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

Details for Product A2M ELISA Kit No. ABIN612742, 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
Mouse (Murine)
Alternatives
Kits with alternative reactivity to:
37
20
9
9
5
4
2
2
1
1
1
Method Type
Sandwich ELISA
Detection Range
0.313-100 ng/mL
Minimum Detection Limit
0.313 ng/mL
Application
ELISA
Options
Supplier
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Supplier Product No.
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Purpose The AssayMax Mouse alpha-2-Macroglobulin ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of mouse alpha-2- macroglobulin in mouse plasma, serum, urine, and cell culture samples. This assay employs a quantitative sandwich enzyme immunoassay technique that measures mouse alpha-2-macroglobulin in approximately 4 hours. A polyclonal antibody specific for mouse alpha-2-macroglobulin has been pre- coated onto a 96-well microplate with removable strips. Mouse alpha-2- macroglobulin in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for mouse alpha-2- macroglobulin, which is recognized by a streptavidin-peroxidase conjugate. All unbound material is washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured.
Brand AssayMax™
Sample Type Cell Culture Cells, Plasma, Serum, Urine
Analytical Method Quantitative
Detection Method Colorimetric
Components Mouse alpha-2-Macroglobulin Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against mouse alpha-2-macroglobulin. Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes that can be cut to fit the format of the individual assay. Mouse alpha-2-Macroglobulin Standard: Mouse alpha-2-macroglobulin in a buffered protein base (100 ng, lyophilized). Biotinylated Mouse alpha-2-Macroglobulin Antibody (50x): A 50-fold concentrated biotinylated polyclonal antibody against mouse alpha-2- macroglobulin (120 l). MIX Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml, 2 bottles). Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (80 l). Chromogen Substrate: A ready-to-use stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution: A 0.5 N hydrochloric acid to stop the chromogen substrate reaction (12 ml).
Material not included Microplate reader capable of measuring absorbance at 405 nm. Pipettes (1-20 µL, 20-200 µL, and multiple channel). Deionized or distilled reagent grade water. Incubator (37 °C)
Alternative Name Alpha-2-Macroglobulin (A2M ELISA Kit Abstract)
Background Alpha-2-macroglobulin (A2m) is a major serum protein with diverse functions, including inhibition of protease activity and binding of growth factors, cytokines, and disease factors (1).
Gene ID 232345
UniProt Q6GQT1
Sample Volume 50 μL
Assay Time 4 h
Plate Pre-coated
Protocol
  • 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 25 minutes.
  • Step 5. Add 50 μL of Stop Solution per well. Read at 450 nm immediately.
Reagent Preparation

Freshly dilute all reagents and bring all reagents to room temperature before use. MIX Diluent Concentrate (10x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute MIX Diluent Concentrate 10-fold with reagent grade water. Store for up to 30 days at 2-8 °C. Mouse alpha-2-Macroglobulin Standard: Reconstitute the 100 ng of Mouse alpha-2-Macroglobulin Standard with 1 mL of MIX Diluent to generate a 100 ng/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 from the standard stock solution (100 ng/mL) 4-fold with MIX Diluent to produce 25, 6.25, 1.563, and 0.391 ng/mL solutions. MIX Diluent serves as the zero standard (0 ng/mL). Any remaining stock solution should be frozen at -20 °C and used within 30 days. Avoid repeated freeze-thaw cycles. Standard Point Dilution [Mouse A2m] (ng/mL) P1 1 part Standard (100 ng/mL) 100.0 P2 1 part P1 + 3 parts MIX Diluent 25.0 P3 1 part P2 + 3 parts MIX Diluent 6.25 P4 1 part P3 + 3 parts MIX Diluent 1.563 P5 1 part P4 + 3 parts MIX Diluent 0.391 P6 MIX Diluent 0.0 Biotinylated Mouse alpha-2-Macroglobulin Antibody (50x): Spin down the antibody briefly and dilute the desired amount of the antibody 50- fold with MIX Diluent. The undiluted antibody should be stored at -20 °C. Wash Buffer Concentrate (20x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute Wash Buffer Concentrate 20-fold with reagent grade water. SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 100-fold with MIX Diluent. The undiluted conjugate should be stored at -20 °C.

Sample Collection 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 plasma. A 50000-fold sample dilution is suggested or within the range of 20000x - 200000x into MIX Diluent, however, user should determine optimal dilution factor depending on application needs. 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. A 50000-fold sample dilution is suggested or within the range of 20000x - 200000x into MIX Diluent, however, user should determine optimal dilution factor depending on application needs. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Urine: Collect urine using sample pot. Centrifuge samples at 800 x g for 10 minutes. A 100-fold sample dilution is suggested or within the range of 5x - 500x into MIX Diluent, however, user should determine optimal dilution factor depending on application needs. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Cell Culture Supernatants: Centrifuge cell culture media at 3000 x g for 10 minutes at 4 °C to remove debris and collect supernatants. Samples can be stored at -20 °C or below. Avoid repeated freeze-thaw cycles. Refer to Sample Dilution Guidelines for further instruction. Guidelines for Dilutions of 100-fold or Greater (for reference only, please follow the insert for specific dilution suggested) 100x 10000x 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. 1000x 100000x 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. 4
Assay Procedure

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 5 securely to minimize exposure to water vapor and store in a vacuum desiccator. Add 50 l of Mouse alpha-2-Macroglobulin Standard or sample per well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. 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 Mouse alpha-2-Macroglobulin Antibody to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Cover wells with a sealing tape and incubate for 1 hour. Wash the microplate as described above. Add 50 l of Streptavidin-Peroxidase Conjugate per well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Cover wells with a sealing tape 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. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Incubate for 25 minutes or till the optimal blue color density develops. Add 50 l of Stop Solution to each well. The color will change from blue to yellow. Gently tap plate to ensure thorough mixing. Break any bubbles that may have formed. 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.

Calculation of Results
  • 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.
Assay Precision Intra-assay and inter-assay coefficients of variation were 5.1 % and 7.0% respectively.
Restrictions For Research Use only
Handling Advice This product is for Research Use Only and is not intended for use in diagnostic procedures. Prepare all reagents (diluent buffer, wash buffer, standard, biotinylated antibody, and SP conjugate) as instructed, prior to running the assay. Prepare all samples prior to running the assay. The dilution factors for the samples are suggested in this insert. However, the user should determine the optimal dilution factor. Spin down the SP conjugate vial and the biotinylated antibody vial before opening and using contents. The Stop Solution is an acidic solution. The kit should not be used beyond the expiration date. 2
Storage 4 °C/-20 °C
Storage Comment Upon arrival, immediately store components of the kit at recommended temperatures up to the expiration date. Store SP Conjugate and Biotinylated Antibody at -20°C. Store Microplate, Diluent Concentrate (10x), Wash Buffer, Stop Solution, and Chromogen Substrate at 2-8°C. Unused microplate wells may be returned to the foil pouch with the desiccant packs and resealed. May be stored for up to 30 days in a vacuum desiccator. Diluent (1x) may be stored for up to 30 days at 2-8°C. Store Standard at 2-8°C before reconstituting with Diluent and at -20°C after reconstituting with Diluent.
Supplier Images
ELISA image for alpha-2-Macroglobulin (A2M) ELISA Kit (ABIN612742) alpha-2-Macroglobulin (A2M) ELISA Kit
Background publications Pineda-Salgado, Craig, Blank, Kessler: "Expression of Panza, an alpha2-macroglobulin, in a restricted dorsal domain of the primitive gut in Xenopus laevis." in: Gene expression patterns : GEP, Vol. 6, Issue 1, pp. 3-10, 2005 (PubMed).

Jensen, Humle Jørgensen, Datta, Sørensen: "Significantly increased fractions of transformed to total alpha2-macroglobulin concentrations in plasma from patients with multiple sclerosis." in: Biochimica et biophysica acta, Vol. 1690, Issue 3, pp. 203-7, 2004 (PubMed).

Yang, Chen: "Glomerular deposition of alpha 2-macroglobulin in glomerular diseases." in: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association, Vol. 12, Issue 3, pp. 465-9, 1997 (PubMed).

Shiota, Umeki, Okano, Kawasaki: "Hepatocyte growth factor and acute phase proteins in patients with chronic liver diseases." in: Journal of medicine, Vol. 26, Issue 5-6, pp. 295-308, 1996 (PubMed).

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