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Assay Time
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4 h
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Plate
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Pre-coated
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Protocol
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- Step 1. Add 50 μL of Standard or Sample per well. Incubate 2 hours.
- Step 2. Wash, then add 50 μL of Biotinylated Antibody per well. Incubate 1 hour.
- Step 3. Wash, then add 50 μL of SP Conjugate per well. Incubate 30 minutes.
- Step 4. Wash, then add 50 μL of Chromogen Substrate per well. Incubate 10 minutes.
- Step 5. Add 50 μL of Stop Solution per well. Read at 450 nm immediately.
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Reagent Preparation
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Freshly dilute all reagents and bring all reagents to room temperature before use. EIA Diluent Concentrate (10x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the EIA 4 Diluent Concentrate 1:10 with reagent grade water. Store for up to 30 days at 2-8 °C. Human Factor IX Standard: Reconstitute the 220 ng (44 mU) of Human Factor IX Standard with 2.2 mL of EIA Diluent to generate a 100 ng/mL (20 mU/mL) standard stock solution. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare duplicate or triplicate standard points by serially diluting the standard stock solution (100 ng/mL) 1:2 with EIA Diluent to produce 50, 25, 12.5, 6.25, 3.125, and 1.563 ng/mL solutions. EIA Diluent serves as the zero standard (0 ng/mL). Any remaining solution should be frozen at -20 °C and used within 30 days. Standard Point Dilution [FIX] (ng/mL) [FIX] (mU/mL) P1 1 part Standard 100.0 20.00 P2 1 part P1 + 1 part EIA Diluent 50.00 10.00 P3 1 part P2 + 1 part EIA Diluent 25.00 5.000 P4 1 part P3 + 1 part EIA Diluent 12.50 2.500 P5 1 part P4 + 1 part EIA Diluent 6.250 1.250 P6 1 part P5 + 1 part EIA Diluent 3.125 0.625 P7 1 part P6 + 1 part EIA Diluent 1.563 0.313 P8 EIA Diluent 0.000 0.000 Biotinylated Human Factor IX Antibody (50x): Spin down the antibody briefly and dilute the desired amount of the antibody 1:50 with EIA Diluent. Any remaining solution should be frozen at -20 °C. Wash Buffer Concentrate (20x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the Wash Buffer Concentrate 1:20 with reagent grade water. SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 1:100 with EIA Diluent. Any remaining solution should be frozen at -20 °C.
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Sample Collection
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Plasma: Collect plasma using one-tenth volume of 0.1 M sodium citrate as an anticoagulant. Centrifuge samples at 3000 x g for 10 minutes and collect supernatants. Dilute samples 1:400 into EIA Diluent and assay. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles (EDTA or Heparin can also be used as an anticoagulant). Serum: Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 3000 x g for 10 minutes, and remove serum. Dilute samples 1:400 into EIA Diluent and assay. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Cell Culture Supernatants: Collect cell culture media and centrifuge at 3000 x g for 10 minutes at 4 °C to remove debris. Collect supernatants and assay. Samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. CSF: Collect cerebrospinal fluid (CSF) using sample pot. Centrifuge samples at 3000 x g for 10 minutes and assay. Samples can be stored at -80 °C for up to 3 months. Avoid repeated freeze-thaw cycles. Refer to Sample Dilution Guidelines below for further instruction. Guidelines for Dilutions of 1:100 or Greater (for reference only, please follow the insert for specific dilution suggested) 1:100 1:10000 A) 4 μL sample: 396 μL buffer(100x) = 100 fold dilution Assuming the needed volume is less than or equal to 400 μL. A) 4 μL sample : 396 μL buffer (100x) B) 4 μL of A : 396 μL buffer (100x) = 10000 fold dilution Assuming the needed volume is less than or equal to 400 μL. 1:1000 1:100000 A) 4 μL sample : 396 μL buffer (100x) B) 24 μL of A : 216 μL buffer (10x) = 1000 fold dilution Assuming the needed volume is less than or equal to 240 μL. A) 4 μL sample : 396 μL buffer (100x) B) 4 μL of A : 396 μL buffer (100x) C) 24 μL of B : 216 μL buffer (10x) = 100000 fold dilution Assuming the needed volume is less than or equal to 240 μL.
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Assay Procedure
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Prepare all reagents, standard solutions, and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20-25 °C). Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccants inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator. 5 Add 50 l of Human Factor IX Standard or sample per well. Cover wells with a sealing tape and incubate for 2 hours. Start the timer after the last addition. Wash five times with 200 l of Wash Buffer manually. Invert the plate each time and decant the contents, hit 4-5 times on absorbent material to completely remove the liquid. If using a machine, wash six times with 300 l of Wash Buffer and then invert the plate, decanting the contents, hit 4-5 times on absorbent material to completely remove the liquid. Add 50 l of Biotinylated Human Factor IX Antibody to each well and incubate for 1 hour. Wash the microplate as described above. Add 50 l of Streptavidin-Peroxidase Conjugate per well and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance. Wash the microplate as described above. Add 50 l of Chromogen Substrate per well and incubate for 10 minutes or till the optimal color density develops. Gently tap the plate to ensure thorough mixing and break the bubbles in the well with pipette tip. Add 50 l of Stop Solution to each well. The color will change from blue to yellow. Read the absorbance on a microplate reader at a wavelength of 450 nm immediately. If wavelength correction is available, subtract readings at 570 nm from those at 450 nm to correct optical imperfections. Otherwise, read the plate at 450 nm only. Please note that some unstable black particles may be generated at high concentration points after stopping the reaction for about 10 minutes, which will reduce the readings.
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Calculation of Results
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- Calculate the mean value of the duplicate or triplicate readings for each standard and sample.
- To generate a standard curve, plot the graph using the standard concentrations on the x-axis and the corresponding mean 450 nm absorbance (OD) on the y-axis. The best-fit line can be determined by regression analysis using log-log or four-parameter logistic curve-fit.
- Determine the unknown sample concentration from the standard curve and multiply the value by the dilution factor.
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Restrictions
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For Research Use only
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