Human Complement C5a ELISA Pair Set

Details for Product No. ABIN2010301, Supplier: Log in to see
Antigen
  • C5
  • LOC565774
  • C5a
  • C5b
  • CPAMD4
  • Hc
  • RGD1561905
  • He
  • complement component 5
  • similar to complement component C5-1
  • hemolytic complement
  • C5
  • LOC565774
  • Hc
Reactivity
Human
Host
Mouse
Conjugate
HRP
Method Type
Sandwich ELISA
Application
ELISA
Options
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Purpose The Human Complement C5a ELISA Pair Set is for the quantitative determination of Human Complement C5a .
Detection Method Colorimetric
Sensitivity 31.25 pg/mL
Characteristics Capture Antibody: 1 mg/mL of mouse anti-Human Complement C5a monoclonal antibody (in PBS, pH 7.4). Dilute to a working concentration of 2 μg/mL in PBS before coating.

Detection Antibody: 0.2 mg/mL of rabbit anti-Human Complement C5a polyclonal antibody conjugated to horseradish-peroxidase (HRP) (in PBS, 50 % HRP-Protector, pH 7.4, store at 4 °C). Dilute to working concentration of 0.25 μg/mL in detection antibody dilution buffer before use.

Standard: Each vial contains 100 ng of recombinant Human Complement C5a . Reconstitute 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 2000 pg/mL is recommended.

Sensitivity: The minimum detectable dose of Human Complement C5a was determined to be approximately 31.25 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
Alternative Name C5
Background C5a is a protein fragment released from complement component C5. This 74 amino acid peptide in humans isgenerated by the cleavage of C5a convertase on the C5 α-chain during the classical, alternative, and lectin pathways ofcomplement activation. The structure of C5a includes a core region consisting of four, anti-parallel alpha-helices heldtogether by three disulfide linkages and a structured C-terminal tail, and C5a is rapidly metabolised bycarboxypeptidase B to a 73 amino acid low activity form, C5a des-Arg. C5a is an extremely potent proinflammatorymediator, as well as a potent chemotactic factor for neutrophils and other leukocytes. It causes histamine release, increases in vascular permeability, induces several cytokines production from leukocytes, enhances neutrophil-endothelial cell adhesion, and augments the humoral and cell-mediated immune response. C5a exerts actions bybinding to G-protein-coupled receptor (C5aR/CD88) on the plasma membrane of target cells. Accordingly, theanaphylatoxin C5a is implicated in a variety of diseases such as rheumatoid arthritis, systemic lupus erythematosus, reperfusion injury, Alzheimer's disease, and sepsis.
Research Area Innate Immunity, Complement Factors
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 Human Complement C5a coated on a 96-well plate. Standards and samples are added to the wells, and any Human Complement C5a present binds to the immobilized antibody. The wells are washed and a horseradish peroxidase conjugated rabbit anti-Human Complement C5a polyclonal 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 Human Complement C5a 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 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. Avoid repeated freeze-thaw cycles.

Detection Antibody: Store at 4°C and protect it from prolonged exposure to light for up to 6 months from date of receipt. DO NOT FREEZE!

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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