beta-2 Microglobulin ELISA Kit

Catalog No. ABIN612797

Product Details beta-2 Microglobulin ELISA Kit

Target: beta-2 Microglobulin (B2M) (beta-2-Microglobulin (B2M))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 0.49-50 ng/mL

Minimum Detection Limit: 0.49 ng/mL

Application: ELISA

Purpose: The AssayMax Human beta-2-Microglobulin ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of human B2M in plasma, serum, milk, saliva, urine, CSF, and cell culture samples. This assay employs a quantitative sandwich enzyme immunoassay technique that measures human B2M in approximately 4 hours. A polyclonal antibody specific for human B2M has been pre-coated onto a 96-well microplate with removable strips. B2M in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for human B2M, which is recognized by a streptavidin-peroxidase (SP) 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, Cerebrospinal Fluid, Milk, Plasma, Saliva, Serum, Urine

Analytical Method: Quantitative

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: Human beta-2-Microglobulin Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human B2M. Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes that can be cut to fit the format of the individual assay. Human beta-2-Microglobulin Standard: Human B2M in a buffered protein base (40 ng, lyophilized). Biotinylated Human beta-2-Microglobulin Antibody (50x): A 50-fold concentrated biotinylated polyclonal antibody against B2M (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). SP Conjugate (100x): A 100-fold concentrate (80 l). Chromogen Substrate (1x): A stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution (1x): A 0.5 N hydrochloric acid solution 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)

Application Details

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 10 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 the MIX Diluent Concentrate 10-fold with reagent grade water to produce a 1x solution. Store for up to 30 days at 2-8 °C. Human beta-2-Microglobulin Standard: Reconstitute the Human beta-2- Microglobulin Standard (40 ng, 2867.2 mIU) with 0.8 mL of MIX Diluent to generate a 50 ng/mL (3584 mIU/mL) standard stock solution. Allow the vial 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 (50 ng/mL) 4-fold with MIX Diluent to produce 12.5, 3.125, 0.781, 0.195, and 0.049 ng/mL solutions. MIX Diluent serves as the zero standard (0 ng/mL). Any remaining stock solution should be stored at -20 °C and used within 30 days. Standard Point Dilution [B2M] (ng/mL) [B2M] (mIU/mL) P1 1 part Standard 50 3584 P2 1 part P1 + 3 parts MIX Diluent 12.5 896 P3 1 part P2 + 3 parts MIX Diluent 3.125 224 P4 1 part P3 + 3 parts MIX Diluent 0.781 56 P5 1 part P4 + 3 parts MIX Diluent 0.195 14 P6 1 part P5 + 3 parts MIX Diluent 0.049 3.5 P7 MIX Diluent 0.0 0.0 5 Biotinylated Human beta-2-Microglobulin Antibody (50x): Spin down the antibody briefly and dilute the desired amount of the antibody 50- fold with MIX Diluent to produce a 1x solution. 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 the Wash Buffer Concentrate 20-fold with reagent grade water to produce a 1x solution. SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 100-fold with MIX Diluent to produce a 1x solution. 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 1000-fold sample dilution is suggested 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 1000-fold sample dilution is suggested 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 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. Saliva: Collect saliva using sample tube. Centrifuge samples at 800 x g for 10 minutes. A 200-fold sample dilution is suggested 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. Milk: Collect milk using sample tube. Centrifuge samples at 800 x g for 10 minutes. A 4000-fold sample dilution is suggested 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. CSF: Collect cerebrospinal fluid (CSF) using sample pot. Centrifuge samples at 3000 x g for 10 minutes. A 1000-fold sample dilution is suggested into MIX Diluent, however, user should determine optimal dilution factor depending on application needs. The undiluted samples can be stored at -80 °C 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.

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 securely to minimize exposure to water vapor and store in a vacuum desiccator. Add 50 l of Human beta-2-Microglobulin Standard or sample 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 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 Human beta-2-Microglobulin 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 SP Conjugate 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 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 to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Incubate for 10 minutes or until the optimal blue color density develops. 6 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 4.9 % and 7.2% respectively.

Restrictions: For Research Use only

Handling

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.

References for beta-2 Microglobulin ELISA Kit (ABIN612797)

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).

Target Details for beta-2 Microglobulin

Target: beta-2 Microglobulin (B2M) (beta-2-Microglobulin (B2M))

Abstract: B2M Products

Synonyms: Ly-m11 ELISA Kit, beta2-m ELISA Kit, beta2m ELISA Kit, bwm ELISA Kit, wu:fa94c05 ELISA Kit, wu:fb10a09 ELISA Kit, b2m-W01 ELISA Kit, b2m-W03 ELISA Kit, b2m-Z01 ELISA Kit, b2m-Z02 ELISA Kit, b2m-Z03 ELISA Kit, B2M ELISA Kit, b2m ELISA Kit, B2MG ELISA Kit, wu:fb66f11 ELISA Kit, wu:fj01f08 ELISA Kit, zgc:64161 ELISA Kit, b-2-m ELISA Kit, beta-2-microglobulin ELISA Kit, beta-2 microglobulin ELISA Kit, beta-2-microglobulin S homeolog ELISA Kit, beta-2-microglobulin, like ELISA Kit, B2M ELISA Kit, B2m ELISA Kit, b2m ELISA Kit, b2m.S ELISA Kit, b2ml ELISA Kit

Background: Beta-2-microglobulin (B2M) 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 a molecular mass of 12 kDa (1-4). B2M is released from the cell surface of HLA class I into the serum and carried to the kidneys for degradation and secretion (5).

Gene ID: 567

UniProt: P61769

Pathways: TCR Signaling, Regulation of Leukocyte Mediated Immunity, Positive Regulation of Immune Effector Process