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Short ELISA protocol

Indirect ELISA - wells are coated with the antigen (protein or peptide) and subsequently incubated with a sample containing antigen-specific antibodies (serum, yolk, affinity purified antibodies or others). This step is followed by incubation with a secondary antibody, coupled to an enzyme such as HRP or AP, for reaction development.
Buffers:
• PBS Tween 20 (0.05 %) for serum samples or PBS Tween 20 (0.5%) for egg yolk

• 0.1 M NaHCO3 (no pH adjustment) (Coating buffer)
• 1 % NaCl + 0.05 % Tween 20 (Washing buffer)
• 2 % nonfat dry milk in PBS + 0.5 % Tween 20 (Blocking buffer)
Other material:
• Maxisorp 96 well pates (Nunc) or other polystyrene plates

• Antigen for coating (protein or peptide)
• HRP conjugated secondary antibody
• Developing solution (One Step or other)

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Procedure

Coating (first trial):
• Protein: 2 µg/ml of coating buffer
• Peptide: 4 µg/ml of coating buffer; info on dissolving a peptide is found here
Pipet 100 µl of protein/peptide solution to each well using multi-channel pipet. Incubate over night at 4°C (cover the plate) or at 30°C for 2 hours.
Blocking (optional):
No washing step needed if blocking directly follows coupling of the antigen onto a plate.
Pipet 200 µl of blocking solution to each well. Incubate at 4°C overnight or 2 hours at 30°C.
Washing:
Very important step. Wash plates at least 3 times, gently.
Sample loading:
Dilution series: from 1: 100 to 1: 100 000. Include a blank (no sample, but secondary antibody added and developing solution).

Yolk: heat to RT and dilute before applying on ELISA plate. More info here.
Incubation time: 1 hour 10 minutes on shaker or 2 hours with no shaking.
Incubation with secondary antibody:
Follow recommendations of secondary antibody producer. Usually the higher dilution the better. At least 1: 10 000. Incubation time: 30 minutes on shaker, 1 hour 30 minutes with no shaking.
Reaction development:
Many different reagents are on the market. Make sure that the reagent contains the right substrate e.g. for HRP or AP, depending what you use. One Step substrates are working fine (no need to prepare them before the assay).
 
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ELISA troubleshooting

No signal or a very weak signal
• Test/find higher affinity primary antibody to use in the system.

• Are there any compounds in the sample which can interfere with the coating of the antigen/antibody to the ELISA plate (e.g. high concentration of urea)?
• Has a key reagent been added?
• Have some steps of the whole procedure been omitted by mistake?
• Has a substrate for the enzyme been prepared correctly?
• Was it the right substrate for the right enzyme?
• Could the detergent concentration in the wash buffer be too high? They are usually used at 0.01-0.1 %. Remove or decrease detergent concentration in the wash buffer.
• Do you have any enzyme inhibitor present in your system? Sodium azide will inhibit peroxidase activity.
• Is the conjugate or substrate still active? Test their activity in another system. Check expiry date of the products.
• Was the reagent incubation time correct? Check the manufacturer's recommendations.
• Was the incubation temperature correct? Check if the system was overheated (>37°C) or too cold (room temperature).
• Was the substrate volume correct? Check your pipet.

High background present
• Non-specific binding of antibodies possible. Modify your blocking conditions. Do not apply extensive washes, since they might contribute to increased variation by denaturation.

• Plate not washed enough. Check that all the wells are filled with buffer during a washing step.
• Conjugate concentration was too high. Check the recommendations made by the antibody manufacturer.
• Consider cross-reactivity of the detection antibody with the coating antibody. Make a control of just coating antibody/antigen and detection antibody with no sample in between.
• Too high temperature during whole procedure (>37°C).
• Albumins can in some cases contribute to increased variability by causing separation of already bound proteins from medium binding surfaces.
• Interference IgGs present in milk powder. Use high purity BSA which is IgG-free.
Non-specific color development on the plate
• Usually results from an incomplete washing of the plate. If you titrate your samples, and wash plates manually during the whole procedure, you can start the wash from the wells with the lowest dilution of your samples.
Strange results
• Check that the ELISA plate used is the correct one for your application (there are different types of plates available).

• Check if all the reagents were prepared correctly added in the right sequence.
• Analyze the whole procedure with the help of the points above.
Poor standard curve obtained
• Washing problem. Wells were not completely aspirated during the wash.

• Plates were stuck on each other during incubations. Keep them separately if they are not rotated.
• Dilution error. Check your pipets and pipetting technique.
• Variable absorption of reagents to the plate. Check pH of your coating buffer, which is usually around pH 7.4 if using PBS, or pH 9.6 if using carbonate-bicarbonate buffer. Consider using another type of ELISA plate.

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Recommended literature about ELISA:


• "ELISA theory and practice" by J.R. Crowther, Methods in Molecular Biology TM 42 1995 Humana Press Inc ISBN 0896032795

• "Immunoassays" by J.P. Gosling, Oxford University Press 2000 ISBN 0-19-963711-3
• Antibody usage in the lab by L.Caponi and P.Migliorini, Springer Lab Manual 1999, ISBN 3-540-65148-9

Useful references:
• Immunoassays by J.P. Gosling, Oxford University Press 2000, ISBN 0-19-963710-5
• Antibody usage in the lab by L.Caponi and P.Migliorini, Springer Lab Manual 1999, ISBN 3-540-65148-9