alpha 2 Macroglobulin ELISA Kit (alpha-2-Macroglobulin)

Details for Product A2M ELISA Kit No. ABIN5563508, Supplier: Log in to see
Antigen
  • A2M
  • LOC733429
  • LOC100221148
  • LOC100224071
  • endod
  • cpamd5
  • fwp007
  • s863-7
  • a2mb
  • endodermin
  • A2MD
  • CPAMD5
  • FWP007
  • S863-7
  • A2mp
  • A2MAC1
  • A2m1
  • A2maa
  • Mam
  • alpha-2-macroglobulin
  • ovostatin
  • pregnancy-zone protein
  • A2M
  • LOC733429
  • AZL_c00450
  • LOC100221148
  • LOC100224071
  • LOC100349077
  • OVST
  • a2m
  • A2m
  • LOC100353095
  • PZP
Reactivity
Human
Alternatives
Kits with alternative reactivity to:
33
19
9
9
5
4
2
2
1
1
1
Method Type
Sandwich ELISA
Detection Range
12.3-3000 ng/mL
Minimum Detection Limit
12.3 ng/mL
Application
ELISA
Options
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Purpose The kit is a sandwich based ELISA for the in vitro quantitative measurement of Alpha-2 M in human serum, plasma, and cell culture media
Sample Type Cell Culture Supernatant, Plasma, Serum
Analytical Method Quantitative
Detection Method Colorimetric
Specificity This assay has high sensitivity and excellent specificity for detection of Alpha-2 M
Cross-Reactivity (Details) No significant cross-activity was observed between this target and other analogues
Sensitivity 5 ng/mL
Components
  1. Human Alpha-2 M Microplate: 96 breakable wells (12strips x 8wells) coated with anti-human Alpha-2 M.
  2. 20x Wash Buffer Concentrate: 1 Vial, 25 ml.
  3. 5x Assay Diluent: 1vial, 15 ml.
  4. Standards: 2 vials, recombinant human Alpha-2 M.
  5. Detection Antibody: 2 vials, biotinylated anti-human Alpha-2 M.
  6. HRP-Streptavidin Concentrate: 1vial.
  7. TBM Substrate solution: 1 Vial, 12 ml.
  8. Stop Solution: 1 Vial, 8 ml of 0.2 M sulfuric acid.
Material not included
  1. Distilled or deionized water 2.Precision pipettes, with disposable plastic tips 3.Beakers, flasks, cylinders necessary for preparation of reagents 4.Microplate washing device (multichannel pipette or automated microplate washer) 5.Microplate shaker 6.Microplate reader capable of reading at 450 nm and 570nm.
Alternative Name A2M (A2M ELISA Kit Abstract)
Gene ID 2
UniProt P01023
Application Notes
  1. All reagents must be at room temperature (18 °C to 25 °C) before running assay.
  2. Do not mix or substitute reagents with those from other lots or other sources.
  3. Do not use kit reagents beyond expiration date on label.
  4. Do not expose kit reagents to strong light during storage or incubation.
  5. Use disposable pipette tips for each transfer to avoid microbial contamination or cross contamination of reagents.
  6. Improper or insufficient washing at any stage of the procedure will result in either false positive or false negative results.
  7. Avoid contact of stop solution with skin or eyes. If contact occurs, immediately flush area with copious amounts of water.
  8. Do not use TMB substrate solution if it has begun to turn blue.
  9. Do not expose bleach to work area during actual test procedure because of potential interference with enzyme activity.
Sample Volume 100 μL
Assay Time 4.5 h
Plate Pre-coated
Protocol The Human Alpha-2 M ELISA kit is a solid phase sandwich ELISA (enzyme-linked immunosorbent assay) for the quantitative measurement of Alpha-2 M in human serum and plasma. An antibody specific for human Alpha-2 M was coated on a 96-well plate. Standards and samples are added to the wells and any Alpha-2 M present binds to the immobilized antibody. The wells are washed and biotinylated anti-human Alpha-2 M antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is added to the wells. The wells are again washed and TMB substrate solution is added, which produces a blue color in direct proportion to the amount of Alpha-2 M present in the initial sample. The Stop Solution changes the color from blue to yellow, and the microwell absorbances are read at 450 nm and 570nm.
Reagent Preparation
  1. Assay diluent: Dilute the concentrated assay diluent 1:5 with distilled water (e.g. 10 mL plus 40ml).2. Wash buffer: Dilute the concentrated wash buffer 1:20 with distilled water (e.g. 20 mL plus 380ml).
  2. Standard a. Briefly spin standard vial before use. Add 500 μL 1x Assay Diluent to prepare a 3000 ng/mL standard. Gently vortex to mix. b. Add 300 μL 1x Assay Diluent to 7 tubes. Label as 1200 ng/mL, 480 ng/mL, 192 ng/mL, 76.8 ng/mL, 30.7 ng/mL, 12.3 ng/mL and the last tube with 1x assay diluent is the blank as 0 ng/mL. c. Perform serial dilutions by adding 200 μL of each standard to the next tube and vortexing between each transfer.
  3. Detection Ab: Briefly spin the Detection Antibody vial before use. Add 100 μL of 1X Assay Diluent into the vial to prepare a detection antibody concentrate. The detection antibody concentrate should be diluted 80-fold with 1X Assay Diluent.
  4. Streptavidin-HRP: Briefly spin the HRP-Streptavidin concentrate vial and pipette up and down to mix gently before use. HRP-Streptavidin concentrate should be diluted 700-fold with 1X Assay Diluent.
  5. Sample: Levels of the target protein may vary among different specimens. Optimal dilution factors for each sample must be determined by the investigator, the recommended dilution for serum and plasma is 1: 2000.
Sample Collection Serum: Use a serum separator tube (SST) and allow samples to clot for two hours at room temperature or overnight at 4 °C before centrifugation for 15 minutes at 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20 °C or -80 °C. Avoid repeated freeze-thaw cycles. Plasma: Collect plasma using EDTA, or citrate or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 x g at 2-8 °C within 30 minutes of collection. Assay immediately or aliquot and store samples at -20 °C or -80 °C. Avoid repeated freeze-thaw cycles. Cell Culture Supernatant: Centrifuge samples for 20 minutes at 1000 x g. Remove particulates and assay immediately or store samples in aliquot at -20 °C or -80 °C for later use. Avoid repeated freeze/thaw cycles.
Sample Preparation
  1. Assay diluent: Dilute the concentrated assay diluent 1:5 with distilled water (e.g. 10 mL plus 40ml).2. Wash buffer: Dilute the concentrated wash buffer 1:20 with distilled water (e.g. 20 mL plus 380ml). 3. Standard a. Briefly spin standard vial before use. Add 500 μL 1x Assay Diluent to prepare a 3000 ng/mL standard. Gently vortex to mix. b. Add 300 μL 1x Assay Diluent to 7 tubes. Label as 1200 ng/mL, 480 ng/mL, 192 ng/mL, 76.8 ng/mL, 30.7 ng/mL, 12.3 ng/mL and the last tube with 1x assay diluent is the blank as 0 ng/mL. c. Perform serial dilutions by adding 200 μL of each standard to the next tube and vortexing between each transfer. 4. Detection Ab: Briefly spin the Detection Antibody vial before use. Add 100 μL of 1X Assay Diluent into the vial to prepare a detection antibody concentrate. The detection antibody concentrate should be diluted 80-fold with 1X Assay Diluent. 5. Streptavidin-HRP: Briefly spin the HRP-Streptavidin concentrate vial and pipette up and down to mix gently before use. HRP-Streptavidin concentrate should be diluted 700-fold with 1X Assay Diluent. 6. Sample: Levels of the target protein may vary among different specimens. Optimal dilution factors for each sample must be determined by the investigator, the recommended dilution for serum and plasma is 1: 2000.
Assay Procedure
  1. All reagents must be brought to room temperature (18-25 °C) prior to use
  2. Prepare all reagents, detection anytibody, standard and samples as directed in the respective sections.
  3. Remove required quantity of test strips/wells, place in well holder.4. Add 100 μL of each standard and sample into appropriate wells. Cover well and incubate for 2.5 hours at room temperature or over night at 4 °C with gentle shaking.
  4. Decant or aspirate contents of wells. Wash wells by filling with at least 300 μL/well prepared wash buffer followed by decanting/aspirating. Repeat wash 4 times for a total of 5 washes. After the last wash, blot plate on absorbent paper to remove residual buffer.
  5. Add 100 μL of 1X prepared biotinylated antibody to each well. Incubate for 1 hour at room temperature with gentle shaking.7. Discard the solution. Repeat the wash as in step
  6. 8. Add 100 μL of prepared Streptavidin solution to each well. Incubate for 45 minutes at room temperature with gentle shaking.
  7. Discard the solution. Repeat the wash as in step
  8. 10. Add 100 μL of TMB One-Step Substrate Reagent to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking.11. Add 50 μL of Stop Solution to each well. Read absorbance at 450nm within 30 minutes of stopping reaction. wavelength correction is available, subtract the optical density readings at 570nm from readings at 450nm.
Calculation of Results

Average the duplicate readings for each standard and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the target antigen concentration versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

Assay Precision intra CV<10%, inter CV <15%
Restrictions For Research Use only
Storage 4 °C,-20 °C
Storage Comment 4°C/-20°C,May be stored at 2-8°C for up to 1 month. For long term storage, please store at -20°C. Try to keep assay plate in a sealed aluminium foil bag and avoid dampness.
Expiry Date 6 months