Influenza A H9N2 Hemagglutinin / HA ELISA Pair Set

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  • Hemagglutinin
  • hemagglutinin
  • temporal expression: early/late
  • ha
  • ACIAD2784
  • HA
  • A56R
Influenza Virus
Method Type
Sandwich ELISA
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Purpose The Influenza A H9N2 Hemagglutinin / HA ELISA Pair Set is for the quantitative determination of Influenza A H9N2 Hemagglutinin / HA.
Detection Method Colorimetric
Sensitivity 156 pg/mL
Characteristics Capture Antibody: 0.5 mg/mL of mouse anti-Influenza A H9N2 Hemagglutinin / HA monoclonal antibody (in PBS, pH 7.4). Dilute to a working concentration of 1 μg/mL in PBS before coating.

Detection Antibody: 0.2 mg/mL of rabbit anti-Influenza A H9N2 Hemagglutinin / HA polyclonal antibody conjugated to horseradish-peroxidase (HRP) (in PBS, 50 % HRP-Protector, pH 7.4, store at 4 °C). Dilute to working concentration of 0.4 μg/mL in detection antibody dilution buffer before use.

Standard: Each vial contains 480 ng of recombinant Influenza A H9N2 Hemagglutinin / HA. 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 10000 pg/mL is recommended.

Sensitivity: The minimum detectable dose of Influenza A H9N2 Hemagglutinin / HA was determined to be approximately 156 pg/mL. This is defined as at least three times standard deviations above the mean optical density of 10 replicates of the zero standard.
  • 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 HA
Background Influenza (flu) is a respiratory infection in mammals and birds. This virus is divided into three main types (A, B and C) . Influenza A is found in a wide variety of bird and mammal species. Influenza B is largely confined to humans and is animportant cause of morbidity. Influenza C infects humans, dogs and pigs, sometimes causing both severe illness andlocal epidemics. Influenza A is further divided into subtypes based on differences in the membrane proteinshemagglutinin (HA) and neuraminidase (NA) . The notation HhNn is used to refer to the subtype comprising the hthdiscovered HA protein and the nth discovered NA protein. The HA is a trimer with a receptor binding pocket on theglobular head of each monomer. Subtypes are further divided into strains. Each genetically distinct virus isolate isusually considered to be a separate strain. H9N2 is a subtype of Influenza A. Hemagglutinin (HA) is a single-pass type I integral membrane glycoprotein from theinfluenza virus, and comprises over 80 % of the envelope proteins present in the virus particle. It is a homotrimer ofdisulfide-linked HA1-HA2. Binding of HA to sialic acid-containing receptors on the surface of its target cell brings aboutthe attachment of the virus particle to the cell and forms a endosome. Low pH in endosomes induce an irreversibleconformational change in HA2, releasing the hydrophobic portion "fusion peptide". After which, virus penetrates the celland pours its contents including the RNA genome into the cytoplasm mediated by fusion of the endocytosed virusparticle's own membrane and the endosomal membrane. Hemagglutinin plays a major role in the determination of hostrange restriction and virulence.
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 Influenza A H9N2 Hemagglutinin / HA coated on a 96-well plate. Standards and samples are added to the wells, and any Influenza A H9N2 Hemagglutinin / HA present binds to the immobilized antibody. The wells are washed and a horseradish peroxidase conjugated rabbit anti-Influenza A H9N2 Hemagglutinin / HA 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 Influenza A H9N2 Hemagglutinin / HA 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
Supplier Images
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Product cited in: Ren, Lu, Wang, Huo, Cui, Zheng, Dai, Chen, Qin, Chen, Yang: "Rapid production of a H? N? influenza vaccine from MDCK cells for protecting chicken against influenza virus infection." in: Applied microbiology and biotechnology, Vol. 99, Issue 7, pp. 2999-3013, 2015 (PubMed).