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Protocol
Type A, H5, and H7 Avian Influenza antigen detection ELISAs
Keywords: avian influenza, ELISA, type A, H7, H5
Describes three ELISA assays to identify type A, H5 and H7 avian influenza viruses using monoclonal antibodies.
This protocol describes three ELISA assays to identify type A, H5 and H7 avian influenza viruses using monoclonal antibodies (Mabs) specific for the nucleoprotein A (NPA), and H5 and H7 haemoagglutinin antigens, respectively. These tests were developed by Partner 7, IZSLER, Brescia.
The principle of the three ELISAs is the same and is based on the use of capture Mabs specific for NPA, H5, and H7 AIVs respectively, coated onto 96 wells ELISA microplates, the addition of samples from allantoic fluids of infected embryonated eggs, and, finally, the addition of the same capture Mabs labelled with horseradish peroxidase (HRP). The wells where AIV antigen has been captured and then reacted with the specific conjugate are identified by mean of a OPD staining.
Monoclonal Antibodies (Mabs) 5F10, 5D8 and 7A4 were produced by IZSLER.
Required Materials
- Glycerol , for storage.
- Propiolactone , (BPL) for inactivation of allatoic fluid.
- 96 well plates
Instructions
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ELISA Buffers (MVA3YJ)
This protocol contains recipies and instructions of how to make the buffers for ELISA assays.
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Coating buffer (MXPE74)
0.05 M carbonate-bicarbonate buffer (pH 9.6).
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Dissolve the reagents in the distilled water.
Store at +4°C.
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PBS (pH 7.4)
- 8 g of NaCl
- 0.2 g KCl
- 1.44 g Na2PO4
- 0.24 g KH2PO4
- H2O (distilled)
Dissolve NaCl, KCl, Na2PO4 and KH2PO4 in 800 ml of distilled water and adjust the pH to 7.4 using HCl.
Adjust the volume to 1 liter and store at room temperature.
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Washing buffer (PBS-Tween)
PBS + 0,05% Tween 20.
Store at room temperature.
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Sera and conjugate diluent buffer
PBS-Tween (washing buffer) + 1% Yeast extract.
Prepare minimum needed volume each day and use it fresh.
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Chromophore/Substrate solution (MXY70G)
Dissolve tablets of OPD (SIGMA) in the appropriate volume of buffer (e.g. one tablet of 60 mg OPD in 120 ml of phosphate-cytrate buffer, pH 5). This solution can be stored at –20°C in appropriate aliquots (6, 12, 18 or 24 ml aliquots, suited respectively for 1, 2, 3 or 4 plates) in the dark. Immediately before use add H2O2 at 0.02% final concentration (e.g. H2O2 30% 4 ?l H2O2 per 6 ml OPD).
Potential carcinogenic agent R45.
Store at 4 °C.
Phosphate-cytrate buffer (pH 5).
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Preparation
Dissolve tablets of OPD (SIGMA) in the appropriate volume of buffer (e.g. one tablet of 60 mg OPD in 120 ml of phosphate-cytrate buffer, pH 5). This solution can be stored at –20°C in appropriate aliquots (6, 12, 18 or 24 ml aliquots, suited respectively for 1, 2, 3 or 4 plates) in the dark. Immediately before use add H2O2 at 0.02% final concentration (e.g. H2O2 30% 4 ?l H2O2 per 6 ml OPD).
Potential carcinogenic agent R45.
Store at 4 °C.
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Blocking solution
Add H2SO4 96% (56 ml) to the distilled water (944 ml).
Store at room temperature.
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Assay procedure
Coating Nunc 96 wells microplates are coated overnight at 4°C with the capture Mabs anti NPA, anti-H5 and anti-H7 respectively, optimally diluted in coating buffer.
Washing Washing the ELISA plates three times (3 minutes/wash) by filling wells with 150-200 ?l/well of washing buffer at appropriate dilution.
Samples Addition of 50 ?l/well of allantoic fluids. Samples have to be tested in duplicate and it is recommended to test two dilutions (undiluted and ½) for each sample. For each plate, a AIV or H5 or H7 positive control and negative control have to be included. Incubate plates for 1 h at 37°C.
Washing washing plates as described above.
Conjugated Mabs Addition of 50 ?l/well of the appropriate dilution of NPA or H5 or H7 specific conjugate respectively. Incubate for 1 h at 37°C.
Washing as above.
Substrate The colorimetric reaction is developed by distributing 50 ?l/well of the substrate solution (OPD 0.5 mg/ml in phosphate-citrate buffer pH 5, 0.02% H2O2); after 10 min. at room temperature, the reaction is stopped by adding 50 ?l/well of H2SO4 2N.
Reading Absorbance values are read at 492 nm..
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Evaluation of results
The reactivity of AIV, H5 and H7 control antigen is expected to be higher than 0.2 OD value.
Allantoic fluid are considered:
- positive when producing reactivity ? 0.2 OD.
- negative when producing reactivity < 0.2 OD.
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Biological Reagents
1) Catching Mabs: The solution of the catching Mabs (Mab anti-NPA: 5F10, Mab anti-H5: 5D8, and Mab anti-H7: 7A4) is a semi-purified concentrated Mab prepared in PBS containing glycerol 50% and stored at -20°C. Final dilution range: 2-5 ?g/ml depending on MAb.
VARNING: Prepare working dilution immediately before use.
2) Conjugated Mabs: Peroxidase-conjugated Mabs anti-NPA (5F10), anti-H5 (5D8) and anti-H7 (7A4). They can be mixed with glycerol 50% and stored at -20°C or pre-diluted in stabilizing buffer and stored at +4°C.
VARNING: Prepare working conjugate dilution immediately before use.
3) Positive controls
Positive AIV control sample: H1N1 infected allantoic fluid inactivated with ß-propiolactone (BPL). Positive H5 control sample: H5N2 infected allantoic fluid inactivated with BPL. Positive H7 control sample: H7N1 infected allantoic fluid inactivated with BPL.
3) Negative control sample: SPF allantoic fluid. Positive and negative control samples should be stored at -20°C in appropriate aliquots (200?l is suitable for one plate).
