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Short ELISA protocol

Indirect ELISA - wells are coated with antigen (protein or peptide) and afterwards incubated with sample containing antigen-specific antibodies (serum, yolk, affinity purified antibodies or others). This step is follwed by incubation with a secondary antibody coupled with enzyme like 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 ug/ml of coating buffer
* Peptide: 4 ug/ml of coating buffer; info on dissolving a peptide is found here
Pipet 100 ul of protein/peptide solution to each well using multi-channel pipet. Incubate over night at 4C (cover the plate) or at 30C for 2 hours.
Blocking (optional):
No washing step needed if blocking follows directly after coupling antigen on a plate.
Pipet 200 ul of blocking solution to each well. Incubate at 4C overnight or 2 hours at 30C.
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: warm it up to RT and dilute it 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 for preparing them before the assay).
 
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ELISA trouble shooting

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 coating of antigen/antibody to the ELISA plate? (like 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 a right substrate for the right enzyme?
* Could a 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 conjugate or substrate still active? Test their activity in another system. Check expiry date of the products.
* Has incubation time with the reagents been correct? Check with the manufacture recommendations.
* Was the incubation temperature correct? Check if the system was not overheated (>37C) 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 incresed variation by denaturation.

* Plate not washed enough. Check that all the wells are actually filled with buffer during a washing step.
* Conjugate concentration was too high. Check with the recommendations of the antibody manufacture.
* Consider cross-reactivity of the detection antibody with 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. Usew 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 ELISA plate used was the right one for your application (there are different types of plates available).

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

* Plates were stuck on each other during incubations. Keep them separately if they are not rotated.
* Dilution error. Check your pipets and pipeting 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 plates.

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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