected.
  • IL-10 (30pg/ml, 20pg/ml, 10pg/ml),
  • TNF- ⁇ (25 ng/ml, 12.5 ng/ml, 6.5 ng/ml),
  • C-reactive protein concentration 50 ng/ml, 25 ng/ml, 12.5 ng/ml
  • Calcitonin (30 pg/ml, 15 pg/ml, 7.5 pg/ml).
  • the cross-reacting agent usually has a higher homology, a similar sequence or a structurally similar epitope with the substance to be tested. It is conceivable that if these cross-reactants are present in the sample, the antibodies may recognize and bind to these cross-reactants, resulting in false positives. Therefore, the cross-reactivity of diagnostic kits is an important indicator. This is very important for the reliability of clinical outcomes. Analysis of results: 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, and TNF- ⁇ concentration is not more than 25ng/ml. There is no significant effect on the IL-6 test results.
  • the components of the kit can be placed at 37 ° C for 6 days and stored at 4 ° C for 3 months.
  • R-H, R-M, and R-L represent: high-value accuracy reference, median accuracy reference, and low-value accuracy reference.
  • the present invention uses a double antibody sandwich method to determine the level of IL-6 in a sample, that is, the coated antibody on the plate and the biotin-labeled antibody and the IL-6 of the test sample form a "coating antibody-antigen-biotin labeled antibody". Sandwich composite structure.
  • the present invention effectively achieves high sensitivity of detection using the avidin-biotin-enzyme complex.
  • the kit of the invention can rapidly and accurately determine the content of IL-6 in serum, and provides reliable clinical reference value for early diagnosis and early treatment of inflammation; on the other hand, the combination of two specific monoclonal antibodies is used. , greatly improved the detection sensitivity of IL-6.
  • the invention has the advantages of simple use, rapid detection, sensitivity, stability, and convenient operation. Applicable to the majority of small and medium hospitals.

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Abstract

一种白介素6检测试剂盒，其包含两株特异性白介素6单克隆抗体。多孔板上包被的单克隆抗体、生物素标记的单克隆抗体、被测样本的白介素6形成"包被抗体-抗原-生物素标记抗体"的夹心复合物结构。抗体上所标记的生物素与HRP标记的链霉亲和素结合，HRP进而与底物反应产生信号，用以进行白介素6定量分析。所述试剂盒能够迅速、灵敏地定量检测样本中的白介素6含量。

Description

白介素6检测试剂盒 技术领域
本发明属于分子免疫学领域，具体涉及一种检测白介素6(IL-6)蛋白含量的试剂盒。
背景技术
机体在受到不同类型的创伤和刺激后会出现一个类似炎症反应的状态，即全身炎症反应综合征(SIRS)。在这一过程中损伤因素刺激机体炎性细胞释放炎症介质。通常情况下，作为防御机制有益于机体创伤后的恢复，并会随着创伤的痊愈而自行消退。然而，如果由于感染、再次损伤等原因使这种炎症反应过度或者反应时间过长，机体合成释放过多的炎症介质，就会引起失控和逐级放大的连锁反应。机体进入一种不良的全身状态，表现出中重度SIRS，随着病情进一步恶化，严重多发伤继发多器官功能不全综合征(MODS)就会序贯发生。因此，创伤、SIRS、MODS在发展上是一个渐进的过程，炎症反应的强度与患者创伤的发展和转归密切相关。
IL-6是降钙素的前体，生理情况下由甲状腺C细胞合成与分泌。正常情况下，体内不能检测到IL-6的存在，在危重病患者的外周血、肺及肝组织中可被检测到，且被认为对全身性感染具有较好的诊断价值和预警作用。
IL-6由T淋巴细胞、B淋巴细胞及内皮细胞产生，它能刺激T细胞、B细胞的增殖，并能促进细胞毒性T细胞分化，提高自然杀伤细胞的活力。创伤后不良者早期血液中即可检测到IL-6的异常升高。在SIRS过程中，促炎细胞因子首先被激活，而后促使机体释放IL-6，后者又正反馈于单核巨噬细胞系统和多形核粒细胞，进一步产生更多的各种炎症介质。因此，IL-6在炎症反应中起核心调节作用，是炎症免疫反应的重要介质。
发明内容
根据一些实施方式，提供了一种检测样本中白介素6的ELISA试 剂盒，其包含：
包被有第一白介素6单克隆抗体的多孔板、
试剂1、
试剂2、
试剂3、
样本稀释液、和
底物液。
在一些实施方案中，包被有第一白介素6单克隆抗体的多孔板中第一白介素6单克隆抗体由保藏号为CGMCC No.8796或8797的杂交瘤细胞所产生。
在一些实施方案中，试剂1也作生物素标记的白介素6单克隆抗体工作液。其包含磷酸氢二钠、磷酸二氢钠、氯化钠，小牛血清、生物素标记的第二白介素6单克隆抗体和防腐剂。在一些实施方案中，第二白介素6单克隆抗体由保藏号为CGMCC No.8797或8796的杂交瘤细胞所产生。第二白介素6单克隆抗体能特异性识别并结合样本中的IL-6，抗体上所标记的生物素与HRP标记的链霉亲和素结合，HRP进而与底物反应产生信号。产生的信号换算成浓度，用于定量分析。在一些实施方案中，试剂1包含5.8g/L磷酸氢二钠、0.593g/L磷酸二氢钠、8.0g/L氯化钠、200ml/L小牛血清、0.5ml/L Proclin-300、和12.5mg/L生物素标记的第二白介素6单克隆抗体。当第一白介素6单克隆抗体由保藏号为CGMCC No.8796的杂交瘤细胞所产生时，第二白介素6单克隆抗体由保藏号为CGMCC No.8797的杂交瘤细胞所产生；或者当第二白介素6单克隆抗体由保藏号为CGMCC No.8796的杂交瘤细胞所产生时，第一白介素6单克隆抗体由保藏号为CGMCC No.8797的杂交瘤细胞所产生。
在一些实施方案中，试剂2用作生物素标记抗体的稀释液、或用作辣根过氧化物酶标记链霉亲和素的稀释液。在一些实施方案中，试剂2包含磷酸氢二钠、磷酸二氢钠、氯化钠，小牛血清、和防腐剂。在具体实施方案中，1000ml的试剂2中包含5.8g磷酸氢二钠、0.593g磷酸二氢钠、8.0g氯化钠、200ml小牛血清和0.5ml Proclin-300。
在一些实施方案中，试剂3用作辣根过氧化物酶标记链霉亲和素 工作液。在一些实施方案中，试剂3包含磷酸氢二钠、磷酸二氢钠、氯化钠、小牛血清、辣根过氧化物酶标记的链霉亲和素、和防腐剂。
在一些实施方案中，样本稀释液包含磷酸氢二钠、磷酸二氢钠、氯化钠、山羊血清、吐温20和防腐剂。在具体实施方案中，1000ml样本稀释液是10×的，其包括58g/L磷酸氢二钠、5.94g/L磷酸二氢钠、180g/L氯化钠、250ml/L山羊血清、10ml/L吐温20和5ml/L防腐剂。样本稀释液可以配制成浓缩型，也可以不是。技术人员可以理解，其它浓缩倍数也包括在本发明范围内。
在一些实施方案中，底物液包含：辣根过氧化物酶底物。在具体实施方案中，底物液包含底物液A和底物液B。底物液A是含有过氧化脲的柠檬酸缓冲液。底物液B是含有TMB的柠檬酸缓冲液。
应当理解，本发明的各试剂可以配制成浓缩型，也可以不是。在一些实施方案中，一些试剂配制成浓缩型，这可以减少运输、储存体积。技术人员可以理解，除了具体实例以外的其它浓缩倍数也包括在本发明范围内。
在一些实施方案中，样本来自哺乳动物，优选来自人类。样本选自全血、血浆、和血清。在具体实施方案中，样本为人血清。
在一些实施方案中，本发明的试剂盒还可以根据需要包括校准品。校准品用于标准曲线的绘制。校准品包含已知浓度的白介素6，白介素6浓度在0-1500pg/ml范围内。在具体实施方案中，校准品中白介素6浓度分别为0、10、25、250、750、1500pg/ml。
在一些实施方案中，本发明的试剂盒还可以根据需要包括质控品。质控品包含已知浓度的白介素6，白介素6浓度在200-1000pg/ml范围内。在具体实施方案中，质控品中白介素6浓度分别为700pg/ml–900pg/ml；200pg/ml–400pg/ml。
在一些实施方案中，校准品或质控品是已知IL-6浓度的血清；血清可以是任何市售血清、或收集自临床机构的人血清；其经灭活处理，并且抗-TP、HBsAg、抗-HCV和抗-HIV均为阴性。在一些实施方案中，还可以使用阴性血清稀释IL-6纯品来制备校准品或质控品。
根据一些实施方案，提供了一种白介素6单克隆抗体，其由保藏号为CGMCC No.8796的杂交瘤细胞所产生。
根据另一些实施方案，提供了一种白介素6单克隆抗体，其由保藏号为CGMCC No.8797的杂交瘤细胞所产生。
根据一些实施方案，提供了本发明的白介素6单克隆抗体在制备ELISA诊断试剂中的用途。在一个具体实施方案中，保藏号为CGMCC No.8796和8797的杂交瘤细胞所产生2株白介素6单克隆抗体共同用于制备ELISA诊断试剂。
根据一些实施方案，提供了保藏号为CGMCC No.8796的杂交瘤细胞。
根据另一些实施方案，提供了保藏号为CGMCC No.8797的杂交瘤细胞。
具体实施方式
为了使本发明易于理解，下面结合具体实施例进一步阐述本发明。以下提供了本发明实施方式中所使用的具体材料及其来源。但是，应当理解的是，这些仅仅是示例性的，并不意图限制本发明，与如下试剂和仪器的类型、型号、品质、性质或功能相同或相似的材料均可以用于实施本发明。
实施例1.IL-6重组蛋白的制备
从NCBI数据库中获得IL-6编码序列，构建原核表达载体pET32-IL6，诱导表达IL-6重组抗原，经Western印迹分析蛋白有免疫反应，镍亲和层析柱纯化大量制备IL-6重组蛋白，该蛋白可以用于抗体的制备。
1.pET28-IL-6表达载体构建
IL-6基因购买于武汉三鹰生物技术有限公司，参照IL-6cDNA序列设计引物：
IL-6-F:cgggatccccagtacccccaggag(SEQ ID No.1)和
IL-6-R:ccgctcgagctacatttgccgaag(SEQ ID No.2)。
PCR扩增IL-6基因，PCR体系：
Figure PCTCN2015077288-appb-000001
Figure PCTCN2015077288-appb-000002
PCR程序：
94℃10min                    1个循环
94℃30s，55℃30s，72℃30s    30个循环
72℃10min                    1个循环
PCR产物使用琼脂糖凝胶回收试剂盒回收后与T载体连接，16℃连接1h获得IL-6-T，转化大肠杆菌DH5α。IL-6-T载体经酶切鉴定后，送公司测序。
测序正确的IL-6-T载体与pET32a表达载体经BanmH I和Xho I酶切3h后，
Figure PCTCN2015077288-appb-000003
使用T4DNA连接酶16℃连接过夜，转化大肠杆菌DH5α；
Figure PCTCN2015077288-appb-000004
将连接产物加到100μl大肠杆菌DH5α感受态细胞中，冰浴20min，在42℃水浴中热击90s，然后迅速置于冰上2-3min，加入900μl新鲜的LB培养基混匀后，37℃摇床培养1h，使DH5α恢复正常生长状态。将转化细胞溶液涂布含有卡那霉素的LB培养基；37℃恒温培养过夜。经酶切鉴定。将构建好的重组质粒转化大肠杆菌BL21。
序列经测序鉴定如下：
ccagtacccccaggagaagattccaaagatgtagccgccccacacagacagccactcacctcttcagaacgaattgacaaacaaattcggtacatcctcgacggcatctcagccctgagaaaggagacatgtaacaagagtaacatgtgtgaaagcagcaaaga ggcactggcagaaaacaacctgaaccttccaaagatggctgaaaaagatggatgcttccaatctggattcaatgaggagacttgcctggtgaaaatcatcactggtcttttggagtttgaggtatacctagagtacctccagaacagatttgagagtagtgaggaacaagccagagctgtgcagatgagtacaaaagtcctgatccagttcctgcagaaaaaggcaaagaatctagatgcaataaccacccctgacccaaccacaaatgccagcctgctgacgaagctgcaggcacagaaccagtggctgcaggacatgacaactcatctcattctgcgcagctttaaggagttcctgcagtccagcctgagggctcttcggcaaatgtag(SEQ ID No.3)。
2.IL-6蛋白诱导表达
构建好的原核表达质粒pET32-IL6转化BL21感受态细胞，挑取单克隆菌落于5ml的LB液体培养基(含氨苄青霉素)中220rpm/min，37℃培养过夜培养。过夜培养菌按1:100转接到新鲜的LB液体培养基中，220rpm/min，37℃培养，待菌液浓度OD600＝0.6时加入IPTG开始诱导(终浓度为0.25mM)；30℃220rpm/min诱导4h收菌；超声破碎菌体，超声条件：每处理3sec间隔7sec，超声5min，输出功率50％。9500rpm，4℃离心20min，分离上清和沉淀。SDS聚丙烯酰胺凝胶电泳鉴定。结果显示，IL-6蛋白大部分为可溶性表达。
3.IL-6蛋白纯化
诱导表达100ml菌液，9500rpm，4℃离心2min，收集菌体。加入预冷的上样缓冲液20ml(300mM NaCl，50mM NaH2PO4，10mM咪唑，10mM Tris碱，pH 8.0)，重悬菌体，超声破碎菌体：每处理3sec间隔7sec，超声20min，输出功率50％。9500rpm，4℃离心20min，分离上清和沉淀。
上样前，要将上清过0.22μm的滤膜。灌制好Ni2+-NTA亲和层析柱，用去离子水进行缓慢洗脱，避免在柱床中引入气泡；用10倍柱体积的上样缓冲液进行预平衡，上样，收集流出液；用20倍柱床体积的冲洗缓冲液(300mM NaCl，50mM NaH2PO4，50mM咪唑，10mM Tris碱，pH 8.0)进行漂洗，收集滤过液。用5倍柱床体积的洗脱缓冲液(300mM NaCl，50mM NaH2PO4，200mM咪唑，10mM Tris碱，pH 8.0)进行洗脱，收集目的蛋白。经BCA蛋白定量试剂盒定量，最终得到3mg，SDS-PAGE电泳分析，蛋白纯度大于95％。
4.Western印迹法鉴定蛋白的免疫反应
纯化后的IL-6抗原，经SDS-PAGE电泳，取出胶板，将胶浸于转移缓冲液中平衡10min；依据胶的大小剪取膜和滤纸4片，放入转移缓 冲液中平衡10min。将PVDF膜需用纯甲醇浸泡饱和3-5秒钟。装配转移三明治：由下到上分别铺：2层滤纸-膜-胶-2层滤纸，每层放好后，用试管赶去气泡。转移，电压设置：24V，转移时间：30min。置膜于15ml封闭缓冲液(1g/100ml BSA)中4℃，过夜。TBS/T洗3次(5min/T)。加入Abcam的抗IL-6单克隆抗体，室温孵育2h。TBS/T洗3次(5min/T)。加入1ml辣根过氧化酶(HRP)标记的羊抗鼠的二抗，室温孵育2h。TBS/T洗3次(5min/T)。15ml TBS洗2次。加入0.5ml化学发光法液，置于凝胶成像系统中显色。结果显示：抗IL-6单克隆抗体可以和IL-6结合反应。
实施例2.单克隆抗体的制备与鉴定
1.免疫Balb/c小鼠
选取6周龄、体重约20g的雌性Balb/c小鼠，初次免疫，分别取上述IL-620-50μg加福氏完全佐剂皮下多点注射，第14和28天分别进行第二次和第三次免疫剂量同上，加福氏不完全佐剂腹腔内注射，融合前3天加强免疫，剂量20-50μg为宜，3天后，取脾融合。
2.细胞融合的步骤
制备饲养细胞层：取一只未免疫的Balb/c小鼠，6周，拉颈处死，浸泡在75％酒精内，5min，用无菌剪刀剪开皮肤，暴露腹膜，用无菌注射器注入6ml预冷的培养液(严禁刺破肠管)，反复冲洗，吸出冲洗液，冲洗液放入10ml离心管，1200rpm/分离6min，用20％(v/v)胎牛血清(FCS)的培养液混悬，调整细胞数至1×105/ml，加入96孔板，100μl/孔，放入37℃CO2孵箱培养。
制备免疫脾细胞：取免疫好的Balb/c小鼠，拉颈处死，无菌取脾脏，用10ml不完全培养液洗一次，脾脏研碎，过200目细胞筛，将脾细胞转移至10ml离心管中，800rpm离心10min，细胞用10ml培养液洗2次，细胞计数，取1×108脾淋巴细胞悬液备用。
制备骨髓瘤细胞SP2/0：取对数生长骨髓瘤细胞离心，用无血清培养液洗2次，计数，取得5×107细胞备用。
细胞融合：将骨髓瘤细胞与脾细胞按1：10的比例混合在一起，在50ml离心管中用无血清不完全培养液洗1次，离心，1200rpm，8min； 弃上清，用吸管吸净残留液体，以免影响聚乙二醇(PEG)浓度。轻轻弹击离心管底，使细胞沉淀略松动。
加入37℃预温的1ml 45g/100ml PEG(分子量4000)溶液，边加边轻微摇动。37℃水浴作用90s。加37℃预温的不完全培养液以终止PEG作用，每隔2min分别加入1ml、2ml、3ml、4ml、5ml和6ml。离心，800rpm,6min。充上清，用含20％(v/v)小牛血清HAT选择培养液重悬。将上述细胞，加到已有饲养细胞层的96孔板内，每孔加100μl。将培养板置37℃、5％CO2培养箱中培养。
3.杂交瘤细胞的筛选
脾细胞和骨髓瘤细胞融合后5天，形成多种细胞的混合体，补加HAT培养基100μl，第10天换HT培养基培养。杂交瘤细胞布满孔底1/5面积时，即可采用间接ELISA法检测培养上清，筛选阳性克隆。以实施例1制备IL-6重组抗原包被酶标板(5μg/ml)100μl/孔,4℃过夜，将酶标板孔中的液体倒尽，加入PBST，重复洗涤三次；加入封闭液200μl/孔进行封闭，置于37℃，1小时。洗涤封闭液，加入100μl细胞培养上清，阳性对照选小鼠的免疫血清，阴性对照选SP2/0培养上清，空白为洗涤液，于37℃静置2h。加入HRP标记羊抗鼠二抗(1:5000)100μl/孔，于37℃静置60min。加入PBST，重复洗涤三次；加入底物反应液100μl/孔，37℃置暗处反应10分钟。加入H2SO4(2mol/L)50μl/孔，终止反应。酶标仪检测450nm吸光度值。结果成功得到2株分泌IL-6单克隆抗体的杂交瘤细胞株。
4.杂交瘤的克隆化
筛选得到的阳性克隆，采用有限稀释法对杂交瘤克隆化，克隆前1天制备饲养细胞层，将要克隆的杂交瘤细胞用不完全培养基从培养孔内轻轻吹干，计数。调整细胞为5个细胞/ml。取准备的饲养细胞层的细胞培养板，每孔加入稀释细胞100μl。孵育于37℃、5％CO2孵箱中。在第7天换液，以后每3天换液1次。9天可见细胞克隆形成，ELISA法检测检测抗体效价。并将最强的阳性克隆再次克隆化，直至细胞阳性率达100％，即可定株；定株的细胞扩大培养。并送保藏中心保藏。
5.杂交瘤细胞的保藏
将杂交瘤细胞株(分类名IL-6杂交瘤细胞株)于2014年3月5 日保藏于中国微生物菌种保藏管理委员会普通微生物中心(北京市朝阳区北辰西路1号院3号)，保藏编号分别为CGMCC No.8796、CGMCC No.8797。
实施例3.单克隆抗体的大量生产
已具备2株单克隆抗体杂交瘤细胞株：取1×107的细胞浓度注射于Balb/c小鼠腹腔，10天后收集腹水。通过以下步骤纯化单克隆抗体：
1.收集所得的腹水以2500rpm离心，取上清。加等体积的PBS(pH7.4)与1/2体积的饱和硫酸铵，4℃、静置30min；
2.以3000rpm，4℃，离心20min；
3.去沉淀，上清加等体积的饱和硫酸铵，静置30min；
4.以3000rpm，4℃，离心20min；
5.取沉淀，加5ml生理盐水与5ml饱和硫酸铵静置30min；
6.以3000rpm，4℃，离心20min；
7.沉淀加5ml生理盐水与5ml 0.02M的PB缓冲液(PH7.4)；
8.加8倍柱体积的PB缓冲液平衡Protein G凝胶柱(购自GE公司)；
9.将第7步中的混合液加到凝胶柱中；
10.使用10倍柱体积的PB缓冲液洗脱杂蛋白；
11.用0.2M pH2.8甘氨酸缓冲液洗脱抗体并收集；
12.使用再生液清洗柱子；
13.加平衡液平衡柱子。
实施例4.本发明IL-6-ELISA检测试剂盒关键步骤的制备
1.制备包被有IL-6单抗的多孔板：
a)包被：采用0.05M、pH值为9.6的碳酸盐缓冲液与适当浓度的实施例3制备的IL-6单克隆抗体(CGMCC No.8796)混合制成包被混合液，并将其加载于微孔板上，4℃包被12h；
碳酸盐缓冲液标准配方：
无水碳酸钠        1.600g
碳酸氢钠          2.940g
pH值9.60-9.80     纯化水定容至1000ml
b)洗板：稀释20倍浓缩洗液至1倍浓度，使用1倍浓度洗液洗板 2次；20倍浓缩洗液标准配方：
Figure PCTCN2015077288-appb-000005
c)封闭：将封闭液(磷酸氢二钠5.8g，磷酸二氢钠0.593g，氯化钠8.0g，山羊血清100ml，Proclin-3000.5ml，纯化水定容至1000ml)加载于微孔板上，室温2小时，甩干，干燥过夜，获得包被有IL-6单抗的多孔板。根据需要，可以将包被板进行包装。
2.试剂1的制备(100×)：
试剂1用作生物素标记的白介素6单克隆抗体工作液。
(1)生物素的标记步骤：
将IL-6单克隆抗体(CGMCC No.8797)用0.1mol/L碳酸氢钠缓冲液(pH 8.0)调整到1mg/ml。用0.1mol/L碳酸氢钠缓冲液(pH 8.0)，对蛋白质充分透析。
用1ml DMSO溶解NHSB(N-羟基琥珀酰亚胺生物素，购自Pierce)1mg，即浓度为1mg/ml。
向1ml透析后的单克隆抗体溶液加入150μl NHSB溶液(即含NHSB 150μg)。
在室温下持续搅拌4小时。
加入6μl 1mol/L NH4Cl(每25μg NHSB加1μl)，在室温下搅拌10分钟。
在4℃，用PBS充分透析过夜，以除去游离的NHSB。
加入50％(v/v)甘油，使生物素标记的白介素6单克隆抗体终浓度为0.5mg/ml，置-20℃保存。
(2)试剂1的配制(100×)的配方：
1000ml中有5.8g/L磷酸氢二钠、0.593g/L磷酸二氢钠、8.0g/L氯化钠、200ml/L小牛血清、0.5ml/L Proclin-300、12.5mg/L生物素标记的第二白介素6单克隆抗体，pH 7.2-7.4。
3.试剂2的制备：
1000ml试剂2中有5.8g/L磷酸氢二钠、0.593g/L磷酸二氢钠、8.0g/L氯化钠、200ml/L小牛血清、0.5ml/L Proclin-300，pH 7.2-7.4。
4.试剂3的制备(100×)：
1000ml中有5.8g/L磷酸氢二钠、0.593g/L磷酸二氢钠、8.0g/L氯化钠、200ml/L小牛血清、0.5ml/L Proclin-300、12mg/L辣根过氧化物酶标记的链霉亲和素(购自life)。
5.样本稀释液的制备(10×)：
58g/L磷酸氢二钠、5.94g/L磷酸二氢钠、180g/L氯化钠、250ml/L山羊血清、10ml/L吐温20和5ml/L防腐剂；pH值6.20-6.60。
6.底物液的制备：
辣根过氧化物酶底物：底物液A为含有0.6‰过氧化脲的10mmol/L柠檬酸缓冲液，避光储存；底物液B为含有0.4‰TMB的5mmol/L柠檬酸缓冲液，避光储存。
7.IL-6校准品的配制：
用1×样品稀释液将高值阳性血清(或IL-6纯品)稀释，使得校准品的IL-6浓度梯度为0、10、25、250、750、1500pg/ml。
根据需要，校准品可溯源至ProSpec公司的参考物质Interleukin-6Human Recombinant(Catalogue Number：CYT-213)，纯度≥97.0％。
8.IL-6质控品的配制：
质控品收集自IL-6临床检测高值的人源样本(血清或血浆)，经灭活处理，经检测其抗-TP、HBsAg、抗-HCV和抗-HIV均为阴性。用1×样本稀释液稀释至规定浓度范围内。高值质控：700-900pg/ml；低值质控：200-400pg/ml。2-8℃保存备用。
测试例
测试例1.本发明IL-6ELISA试剂盒的使用方法
样本要求：标本无需特殊处理，采用常规医用技术收集全血标本，离心分离后吸取血清或血浆用于检测。全血标本请于室温放置2小时或4℃过夜后于1000g离心20分钟，取上清即可检测，或将标本放于-20℃或-80℃保存，但应避免反复冻融。请不要使用严重溶血、脂血或黄疸标本。
1.实验前准备
1.1.自4℃冰箱中取出试剂盒，均应平衡到室温(18-25℃)。
1.2. 20倍浓缩洗涤液用纯化水或蒸馏水稀释20倍后使用。
1.3. 10倍样品稀释液用纯化水或蒸馏水稀释10倍后使用。
2.试验方法
2.1.加校准品和待测样本：取足够数量的包被板，固定于框架上，分别设置校准品孔、待测样本孔和空白对照孔，记录各孔位置，除空白对照孔外，在校准品孔中加入稀释后的校准品100μl，校准品零点孔加入IL-6样品稀释液100μl，待测样本孔中加入待测样本100μl；同时加入加生物素标记IL-6抗体工作液每孔50μl，二者混合加入室温反应120min。
2.2.洗板：弃去液体，吸水纸上拍干，每孔加满洗涤液，静置5-10s，甩去洗涤液，吸水纸上拍干，洗5次。
2.3.辣根酶标记亲和素：每孔加入稀释后的辣根酶标记亲和素工作液100μl，(空白对照孔不加)室温反应60min。
2.4.洗板：弃去液体，吸水纸上拍干，每孔加满洗涤液，静置5-10s，甩去洗涤液，吸水纸上拍干，洗5次。
2.5.显色：每孔加入50μl底物液A和50μl底物液B，充分混匀，室温避光反应15min。
2.6.终止：显色完毕后，每孔加入50μl终止液，充分混匀。
2.7.测定：终止反应后，立即置酶标仪450nm波长(以空白孔调零)或双波长450nm/630nm下测定OD值。
2.8.计算：根据校准品的浓度及对应的OD值，使用双对数线性拟合方式(log(X)-log(Y))计算结果，结果乘以稀释倍数，即为样本最终浓度。
测试例2.本发明IL-6的ELISA试剂盒的方法学指标
1.准确度：回收率应在95％～105％之间；
2.空白检出限：应不高于4.6pg/ml；
3.测量系统的线性：在4.6pg/ml-1500pg/ml范围内线性相关系数r≥0.9900。
4.重复性：批内变异系数：CV批内应不超于5％；
5.批间差：批间变异系数：CV批间应不超于10％；
表1.准确性、最低检出限、系统线性、重复性检测结果
Figure PCTCN2015077288-appb-000006
6.分析特异性：
分别检测IL-6阴性样本N1、阳性样本P1血液标本。
在N1、P1样本中分别添加三个浓度梯度的交叉反应原，检测交叉反应原对IL-6抗体检测的交叉反应情况，实验结果见表4：
IL-10(30pg/ml、20pg/ml、10pg/ml)、
降钙素原(400pg/ml、200pg/ml、100pg/ml)、
TNF-α(25ng/ml、12.5ng/ml、6.5ng/ml)、
C反应蛋白浓度(50ng/ml、25ng/ml、12.5ng/ml)、
降钙素(30pg/ml、15pg/ml、7.5pg/ml)。
交叉反应原通常和待测物质具有较高的同源性、序列相似或者具有结构相似的表位。可以想象，如果样本中存在这些交叉反应原时，抗体可能会识别并结合这些交叉反应原，导致假阳性。因此，诊断试剂盒的交叉反应性是一项重要的指标。这对于临床结果的可靠性非常重要。结果分析：白介素-10不大于30pg/ml、降钙素不大于30pg/ml、降钙素原不大于400pg/ml、C反应蛋白不大于50ng/ml、TNF-α浓度不大于25ng/ml时，对IL-6检测结果没有明显影响。
7.稳定性：试剂盒各组分37℃可放置6天，4℃保存3个月。
表2. 37℃存放稳定性实验结果
Figure PCTCN2015077288-appb-000007
Figure PCTCN2015077288-appb-000008
表3. 4℃保存条件下稳定性实验结果
Figure PCTCN2015077288-appb-000009
R-H、R-M、R-L分别代表：高值准确度参考品、中值准确度参考品、低值准确度参考品。
8.干扰物质分析：
8.1血红蛋白：
实验结果见表5。结论：当轻度溶血标本血红蛋白≤200mg/ml时，对结果基本无影响，当严重溶血时检测结果可能导致假阳性，故不宜用严重溶血标本。
8.2甘油三酯：
实验结果见表6。结论：当甘油三酯在1000mg/dl内，对IL-6检测结果没有显著性影响。
8.3胆红素：
实验结果见表7。结果分析：当胆红素浓度不大于20mg/dl时对IL-6检测结果无明显影响，但样本中胆红素高于20mg/dl时，可能会引起假阴性。
8.4肝素钠：
实验结果见表8。结果分析：当肝素钠浓度不大于40U/ml时对IL-6检测结果无明显影响。
8.5EDTA.K2：
实验结果见表9。结果分析：当EDTA.K2不大于600μg/ml时对IL-6检测结果没有明显影响。
8.6生物素：
实验结果见表10。结果分析：当生物素浓度不大于20ng/ml时对IL-6检测结果没有明显影响。
本发明采用双抗体夹心法测定样本中IL-6水平，即酶标板上包被的抗体与生物素标记抗体和被测样品的IL-6形成“包被抗体-抗原-生物素标记抗体”的夹心复合物结构。另外本发明有效地采用亲和素-生物素-酶复合物实现了检测的高灵敏度。
本发明的试剂盒一方面可以快速、准确地测定血清中IL-6的含量，对于炎症的早期诊断、早期治疗提供可靠的临床参考价值；另一方面由于使用两株特异性单克隆抗体的组合，大大提高了IL-6的检测灵敏度。
本发明具有使用简便、检测快速、灵敏、稳定、操作简便等优点。适用于广大中小医院使用。
Figure PCTCN2015077288-appb-000010
Figure PCTCN2015077288-appb-000011
Figure PCTCN2015077288-appb-000012
Figure PCTCN2015077288-appb-000013

Claims (8)

  1. 一种检测样本中白介素6的ELISA试剂盒，其包含：
    包被有第一白介素6单克隆抗体的多孔板、
    试剂1、
    试剂2、
    试剂3、
    样本稀释液、和
    底物液；
    其中：
    试剂1包含：磷酸氢二钠、磷酸二氢钠、氯化钠、小牛血清、生物素标记的第二白介素6单克隆抗体、和防腐剂；
    试剂2包含：磷酸氢二钠、磷酸二氢钠、氯化钠、小牛血清、和防腐剂；
    试剂3包含：磷酸氢二钠、磷酸二氢钠、氯化钠、小牛血清、辣根过氧化物酶标记的链霉亲和素、和防腐剂；
    样本稀释液包含：磷酸氢二钠、磷酸二氢钠、氯化钠、山羊血清、吐温20和防腐剂；
    底物液包含：辣根过氧化物酶底物；
    当第一白介素6单克隆抗体由保藏号为CGMCC No.8796的杂交瘤细胞所产生时，第二白介素6单克隆抗体由保藏号为CGMCC No.8797的杂交瘤细胞所产生；或者当第二白介素6单克隆抗体由保藏号为CGMCC No.8796的杂交瘤细胞所产生时，第一白介素6单克隆抗体由保藏号为CGMCC No.8797的杂交瘤细胞所产生；
    样本来自哺乳动物，优选人类，样本选自全血、血浆和血清。
  2. 根据权利要求1所述的检测样本中白介素6的ELISA试剂盒，还包括：
    校准品，校准品包含已知浓度的白介素6，白介素6浓度在0-1500pg/ml范围内。
  3. 根据权利要求1所述的检测样本中白介素6的ELISA试剂盒，还包括质控品，质控品包含已知浓度的白介素6，白介素6浓度在200-1000pg/ml范围内。
  4. 一种检测样本中白介素6的ELISA试剂盒，其包含：
    包被有第一白介素6单克隆抗体的多孔板、
    试剂1、
    试剂2、
    试剂3、
    样本稀释液、和
    底物液；
    其中：
    试剂1包含：5.8g/L磷酸氢二钠、0.593g/L磷酸二氢钠、8.0g/L氯化钠、200ml/L小牛血清、0.5ml/L Proclin-300、和12.5mg/L生物素标记的第二白介素6单克隆抗体；
    试剂2包含：5.8g/L磷酸氢二钠、0.593g/L磷酸二氢钠、8.0g/L氯化钠、200ml/L小牛血清、和0.5ml/L Proclin-300；
    试剂3包含：5.8g/L磷酸氢二钠、0.593g/L磷酸二氢钠、8.0g/L氯化钠、200ml/L小牛血清、0.5ml/L Proclin-300、和12mg/L辣根过氧化物酶标记的链霉亲和素；
    样本稀释液包含：58g/L磷酸氢二钠、5.94g/L磷酸二氢钠、180g/L氯化钠、250ml/L山羊血清、10ml/L吐温20和5ml/L防腐剂；
    底物液由底物液A和底物液B组成，底物液A含有过氧化脲和柠檬酸缓冲液，底物液B含有四甲基联苯胺和柠檬酸缓冲液；
    当第一白介素6单克隆抗体由保藏号为CGMCC No.8796的杂交瘤细胞所产生时，第二白介素6单克隆抗体由保藏号为CGMCC No.8797的杂交瘤细胞所产生；或者当第二白介素6单克隆抗体由保藏号为CGMCC No.8796的杂交瘤细胞所产生时，第一白介素6单克隆抗体由保藏号为CGMCC No.8797的杂交瘤细胞所产生。
  5. 一种白介素6单克隆抗体，其由保藏号为CGMCC No.8796的 杂交瘤细胞所产生。
  6. 一种白介素6单克隆抗体，其由保藏号为CGMCC No.8797的杂交瘤细胞所产生。
  7. 如下两株白介素6单克隆抗体的组合在制备ELISA诊断试剂中的用途：
    由保藏号为CGMCC No.8796的杂交瘤细胞所产生的白介素6单克隆抗体、以及由保藏号为CGMCC No.8797的杂交瘤细胞所产生的白介素6单克隆抗体。
  8. 一种杂交瘤细胞，其选自保藏号为CGMCC No.8796、和CGMCC No.8797的杂交瘤细胞。
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