Human MICB / MIC-B ELISA Pair Set

Details for Product No. ABIN2010453, Supplier: Log in to see
Target Name (Antigen)
  • PERB11.2
  • MHC class I polypeptide-related sequence B
  • MHC class I-related protein-like
  • MICB
  • LOC512825
Reactivity
Human
Application
ELISA
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Purpose The ELISA Pair Set is a solid phase sandwich ELISA (Enzyme-Linked Immunosorbent Assay). It utlizes monoclonal antibody specific for MICB coated on a 96-well plate. Standards and samples are added to the wells, andany MICB present binds to the immobilized antibody. The wells are washed and a biotinylated rabbit anti-MICBpolyclonal antibody is then added, producing an antibody-antigen-antibody "sandwich". To produces color in proportionto the amount of MICB present in the sample strepavidin-HRP and TMB substrate solution are loaded. Theabsorbances of the microwell are read at 450 nm.
Sensitivity The minimum detectable dose of human MICB was determined to be approximately 62.5 pg/mL. This is defined as at least three times standard deviations above the mean optical density of 10 replicates of the zero standard.
Characteristics Pair Set
Components Capture Antibody - 0.5 mg/mL of mouse anti-MICB monoclonal antibody. Dilute to a workingconcentration of 0.5 μg/mL in CBS before coating.
Detection Antibody - Each vial contains 70 μg biotinylated rabbit anti-MICB polyclonal antibody.Reconstitute with 1 mL detection antibody dilution buffer. After reconstitution, store at -20 °C to -70 °Cin a manual defrost freezer. Dilute to a working concentration of 0.5 μg/mL in detection antibodydilution buffer before use.
Standard - Each vial contains 50ng of recombinant MICB. Reconstitute vith 1 mL detectionantibody dilution buffer. After reconstitution, store at -20 °C to -70 °C in a manual defrost freezer. Aseven-point standard curve using 2-fold serial dilutions in sample dilution buffer, and a highstandard of 4ng/mL is recommended.
Streptavidin-HRP - 50 μL of streptavidin conjugated to horseradish-peroxidase. 1:2000 Dilution indetection antibody dilution buffer before use.
Material not included CBS - 0.05 M
Na2CO3 - 0.05 M
NaHCO3, pH 9.6, 0.2 μm filtered
TBS - 25 mM
Tris, adjust pH to 7.4 by HCl
Wash Buffer - 0.05 % Tween20 in TBS, pH 7.2 - 7.4
Blocking Buffer - 5 % BSA in Wash Buffer
Sample dilution buffer - 0.5 % BSA in wash buffer, pH 7.2 - 7.4, 0.2 μm filtered
Detection antibody dilution buffer - 0.5 % BSA in wash buffer, pH 7.2 - 7.4, 0.2 μm filtered
Substrate Solution: To achieve best assay results, fresh substrate solution is recommended Substrate stock solution - 10 mg/mL TMB (Tetramethylbenzidine ) in DMSO Substrate dilution buffer - 0.05 M Na2HPO4 and 0.025 M citric acid , adjust pH to 5.5
Substrate working solution - For each plate dilute 250 μL substrate stock solution in 25 mL substrate dilutionbuffer and then add 80 μL 0.75 % H2O2 , mix it well
Stop Solution - 2 N H2SO4
Alternative Name MICB
Background MHC class I polypeptide-related sequence B, also known as MICB, is a heavily glycosylated protein serving as a ligandfor the type II receptor NKG2D. MICB is widely expressed with the exception of the central nervous system where it isabsent. MICB is expressed in many, but not all, epithelial tumors of lung, breast, kidney, ovary, prostate and colon. Inhepatocellular carcinomas, it is expressed in tumor cells but not in surrounding non-cancerous tissue. MICB shares85 % amino acid identity with MICA, a closely related protein, both of which contain three extracellular immunoglobulin-like domains, but without capacity to bind peptide or interact with beta-2-microglobulin. Acting as a stress-induced self-antigen, binding of MICB to the NKG2D receptor activates the cytolytic response of natural killer (NK) cells, CD8+αβ Tcells, and γδ T cells on which the receptor is expressed. MICA, a ligand of the activating immunoreceptor NKG2D, isreleased by tumor cells in a soluble form and can be detected in sera of tumor patients at significant levels. MICA/B areminimally expressed on normal cells, but are frequently expressed on epithelial tumors and can be induced by bacterialand viral infections. MICA/B recognition thus is involved in tumor surveillance, viral infections, and autoimmunediseases. Unlike classical MHC class I molecules, MICB does not form a heterodimer with beta-2-microglobulin. It binds as amonomer to a KLRK1 / NKG2D homodimer. KLRK1 forms a complex with HCST / DAP10 in which KLRK1 binds MICBwhile HCST acts as an adapter molecule which enables signal transduction. Receptor-ligand interaction inducesclustering of both proteins in ordered structures called immune synapses and also leads to their intercellular transfer. This is associated with a reduction in the cytotoxicity of KLRK1-expressing cells. MICB binds to human cytomegalovirusglycoprotein UL16 which causes sequestration of MICB in the endoplasmic reticulum and increases resistance toKLRK1-mediated cytotoxicity.
Application Notes Optimal working dilution should be determined by the investigator.
Comment

The human MICB ELISA Pair Set is for the quantitative determination of human MICB.This ELISA Pair Set contains the basic components required for the development of sandwich ELISAs.

Reagent Preparation
  1. Dilute the capture antibody to the working concentration in CBS. Immediately coat a 96-well microplate with 100 μL per well of the diluted capture antibody. Seal the plate and incubate overnight at 4 °C.
    2. Aspirate each well and wash with at least 300 μL wash buffer, repeating the process two times for a total of three washes. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining wash buffer by inverting the plate and blotting it against clean paper towels.
    3. Block plates by adding 300 μL of blocking buffer to each well. Incubate at room temperature for a minimum of 1 hour.
    4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
Assay Procedure
  1. Add 100 μL of sample or standards in sample dilution buffer per well. Seal the plate and incubate 2 hours at roomtemperature.
    2. Repeat the aspiration/wash as in step 2 of plate preparation.
    3. Add 100 μL of the detection antibody, diluted in antibody dilution buffer, to each well. Seal the plate and incubate 1hour at room temperature.
    4. Repeat the aspiration/wash as in step 2 of plate preparation.
    5. Add 100 μL of Streptavidin-HRP to each well. Incubate for 1 hour at room temperature.
    6. Repeat the aspiration/wash as in step 2 of plate preparation.
    7. Add 200 μL of substrate solution to each well. Incubate for 20 minutes at room temperature (if substrate solutionis not as requested, the incubation time should be optimized). Avoid placing the plate in direct light.
    8. Add 50 μL of stop solution to each well. Gently tap the plate to ensure thorough mixing.9. Determine the optical density of each well immediately, using a microplate reader set to 450 nm.
Calculation of Results

Calculate the mean absorbance for each set of duplicate standards, controls and samples. Subtract the mean zero standard absorbance from each. Construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. To determine the concentration of the unknowns, find the unknowns' mean absorbance value on the y-axis and draw a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the x-axis and read the concentration. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. Alternatively, computer-based curve-fitting statistical software may also be employed to calculate the concentration of the sample.

Restrictions For Research Use only
Format Lyophilized
Precaution of Use The Stop Solution suggested for use with this Pair Set is an acid solution. Wear eye, hand, face, andclothing protection when using this material.
Handling Advice Avoid repeated freeze-thaw cycles.
Storage 4 °C/-20 °C/-80 °C
Storage Comment Capture Antibody: Aliquot and store at -20°C to -80°C for up to 6 months from date of receipt. Detection Antibody: Aliquot and store at -20°C to -80°C for up to 6 months from date of receipt. Standard: Store lyophilized standard at -20°C to -80°C for up to 6 months from date of receipt. Aliquot and store the reconstituted Standard at -80°C for up to 1 month. Streptavidin-HRP: Store at 4°C and protect it from prolonged exposure to light. DO NOT FREEZE! It is stable for up to 6 months from date of receipt.
Expiry Date 6 months
Supplier Images
ELISA image for Human MICB / MIC-B ELISA Pair Set (ABIN2010453) Human MICB ELISA Pair Set Standard Curve 13867