Human P53-Ab ELISA Kit For the quantitative in vitro determination of Human P53 autoantibodies concentrations in serum - plasma - celiac fluid - tissue homogenate - body fluid FOR LABORATORY RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. This package insert must be read in The ELISA kit is intended to be used in diagnostic or therapeutic procedures. The Stop Solution changes the color from blue to yellow and the The intensity of the color is measured at 450 nm using a spectrophotometer. In order to measure the concentration of P53-Ab in the sample, this P53-Ab ELISA Kit includes a set of calibration standards. The calibration standards are assayed at the same time as The samples and allow the operator to produce a standard curve of Optical Density versus P53-Ab concentration. The concentration of P53-Ab in the samples is then Determined by comparing the OD of the samples to the standard curve. SAMPLE COLLECTION AND STORAGESSerum - Use a serum separator tube and allow samples to clot for 2 hours at room temperature or overnight at 4Â°C before centrifugation for 20 minutes at approximately 2000Ã—g. Remove serum and assay immediately or aliquot and store samples at -20Â°C. Avoid repeated freeze-thaw cyclesPlasma - Collect plasma using heparin as an anticoagulant. Centrifuge samples for 30 minutes at 2000Ã—g at 2-8Â°C within 30 minutes of collection. Store samples at -20Â°C. Avoiding 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 -20Â°C. : The samples shoule be centrifugated dequately and no hemolysis or granule was allowed.MATERIALS REQUIRED BUT NOT SUPPLIED1. 37 Â°C incubator2. Standard microplate reader capable of measuring absorb ANCE at 450 nm3. Precision pipettes, disposable pipette tips and Absorbent paper4. Distilled or deionized waterREAGENTS PROVIDED All reagents provided are stored at 2-8Â°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 6.0ml 3.0mlHRP-Conjugate reagent 10.0ml 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 2 User manual 1 1Sealed bags 1 1Note: 1. Standard concentration was followed by: 500,250,125,61.5,31.2,15.6 pg/mL.2. If samples generate values â€‹â€‹higher than the highest standard, please dilute the samples with Sample Diluent and repeat the Standard. conjugate and microtiter plates are matched for optimal performance. Use only the reagents supplied by manufacturer. 2. Allow kit reage Nts and materials to reach room temperature (20-25Â°C) before use. Do not use water baths to thaw samples or reagents. 3. Do not use kit components beyond their expiration date. 4. Use only deionized or distilled water to dilute 5. Do not remove microtiter plate from the storage bag until needed. Unused strips should be stored at 2-8Â°C in their pouch with the desiccant provided. 6. Use fresh disposable pipette tips for each transfer to avoid contamination. Disposable knuckle must be treated as potentially hazardous and capable of transmitting disease. Disposable knuckle must be during the assay procedure, since no known test method can offer complete assurance that products derived from Human blood will not transmit infectious agents. Therefore, all blood derivatives should be considered potentially infectious and good applicant practices should be followed. 9. All samples should be disposed of in a manner that wi 11. Liquid Waste: Add sodium hypochlorite to a final concentration of 1.0%. The waste should be allowed to stand for a minimum of 30 minutes to inactivate the viruses before disposal. 11. Substrate Solution is easily contaminated. If bluish 12. Substrate B contain 20% acetone, keep this reagent away from sources of heat or flame. 13. Remove all kit reagents from refrigerator and allow them to reach room temperature ( 20-25Â°C) .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 2-8Â°C. ASSAY PROCEDURE1. Prepare all reagents before starting Iso recommended procedure. It is recommended that all Standards and Samples be added in duplicate to the Microelisa Stripplate.2. Add 50Î¼l of Standard or Sample to the appropriate wells. Blank well doesn't add anyting.3. Add 100Î¼l of HRP-conjugate reagent To standard wells and sample w Ells except the blank well, cover with an adhesive strip and incubate for 60 minutes at 37Â°C.4. Wash the Microtiter Plate 4 times.Manual 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 Note: Hold the paper of the plate frame firm when washing the plate to assure that all strips remain securely in frame. Automated Washing - Aspirate all wells, then wash plates four times Using Washer Buffer (1X). Always adjust your brush to aspirate as much liquid as possible and set fill volume at 350Î¼L/well/wash. After final wash, invert plate, and blot dry by hitting plate mount absorbent paper or paper blanket until no Add chromogen solution A 50Î¼l and chromogen solution B 50Î¼l to each well. Gently mix and incubate for 15 minutes at 37Â°C. Protect from light.6. Add 50Î¼l Stop Solution to each well. The color in the wells Read the Optical Density (OD) at 450 nm using a microtiter plate reader The standard curve is generated by plotting the average OD (450 nm) obtained for each of the six standard concentrations on the vertical (X) 2. First, calculate the mean OD value for each standard and sample. All OD Values â€‹â€‹are subtracted by the mean value of the balnk well before result interpretation. Construct the standard curve using Graph To determine the amount in each sample, first locate the OD value on the Y-axis and extend a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the X-axis 4. Any variation in operator, pipetting and washing technique, incubation time or temperature, and kit age can cause variation in result. Each user should obtain their own standard curve.5. Intra-assay CV(%) And Inter-assay CV (%) are less than 15%.6. Assay range: 15.6 pg/mL â€“ 500 pg/mL.7. Sensitivity: The minimum detectable dose of Human P53-Ab is typically less than 10 pg/mL .8. Cross-reactivity: This assay recognizes recombinant and natural Human P53-Ab. No significant cross-reactivity or interference was observed. 9. Storage: 2-8 Â° C (Use frequently); six months (-20 Â° C). 10. QUOTE READ ONH;
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