Short ELISA protocol

Indirect ELISA - wells are coated with antigen (protein or peptide) and afterwards indubated with sample containing antigen-specific antibodies (serum or yolk or affinity purified antibodies or others). This step is follwed by incubation with a secondary antibody coupled with enzyme like HRP or AP, for later 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)

Procedure:

Coating (first trial):

• Protein: 2 ug/ml of coating buffer
• Peptide: 4 ug/ml of coating buffer; info on dissolving a peptide is here

Pipet 100 ul of protein/peptide solution to each well using multi-chanell 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, genlty.

Sample loading: Dilution series: from 1: 100 to 1: 100 000. Include a blank (no sample, but secondary antibody added and reaction developed).
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 recomendations of secondary antibody producer. Usually the higher dilution the better. At least 1: 10 000. Incubation time: 30 min. on shaker, 1 hour 30 minutes with no shaking.

Reaction development: Many diferent 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).

Problems with procol developement? Please, check: ELISA troubleshooting

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