IgM ELISA Kit

Catalog No. ABIN612720

Product Details IgM ELISA Kit

Target: IgM

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Minimum Detection Limit: 1.5 ng/mL

Application: ELISA

Purpose: The AssayMax Human Immunoglobulin M (IgM) ELISA kit is designed for detection of human IgM in plasma, serum, cell culture media and urine samples

Brand: AssayMax

Sample Type: Plasma, Cell Culture Supernatant

Analytical Method: Quantitative

Specificity: Immunoglobulins: IgM, IgA, IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, IgD 2, IgE

Components: IgM Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against IgM Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay. IgM Standard: Human IgM in a buffered protein base (200 ng, lyophilized) Biotinylated Human IgM Antibody (50x): A 50-fold concentrated biotinylated polyclonal antibody against human IgM (140µl). 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 (80µl). 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, 200-1000µLand multiple channel pipettes) Deionized or distilled reagent grade water

Application Details

Assay Time: < 4 h

Plate: Pre-coated

Protocol: This assay employs a quantitative sandwich enzyme immunoassay technique that measures human IgM in less than 4 hours. A polyclonal antibody specific for human IgM has been pre-coated onto a 96-well microplate with removable strips. IgM in standards and samples is sandwiched by the immobilized polyclonal antibody and biotinylated polyclonal antibody specific for human IgM, 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. MIx Diluent Concentrate (10x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the MIx Diluent 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. Standard Curve: Reconstitute the 200 ng of IgM Standard with 2 ml of MIx Diluent to generate a 100 ng/ml 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 the standard solution (100 ng/ml) 1:2 with MIx Diluent to produce 50, 25, 12.5, 6.25, 3.125, and 1.563 ng/ml solutions. MIx Diluent serves as the zero standard (0 ng/ml). Any remaining solution should be frozen at

Sample Collection: 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. 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. Dilute samples 1:60000 into MIx Diluent. If necessary dilute samples within the range of 1:30000 to 1:120000. 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 1:60000 into MIx Diluent. If necessary dilute samples within the range of 1:30000 to 1:120000. The undiluted samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. 2 Urine: Collect urine using sample pot. Centrifuge samples at 600 x g for 10 minutes. Dilute samples 1:20 into MIx Diluent. If necessary dilute samples within the range of 1:10 to 1:40. Store samples at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Saliva: Collect saliva using sample tube. Centrifuge samples at 600 x g for 10 minutes. Dilute samples 1:200 into MIx Diluent. If necessary dilute samples within the range of 1:100 to 1:400.Store samples at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Milk: Collect milk using sample tube. Centrifuge samples at 600 x g for 10 minutes Dilute samples 1:2000 into MIx Diluent. If necessary dilute samples within the range of 1:1000 to 1:4000. Store samples 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. 3 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. 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 complete remove liquid at each step. Add 50 µL of Biotinylated Human IgM Antibody to each well and incubate for one hour. Wash the microplate as described above. 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 the 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 7.3% respectively.

Restrictions: For Research Use only

Handling

Handling Advice: Prepare all reagents (working diluent buffer, wash buffer, standards, 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 protocol. 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 kit should not be used beyond the expiration date.

Storage: 4 °C/-20 °C

Storage Comment: Store components of the kit at 2-8°C or -20°C upon arrival 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 Opened unused microplate wells may be returned to the foil pouch with the desiccant packs. Reseal along zip-seal. May be stored for up to 1 month in a vacuum desiccator. Diluent (1x) may be stored for up to 1 month at 2-8°C. Store Standard at 2-8°C before reconstituting with Diluent and at -20°C after reconstituting with Diluent.

References for IgM ELISA Kit (ABIN612720)

Doppler, Appeltshauser, Krämer, Ng, Meinl, Villmann, Brophy, Dib-Hajj, Waxman, Weishaupt, Sommer: "Contactin-1 and Neurofascin-155/-186 Are Not Targets of Auto-Antibodies in Multifocal Motor Neuropathy." in: PLoS ONE, Vol. 10, Issue 7, pp. e0134274, (2015) (PubMed).

Vangelista, Cesco-Gaspere, Lorenzi, Burrone: "A minimal receptor-Ig chimera of human FcepsilonRI alpha-chain efficiently binds secretory and membrane IgE." in: Protein engineering, Vol. 15, Issue 1, pp. 51-7, (2002) (PubMed).

Target Details for IgM

Target: IgM

Abstract: IgM Products

Synonyms: IgM constant region ELISA Kit, IGM ELISA Kit

Target Type: Antibody

Background: Human Immunoglobulin M (IgM) is a mushroom-shaped largest antibody against A and B antigens on red blood cell and is produced by B cells. It forms a pentamer or a hexamer in serum, and also a monomer on B cell surface. Each of the five monomers has a molecular mass of 180 kDa, consists of two light and two heavy chains, and a joining J chain required for the synthesis of the pentamer (2-3). Upon an exposure to an acute infection, IgM is the predominant antibody produced to fight the foreign red blood cell antigen. It activates complement and agglutinates red blood cells. IgM is the first immunoglobulin made by the fetus and by B cells when stimulated by antigen (4-5). It does not pass across the human placenta due to its large size. Elevated IgM indicates viral hepatitis infection and primary biliary cirrhosis (6-8). IgM is a useful tool in the diagnosis of infectious diseases.