上海优选生物科技有限公司主要经营各种品牌档次的ELISA试剂盒，品质保证，售后服务完善。并提供免费代检测。服务于高校及免疫学科研单位。技术人员更好的为您服务。（注：本试剂只用于科研，欢迎来电咨询!）如需订购请联系24小时为您服务。24小时服务热线:13818340512QQ:985021598 404358493邮箱:info@sinobestbio.com    INTENDED USEFor the quantitative determination of activated objective concentrations in serum, plasma, tissue homogenate, cell culture supernates and other biological fluids.For Research use only and is not for use in diagnostic or therapeutic procedures. If you have any questions, please contact the Sino Bestbio Co.,Ltd. PRINCIPLE OF THE ASSAYThe kit uses a doubleantibody sandwich enzymelinked immunosorbent assay (ELISA) to analyze the level of objective in samples. Add standard and sample to wells precoated with one objective antibody at the same time add second HRPconjugated objective antibody to bind the analyte, followed by incubation and washing procedures to remove unbound substance. Finally, HRP substrates are added, incubated for detection, and a blue color is developed. Reaction is stopped and color turns to yellow when Stopping Solution (acidic) is added. The yellow color intensity proportionally correlates to the concentration of the concentration of the objective in samples. SAMPLE COLLECTION AND STORAGESSerum - Use a serum separator tube and allow samples to clot for 2 hours at room temperature or overnight at 4℃ before centrifugation for 20 minutes at approximately 2000×g. Remove serum and assay immediately or aliquot and store samples at-20℃. Avoid repeated freeze-thaw cyclesPlasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 30 minutes at 2000×g at 2-8℃within 30 minutes of collection. Store samples at -20℃. Avoid repeated freeze-thaw cycles.Cell culture supernates, tissue homogenate and other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20℃. Avoid repeated freeze-thaw cycles.Sample preparation - Typically, serum, plasma and other biological fluids samples do not require dilution. If samples generate values higher than the highest standard, please dilute the samples with Sample Diluent and repeat the assay.Notes: Serum and plasma to be used within 7 days may be stored at 2-8℃, otherwise samples must be stored at -20 or -80℃ to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles. When performing the assay slowly bring samples to room temperature. The samples shoule be centrifugated dequately and no hemolysis or granule was allowed. REAGENTS PROVIDEDAll reagents provided are stored at 2-8°C. Refer to the expiration date on the label.Name 96 determinations 48 determinationsMicroelisa stripplate 12*8strips 12*4stripsStandard（6 vial） 0.5ml/vial 0.5ml/vialSample diluent 10ml 10mlHRP-Conjugate reagent 10ml 5.0ml20X Wash solution 25ml 15mlChromogen Solution A 6.0ml 3.0mlChromogen Solution B 6.0ml 3.0mlStop Solution 6.0ml 3.0mlClosure plate membrane 2 2User manual 1 1Sealed bags 1 1 MATERIALS REQUIRED BUT NOT SUPPLIED1. Standard microplate reader capable of measuring absorbance at 450 nm2. Automated Washing3. 37℃ incubator4. Clean tube and Eppendorf tube5. Distilled or deionized water6. Precision pipettes, disposable pipette tips, multi-channel pipettes  and Absorbent paper PRECAUTIONS1. The operation should be carried out in strict accordance with the provided instructions.2. To preserve unused strip-wells, it should be stored in the sealed bag.3. Always avoid foaming when mixing or reconstituting protein solutions.4. Pipette reagents and samples into the center of each well.5. The samples should be transferred into the assay wells within 15 minutes of dilution.6. We recommended that all standard, testing samples are tested in duplicate to minimize the test errors.7. If the blue color too shallow after 15 minutes incubation with the substrates, it may be appropriate to extend the incubation time.8. Avoid crosscontamination by changing tips, using separate reservoirs for each reagent, avoid using the suction head without extensive wash.9. Do not mix the reagents from different batches10. Stop Solution should be added in the same order of the Substrate solution.11. Chromogenic Substrate B is light-sensitive, please avoid prolonged exposure to light.12. The kit should be kept at 2-8℃ and cannot be used after expiration date. The standards should be kept at -20℃after receiving. WASHING METHODManual Washing - Remove incubation mixture by aspirating contents of the plate into a sink or proper waste container. Using a squirt bottle, fill each well completely with Wash Solution (1X), then aspirate contents of the plate into a sink or proper waste container. Repeat this procedure for a total of four times. After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears. Note: Hold the sides of the plate frame firmly when washing the plate to assure that all strips remain securely in frame.Automated Washing - Aspirate all wells, then wash plates four times using Wash Solution (1X). Always adjust your washer to aspirate as much liquid as possible and set fill volume at 350μL/well/wash. After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears. REAGENT PREPARATION AND STORAGEWash Solution (1X) - Dilute 1 volume of Wash solution (20X) with 19 volumes of deionized or distilled water. Wash Solution is stable for 1 month at 2-8℃. ASSAY PROCEDURE1.    First, secure the desired number of coated wells in the holder. Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.2.    Add 50μl of Standard or Sample to the appropriate wells. Blank well doesn’t add anyting.3. Add 100μl of HRP-conjugate reagent to standard wells and sample wells except the blank well, cover with an adhesive strip and incubate for 60 minutes at 37°C.4.  Wash the Microtiter Plate 4 times.(See WASHING METHOD)5.    Add chromogen solution A 50μl and chromogen solution B 50μl to each well. Gently mix and incubate for 15 minutes at37°C. Protect from light.6.    Add 50μl Stop Solution to each well. The color in the wells should change from blue to yellow. If the color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure thorough mixing.7.  Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes. CALCULATION OF RESULTSThe standard curve is generated by plotting the average O.D. (450nm) obtained for each of the standard O.D.values on the vertical (X) axis versus the corresponding concentration on the horizontal (Y) axis. Calculate the mean O.D. value for each standard and sample. All O.D. values, are subtracted by the mean value of the zero standard (Blank well) before result interpretation. Construct the standard curve using graph paper or statistical software. Remember to multiply total dilution timeswhen calculation.Typical standard curve data( reference in general, not for this kit in particular )Standard Concentration Mean OD450 Adjusted0 0.043 none31.2 0.121 0.07862.5 0.236 0.193125 0.355 0.312250 0.541 0.498500 1.198 1.1551000 2.169 2.126 PROCEDURES IN SUMMARYPrewarm all reagents to room temperature before assay.Prepare reagents, samples in clean tubes or 96-well plate.Transfer standard and samples to assay plate/strip, and add HRP-Conjugate reagent and incubate 60 minutes at 37℃.Plate-wash four times, add Substrate A and B, incubate 15 minutes at 37℃ Add stop solution.Measure within 15 minutes ORDERING INFORMATIONA. To place an orderFor Chinese Customers:Please provide the following information to our customer service department to expedite your telephone, fax or mail order:  1.  Your name, telephone and/or fax number  2.  Customer account number  3.  Shipping and billing address  4.  Purchase order number  5.  Catalog number and description of product  6.  Quantity and product size     TELEPHONE/FAX ORDERS: 400-617-3884 (China);     QQ CUSTOMER ORDERS: 404358493; 985021598     EMAIL ORDERS: info@sinobestbio.com         For International Customers:To best serve our international customers, it is Sino Bestbio’s policy to sell our products through a network of distributors. To place an order or to obtain additional information about Sino Bestbio products, please contact your local distributor. B. Conditions of SaleAll products are for research or manufacturing use only. They are not intended for use in clinical diagnosis or for administration to human or animals. All products are intended for in vitro use only. C. Material Safety Data Sheets (MSDS)Material safety data sheets for Sino Bestbio  products may be ordered by fax or phone. See Section A above for details on ordering.