Human Complement Component C2 ELISA Pair Set

Details for Product No. ABIN2010303, Supplier: Log in to see
Antigen
  • CO2
  • complement component 2
  • complement component 2 (within H-2S)
  • C2
Reactivity
Human
Host
Rabbit
Conjugate
HRP
Method Type
Sandwich ELISA
Application
ELISA
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Purpose The Human Complement Component C2 ELISA Pair Set is for the quantitative determination of Human Complement Component C2.
This ELISA Pair Set contains the basic components required for the development of sandwich ELISAs.
Detection Method Colorimetric
Sensitivity 78.125 pg/mL
Characteristics Capture Antibody: 0.5 mg/mL of rabbit anti-Complement Component C2 monoclonal antibody. Dilute to a working concentration of 2.0 μg/mL in CBS before coating.

Detection Antibody: 0.5 mg/mL mouse anti-Complement Component C2 monoclonal antibody conjugated to horseradish-peroxidase (HRP). Dilute to working concentration of 1 μg/mL in detection antibody dilution buffer before use.

Standard: Each vial contains 240 ng of recombinant Complement Component C2. Reconstitute standard powder with 1 mL detection antibody dilution buffer. After reconstitution, store at -20 °C to -80 °C in a manual defrost freezer. A seven-point standard curve using 2-fold serial dilutions in sample dilution buffer, and a high standard of 5000 pg/mL is recommended.

Sensitivity: The minimum detectable dose of Human Complement Component C2 was determined to be approximately 78.125 pg/mL. This is defined as at least three times standard deviations above the mean optical density of 10 replicates of the zero standard.
Components
  • Capture Ab
  • Detection Ab
  • Standard
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
Background Complement component C2 is part of the classical complement pathway which plays a major role in innate immunityagainst infection. C2 is a glycoprotein synthesized in liver hepatocytes and several other cell types in extrahepatictissues. This pathway is triggered by a multimolecular complex C1, and subsequently the single-chain form of C2 iscleaved into two chains referred to C2a and C2b by activated C1. C2a containing a serine protease domain combineswith complement component C4b to form the C3 convertase C4b2a which is responsible for C3 activation, and leads tothe stimulation of adaptive immune responses via Lectin pathway. Deficiency of C2 (C2D) is the most common geneticdeficiency of the complement system, and two types of C2D have been recognized in the context of specific MHChaplotypes. C2D in human is reported to increase susceptibility to infection, and is associated with certain autoimmunediseases, such as rheumatological disorders.
Research Area Immunology, Innate Immunity
Application Notes Optimal working dilution should be determined by the investigator.
Protocol This ELISA Pair Set is a solid phase sandwich ELISA (Enzyme-Linked Immunosorbent Assay). It utilizes a monoclonal antibody specific for Complement Component C2 coated on a 96-well plate. Standards and samples are added to the wells, and any Complement Component C2 present binds to the immobilized antibody. The wells are washed and a horseradish peroxidase conjugated mouse anti-Complement Component C2 monoclonal antibody is then added, producing an antibody-antigen-antibody "sandwich". The wells are again washed and TMB substrate solution is loaded, which produces color in proportion to the amount of Complement Component C2 present in the sample. To end the enzyme reaction, the stop solution is added and absorbances of the microwell are read at 450 nm.
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 Liquid
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. Avoid repeated freeze-thaw cycles.

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. Avoid repeated freeze-thaw cycles.

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. Avoid repeated freeze-thaw cycles.
Expiry Date 6 months
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