Protein analysis

Protein assay: ELISA

Enzyme-linked immunosorbent assay (ELISA) is a method that is analogous to immunodetection of proteins on a membrane, and is used for the quantitative assay of proteins in solution. In an ELISA, proteins are immobilized on a solid support (e.g., the wells of a 96-well plate) and used as capture molecules to bind the protein that is being assayed. After a wash step to remove nonspecifically bound material, a secondary antibody — specific for the protein being assayed — is added. This secondary antibody is usually conjugated to an enzyme that allows its detection by chromogenic or chemiluminescent methods.

In one type of ELISA assay, an antibody that binds an epitope on a target protein is immobilized, and a test solution added. The immobilized antibody will capture any target protein present in the sample. A wash step removes nonspecifically bound material, and subsequently a second antibody is added that reacts with a second epitope on the protein. Alternatively, a protein can be immobilized on a solid support, and antibodies reacting with the protein can be detected and quantitated in a test solution by the addition of a secondary antibody that reacts with the primary antibody (see figure Two common types of ELISA).

Two common types of ELISA

This procedure is used to immobilize proteins onto the inner surfaces of 96-well microplates. The proteins can then be assayed using a primary and secondary antibody in a process analogous to that used for detection of proteins on western blots.

Important notes before starting

  • The ease with which proteins bind to polystyrene plates is very much dependent on the particular protein. Optimization of binding conditions is necessary. Refer to the manufacturer’s instructions.
  • As a starting point, three buffers at different pH values should be compared.
  • Binding may be carried out at 4–37°C. Successful binding may depend on the stability of the protein

Materials required

  • Suitable 96-well microplates

Coating buffers

PBS, pH 7.2 (see table PBS, pH 7.2) 
50 mM sodium carbonate, pH 9.6 (see table Sodium carbonate, pH 9.6) 
50 mM sodium carbonate, pH 10.6 (see table Sodium carbonate, pH 10.6) 
Microplate blocking buffer(see table Microplate blocking buffer)  

PBS, pH 7.2
Composition of working solution Component   Amount per liter
50 mM potassium phosphate 0.5 M K2HPO2
0.5 M K2HPO2
71.7 ml
28.3 ml
150 mM NaCl NaCl 8.8 g

50 mM sodium carbonate, pH 9.6
Composition of working solution Component Amount per liter
50 mM Na2CO3  Na2CO3·H2O 6.2 g

50 mM sodium carbonate, pH 10.6
Composition of working solution Component Amount per liter
50 mM Na2CO3 Na2CO3·H2O 6.2 g

Microplate blocking buffer
Composition of working solution Component Amount per liter
2% sucrose Sucrose 20 g
0.1% BSA BSA 1 g
0.9% NaCl NaCl  

  1. Serially dilute the protein to be immobilized in coating buffer(s).
  2. Add 200 µl of the protein solution to each well, and incubate overnight at 4°C.
  3. Wash wells 4 times with PBS. Soak wells for 10–60 s per wash, and dry the wells by tapping the plate on paper towels.
  4. Block wells with 250 µl of microplate blocking buffer for 2 h at room temperature (20–25°C) on a shaker platform.

    Tip: After blocking, plates can be dried overnight at 20–25°C, but sensitivity of the assay will be reduced.

    Tip: After drying, it may be possible to store the plates at 4°C for a period of time before use, but this will depend on the specific protein to be assayed.

  5. Wash wells 4 times with PBS. Soak wells for 10–60 s per wash, and dry the wells by tapping the plate on paper towels.
  6. Proceed with the protocol Assay of proteins with a protein specific antibody.

Important notes before starting

  • Binding of detection antibodies should be carried out for at least 1 h at room temperature. If the concentration of the protein to be detected is very low or if the epitope is partly hidden, incubation times of 2–4 h or overnight may increase sensitivity.
  • Best results will be obtained if all steps are carried out on a shaker. If there is no shaker available, incubation times should be increased (up to 2–3 h at room temperature or overnight at 4°C) or the incubation temperature should be raised to allow sufficient diffusion of molecules.
  • Antibody dilution depends on the individual antibody used. Please refer to manufacturer’s recommendations or begin at concentrations useful for western-blot or dot-blot analyses and try further dilutions. Usually primary monoclonal antibody at 0.1 µg/ml to 1 µg/ml will yield satisfactory results. Each antibody should be titrated over this range of concentrations to determine the optimal dilution.
  • Suitable negative controls are essential. Assays should always be performed in parallel with samples without any proteins (lysis/dilution buffer alone, reagent blank) and with samples similar to those assayed but lacking the target protein (e.g., lysate from E. coli transformed with vector lacking the protein-encoding insert). These controls should be incubated with antibodies and the remaining assay components. Note: This protocol is intended to be used as an example. Optimal conditions for each individual protein and antibody should be determined.
  • If establishing a new assay system, the binding of the protein or antibody to the solid support should be optimized first (incubation time and amounts of protein). Primary antibody or other secondary components of the assay should be optimized afterwards.

Materials required

  • 96-well microplates coated with protein (see “Coating 96-well microplates with protein for ELISA”)
  • PBS/BSA (see table PBS/BSA)
  • PBS (see table“PBS, pH 7.2)
  • Anti-target–protein antibody
  • Secondary-antibody conjugate
  • Substrate for alkaline phosphatase or horseradish peroxidase or one of the alternative substrates for horseradish peroxidase as described in tables Phosphate–citrate buffer, pH 5.0, Substrate for alkaline phosphatase: p-Nitrophenyl Phosphate (NPP), Substrate for horseradish peroxidase: 2,2’Azino-bis[3-Ethylbenz-thiazoline-6-Sulfonic Acid] ABTS, Alternative substrate for horseradish peroxidase: o-Phenylenediamine (OPD), and Alternative substrate for horseradish peroxidase: 3,3’,5,5’-Tetramethylbenzidine (TMD). Further details about substrates can be found in the table Details of protein substrates for protein assay procedures.
PBS/BSA*
Composition of working solution Component   Amount per liter
0.2% BSA in PBS BSA dissolved in PBS 2 g

Phosphate–citrate buffer, pH 5.0
Component Volume
0.2 M Na2HPO4 251.5 ml
0.1 M citric acid 48.5 ml

Substrate for alkaline phosphatase: p-nitrophenyl phosphate (pNPP)
Component Amount
 pNPP pNPP
1 M diethanol-amine; 0.01% MgCl2·6 H2O, pH 9.8 10 ml

Substrate for horseradish peroxidase: 2,2'azino-bis[3-ethylbenz-thiazoline-6-sulfonic acid] ABTS
Component Amount 
ABTS 10 mg
Phosphate–citrate buffer 10 ml
Immediately before use add: 30% H2O2 2 µl

Alternative substrate for horseradish peroxidase: o-phenylenediamine (OPD)*
Component Amount
OPD 10 mg
Phosphate–citrate buffer 10 ml
Immediately before use add: 30% H2O2 2 µl

Alternative substrate for horseradish peroxidase: 3,3',5,5'-Tetramethylbenzidine (TMB)
Component Amount
TMB 10 mg
DMSO 1 ml
Dissolve, then add phosphate–citrate buffer 9 ml

Details of protein substrates for protein assay procedures
Substrate Wavelength for monitoring
color development
Stopping reagent*   Wavelength for determining
stopped product 
pNPP 405 nm 3 M NaOH 405 nm
ABTS 415 nm 1% SDS 415 nm
OPD 450 nm 3 M HCl or
3 M H2 SO4
492 nm
TMB 370 nm or 650 nm 2 M H2SO4 450 nm

  1. Add 200 µl of anti-target–protein antibody diluted 1/2000 in PBS/BSA. Cover plate, and incubate for 1–2 h at RT.
    For higher sensitivity, antibody binding can be performed overnight at 4°C.
  2. Wash wells 4 times with PBS-Tween. Soak wells for 10–60 s per wash, and dry the wells by gently tapping the plate on paper towels after the wash.
  3. Dilute secondary antibody in PBS/BSA according to the manufacturer’s recommendations. Add 200 µl of the diluted antibody to each well, and incubate at room temperature for 45 min.
  4. Wash wells 4 times with PBS-Tween. Soak wells for 10–60 s per wash, and dry the wells by gently tapping the plate on paper towels.
  5. Add 200 µl of substrate solution, and monitor color development in a microplate reader.

    Note: Substrate solution should always be prepared immediately before use.

    Tip: Monitor color development over a period of 45 min, or add 50 µl stopping reagent after a specific time and measure product. When testing a new assay system, a time-course of color development should be carried out to determine optimal development time and temperature.

    Tip: If the reaction is stopped the signal will increase slightly, depending on the substrate used, and the color will be stable for a certain period of time.
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