Biotechnology Reagents (IGFs) >> Product Support >> Protocols >> #3020 General Procedure for Microplate Enzyme Immunoassay (EIA)

Procedure for Microplate Enzyme Immunoassay (EIA)

The following protocol is based on the absorption of the analyte directly to the solid phase (ELISA wells), its detection by an unlabelled specific antibody in conjunction with an enzyme-labelled secondary antibody. This method has been used to detect peptides derived from various sources such as bodily fluids, mammalian or prokaryotic cell culture media or cellular extracts, and at various points in the down-stream processing of recombinantly-produced proteins.

Equipment and reagents required:

Equipment:
EIA-grade 96-well immunoassay plates
squeeze bottle for plate washing
multichannel pipette (optional)
multichannel plate washer (optional)
microplate shaker (optional)
microplate reader (optional)

Reagents:
Antibodies:

Primary antibody: against the target peptide; monoclonal or polyclonal

Secondary antibody: enzyme-labelled directed against the species of the primary antibody.

Buffers and solutions:

Bicarbonate coating buffer
1.59 g Na₂CO₃
2.93 g NaHCO₃
H₂O to 1 litre
pH should be 9.6 without adjustment

Phosphate buffered saline with 0.05%Tween-20 (PBS-T)
8 g NaCl
0.2 g KH₂PO₄
1.15 g Na₂HPO₄ anhydrous (or 2.9 g Na₂HPO₄.12H₂O)
0.2 g KCl
0.5 ml Tween 20
H₂O to 1 litre
pH should be 7.2-7.4 without adjustment

Blocking solution:
PBS containing 1% bovine serum albumin.
(Do not add Tween to this solution)

Stopping solution for OPD substrate: 1M H₂SO₄
5.5 ml of concentrated H₂SO₄ added to 94.5 ml of H₂O

Stopping solution for p-NPP substrate: 0.2 M EDTA
5.8 g ethylenediaminetetracetic acid
H₂O to 100 ml
Adjust pH to 8.0 with NaOH to dissolve EDTA

Substrates and substrate buffers:

p-nitophenyl phosphate (p-NPP) (alkaline phosphatase substrate)
1mg/ml p-nitrophenylphosphate in diethanolamine buffer

diethanolamine buffer
48.5 ml diethanolamine
400 ml H₂O
pH to 9.8 with conc. HCl
add MgCl₂ to 0.5 mM
final volume to 500 ml with H₂O

ortho-phenylene diamine (OPD) (horse radish peroxidase substrate)
1 mg/ml o-phenylamine diamine in citrate buffer, pH 5.0

citrate-phosphate buffer
3.65 g citric acid
4.76 g Na₂HPO₄
500 ml H₂0; pH should be 5.0 without adjustment

Add 1 µl 30 %H₂O₂ per ml buffer just before use

Method:
1. If possible, dissolve or dilute standards or sample in bicarbonate coating buffer.

2. Add 100 µl of the diluted standard or sample to empty well(s) of microplate. Cover and leave in a moist environment overnight (16 hours).

3. Remove peptide solution from plate by “flicking” or aspirating the wells.

4. Add 150 µl of blocking solution to each well of the plate and leave on the bench (not shaking) for 30 minutes.

5. Flick out the plate and wash twice by filling the wells with PBS-T and flicking the plate out immediately. Tap out residual on absorbent paper, cover and keep moist.

6. Add 100 µl of an appropriate dilution of anti-peptide antibody in PBS-T to the “coated” wells. Incubate on plate shaker (approximately 200 oscillations per minute) for 1 hour at room temperature.

7. Wash plate 3 to 5 times by filling the wells with PBS-T and aspirating the wells after one minute. Tap out the plate on absorbent paper.

8. Add 100 µl of an appropriate dilution of enzyme-labelled antibody in PBS-T to the wells. Incubate on plate shaker for 1 hour at room temperature.

9. Wash plate 3 to 5 times by filling the wells with PBS-T and aspirating the wells after one minute. Tap out the plate on absorbent paper.

10. Add 100 µl of appropriate substrate solution to each well.

a. For HRP/OPD reactions, incubate at room temperature on shaker for 10-15 minutes and stop reaction by adding 25 µl per well of 1M H₂SO₄.

b. For alkaline phosphatase/p-NPP reactions, incubate on shaker for 20-40 minutes and stop reaction by adding 25 µl per well of 0.2 M EDTA.

11. Record results by measuring absorbance at 490-492 nm for OPD and 405 nm for pNPP.

General Notes on Procedure:

a. We recommend a high-binding capacity EIA grade plate such as Nunc MaxiSorp, Greiner Microlon 600, or Costar High Binding Surface plates. These plates have a binding capacity of approximately 500 ng per well when filled with 100 µl of solution.

b. The bicarbonate coating buffer provides a pH where most proteins are charged. It is not essential to use this buffer for attachment of proteins to the plastic wells, and proteins can usually be bound from a variety of solutions including tissue culture supernatants. If column chromatography fractions are to be assayed, it is advised to dilute the samples in coating buffer before application to the plates.

c. Coating with many charged molecules occurs rapidly and may reach near maximum levels after a few hours at room temperature; overnight coating is not essential. However, longer coating times, such as “over the weekend” are normally not detrimental.

d. Plate shaking is not essential for the assay, but it does markedly decrease the required incubation times. For unshaken assays, increase incubation times by 100 %.

e. Primary Antibody: Unless otherwise advised by the manufacturer, try starting with a 1/1,000 dilution.

f. Secondary antibody: This should be chosen for its high activity both in terms of enzyme activity and target sensitivity. Most commercial products are of good quality and can be used at the manufacturer’s recommended dilution. The secondary antibody need not be species specific; however, it may be required not to react with certain species of immunoglobulin (or other proteins) which may contaminate the target protein preparation (for example bovine IgG present in culture media containing fetal calf serum).

g. Avoid “flicking” plates which contain biological hazards such as infectious material (eg, from human sources) or recombinant DNA. Wells should be aspirated by vacuum and the aspirate disinfected and disposed of via protocols recommended for such substances.

h. BSA may not be required in the blocking step. We often find that a 30 minute incubation at room temperature with wells filled with PBS-T is sufficient.

i. If a “plate reader” is not available, the HRP/OPD enzyme/substrate combination is preferable as colour of the reaction product can be easily graded by eye and ranges from clear, to pale straw, to a deep brown as the amount of bound antibody increases.


Hazards:
OPD is a suspected carcinogen
Catalysed pNPP contains phenol.


© Novozymes GroPep Limited
28 Dalgleish Street
Thebarton SA 5031
PO Box 10065 BC
Adelaide SA AUSTRALIA
Telephone +61 (0)8 8354 7700
Facsimile +61 (0)8 8354 7788
Email: AUAD-bioreagents@novozymes.com
Website: www.gropep.com.au