人白介素18(IL-18)ELISA分析检测试剂盒使用说明书
白细胞介素18
白细胞介素18白细胞介素18（Interleukin-18）是一种细胞因子，属于白细胞介素家族的一员。
它被认为在免疫应答中起着重要角色，并且与很多疾病的发展和进展有关。
本文将详细介绍白细胞介素18的功能、调控机制以及其在某些疾病中的作用。
白细胞介素18最初是在1995年被发现的，它由肝脏和肝炎病毒感染的肝细胞产生，并被发现能够刺激白细胞产生干扰素-γ（IFN-γ）。
进一步的研究发现，白细胞介素18的产生受到多个因素的调控，包括细胞因子、信号通路和转录因子。
白细胞介素18在免疫应答中的功能是多样的。
首先，它能够刺激NK细胞的活化和增殖，进而促进它们产生INF-γ和其他促炎因子。
其次，白细胞介素18还能够激活巨噬细胞，并增强它们对细菌等外源性病原体的杀伤能力。
此外，白细胞介素18也参与了免疫细胞的极化和调控，对炎症过程的进展具有重要的影响。
白细胞介素18的调控机制非常复杂，它的产生受到多种信号通路的调控。
一些细胞因子，如肿瘤坏死因子（TNF）和白细胞介素1（IL-1）等，能够刺激白细胞介素18的产生。
另外，TLR（Toll-like receptor）信号通路和NLRP3炎症小体也参与了白细胞介素18的调控。
此外，一些转录因子，如NF-κB和AP-1，也能够直接或间接地影响白细胞介素18的产生。
白细胞介素18在多种疾病的发展和进展中发挥着重要作用。
首先，它在免疫性疾病中的作用已经得到了大量的研究。
例如，炎症性肠病（IBD）和风湿性关节炎等疾病中，白细胞介素18的高表达与炎症的严重程度和病情的进展有关。
其次，白细胞介素18还参与了一些肿瘤的发展。
研究发现，白细胞介素18能够促进肿瘤细胞的侵袭和转移，并且与肿瘤的预后密切相关。
此外，白细胞介素18还被发现参与了心血管疾病、神经退行性疾病和肾脏疾病等疾病的发展。
在药物治疗方面，研究人员已经尝试使用白细胞介素18作为治疗免疫性疾病和肿瘤的靶点。
一些抗体和小分子化合物已经被开发出来，以抑制白细胞介素18的活性。
elisa说明书
ELISA KitCatalog # KRC2341 (96 tests)KRC2342 (192 tests)RatIL-18Invitrogen Corporation542 Flynn Road, Camarillo, CA 93012Tel: 800-955-6288E-mail: techsupport@Table of ContentsTable of Contents (3)Contents and Storage (4)Introduction (5)Purpose (5)Principle of the Method (5)Background Information (5)Methods (7)Materials Needed But Not Provided (7)Procedural Notes (7)Preparation of Reagents (8)Assay Procedure (9)Typical Data (10)Performance Characteristics (11)Sensitivity (11)Precision (11)Linearity of Dilution (11)Recovery (12)Specificity (12)Expected Values (12)Stimulation Protocols (12)Limitations of the Procedure (13)Appendix (14)Troubleshooting Guide (14)Technical Support (15)References (16)Citations (16)Contents and Storage Storage Store at 2 to 8°C.ContentsReagents Provided96Test Kit192Test KitRt IL-18 Standard, lyophilized, recombinant baculovirus Rt IL-18.Refer to vial label for quantity and reconstitution volume.2 vials 4 vialsStandard Diluent Buffer. Contains 8 mM sodium azide; 25 ml perbottle.1 bottle2 bottles Incubation Buffer. Contains 8 mM sodium azide; 11 ml per bottle. 1 bottle 1 bottle Rt IL-18 High and Low Control, lyophilized, recombinant baculovirusRt IL-18. Refer to vial label for reconstitution volume and range.2 vials 4 vials Rt IL-18 Antibody-Coated Wells, 96 wells per plate. 1 plate 2 plates Rt IL-18 Biotin Conjugate (Biotin-labeled anti-IL-18). Contains 8 mMsodium azide; 11 ml per bottle.1 bottle2 bottles Streptavidin-Peroxidase (HRP), (100x) concentrate. Contains 3.3 mMthymol; 0.125 ml per vial.1 vial2 vialsStreptavidin-Peroxidase (HRP) Diluent. Contains 3.3 mM thymol;25 ml per bottle.1 bottle 1 bottle Wash Buffer Concentrate (25X); 100 mL per bottle. 1 bottle 1 bottle Stabilized Chromogen, Tetramethylbenzidine (TMB); 25 mL per bottle. 1 bottle 1 bottle Stop Solution; 25 mL per bottle. 1 bottle 1 bottle Plate Covers, adhesive strips. 4 6Disposal Note This kit contains materials with small quantities of sodium azide. Sodium azide reacts with lead and copper plumbing to form explosive metal azides. Upon disposal, flush drains with a large volume of water to prevent azide accumulation. Avoid ingestion and contact with eyes, skin and mucous membranes. In case of contact, rinse affected area with plenty of water. Observe all federal, state and local regulations for disposal.Safety All blood components and biological materials should be handled as potentially hazardous. Follow universal precautions as established by the Centers forDisease Control and Prevention and by the Occupational Safety and HealthAdministration when handling and disposing of infectious agents.IntroductionPurpose The Invitrogen Rat Interleukin-18 (Rt IL-18) ELISA is to be used for the quantitative determination of IL-18 in rat serum, EDTA plasma, buffered solution,or cell culture medium. The assay will recognize both natural and recombinant RtIL-18.For Research Use Only. CAUTION: Not for human or animal therapeutic ordiagnostic use.Principle of the Method The Invitrogen Rt IL-18 kit is a solid phase sandwich Enzyme Linked-Immuno-Sorbent Assay (ELISA). A polyclonal antibody specific for Rt IL-18 has been coated onto the wells of the microtiter strips provided. Samples, including standards of known Rt IL-18 content, control specimens, and unknowns, are pipetted into these wells.During the first incubation, the Rt IL-18 antigen binds to the immobilized (capture) antibody on one site. After washing, a biotinylated monoclonal antibody specific for Rt IL-18 is added. During the second incubation, this antibody binds to the immobilized Rt IL-18 captured during the first incubation.After removal of excess second antibody, Streptavidin-Peroxidase (enzyme) is added. This binds to the biotinylated antibody to complete the four-member sandwich. After a second incubation and washing to remove all the unbound enzyme, a substrate solution is added, which is acted upon by the bound enzyme to produce color. The intensity of this colored product is directly proportional to the concentration of Rt IL-18 present in the original specimen.Background Information IL-18, also known as Interferon-gamma Inducing Factor (IGIF), is a cytokine with Mr=18 kDa (157 amino acid residues) produced by macrophages and monocytes, Kuppfer cells, keratinocytes, intestinal epithelial cells, osteoblasts, mouse diencephalon, and adrenal cortical cells of reserpine-treated rats. IL-18 is synthesized as an inactive precursor molecule with Mr=24 kDa which lacks a signal peptide. The IL-18 precursor is cleaved by IL-1 converting enzyme (ICE, Caspase-1), producing the bioactive, mature form. Only the mature, 18 kDa, form of IL-18 is secreted. Cells that respond to IL-18 include Th1-type cells and NK cells.IL-18 exerts several effects on Th1-like cells. IL-18 stimulates Th1 cell proliferation, Fas ligand expression and IL-2R alpha chain expression.IL-18 also works in combination with IL-12 to induce the production of IFN-γ, GM-CSF, and IL-2 by Th1-type cells. Standard bioassays for mIL-18 measure dose dependent IFN-γ production by IL-18 target cells, such as mouse IL-18 receptor transfected KG-1 cells (human myelomonocyte: ATCC CCL246). Immunomodulatory pathways, which include IL-18 stimulation of IFN-γproduction, are under investigation. IFN-γ production by Th1-type cells and NK cells is important in many immune functions, including defense against viral and parasitic infections, enhancement of NK activity, activation of macrophages, enhancement of B cell function including B cell maturation, proliferation and immunoglobulin secretion, enhancement of MHC class I and class II antigen expression, and inhibition of osteoclast activation.MethodsMaterials Needed But Not Provided •Microtiter plate reader (at or near 450 nm) with software•Calibrated adjustable precision pipettes•Distilled or deionized water•Plate washer: automated or manual (squirt bottle, manifold dispenser, etc.) •Glass or plastic tubes for diluting solutions•Absorbent paper towels•Calibrated beakers and graduated cylindersProcedural Notes 1. When not in use, kit components should be refrigerated. All reagents should bewarmed to room temperature before use.2. Microtiter plates should be allowed to come to room temperature beforeopening the foil bags. Once the desired number of strips has been removed, immediately reseal the bag and store at 2 to 8°C to maintain plate integrity.3. Samples should be collected in pyrogen/endotoxin-free tubes.4. Samples should be frozen if not analyzed shortly after collection. Avoid multiplefreeze-thaw cycles of frozen samples. Thaw completely and mix well prior to analysis.5. When possible, avoid use of badly hemolyzed or lipemic sera. If large amountsof particulate matter are present, centrifuge or filter prior to analysis.6. It is recommended that all standards, controls and samples be run in duplicate.7. When pipetting reagents, maintain a consistent order of addition fromwell-to-well. This ensures equal incubation times for all wells.8. Do not mix or interchange different reagent lots from various kit lots.9. Do not use reagents after the kit expiration date.10. Absorbances should be read immediately, but can be read up to 2 hours afterassay completion. For best results, keep plate covered in the dark.11. In-house controls or kit controls, if provided, should be run with every assay. Ifcontrol values fall outside pre-established ranges, the accuracy of the assay is suspect.12. All residual wash liquid must be drained from the wells by efficient aspiration orby decantation followed by tapping the plate forcefully on absorbent paper.Never insert absorbent paper directly into the wells.13. Because Stabilized Chromogen is light sensitive, avoid prolonged exposure tolight. Avoid contact between chromogen and metal, or color may develop.Directions for Washing •Incomplete washing will adversely affect the test outcome. All washing must be performed with the Wash Buffer Concentrate (25X) provided. •Washing can be performed manually as follows: completely aspirate the liquid from all wells by gently lowering an aspiration tip into the bottom of each well.Take care not to scratch the inside of the well. After aspiration, fill the wells with at least 0.4 ml of diluted Wash Buffer. Let soak for 15 to 30 seconds, then aspirate the liquid. Repeat as directed under Assay Procedure. After the washing procedure, the plate is inverted and tapped dry on absorbent tissue. •Alternatively, the diluted Wash Buffer may be put into a squirt bottle. If a squirt bottle is used, flood the plate with the diluted Wash Buffer, completely filling all wells. After the washing procedure, the plate is inverted and tapped dry on absorbent tissue.•If using an automated washer, follow the washing instructions carefully.Preparation of ReagentsDilution of Standard Note Note: Either glass or plastic tubes may be used for standard dilutions.The Rt IL-18 standard was calibrated against a highly purified recombinant baculovirus protein.1. Reconstitute standard to 5,000 pg/ml with Standard Diluent Buffer. Refer tostandard vial label for instructions. Swirl or mix gently and allow to sit for10 minutes to ensure complete reconstitution. It is recommended thatstandard be used within 1 hour of reconstitution.2. Add 0.1 ml of the reconstituted standard to a tube containing 0.400 mlStandard Diluent bel as 1,000 pg/ml Rt IL-18. Mix.3. Add 0.250 ml of Standard Diluent Buffer to each of 6 tubes labeled 500, 250,125, 62.5, 31.3 and 15.6 pg/ml Rt IL-18.4. Make serial dilutions of the standard as described in the following dilutiondiagram. Mix thoroughly between steps.Remaining reconstituted standard should be discarded. Return the Standard Diluent Buffer to the refrigerator.Preparing SAV-HRP Note: Prepare within 15 minutes of usage. The Streptavidin-HRP (100x concentrate) is in 50% glycerol, which is viscous. To ensure accurate dilution, allow Streptavidin-HRP concentrate to reach room temperature. Gently mix. Pipette Streptavidin-HRP concentrate slowly. Remove excess concentrate solution from pipette tip by gently wiping with clean absorbent paper.1. Dilute 10 µl of this 100x concentrated solution with 1 ml ofStreptavidin-HRP Diluent for each 8-well strip used in the assay. Label asStreptavidin-HRP Working Solution.2. Return the unused Streptavidin-HRP concentrate to the refrigerator.# of 8-Well StripsVolume of Streptavidin-HRPConcentrateVolume of Diluent2 20 µl solution 2 ml4 40 µl solution 4 ml6 60 µl solution 6 ml8 80 µl solution 8 ml10 100 µl solution 10 ml12 120 µl solution 12 ml5,000pg/ml1,000pg/ml500pg/ml250pg/ml125pg/ml62.5pg/ml31.3pg/ml15.6pg/mlpg/ml 100 µlDilution of Wash Buffer 1. Allow the Wash Buffer Concentrate (25X) to reach room temperature and mixto ensure that any precipitated salts have redissolved. Dilute 1 volume of the Wash Buffer Concentrate (25X) with 24 volumes of deionized water (e.g.,50 ml may be diluted up to 1.25 liters, 100 ml may be diluted up to 2.5 liters).Label as Working Wash Buffer.2. Store both the concentrate and the Working Wash Buffer in the refrigerator.The diluted buffer should be used within 14 days.Assay Procedure Be sure to read the Procedural Notes section before carrying out the assay. Allow all reagents to reach room temperature before use. Gently mix all liquid reagents prior to use.Note: A standard curve must be run with each assay.1. Determine the number of 8-well strips needed for the assay. Insert these inthe frame(s) for current use. (Re-bag extra strips and frame. Store these in the refrigerator for future use.)2. Add 100 µl of the Standard Diluent Buffer to the zero standard wells. Well(s)reserved for chromogen blank should be left empty.3. Add 100 µl of standards, samples or controls to the appropriate microtiterwells. (See Preparation of Reagents.)4. Add 50 µl of Incubation Buffer to the zero standard wells and to the wellscontaining standards and serum/plasma samples, or 50 µl of Standard Diluent Buffer to the wells containing cell culture samples and controls.Well(s) reserved for chromogen blank should be left empty. Tap gently on the side of the plate to mix.5. Cover plate with plate cover and incubate for 2hours at roomtemperature.6. Thoroughly aspirate or decant solution from wells and discard the liquid.Wash wells 4 times. See Directions for Washing.7. Pipette 100 µl of biotinylated Rt IL-18Biotin Conjugate solution into eachwell except the chromogen blank(s). Tap gently on the side of the plate to mix.8. Cover plate with plate cover and incubate for 1hour at room temperature.9. Thoroughly aspirate or decant solution from wells and discard the liquid.Wash wells 4 times. See Directions for Washing.10. Add 100 µl Streptavidin-HRP Working Solution to each well except thechromogen blank(s). See Preparation of Reagents.11. Cover plate with the plate cover and incubate for 30minutes at roomtemperature.12. Thoroughly aspirate or decant solution from wells and discard the liquid.Wash wells 4 times. See Directions for Washing.13. Add 100 µl of Stabilized Chromogen to each well. The liquid in the wells willbegin to turn blue.14. Incubate for 30minutes at room temperature and in the dark. Note: Donot cover the plate with aluminum foil or metalized mylar. The incubation time for chromogen substrate is often determined by the microtiter plate reader used. Many plate readers have the capacity to recorda maximum optical density (O.D.) of 2.0. The O.D. values should bemonitored and the substrate reaction stopped before the O.D. of the positive wells exceed the limits of the instrument. The O.D. values at 450 nm can only be read after the Stop Solution has been added to each well. If using a reader that records only to 2.0 O.D., stopping the assay after 20 to25 minutes is suggested.15. Add 100 µl of Stop Solution to each well. Tap side of plate gently to mix. Thesolution in the wells should change from blue to yellow.16. Read the absorbance of each well at 450 nm having blanked the platereader against a chromogen blank composed of 100 µl each of Stabilized Chromogen and Stop Solution. Read the plate within 2 hours after adding the Stop Solution.17. Use a curve fitting software to generate the standard curve. A fourparameter algorithm provides the best standard curve fit.18.Read the concentrations for unknown samples and controls from thestandard curve. (Samples producing signals greater than that of the highest standard should be diluted in Standard Diluent Buffer for serum/plasma samples or corresponding medium for cell culture samples and reanalyzed, multiplying the concentration found by the appropriate dilution factor.)Typical Data (Example) The following data were obtained for the various standards over the range of 0 to 1,000 pg/ml Rt IL-18.StandardRt IL-18 (pg/ml)Optical Density(450 nm)1,000 3.03500 1.94250 1.13125 0.6462.5 0.3831.3 0.2215.6 0.150 0.07Performance CharacteristicsSensitivity The minimum detectable dose of Rt IL-18 is < 4 pg/ml. This was determined by adding two standard deviations to the mean O.D. obtained when the zerostandard was assayed 30 times.Precision 1. Intra-Assay PrecisionSamples of known Rt IL-18 concentration were assayed in replicates of 22 todetermine precision within an assay.Sample 1 Sample 2 Sample 3Mean (pg/ml) 93 387 877SD 3.4 17.3 30.4%CV 3.7 4.5 3.5SD = Standard DeviationCV = Coefficient of Variation2. Inter-Assay PrecisionSamples were assayed 22 times in multiple assays to determine precisionbetween assays.Sample 1Sample 2 Sample 3Mean (pg/ml) 89 386 850SD 6.0 17.1 38.4%CV 6.7 4.4 4.5SD = Standard DeviationCV = Coefficient of VariationLinearity of Dilution Rat serum and cell culture samples were serially diluted in Standard Diluent Buffer or RPMI containing 1% fetal bovine serum,respectively,over the range of the assay. Linear regression analysis of samples versus the expected concentration yielded an average correlation coefficient of 0.99.Serum Cell CultureDilutionMeasured(pg/ml)Expected(pg/ml)%ExpectedMeasured(pg/ml)Expected(pg/ml)%Expectedneat 180 - 194 -1/2 87 90 97 90 97 931/4 43 45 96 44 48.5 911/8 28 22.5 124 25.7 24.3 1051/16 14 11 124 13.5 12.1 111Recovery The recovery of Rt IL-18 added to rat serum averaged 90%. The recovery of Rt IL-18 added to EDTA plasma averaged 95%. The recovery of Rt IL-18 added totissue culture medium containing 1% fetal bovine serum averaged 101%, whilethe recovery of Rt IL-18 added to tissue culture medium containing 10% fetalbovine serum averaged 97%.Specificity Buffered solutions of a panel of substances at 100 ng/ml were assayed with the Invitrogen Rt IL-18 kit. The following substances were tested and found to haveno cross-reactivity: human IL-18, rat IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12p70, IL-13,MIP-2, TNF-α, CINC-2β, VEGF, GM-CSF; mouse IL-18. Both E. Coli andbaculovirus derived Rt IL-18 were detectable with this kit.Expected Values Four pools of rat serum and one pool of rat EDTA plasma were evaluated in this assay. The following concentrations were detected:Sample ConcentrationsPool serum 1 23 pg/mlPool serum 2 51 pg/mlPool serum 3 149 pg/mlPool serum 4 7 pg/mlPool EDTA plasma 6.5 pg/mlStimulation Protocols Cell culture supernatants were evaluated in this assay.Rat Whole Blood (WB) cells were cultured in RPMI for 24, 48 or 72 hours either without stimulus or with a blend of LPS (25 mg/ml) and PHA (5 mg/ml), or with a blend of ionomycin (100 ng/ml) and PMA (100 ng/ml). Results are shown below.IL-18 (pg/ml)Stimulus Cell type24hrs 48hrs72hrsNone WB cells <4 <4 <4LPS + PHA WB cells 8 13 20PMA + ionomycin WB cells 8 22 27Rat splenocytes were cultured at different cellular concentrations in RPMI supplemented with 5% FCS for 24, 48, 72 or 96 hours with a blend of LPS (25 mg/ml) and PHA (5 mg/ml). Results are shown below.IL-18 (pg/ml)CellconcentrationCell type24hrs48hrs72hrs96hrs0.25 x 106 cells/ml Splenocytes17.4 27 36 450.8 x 106 cells/ml Splenocytes49 ND 75 892.5 x 106 cells/ml Splenocytes167 212 238 220Limitations of the Procedure Do not extrapolate the standard curve beyond the top standard point; the dose-response is non-linear in this region and accuracy is difficult to obtain. Dilute all samples above the top standard point with Standard Diluent Buffer; reanalyze these and multiply results by the appropriate dilution factor.The influence of various drugs, aberrant sera (hemolyzed, hyperlipidemic, jaundiced, etc.) and the use of biological fluids in place of serum samples have not been thoroughly investigated. The rate of degradation of native Rt IL-18in various matrices has not been investigated. The immunoassay literature contains frequent references to aberrant signals seen with some sera, attributed to heterophilic antibodies. Though such samples have not been seen to date, the possibility of this occurrence cannot be excluded.Appendix Troubleshooting GuideElevated background Cause: Insufficient washing and/or draining of wells after washing. Solution containing either biotin or SAV-HRP can elevate the background if residual is left in the well.Solution: Wash according to the protocol. Verify the function of automated plate washer. At the end of each washing step, invert plate on absorbent tissue on countertop and allow to completely drain, tapping forcefully if necessary to remove residual fluid.Cause: Contamination of substrate solution with metal ions or oxidizing reagents. Solution: Use distilled/deionized water for dilution of wash buffer and use plastic equipment. DO NOT COVER plate with foil.Cause: Contamination of pipette, dispensing reservoir or substrate solution with SAV-HRP conjugate.Solution: Do not use chromogen that appears blue prior to dispensing onto the plate. Obtain new vial of chromogen.Cause: Incubation time is too long or incubation temperature is too high. Solution: Reduce incubation time and/or temperature.Elevated sample/ standard ODs Cause: Incorrect dilution of standard stock solution; intermediary dilutions not followed correctly.Solution: Follow the protocol instructions regarding the dilution of the standard. Cause: Incorrect dilution of the SAV-HRP conjugate.Solution: Warm solution of SAV-HRP concentrate to room temperature, draw up slowly and wipe tip with kim-wipe to remove excess. Dilute ONLY in SAV diluent provided.Cause: Incubation times extended.Solution: Follow incubation times outlined in protocol.Cause: Incubations carried out at 37°C when RT is dictated.Solution: Perform incubations at RT (= 25 ± 2°C) when instructed in the protocol.Poor standard curve Cause: Improper preparation of standard stock solution.Solution: Dilute lyophilized standard as directed by the vial label only with the standard diluent buffer or in a diluent that most closely matches the matrix of your sample.Cause: Reagents (lyophilized standard, standard diluent buffer, etc.) from different kits, either different cytokine or different lot number, were substituted. Solution: NEVER substitute any components from another kit.Cause: Errors in pipetting the standard or subsequent steps.Solution: Always dispense into wells quickly and in the same order. Do not touch the pipette tip on the individual microwells when dispensing. Use calibrated pipettes and the appropriate tips for that device.Weak/no color developsCause: Reagents not at RT (25 ± 2°C) at start of assay.Solution: Allow ALL reagents to warm to RT prior to commencing assay. Cause: Incorrect storage of components, e.g., not stored at 2 to 8°C.Solution: Store all components exactly as directed in protocol and on labels. Cause: Working SAV-HRP solution made up longer than 15 minutes before use in assay.Solution: Use the diluted SAV-HRP within 15 minutes of dilution.Cause: TMB solution lost activity.Solution 1: The TMB solution should be clear before it is dispensed into the wells of the microtiter plate. An intense aqua blue color indicates that the product is contaminated. Please contact Technical Support if this problem is noted. To avoid contamination, we recommend that the quantity required for an assay be dispensed into a disposable trough for pipetting. Any TMB solution left in the trough should be discarded.Solution 2: Avoid contact of the TMB solution with items containing metal ions. Cause: Attempt to measure analyte in a matrix for which the ELISA assay has not been optimized.Solution: Please contact Technical Support for advice when using nonvalidated sample types.Cause: Wells have been scratched with pipette tip or washing tips.Solution: Use caution when dispensing and aspirating into and out of microwells.Poor PrecisionCause: Errors in pipetting the standards, samples or subsequent steps.Solution: Always dispense into wells quickly and in the same order. Do not touch the pipette tip on the individual microwells when dispensing. Use calibratedpipettes and the appropriate tips for that device. Check for any leaks in the pipette tip.Cause: Repetitive use of tips for several samples or different reagents. Solution: Use fresh tips for each sample or reagent transfer.Cause: Wells have been scratched with pipette tip or washing tips.Solution: Use caution when dispensing and aspirating into and out of microwells.Technical SupportContact Us For more troubleshooting tips, information, or assistance, please call, email, or goonline to /ELISA.USA: Invitrogen Corporation 542 Flynn RoadCamarillo, CA 93012 Tel: 800-955-6288E-mail: techsupport@Europe:Invitrogen LtdInchinnan Business Park 3 Fountain DrivePaisley PA4 9RF, UK Tel: +44 (0) 141 814 6100 Fax: +44 (0) 141 814 6117 E-mail: eurotech@References 1. Dinarello, C.A., et al. (1998) Overview of Interleukin-18: more than an interferon-gamma inducing factor. J. Leukoc. Biol. 63(6):658-664.2. Okamura, H., et al. (1995) Cloning of a new cytokine that induces IFN-gammaproduction by T cells. Nature 378(6552):88-91.3. Ushio, S., et al. (1996) Cloning of the cDNA for human IFN-gamma-inducing factor,expression in Escherichia coli, and studies on the biologic activities of the protein. J.Immunol. 156(11):4274-4279.4. Micallef, M.J., et al. (1996) Interferon-gamma-inducing factor enhances T helper 1cytokine production by stimulated human T cells: synergism with interleukin-12 forinterferon-gamma production. Eur. J. Immunol. 26(7):1647-1651.5. Dao, T., et al. (1996) Interferon-gamma-inducing factor, a novel cytokine, enhancesFas ligand-mediated cytotoxicity of murine T helper 1 cells. Cell. Immunol.173(2):230-235.6. Jordan, J.A., et al. (2001) Role of IL-18 in acute lung inflammation. J. Immunol.167(12):7060-7068.Citations 1. Rana, S., et al. (2005) J. Leukoc. Biol. 77(5):719-28.2. Rosenthal, L.A., et al. (2004) Am. J. Respir. Cell. Mol. Biol. 30: 702-709.For an up-to-date and complete list, visit /ELISA or contact TechnicalSupport.Limited Warranty Invitrogen is committed to providing our customers with high-quality goods and services. Our goal is to ensure that every customer is 100% satisfied with our products and our service. If you should have any questions or concerns about an Invitrogen product or service, please contact our Technical Support Representatives. Invitrogen warrants that all of its products will perform according to the specifications stated on the Certificate of Analysis. The company will replace, free of charge, any product that does not meet those specifications. This warranty limits Invitrogen Corporation’s liability only to the cost of the product. No warranty is granted for products beyond their listed expiration date. No warranty is applicable unless all product components are stored in accordance with instructions. Invitrogen reserves the right to select the method(s) used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order. Invitrogen makes every effort to ensure the accuracy of its publications, but realizes that the occasional typographical or other error is inevitable. Therefore Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation. If you discover an error in any of our publications, please report it to our Technical Support Representatives. Invitrogen assumes no responsibility or liability for any special, incidental, indirect or consequential loss or damage whatsoever. The above limited warranty is sole and exclusive. No other warranty is made, whether expressed or implied, including any warranty of merchantability or fitness for a particular purpose.Licensing Information These products may be covered by one or more Limited Use Label Licenses (see the Invitrogen Catalog or our website, ). By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses. Unless otherwise indicated, these products are for research use only and are not intended for human or animal diagnostic, therapeutic or commercial use.。
人白介素1β (IL-1β)酶联免疫吸附测定试剂盒使用说明书
2022年修订第一版（本试剂盒仅供体外研究使用，不用于临床诊断!）产品货号：E-EL-H0149c产品规格：96T/48T/24T/96T*5Elabscience 人白介素1β(IL-1β)酶联免疫吸附测定试剂盒使用说明书Human IL-1β(Interleukin 1 Beta) ELISA Kit使用前请仔细阅读说明书。
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Copyright ©2021-2022 Elabscience Biotechnology Co.,Ltd. All Rights Reserved目录用途 (3)基本性能 (3)检测原理 (3)试剂盒组成及保存 (4)试验所需自备物品 (5)样品收集方法 (5)注意事项 (6)■ 试剂盒注意事项 (6)■ 样品注意事项 (6)样本稀释方案 (6)检测前准备工作 (7)操作步骤 (8)结果判断 (10)技术资源 (10)典型数据 (10)性能 (11)■ 精密度 (11)■ 回收率 (11)■ 线性 (11)声明 (12)Intended use (13)Character (13)Test principle (13)Kit components & Storage (14)Other supplies required (15)Sample collection (15)Note (16)■ Note for kit (16)■ Note for sample (16)Dilution Method (17)Reagent preparation (17)Assay procedure (18)Calculation of results (20)Technical resources (20)Typical data (20)Performance (21)■ Precision (21)■ Recovery (21)■ Linearity (21)Declaration (22)用途该试剂盒用于体外定量检测人 血清、血浆或其他相关生物液体中IL-1β浓度。
人白细胞介素6(IL-6)定量检测试剂盒(ELISA)
仅供科研使用，不得用于临床检验。
人白细胞介素6（IL-6）定量检测试剂盒（ELISA）说明书【产品名称】通用名称：人白细胞介素6（IL-6）定量检测试剂盒（ELISA）英文名称：Human Interleukin-6（IL-6）ELISA KIT【包装规格】48人份/盒，96人份/盒【预期用途】仅供科研使用，定量检测血清、血浆、细胞培养上清液中人白细胞介素6（IL-6）的浓度。
【检验原理】本试剂盒采用双抗体夹心酶联免疫吸附试验（ELISA）。
在预包被抗人白细胞介素6（IL-6）抗体（固相抗体）的微孔酶标板中，加入人白细胞介素6（IL-6）校准品和待测样本，再加入另一株HRP标记的抗人白细胞介素6（IL-6）抗体（酶标抗体），经过温育与充分洗涤，去除未结合的组分，在微孔板固相表面形成固相抗体-抗原-酶标抗体的夹心复合物。
加底物A 和B，底物在HRP催化下，产生蓝色产物，在终止液（2M 硫酸）作用下，最终转化为黄色，在酶标仪上测定吸光度（OD值），吸光度（OD值）与待测样品中人白细胞介素6（IL-6）的浓度正相关。
拟合校准品曲线，可以计算出样本中人白细胞介素6（IL-6）的浓度。
【主要组成成分】主要成分校准品浓度依次为：320、160、80、40、20、0 pg/ml。
校准品已经通过测试，结果表明HBs抗原阴性，HIV1、HIV2和HCV抗体阴性，由于不存在一种试验方法能够完全保证没有这些物质，本品必须按照具有潜在的感染性进行处理，处理过程应当遵循通用的安全措施。
需要但未提供的材料及耗材1、酶标仪2、精密移液器及一次性吸头3、蒸馏水4、洗瓶或者自动洗板机5、37℃水浴锅或恒温箱6、500ml量筒7、无粉一次性乳胶手套8、质控品（可从蓝图生物科技产品研发系统中选择）【储存条件及有效期】1、2-8℃保存，切勿冷冻，有效期6个月。
2、开封使用后，包被微孔板放入带有干燥剂的自封袋中，密闭自封袋，并将全部试剂放回2-8℃冰箱。
人白细胞介素 8（IL-8）定量检测试剂盒（ELISA） 说明书
本试剂盒只能用于科学研究，不得用于医学诊断。
人白细胞介素8（IL-8）定量检测试剂盒（ELISA）使用说明书【试剂盒名称】人白细胞介素8（IL-8）定量检测试剂盒（ELISA）【试剂盒用途】定量检测人血清、血浆及相关液体样本中白细胞介素8（IL-8）的含量。
【检测原理】本试剂盒采用双抗体两步夹心酶联免疫吸附法（ELISA）。
将标准品、待测样本加入到预先包被人白细胞介素8（IL-8）单克隆抗体透明酶标包被板中，温育足够时间后，洗涤除去未结合的成分，再加入酶标工作液，温育足够时间后，洗涤除去未结合的成分。
依次加入底物A、B，底物（TMB）在辣根过氧化物酶（HRP）催化下转化为蓝色产物，在酸的作用下变成黄色，颜色的深浅与样品中人白细胞介素8（IL-8）浓度呈正相关，450nm波长下测定OD值，根据标准品和样品的OD值，计算样本中人白细胞介素8（IL-8）含量。
【试剂盒组成】1酶标包被板12孔×8条7显色剂A液6mL2标准品0.3mL×6管8显色剂B液6mL320倍浓缩洗涤液25mL9终止液6mL4样本稀释液6mL10说明书1份5特殊稀释液6mL11封板膜2张6酶标试剂6mL12密封袋1个备注：标准品（1号→6号）浓度依次为：120、60、30、15、7.5、3.75pg/mL.【需要而未提供的试剂和器材】1、37℃恒温箱2、标准规格酶标仪3、精密移液器及一次性吸头4、蒸馏水5、一次性试管6、吸水纸【操作步骤】1、准备：从冰箱取出试剂盒，室温复温平衡30分钟。
2、配液：用蒸馏水将20倍浓缩洗涤液稀释成原倍的洗涤液。
3、加标准品和待测样本：取足够数量的酶标包被板，固定于框架上，分别设置标准品孔、待测样本孔和空白对照孔，记录各孔位置，在标准品孔中加入标准品50μL；待测样本孔中先加入待测样本10μL，再加样本稀释液40μL（即样本稀释5倍）；空白对照孔不加。
4、温育：37℃水浴锅或恒温箱温育30min。
人白介素试剂盒检测基本方法原理步骤专项说明书
人IL-10定量分析酶联免疫检测试剂盒IL-10简介：人IL-10 是由160个氨基酸构成4个α螺旋构造旳旳细胞因子，分子量为18.5 kDa。
在水溶液中以二聚体形式存在，分子量约为39 kDa。
人IL-10是个多效性旳因子，可以对多种细胞起作用，重要体现为免疫克制和免疫刺激两方面旳作用，特别是在调节淋巴系和髓系旳细胞功能方面扮演重要旳作用。
IL-10 也具有单核细胞/巨噬细胞依赖、抗原刺激引起旳旳PBMNC 和 NK 细胞合成其他细胞因子旳克制作用。
巨噬细胞膜介导旳共刺激引起旳T细胞和NK细胞活化后旳产物及功能也可被IL-10所克制。
IL-10是单核细胞/巨噬细胞功能旳强有力旳调节物。
IL-10作为细胞介导旳免疫反映旳克制物，可以克制像前列腺素E2、TNF-α、IL-1、IL-10和 IL-8等许多引起炎症旳细胞因子。
IL-10 可以增进可溶性TNF受体旳释放和ICAM－1和B7在细胞表面旳体现。
IL-10还参与B细胞、柱状细胞及胸腺细胞旳增殖和分化旳调控。
人旳IL-10与鼠类旳IL-10在DNA序列和氨基酸序列上高度同源，其DNA序列中一种开放旳基因框与艾伯斯坦-巴尔病毒基因组上基因框BCRF1高度同源，这也许在病毒感染中扮演重要角色旳因素。
在许多与寄生虫感染旳报道中，IL－10体现也会增长，如曼森氏血吸虫感染、利什曼原虫感染、弓形虫感染、锥虫感染等。
分枝杆菌旳感染如麻风分枝杆菌、结核杆菌、鸟分枝杆菌等旳感染也会引起IL－10旳体现升高。
检测原理:本试剂盒采用双抗体夹心ELISA法检测样本中IL-10 旳浓度。
IL-10 捕获抗体已预包被于酶标板上，当加入标本或参照品时，其中旳IL-10 会与捕获抗体结合，其他游离旳成分通过洗涤旳过程被除去。
当加入生物素化旳抗人IL-10 抗体后，抗人IL-10 抗体与IL-10 接合，形成夹心旳免疫复合物，其他游离旳成分通过洗涤旳过程被除去。
随后加入辣根过氧化物酶标记旳亲合素。
小儿过敏性紫癜血清白介素18含量的检测分析
小儿过敏性紫癜血清白介素18含量的检测分析张国华【摘要】目的:检测并分析过敏性紫癜患儿白介素18的含量.方法:过敏性紫癜患儿58例,根据临床尿检结果分为单纯性紫癜组30例和紫癜性肾炎组28例,另设健康对照组20例;采用双抗体夹心酶联免疫吸附法,检测患儿白介素18的血清水平,并统计进行分析.结果:单纯性紫癜组及紫癜性肾炎组白介素18的检出值均明显高于健康对照组;紫癜性肾炎组的血清白介素18水平高于单纯性紫癜组,差异均有统计学意义.结论:过敏性紫癜患儿血中有较高的白介素18表达;白介素18的表达与患儿的肾脏损害相关.【期刊名称】《中国民族民间医药》【年(卷),期】2013(022)022【总页数】1页(P63-63)【关键词】过敏性紫癜;白介素18;肾脏损害;检测分析【作者】张国华【作者单位】南方医科大学附属佛山妇幼保健院,广州,佛山,528000【正文语种】中文【中图分类】R725.2过敏性紫癜（HSP）多发于儿童，是以坏死性小血管炎为主要病理改变的全身性疾病。
本文检测了HSP患儿血清中白细胞介素18（IL－18）的表达水平。
旨在比较分析IL－18在小儿HSP病程中的表达情况，并探讨它们与肾脏损害的关联性，现报告如下。
1.1 材料选取2009年至2011年佛山市多家医院儿科确诊为HSP的住院患儿58例为研究对象，根据尿检结果分为单纯性紫癜组30例和紫癜性肾炎组28例。
58例患儿中，男25例，女33例；年龄：单纯性紫癜组8.37±2.26岁，紫癜性肾炎组7.89±3.38岁；另设健康对照组20例。
1.2 具体分组及入选标准为1.2.1 健康对照组20例，为本院常规性体检小儿，均无肾脏、风湿及哮喘疾病史。
1.2.2 单纯性紫癜组30例，包含皮肤型、腹型、关节型以及混合型。
诊断标准按《实用儿科学》第7版所列1990年美国风湿病协会制定标准［1］。
1.2.3 紫癜性肾炎组28例，即过敏性紫癜病程中伴有血尿和（或）蛋白尿。
白介素18
白细胞介素---18(IL---18)高峰生物技术201007040018IL---18原名IFN--γ诱导因子，是从小鼠及人的肝细胞中克隆得到的一种新的细胞因子，1996年正式命名为IL--18.鼠IL--18前体蛋白由192个氨基酸组成，相对分子质量2.4*104。
N端有一结构特殊的35个氨基酸前导序列，缺乏N--糖基化位点和疏水信号肽样位点。
天然蛋白质由157个氨基酸组成，相对分子质量为1.8*104。
人的IL---18基因编码193个氨基酸前体蛋白，与鼠的IL---18有65%同源性。
SDS--PAGE显示人的IL---18相对分子质量为18.3*103。
人的IL---18主要由单核--巨噬细胞产生，小鼠的IL---18主要由枯否细胞和活化的巨噬细胞产生。
1、IL-18的来源在多种器官、组织和细胞中可检测到IL-18的mRNA，如胸腺、肝脏、脾脏、肾脏、胰腺、枯否细胞和活化的巨噬细胞。
未经处理的小鼠表皮细胞恒定地表达低水平的IL-18mRNA。
体内实验表明，经接触性过敏原刺激后，表皮细胞中IL-18mRNA的表达量在刺激早期就明显上调，在刺激后4～6h 达到峰值。
进一步的实验表明角质形成细胞是表皮中IL-18主要的来源［3］。
成骨细胞基质细胞表达IL-18。
在不支持破骨细胞样多核细胞（OCL）形成的基质细胞系细胞中IL-18的mRNA的表达量较高，在支持OCL形成的基质细胞系的细胞中表达较低［7］。
利血平处理和急性的冷应激都可以使大鼠肾上腺表达IL-18mRNA。
原位杂交显示IL-18的mRNA主要集中在肾上腺皮质，尤其是在产生糖皮质激素的网状带和束状带。
在神经垂体也可检测到IL-18的mRNA，应激对于该部位的IL-18诱生作用并不明显。
这些结果显示IL-18可能是一种神经免疫调节子在免疫系统参与应激过程中起重要的作用［4］。
在胚胎和成体的小肠中测得的IL-18表达量相对于其它器官组织是最高的。
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人白介素18（IL-18）ELISA分析检测试剂盒使用说明书
本试剂仅供研究使用
目的：本试剂盒用于测定小鼠血清，血浆及相关液体样本中转化生长因子（IL-18）的含量。
实验原理：
本试剂盒应用双抗体夹心法测定标本中人白介素18（IL-18）水平。
用纯化的转化生长因子（IL-18）抗体包被微孔板，制成固相抗体，往包被单抗的微孔中依次加入转化生长因子（IL-18），再与HRP标记的羊抗鼠抗体结合，形成抗体-抗原-酶标抗体复合物，经过彻底洗涤后加底物TMB显色。
TMB在HRP 酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。
颜色的深浅和样品中的转化生长因子（IL-18）呈正相关。
用酶标仪在450nm波长下测定吸光度（OD 值），通过标准曲线计算样品中人白介素18（IL-18）浓度。
试剂盒内容及其配制：
试剂盒成份96孔配置48孔配置
96/48人份酶标板1块板
（96T）
半块板
（48T）
塑料膜板盖1块半块
标准品：100ng/ml 1瓶
（1.0ml）
1瓶
（0.5ml）
空白对照1瓶
（1.0ml）
1瓶
（0.5ml）
标准品稀释缓冲液1瓶
（8.0ml）
1瓶
（4.0ml）
生物素标记的抗OT抗体1瓶
（8.0ml）
1瓶
（4.0ml）
亲和链酶素-HRP 1瓶
（12ml）
1瓶（5ml）
洗涤缓冲液1瓶
（20ml）
1瓶（10ml）
底物A 1瓶
（6.0ml）
1瓶
（3.0ml）
底物B 1瓶
（6.0ml）
1瓶
（3.0ml）
终止液1瓶
（6.0ml）
1瓶
（3.0ml）
试剂盒组成：
试剂盒组成48孔配置96孔配置保存
说明书1份1份
封板膜2片（48）2片（96）
密封袋1个1个
酶标包被板1×48 1×96 2-8℃保存标准品：45μ
mol/L
0.5ml×1瓶0.5ml×1瓶2-8℃保存标准品稀释液 1.5ml×1瓶 1.5ml×1瓶2-8℃保存酶标试剂 3 ml×1瓶 6 ml×1瓶2-8℃保存样品稀释液 3 ml×1瓶 6 ml×1瓶2-8℃保存显色剂A液 3 ml×1瓶 6 ml×1瓶2-8℃保存显色剂B液 3 ml×1瓶 6 ml×1瓶2-8℃保存终止液3ml×1瓶6ml×1瓶2-8℃保存
浓缩洗涤液（20ml×20倍）×1
瓶
（20ml×30倍）
×1瓶
2-8℃保存
自备材料：
1. 蒸馏水。
2. 加样器：5ul、10ul 、50ul 、100ul 、200ul 、500ul、1000ul。
3. 振荡器及磁力搅拌器等。
样品收集、处理及保存方法：
1、血清……操作过程中避免任何细胞刺激。
使用不含热原和内毒素的试管。
收集血液后，1000×g离心10分钟将血清和红细胞迅速小心地分离。
2、血浆……EDTA、柠檬酸盐、肝素血浆可用于检测。
1000 ×g离心30分钟去除颗粒。
3、细胞上清液……1000 ×g离心10分钟去除颗粒和聚合物。
4、组织匀浆……将组织加入适量生理盐水捣碎。
1000 ×g离心10分钟，取上清液。
5、保存……如果样品不立即使用，应将其分成小部分-70℃保存，避免反复冷冻。
尽可能的不要使用溶血或高血脂血。
如果血清中大量颗粒，检测前先离心或过滤。
不要在37℃或更高的温度加热解冻。
应在室温下解冻并确保样品均匀地充分解冻。
人白介素18ELISA试剂盒操作注意事项：
●试剂应按标签说明书储存，使用前恢复到室温。
稀稀过后的标准品应丢弃，不可保存。
●实验中不用的板条应立即放回包装袋中，密封保存，以免变质。
●不用的其它试剂应包装好或盖好。
不同批号的试剂不要混用。
保质前使用。
●使用一次性的吸头以免交叉污染，吸取终止液和底物A、B液时，避免使用带金属部分的加样器。
●使用干净的塑料容器配置洗涤液。
使用前充分混匀试剂盒里的各种成份及样品。
●底物A应挥发，避免长时间打开盖子。
底物B对光敏感，避免长时间暴露于光下。
避免用手接触，有毒。
实验完成后应立即读取OD值。
●加入试剂的顺序应一致，以保证所有反应板孔温育的时间一样。
●按照说明书中标明的时间、加液的量及顺序进行温育操作。
安全性：
1. 避免直接接触终止液和底物A、B，一旦接触到这些液体，请尽快用水冲洗。
2. 实难中不要吃喝、抽烟或使用化妆品。
3. 不要用嘴吸取试剂盒里的任何成份。
试剂的准备：
1. 标准品：标准品的系列稀释应在实验时准备，不能储存。
稀释前将标准品振荡混匀。
稀释比例按下表中进行：
100 ng/ml （6号标
准品）
原倍浓度不用稀释直
接加入50ul
50
ng/ml （5号标
准品）
100ul的原倍标准品
加入100ul的标准品
稀释液
25
ng/ml （4号标
准品）
100ul的5号标准品加
入100ul的标准品稀
释液
12.5 ng/ml （3号标
准品）
100ul的4号标准品加
入100ul的标准品稀
释液
6.25 ng/ml （2号标
准品）
100ul的3号标准品加
入100ul的标准品稀
释液
3.12 ng/ml （1号标
准品）
100ul的2号标准品加
入100ul的标准品稀
释液
ng/ml （空白对
照）
原倍浓度不用稀释直
接加入50ul
2. 洗涤缓冲液（50×）的稀释：蒸馏水50倍稀释。
试剂盒性能：
1. 灵敏度：最小的检测浓度小于1号标准品。
稀释度的线性。
样品线性回归与预期浓度相关系数R值为0.990。
2. 特异性：不与其它细胞因子反应。
3. 重复性：板内、板间变异系数均小于10%。
人白介素18ELISA检测试剂盒操作步骤：
1. 使用前，将所有试剂充分混匀。
不要使液体产生大量的泡沫，以免加样时加入大量的气泡，产生加样上的误差。
2. 根据待测样品数量加上标准品的数量决定所需的板条数。
每个标准品和空白孔建议做复孔。
每个样品根据自己的数量来定，能使用复孔的尽量做复孔。
3. 加入稀释好后的标准品50ul 于反应孔、加入待测样品50 ul 于反应孔内。
立即加入50 ul的生物素标记的抗体。
盖上膜板，轻轻振荡混匀，37℃温育45分钟。
4. 甩去孔内液体，每孔加满洗涤液，振荡30秒，甩去洗涤液，用吸水纸拍干。
重复此操作4次。
如果用洗板机洗涤，洗涤次数增加一次。
5. 每孔加入100ul的亲和链酶素-HRP，轻轻振荡混匀，37℃温育30分钟。
6. 甩去孔内液体，每孔加满洗涤液，振荡30秒，甩去洗涤液，用吸水纸拍干。
重复此操作4次。
如果用洗板机洗涤，洗涤次数增加一次。
7. 每孔加入底物A、B各50ul，轻轻振荡混匀，37℃温育5分钟。
避免光照。
8. 取出酶标板，迅速加入50ul终止液，加入终止液后应立即测定结果。
9. 在450nm波长处测定各孔的OD值。
结果判断与分析：
1、仪器值：于波长450nm的酶标仪上读取各孔的OD值
2、以吸光度OD值为纵坐标（Y ），相应的OT 标准品浓度为横坐标（X ），做得相应的曲线，样品的OT 含量可根据其OD 值由标准曲线换算出相应的浓度，再乘以稀释倍数；或用标准物的浓度与OD值计算出标准曲线的回归方程式，将样品的OD值代入方程式，计算出样品浓度，再乘以稀释倍数，即为样品的实际浓度。
3、检测值范围： 0-100ng/ml
4、敏感度：0.39ng/ml。