alpha 2 Macroglobulin ELISA Kit

Catalog No. ABIN956039

Product Details alpha 2 Macroglobulin ELISA Kit

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

Reactivity: Rat

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Purpose: The Rat Alpha 2-Macroglobulin ELISA is a highly sensitive two-site enzyme-linked immunoassay (ELISA) for the quantitative determination of alpha 2-macroglobulin in rat biological samples.

Analytical Method: Quantitative

Characteristics: This kit is specific to the alpha 2-macroglobulin and will not cross-react with the closely related alpha 1-macroglobulin.In this assay the alpha 2-macroglobulin present in samples reacts with the anti-A2M antibodies, which have been adsorbed to the surface of polystyrene microtiter wells. After the removal of unbound proteins by washing, anti-A2M antibodies conjugated with horseradish peroxidase (HRP), are added. This HRP-conjugated antibody forms a complex with the previously bound alpha 2-macroglobulin. Following another washing step, the enzyme bound to the immunosorbent is assayed by the addition of a chromogenic substrate, 3,3',5,5'-tetramethylbenzidine (TMB). The quantity of bound enzyme is proportional to the concentration of alpha 2-macroglobulin in the sample tested, thus, the absorbance, at 450 nm, is a measure of the concentration of alpha 2-macroglobulin in the test sample. The quantity of alpha 2-macroglobulin in the test sample can be interpolated from the calibration curve constructed from the calibrators, and corrected for sample dilution.

Components: 1. Diluent Concentrate: One bottle containing 50 mL of a 5X concentrated diluent running buffer.
2. Wash Solution Concentrate: One bottle containing 50 mL of a 20X concentrated wash solution.
3. Enzyme-Antibody Conjugate Concentrate: One vial containing 150 µL of a 100X concentrated affinity-purified anti-rat alpha 2-macroglobulin antibody conjugated with HRP in a stabilizing buffer.
4. TMB Substrate Solution: One vial containing 12 mL of TMB and hydrogen peroxide in citric acid buffer at pH 3.3.
5. Stop Solution: One vial containing 12 mL of 0.3 M sulfuric acid. WARNING: Avoid contact with skin.
6. Microtiter Plate: Twelve removable eight-well strips in well holder frame. Wells are coated with affinity-purified anti-rat alpha 2- macroglobulin.
7. Rat Alpha 2-Macroglobulin Calibrator: One vial containing a lyophilized Rat Alpha 2-Macroglobulin Calibrator.
8. Positive Control: One vial containing 50 µL of serum with 0.1% sodium azide. See the Control Certificate for the concentration.

Material not included: Test tubes
Precision pipettes (2 µL to 200 µL) for making and dispensing dilutions
Microplate washer/aspirator
Distilled or de-ionized H2O Microplate reader
Assorted glassware for the preparation of reagents and buffer solutions
Timer

Application Details

Plate: Pre-coated

Reagent Preparation:

1. Diluent Concentrate: The Diluent solution supplied is a 5X concentrate and must be diluted 1:5 with distilled or de-ionized water.
   2. Wash Solution Concentrate: The Wash Solution supplied is a 20X concentrate and must be diluted 1:20 with distilled or de-ionized water. Crystal formation in the concentrate is not uncommon when storage temperatures are low. Warming of the concentrate to 30-35°C before dilution can dissolve crystals.
   3. Enzyme-Antibody Conjugate Concentrate: Calculate the required amount of working conjugate solution for each microtiter plate test strip by adding 10 µL Enzyme-Antibody Conjugate to 990 µL of 1X Diluent for each test strip to be used for testing. Mix uniformly, but gently. Avoid foaming.
   4. TMB Substrate Solution: Ready to use as supplied.
   5. Stop Solution: Ready to use as supplied.
   6. Microtiter Plate: Ready to use as supplied. Unseal Microtiter Pouch and remove plate from pouch. Remove all strips and wells that will not be used in the assay and place back in pouch and re-seal along with desiccant.
   7. Rat Alpha 2-Macroglobulin Calibrator: Add 1.0 mL of distilled or de-ionized water to the lyophilized Rat Alpha 2-Macroglobulin Calibrator and mix gently until dissolved. The calibrator is now at a concentration of 1.811 µg/mL (the reconstituted calibrator should be aliquoted and frozen if future use is intended). Prepare the Rat Alpha 2-Macroglobulin Calibrators immediately prior to use according to the table below. Mix well between each step. Avoid foaming.
   8. Positive Control: The concentration and recommended dilution are provided on the Control Certificate. Before use, briefly centrifuge the Positive Control to allow all of the liquid to collect in the bottom of the vial.

Calculation of Results:

1. Subtract the average background value from the test values for each sample.
   2. Using the results observed for the calibrators construct a calibration curve. The appropriate curve fit is that of a four- parameter logistics curve. A second order polynomial (quadratic) or other curve fits may also be used.
   3. Interpolate test sample values from the calibration curve. Correct for sample dilution factor to arrive at alpha 2- macroglobulin concentration in original sample.
   
   1. Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete understanding of the information contained in the package insert instructions and with adherence to good laboratory practice.
   2. Factors that might affect the performance of the assay include proper instrument function, cleanliness of glassware, quality of distilled or de-ionized water, and accuracy of reagent and sample pipettings, washing technique, incubation time or temperature.

Restrictions: For Research Use only

Handling

Storage: 4 °C

Storage Comment: 1. Complete Kit: The expiration date for the kit is stated on the outer label. The recommended storage temperature is 4°C. Note: See long-term storage recommendations below for the Rat Alpha 2-Macroglobulin Calibrator and Positive Control.
2. Diluent: The 5X Diluent Concentrate is stable until the expiration date. The 1X working solution is stable for at least one week from the date of preparation. Both solutions should be stored at 4°C.
3. Wash Solution: The 20X Wash Solution Concentrate is stable until the expiration date. The 1X working solution is stable for at least one week from the date of preparation. Both solutions can be stored at room temperature (RT, 16-25°C) or at 4°C.
4. Enzyme-Antibody Conjugate: Undiluted anti-A2M-HRP conjugate should be stored at 4°C and diluted immediately prior to use. The working conjugate solution is stable for up to 1 hour when stored in the dark.
5. TMB Substrate Solution: The TMB Substrate Solution should be stored at 4°C and is stable until the expiration date.
6. Stop Solution: The Stop Solution should be stored at 4°C and is stable until the expiration date.
7. Microtiter Plate: Anti-rat alpha 2-macroglobulin coated wells are stable until the expiration date and should be stored at 4°C in the sealed foil pouch with a desiccant pack.
8. Rat Alpha 2-Macroglobulin Calibrator: The lyophilized Rat Alpha 2-Macroglobulin Calibrator should be stored at 4°C or frozen until reconstituted. The reconstituted calibrator should be aliquoted and stored frozen. Avoid multiple freeze/thaw cycles. The working calibrator solutions should be prepared immediately prior to use and are stable for up to 8 hours.
9. Positive Control: For storage longer than 7 days keep frozen until the expiration date. Storage less than 7 days can be at 4°C. Avoid multiple freeze/thaw cycles.

Expiry Date: The expiry date is stated on the label.

Target Details for alpha 2 Macroglobulin

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

Alternative Name: Alpha-2 Macroglobulin (A2M Products)

Synonyms: A2MD ELISA Kit, CPAMD5 ELISA Kit, FWP007 ELISA Kit, S863-7 ELISA Kit, A2MAC1 ELISA Kit, A2m1 ELISA Kit, A2maa ELISA Kit, Mam ELISA Kit, A2mp ELISA Kit, A2M ELISA Kit, LOC733429 ELISA Kit, endod ELISA Kit, cpamd5 ELISA Kit, fwp007 ELISA Kit, s863-7 ELISA Kit, a2mb ELISA Kit, endodermin ELISA Kit, Alpha-2-M ELISA Kit, alpha-2-macroglobulin ELISA Kit, pregnancy-zone protein L homeolog ELISA Kit, alpha-2-macroglobulin S homeolog ELISA Kit, pregnancy zone protein ELISA Kit, alpha-2-macroglobulin-like ELISA Kit, A2M ELISA Kit, A2m ELISA Kit, LOC477699 ELISA Kit, pzp.L ELISA Kit, AZL_c00450 ELISA Kit, LOC100349077 ELISA Kit, a2m ELISA Kit, LOC100061656 ELISA Kit, LOC100090399 ELISA Kit, a2m.S ELISA Kit, LOC100469973 ELISA Kit, LOC100595735 ELISA Kit, PZP ELISA Kit, LOC100353095 ELISA Kit, LOC101122940 ELISA Kit, LOC101801552 ELISA Kit, LOC100911545 ELISA Kit

Background: Alpha 2-Macroglobulin (A2M) is a major protease inhibitor in serum and an acute phase protein which increases significantly in concentration in the rat as a result of inflammation. The major pathophysiological role for rat alpha 2- macroglobulin has yet to be conclusively defined.

Pathways: Lipid Metabolism