alpha 2 Macroglobulin ELISA Kit

Catalog No. ABIN612730

Human alpha 2 Macroglobulin ELISA Kit, Colorimetric assay for quantification of Human alpha 2 Macroglobulin.

Quick Overview for alpha 2 Macroglobulin ELISA Kit (ABIN612730)

Target: alpha 2 Macroglobulin (A2M) (alpha-2-Macroglobulin (A2M))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Competition ELISA

Application: ELISA

Sample Type: Plasma

Product Details alpha 2 Macroglobulin ELISA Kit

Minimum Detection Limit: 1 µg/mL

Purpose: The AssayMax Human alpha-2-Macroglobulin ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of human alpha-2-Macroglobulin in plasma, and serum

Brand: AssayMax™

Analytical Method: Quantitative

Specificity: Reference Value: The normal blood levels of alpha-2-Macroglobulin range from 1.49-1.79 g/L.

Components: Alpha-2-Macroglobulin Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against alpha-2-Macroglobulin. Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay. Alpha-2-Macroglobulin Standard: Human alpha-2-Macroglobulin in a buffered protein base (80 µg, lyophilized). Biotinylated Alpha-2-Macroglobulin: 1 vial, lyophilized. EIA Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml). Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (80µl). 1 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 450 nm. Pipettes (1-20 µL, 20-200 µL, 200-1000µLand multiple channel). Deionized or distilled reagent grade water

Application Details

Sample Volume: 25 μL

Assay Time: < 3 h

Plate: Pre-coated

Protocol: This assay employs a quantitative competitive enzyme immunoassay technique that measures human alpha- 2-Macroglobulin in less than 3 hours. A polyclonal antibody specific for human alpha-2- Macroglobulin has been pre-coated onto a 96-well microplate with removable strips. Alpha-2- Macroglobulin in standards and samples is competed by a biotinylated alpha-2-Macroglobulin sandwiched by the immobilized antibody and streptavidin-peroxidase conjugate. All unbound material is then washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured.

Reagent Preparation:

Freshly dilute all reagents and bring all reagents to room temperature before use. If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. EIA Diluent Concentrate (10x): Dilute EIA Diluent Concentrate 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. Standard Curve: Reconstitute the 80 g of Human alpha-2-Macroglobulin Standard with 2 ml of EIA Diluent to generate a stock solution of 40 g/ml. 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 alpha-2-Macroglobulin standard solution (40 g/ml) 1:2 with EIA Diluent to produce 20, 10, 5, 2.5 and 1.25 g/ml solutions. EIA Diluent serves as the zero standard (0 g/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [Alpha-2-Macroglobulin] ( g/ml) 1 part Standard (40 g/ml) P1 40.00 P2 1 part P1 + 1 part EIA Diluent 20.00 P3 1 part P2 + 1 part EIA Diluent 10.00 P4 1 part P3 + 1 part EIA Diluent 5.00 P5 1 part P4 + 1 part EIA Diluent 2.50 P6 1 part P5 + 1 part EIA Diluent 1.25 P7 EIA Diluent 0.00 Biotinylated Alpha-2-Macroglobulin (2x): Dilute Biotinylated alpha-2-Macroglobulin with 4 ml EIA Diluent to produce a 2-fold stock solution. Allow the biotin to sit for 10 minutes with gentle agitation prior to making dilutions. The stock solution should be further diluted 1:2 with EIA Diluent. Any remaining solution should be frozen at -20°C. Wash Buffer Concentrate (20x): Dilute 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.

Sample Collection: Plasma: Collect plasma using one-tenth volume of 0.1 M sodium citrate as an anticoagulant. Centrifuge samples at 2000 x g for 10 minutes and assay. Dilute samples 1:400 with EIA Diluent. 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 anticoagulant.) Serum: Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 2000 x g for 10 minutes. Remove serum and assay. Dilute samples 1:400 into EIA Diluent. The undiluted samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles.

Assay Procedure:

Prepare all reagents, working standards and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20 - 30 °C). Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccant inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator. Add 25 µL of standard or sample per well, and immediately add 25 µL of Biotinylated alpha- 2-Macroglobulin to each well (on top of the Standard or sample) and mix gently. Cover wells with a sealing tape and incubate for two hours. Start the timer after the last sample addition. Wash five times with 200 µL of Wash Buffer manually. Invert the plate each time and decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid. If using a machine wash six times with 300 µL of Wash Buffer and then invert the plate, decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid. Add 50 µL of Streptavidin-Peroxidase Conjugate to each well and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance. Wash a microplate as described above. Add 50 µL of Chromogen Substrate per well and incubate for about 10 minutes or till the optimal blue color density develops. Gently tap plate to ensure thorough mixing and break the bubbles in the well with pipette tip. Add 50 µL 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.

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 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. Standard Curve The curve is provided for illustration only. A standard curve should be generated each time the assay is performed.

Assay Precision: Intra-assay and inter-assay coefficients of variation were 4.9 % and 7.1% respectively.

Restrictions: For Research Use only

Handling

Handling Advice: The kit should not be used beyond the expiration date.

Storage: 4 °C/-20 °C

Storage Comment: Store kit at 2-8°C or -20°C upon arrival up to the expiration date. Opened EIA Diluent may be stored for up to 1 month at 2-8°C. Store reconstituted reagents at -20°C or below. Opened unused strip wells may return to the foil pouch with the desiccant pack, reseal along zip-seal. May be stored for up to 1 month in a vacuum desiccator.

Target Details for alpha 2 Macroglobulin

Target: alpha 2 Macroglobulin (A2M) (alpha-2-Macroglobulin (A2M))

Alternative Name: alpha-2-Macroglobulin

Background: Alpha-2-Macroglobulin is a major serum protein with diverse functions, including inhibition of protease activity and binding of growth factors, cytokines, and disease factors. Increased serum alpha-2-Macroglobulin has been suggested to be associated with multiple sclerosis (MS) , glomerular disease , and with liver diseases.

Pathways: Lipid Metabolism