2-Microglobulin ELISA Kit

Details for Product No. ABIN612797, Supplier: Log in to see
Method Type
Sandwich ELISA
Minimum Detection Limit
0.7 ng/mL
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Purpose The AssayMax Human 2-Microglobulin ELISA kit is designed for detection of human 2- Microglobulin in plasma, serum, urine and cell culture supernatants
Brand AssayMax
Sample Type Plasma, Cell Culture Supernatant
Analytical Method Quantitative
Detection Method Colorimetric
Specificity The normal serum levels of b2M is less than 2.7 ug/ml, and urine levels of b2M is less than 200 ng/ml.
Components 2-Microglobulin Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human 2-Microglobulin. Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes, which can be cut to fit the format of the individual assay. 2-Microglobulin Standard: human 2-Microglobulin in a buffered protein base (200 ng, lyophilized). 1 Biotinylated 2-Microglobulin Antibody (100x): A 100-fold biotinylated polyclonal antibody against 2-Microglobulin (80µl). 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). Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (90µl). 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.
Background 2-Microglobulin ( 2M) is a small serum protein that constitutes the light chain of the major histocompatibility class I human leukocyte antigen (HLA class I), an integral membrane protein involved in the immune response. The protein is 99 amino acid residues in length and has molecular mass of 12 kDa (1 - 4). 2M is released from the cell surface of HLA class I into the serum and carried to the kidneys for degradation and secretion. In chronic renal failure, 2M accumulates as insoluble amyloid aggregates and causes arthralgias, destructive osteoarthropathies, carpal tunnel syndrome and dialysis-related amyloidosis (6 - 8). Elevated serum 2M levels are associated with poor prognosis in multiple myeloma and lymphoma (9 - 12).
Sample Volume 50 μL
Assay Time 4 h
Plate Pre-coated
Protocol This assay employs a quantitative sandwich enzyme immunoassay technique that measures 2-Microglobulin in 4 hours. A polyclonal antibody specific for 2-Microglobulin has been pre-coated onto a microplate. 2-Microglobulin in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for 2-Microglobulin, which is recognized by a 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. MIx Diluent Concentrate (10x): Dilute the MIx Diluent Concentrate 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. 2-Microglobulin Standard: Reconstitute the 200 ng of human 2-Microglobulin Standard with 1 ml of MIx Diluent to generate a standard solution of 200 ng/ml. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare triplicate standard points by serially diluting the Standard solution (200 ng/ml) 1:4 with MIx Diluent to produce 50, 12.5, 3.13 and 0.78 ng/ml. MIx Diluent serves as the zero standard (0 ng/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [b2M] (ng/ml) P1 1 part Standard (200 ng/ml) 200.00 P2 1 part P1 + 3 part MIx Diluent 50.00 P3 1 part P2 + 3 part MIx Diluent 12.50 P4 1 part P3 + 3 part MIx Diluent 3.13 P5 1 part P4 + 3 part MIx Diluent 0.78 P6 MIx Diluent 0.00 Biotinylated 2-Microglobulin Antibody (100x): Spin down the biotinylated antibody briefly and dilute the desired amount of the antibody 1:100 with MIx Diluent. Any remaining solution should be frozen at -20°C. Wash Buffer Concentrate (20x): Dilute the 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 MIx 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 1:2000 into MIx Diluent. Add 5 µl of sample to 495 µl of MIx Diluent (1:100) to make Solution A, then add 25 µl of Solution A to 475 µl of MIx Diluent (1:20) to make a final working solution (1:2000). 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 1:2000 into MIx Diluent. Add 5 µl of sample to 495 µl of MIx Diluent (1:100) to make Solution A, then add 25 µl of Solution A to 475 µl of MIx Diluent (1:20) to make a final working solution (1:2000). 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 2000 x g for 10 minutes to remove debris. Collect supernatants and assay. Store samples at -20°C or below. Avoid repeated freeze-thaw cycles. Urine: Collect urine using sample pot. Centrifuge samples at 600 x g for 10 minutes and assay. Dilute samples 1:100 into MIx Diluent. Store samples at -20°C or below for up to 1 months. Avoid repeated freeze-thaw cycles. 2
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 50 µL of standard or sample per well. 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. Invert the plate and decant the contents, and hit it 4-5 times on absorbent paper towel to completely remove liquid at each step. Add 50 µL of Biotinylated 2-Microglobulin Antibody to each well and incubate for one hour. Wash five times with 200 µL of Wash Buffer. Add 50 µL of Streptavidin-Peroxidase Conjugate per well and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance. Wash five times with 200 µL of Wash Buffer. 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. 3 Read the absorbance on a microplate reader at a wavelength of 450 nm immediately. 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 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 7.2% respectively.
Restrictions For Research Use only
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 MIx 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.
Supplier Images
ELISA image for 2-Microglobulin ELISA Kit (ABIN612797) 2-Microglobulin ELISA Kit
Background publications Eakin, Berman, Miranker: "A native to amyloidogenic transition regulated by a backbone trigger." in: Nature structural & molecular biology, Vol. 13, Issue 3, pp. 202-8, 2006 (PubMed).

Corlin, Sen, Ladefoged, Lund, Nissen, Heegaard: "Quantification of cleaved beta2-microglobulin in serum from patients undergoing chronic hemodialysis." in: Clinical chemistry, Vol. 51, Issue 7, pp. 1177-84, 2005 (PubMed).

Trinh, Smith, Kalverda, Phillips, Radford: "Crystal structure of monomeric human beta-2-microglobulin reveals clues to its amyloidogenic properties." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 99, Issue 15, pp. 9771-6, 2002 (PubMed).

Kantarjian, Smith, Estey, Polyzos, OBrien, Pierce, Beran, Feldman, Keating: "Prognostic significance of elevated serum beta 2-microglobulin levels in adult acute lymphocytic leukemia." in: The American journal of medicine, Vol. 93, Issue 6, pp. 599-604, 1993 (PubMed).

Saper, Bjorkman, Wiley: "Refined structure of the human histocompatibility antigen HLA-A2 at 2.6 A resolution." in: Journal of molecular biology, Vol. 219, Issue 2, pp. 277-319, 1991 (PubMed).

Güssow, Rein, Ginjaar, Hochstenbach, Seemann, Kottman, Ploegh: "The human beta 2-microglobulin gene. Primary structure and definition of the transcriptional unit." in: Journal of immunology (Baltimore, Md. : 1950), Vol. 139, Issue 9, pp. 3132-8, 1987 (PubMed).

Bataille, Magub, Grenier, Donnadio, Sany: "Serum beta-2-microglobulin in multiple myeloma: relation to presenting features and clinical status." in: European journal of cancer & clinical oncology, Vol. 18, Issue 1, pp. 59-66, 1982 (PubMed).

Späti, Child, Kerruish, Cooper: "Behaviour of serum beta 2-microglobulin and acute phase reactant proteins in chronic lymphocytic leukaemia. A multicentre study." in: Acta haematologica, Vol. 64, Issue 2, pp. 79-86, 1981 (PubMed).

Krangel, Orr, Strominger: "Assembly and maturation of HLA-A and HLA-B antigens in vivo." in: Cell, Vol. 18, Issue 4, pp. 979-91, 1980 (PubMed).

Product cited in: Gattoni-Celli, Kirsch, Timpane, Isselbacher: "Beta 2-microglobulin gene is mutated in a human colon cancer cell line (HCT) deficient in the expression of HLA class I antigens on the cell surface." in: Cancer research, Vol. 52, Issue 5, pp. 1201-4, 1992 (PubMed).