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  • 產品名稱：MouseMEP1AELISAPairSet

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MouseMEP1AELISAPairSet
詳情介紹：
Purpose The ELISA Pair Set is a solid phase sandwich ELISA (Enzyme-Linked Immunosorbent Assay). It utlizes monoclonal antibody specific for MEP1A coated on a 96-well plate. Standards and samples are added to the wells,and any MEP1A present binds to the immobilized antibody. The wells are washed and a horseradish peroxidaseconjugated rabbit anti-MEP1A monoclonal antibody is then added, producing an antibody-antigen-antibody "sandwich".The wells are again washed and TMB substrate solution is loaded, which produces color in proportion to the amount ofMEP1A present in the sample. To end the enzyme reaction, the stop solution is added and absorbances of themicrowell are read at 450 nm.
Sensitivity The minimum detectable dose of Mouse MEP1A was determined to be approximately 78.125 pg/mL. This is defined as at least three times standard deviations above the mean optical density of 10 replicates of the zero standard.
Characteristics Pair Set
Components Capture Antibody - 1.0 mg/mL of rabbit anti-mouse MEP1A monoclonal antibody. Dilute to aworking concentration of 2.0 μg/mL in CBS before coating.
Detection Antibody - 0.25 mg/mL rabbit anti-mouse MEP1A polyclonal antibody conjugated tohorseradish-peroxidase (HRP) . Dilute to working concentration of 0.5 μg/mL in detection antibodydiluteion buffer before use.
Standard - Each vial contains 170 ng of recombinant mouse MEP1A. Reconstitute standardpowder with 1mL detection antibody dilution buffer. After reconstitution, store at -20 °C to -70 °C in amanual defrost freezer. A seven-point standard curve using 2-fold serial dilutions in sample dilutionbuffer, and a high standard of 5000 pg/mL is recommended.
Material not included CBS - 0.05 M
Na2CO3 - 0.05 M
NaHCO3, pH 9.6, 0.2 μm filtered
TBS - 25 mM
Tris, adjust pH to 7.4 by HCl
Wash Buffer - 0.05 % Tween20 in TBS, pH 7.2 - 7.4
Blocking Buffer - 5 % BSA in Wash Buffer
Sample dilution buffer - 0.5 % BSA in wash buffer, pH 7.2 - 7.4, 0.2 μm filtered
Detection antibody dilution buffer - 0.5 % BSA in wash buffer, pH 7.2 - 7.4, 0.2 μm filtered
Substrate Solution: To achieve best assay results, fresh substrate solution is recommended Substrate stock solution - 10 mg/mL TMB (Tetramethylbenzidine ) in DMSO Substrate dilution buffer - 0.05 M Na2HPO4 and 0.025 M citric acid , adjust pH to 5.5
Substrate working solution - For each plate dilute 250 μL substrate stock solution in 25 mL substrate dilutionbuffer and then add 80 μL 0.75 % H2O2 , mix it well
Stop Solution - 2 N H2SO4
Alternative Name MEP1A
Background Meprin A subunit alpha, also known as MEP1A, and Endopeptidase-2, is a single-pass type I membrane protein whichbelongs to the peptidase M12A family. MEP1A contains one EGF-like domain, one MAM domain, and one MATHdomain. Meprins are unique plasma membrane and secreted metalloproteinases that are highly regulated at thetranscriptional and post-translational levels. Meprin alpha and beta subunits are abundantly expressed in kidney andintestinal epithelial cells, are secreted into the urinary tract and intestinal lumen, and are found in leukocytes and cancercells under certain conditions. Meprins are capable of proteolytically degrading extracellular matrix proteins, proteolytically processing bioactive proteins, and play a role in inflammatory processes. Meprin A and B are highlyregulated, secreted and cell-surface homo- and hetero-oligomeric enzymes. Meprins are abundantly expressed inkidney and intestine. The multidomain alpha and beta subunits have high sequence identity. They have very differentsubstrate specificities, oligomerization potentials and are differentially regulated. Meprin A appears to be an importanttherapeutic target and urinary excretion appears to be a potential biomarker of acute kidney injury ( AKI) .
Application Notes Optimal working dilution should be determined by the investigator.
Comment

The Mouse MEP1A ELISA Pair Set is for the quantitative determination of Mouse MEP1A.This ELISA Pair Set contains the basic components required for the development of sandwich ELISAs.
Reagent Preparation
  1. Dilute the capture antibody to the working concentration in CBS. Immediately coat a 96-well microplate with 100 μL per well of the diluted capture antibody. Seal the plate and incubate overnight at 4 °C.
    2. Aspirate each well and wash with at least 300 μL wash buffer, repeating the process two times for a total of three washes. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining wash buffer by inverting the plate and blotting it against clean paper towels.
    3. Block plates by adding 300 μL of blocking buffer to each well. Incubate at room temperature for a minimum of 1 hour.
    4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
Assay Procedure
  1. Add 100 μL of sample or standards in sample dilution buffer per well. Seal the plate and incubate 2 hours at roomtemperature.
    2. Repeat the aspiration/wash as in step 2 of plate preparation.
    3. Add 100 μL of the detection antibody, diluted in antibody dilution buffer, to each well. Seal the plate and incubate 1hour at room temperature.
    4. Repeat the aspiration/wash as in step 2 of plate preparation.
    5. Add 200 μL of substrate solution to each well. Incubate for 20?minutes at room temperature (if substrate solutionis not as requested, the incubation time should be optimized). Avoid placing the plate in direct light.
    6. Add 50 μL of stop solution to each well. Gently tap the plate to ensure thorough mixing.
    7. Determine the optical density of each well immediately, using a microplate reader set to 450 nm.
Calculation of Results

Calculate the mean absorbance for each set of duplicate standards, controls and samples. Subtract the mean zero standard absorbance from each. Construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. To determine the concentration of the unknowns, find the unknowns' mean absorbance value on the y-axis and draw a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the x-axis and read the concentration. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. Alternatively, computer-based curve-fitting statistical software may also be employed to calculate the concentration of the sample.
Restrictions For Research Use only
Format Lyophilized
Handling Advice Avoid repeated freeze-thaw cycles.
Storage -20 °C/-80 °C
Storage Comment Capture Antibody: Aliquot and store at -20°C to -80°C for up to 6 months from date of receipt. Detection Antibody: Protect it from prolonged exposure to light. Aliquot and store at -20°C to -80°C and for up to 6 months from date of receipt. Standard: Store lyophilized standard at -20°C to -80°C for up to 6 months from date of receipt. Aliquot and store the reconstituted Standard at -80°C for up to 1 month.
Expiry Date 6 months
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