Human Cystatin B / CSTB ELISA Pair Set

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Target Name (Antigen)
  • CST6
  • EPM1
  • EPM1A
  • PME
  • STFB
  • ULD
  • CSTB
  • Cyb
  • Epm1
  • Stfb
  • MGC189005
  • pme
  • cst6
  • epm1
  • stfb
  • AA960480
  • ATCYS6
  • cystatin B
  • LOC100008600
  • LOC100136235
  • CPI1
  • cystatin B
  • cystatin B (stefin B)
  • cystatin 14a, tandem duplicate 2
  • cystatin-B
  • CSTB
  • Cstb
  • cstb
  • cst14a.2
  • CYSB
  • cpi1
  • LOC101123265
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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 Cystatin B / CSTB coated on a 96-well plate. Standards and samples are added tothe wells, and any Cystatin B / CSTB present binds to the immobilized antibody. The wells are washed and ahorseradish peroxidase conjugated mouse anti-Cystatin B / CSTB monoclonal antibody is then added, producing anantibody-antigen-antibody "sandwich". The wells are again washed and TMB substrate solution is loaded, whichproduces color in proportion to the amount of Cystatin B / CSTB present in the sample. To end the enzyme reaction, the stop solution is added and absorbances of the microwell are read at 450 nm.
Sensitivity The minimum detectable dose of Human Cystatin B / CSTB was determined to be approximately 9.4 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 - 0.2 mg/mL of mouse anti-CSTB monoclonal antibody. Dilute to a workingconcentration of 2 μg/mL in CBS before coating.
Detection Antibody - 0.5 mg/mL mouse anti-CSTB monoclonal antibody conjugated tohorseradish-peroxidase (HRP) . Dilute to working concentration of 0.5 μg/mL in detection antibodydiluteion buffer before use.
Standard - Each vial contains 24 ng of recombinant CSTB. Reconstitute with 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 0.6 ng/mL is recommended.
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 CSTB
Background Cystatin-B, also known as CPI-B, Liver thiol proteinase inhibitor, Stefin-B, CSTB and CST6, is a cytoplasm and nucleusprotein which belongs to the cystatin family. Cystatin-B / CSTB is an intracellular thiol proteinase inhibitor. Tightlybinding reversible inhibitor of cathepsins L, H and B. Cystatin-B / CSTB is able to form a dimer stabilized bynoncovalent forces, inhibiting papain and cathepsins l, h and b. Cystatin-B / CSTB is also thought to play a role inprotecting against the proteases leaking from lysosomes. Defects in Cystatin-B / CSTB are the cause of progressivemyoclonic epilepsy type 1 (EPM1) which is an autosomal recessive disorder characterized by severe, stimulus-sensitivemyoclonus and tonic-clonic seizures. The cystatins are a family of cysteine protease inhibitors with homology to chickencystatin. Cystatins are physiological inhibitors of cysteine proteinases which are widely distributed in human tissues andfluids. Cystatins typically comprise about 115 amino acids, are largely acidic, contain four conserved cysteine residuesknown to form two disulfide bonds. Cystatins may be glycosylated and / or phosphorylated, with similarity to fetuins, kininogens, stefins, histidine-rich glycoproteins and cystatin-related proteins. Some of the members are active cysteineprotease inhibitors, while others have lost or perhaps never acquired inhibitory activity. Cystatins mainly inhibitpeptidases belonging to peptidase families C1 (papain family) and C13 (legumain family) .
Application Notes Optimal working dilution should be determined by the investigator.

The Human Cystatin B / CSTB ELISA Pair Set is for the quantitative determination of Human Cystatin B / CSTB.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 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.
    6. Add 50 μL of stop solution to each well. Gently tap the plate to ensure thorough mixing.
    7. 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
Handling Advice Avoid repeated freeze-thaw cycles.
Storage -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: Protect it from prolonged exposure to light. Aliquot and store at -20°C to -80°C and 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.
Expiry Date 6 months
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