Human BNP ELISA KitFor the quantitative in vitro determination of Human brain natriuretic peptideconcentrations inserum - plasma - celiac fluid - tissue homogenate - body fluidFOR LABORATORY RESEARCH USE ONLY.NOT FOR USE IN DIAGNOSTIC PROCEDURES.This package insert must be read in its entirety before using thisproduct.ELISAENZYME LINKED IMMUNOSORBENT ASSAYINTENDED USE AND TEST PRINCIPLEThis BNP ELISA kit is intended Laboratory for Research use only and is not for use in diagnosticor therapeutic procedures. The Stop Solution changes the color from blue to yellow and theintensity of the color is measured at 450 nm using a spectrophotometer. In order to measure theconcentration of BNP in the sample, this BNP ELISA Kit includes a set of calibration standards.The calibration standards are assayed at the same time as the samples and allow the operator toproduce a standard curve of Optical Density versus BNP concentration. The concentration of BNPin the samples is then determined by comparing the O.D. 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 temperatureor overnight at 4℃ before centrifugation for 20 minutes at approximately 2000×g. Removeserum and assay immediately or aliquot and store samples at -20℃. Avoid repeated freezethawcyclesPlasma - Collect plasma using heparin as an anticoagulant. Centrifuge samples for 30 minutes at2000×g at 2-8℃ within 30 minutes of collection. Store samples at -20℃. Avoid repeatedfreeze-thaw cycles.Cell culture supernates, tissue homogenate and other biological fluids - Removeparticulates by centrifugation and assay immediately or aliquot and store samples at -20℃.Avoid repeated freeze-thaw cycles.Note: The samples shoule be centrifugated dequately and no hemolysis or granule wasallowed.MATERIALS REQUIRED BUT NOT SUPPLIED1. 37 ℃ incubator2. Standard microplate reader capable of measuring absorbance at 450 nm3. Precision pipettes, disposable pipette tips and Absorbent paper4. Distilled or deionized waterREAGENTS PROVIDED1All 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 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 2User manual 1 1Sealed bags 1 1Note:1. Standard concentration was followed by: 4000、2000、1000、500、250、125 pg/mL.2. If samples generate values higher than the highest standard, please dilute thesamples with Sample Diluent and repeat the assay.PRECAUTIONS1. Do not substitute reagents from one kit lot to another. Standard, conjugate and microtiterplates are matched for optimal performance. Use only the reagents supplied by manufacturer.2. Allow kit reagents and materials to reach room temperature (20-25°C) before use. Do not usewater 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 reagents.5. Do not remove microtiter plate from the storage bag until needed. Unused strips should bestored at 2-8°C in their pouch with the desiccant provided.6. Use fresh disposable pipette tips for each transfer to avoid contamination.7. Do not mix acid and sodium hypochlorite solutions.28. Serum and plasma should be handled as potentially hazardous and capable of transmittingdisease. Disposable gloves must be worn during the assay procedure, since no known test methodcan offer complete assurance that products derived from Rat blood will not transmit infectiousagents. Therefore, all blood derivatives should be considered potentially infectious and goodlaboratory practices should be followed.9. All samples should be disposed of in a manner that will inactivate viruses.10. Liquid Waste: Add sodium hypochlorite to a final concentration of 1.0%. The waste shouldbe allowed to stand for a minimum of 30 minutes to inactivate the viruses before disposal.11. Substrate Solution is easily contaminated. If bluish prior to use, do not use.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 ( 20-25°C).REAGENT PREPARATION AND STORAGEWash Solution (1X) - Dilute 1 volume of Wash solution (20X) with 19 volumes of deionized ordistilled water. Wash Solution is stable for 1 month at 2-8°C.ASSAY PROCEDURE1. Prepare all reagents before starting assay procedure. It is recommended that all Standards andSamples be added in duplicate to the Microelisa Stripplate.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.Manual Washing - Remove incubation mixture by aspirating contents of the plate into a sink orproper 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 fora total of four times. After final wash, invert plate, and blot dry by hitting plate onto absorbentpaper or paper towels until no moisture appears. Note: Hold the sides of the plate frame firmly3when washing the plate to assure that all strips remain securely in frame.Automated Washing - Aspirate all wells, then wash plates four times using Wash Buffer (1X).Always adjust your washer to aspirate as much liquid as possible and set fill volume at350μL/well/wash. After final wash, invert plate, and blot dry by hitting plate onto absorbent paperor paper towels until no moisture appears.5. Add chromogen solution A 50μl and chromogen solution B 50μl to each well. Gently mix andincubate for 15 minutes at 37°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 plateto ensure thorough mixing.7. Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes.CALCULATION OF RESULTS1. This standard curve is used to determine the amount in an unknown sample. The standardcurve is generated by plotting the average O.D. (450 nm) obtained for each of the six standardconcentrations on the vertical (X) axis versus the corresponding concentration on the horizontal(Y) axis.2. First, calculate the mean O.D. value for each standard and sample. All O.D. Values aresubtracted by the mean value of the balnk well before result interpretation. Construct the standardcurve using graph paper or statistical software.3. To determine the amount in each sample, first locate the O.D. value on the Y-axis and extenda horizontal line to the standard curve. At the point of intersection, draw a vertical line to the Xaxisand read the corresponding concentration.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: 125 pg/mL – 4000 pg/mL.7. Sensitivity: The minimum detectable dose of Human BNP is typically less than100 pg/mL.8. Cross-reactivity: This assay recognizes recombinant and natural Human BNP. No significantcross-reactivity or interference was observed.49. Storage: 2-8℃ (Use frequently); six months (-20℃)。10. Standard curveFOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTICAPPLICATIONS! PLEASE READ THROUGH ENTIRE PROCEDUREBEFORE BEGINNING!5