ELISA

Overview

Enzyme-linked Immunosorbent Assay (shortened as ELISA) is used to identify peptides, proteins, antibodies and hormones. Also, called as enzyme immunoassay (EIA), ELISA finds use in the fields of biotechnology and medicine as a diagnostic tool. Mainly, antibodies and color changes are used to identify target substances. Also, ELISAs are useful in measuring antigen and antibody concentration.

Development and brief history

Before the advent of ELISA, radioimmunoassay employing radioactively-labelled antigens and antibodies were used. Radioactivity served as the reporter signal indicating specific antigen or antibody. As radioimmunoassay posed significant health risks to researchers, alternatives were sought.

In 1960s, Enzyme linking process was developed by two different teams spearheaded by Stratis Avrameas and G. B. Pierce. In the same period, immunosorbent preparation technique was published by Wide and Jerker Porath. Independent research papers published in 1971 by Peter Perlmann and Eva Engvall at Stockholm University in Sweden, and Anton Schuurs and Bauke van Weemen in the Netherlands produced the knowledge that go into making ELISA.

The conventional ELISA involves usage of chromogenic reporters and substrates to produce color changes to indicate the presence of specific antigen or an analyte. Newer Assay techniques make use of fluorogenic, electrochemiluminescent, and quantitative PCR reporters to create quantifiable signals. The advantage of using advanced reporters help in measuring multiple analytes in a single or cycle of assays (Multiplexing) and higher sensitivities (specificity and sensitivity)

Technically, newer assays use reporters that are not enzymes in most cases, nonetheless the underlying principles of the assays are similar. Therefore, these assays are grouped as ELISAs.

Principle

ELISA works by coupling antibody or antigen to assay enzyme. The assay combines the specificity of antibody and sensitivity of assay enzymes to primarily detect antigens through assay antibody or antibodies through assay antigens. The sensitivity and precision of the assay is enhanced by coating the plate with high-affinity antibodies.

Types of ELISA

Direct ELISA

Quicker of all the ELISA, the direct assay is used in detection of antigens coated to the multiwell plate by an antibody conjugated to an enzyme. The assay is less consuming in terms of time, steps and reagents.

Simple Procedure
  1. The antigen is diluted to a final concentration of 20 μg/ml in PBS. The wells of a PVC microtiter plate are coated with the antigen of 50 μl in the top wells of the plate.
  2. The plate is covered with adhesive plastic and incubated for 2 hours at room temperature.
  3. The coating solution is discarded and completely removed by flicking the plate over a sink and the remaining drops are removed by patting the plate on a paper towel.
  4. The remaining protein-binding sites in the coated wells are blocked by adding 200 μl blocking buffer.
  5. The plate is covered with an adhesive plastic and incubated for at least 2 hours at room temperature.
  6. The plate is washed twice with PBS.
  7. 100 μl of the antibody is added to the blocking buffer before use.
  8. The plate is covered with an adhesive plastic and incubated for 2 hours.
  9. The plate is washed four times with PBS.
  10. the substrate solution is dispensed in each well.
  11. Post color development, stop solution is added to the wells.
  12. Absorbance of each well is studied against a plate reader.

Indirect ELISA

The assay is usually carried out in two stages. Like Direct ELISA, the antigen is coated to a polystyrene multiwell plate. In the first stage, unlabeled primary antibody is introduced into the well which is specific to an antigen. In the second stage, enzyme labeled secondary antibody (often polyclonal antibody) is introduced to the well.

The advantages of Indirect ELISA include enhanced sensitivities since more than one labeled antibodies are used for bounding with primary antibody. The experimental procedure can be made flexible as per the demands of the study given the stages in Indirect ELISA.

Simple Procedure
  1. The antigen is diluted to a concentration of 20 µg/ml in PBS. The wells of a PVC microtiter plate are coated with the antigen by pipetting 50 µl of the antigen dilution in the top wells of the plate.
  2. 200 µl blocking buffer is added to block the remaining protein-binding sites in the coated wells with 5% non-fat dry milk.
  3. The plate is covered with adhesive plastic and incubated for at least 2 hr at room temperature.
  4. The plate is washed twice with PBS.
  5. 100 µl of diluted primary antibody is added to each well.
  6. The plate is covered with an adhesive plastic and incubated for 2 hours at room temperature.
  7. The plates should be washed with PBS four times
  8. 100 µl of conjugated secondary antibody diluted at optimal concentration in blocking buffer is added.
  9. The plate is covered with adhesive plastic and incubated for 1 – 2 hours at room temperature.
  10. Again, the plates are washed with PBS four times.

Sandwich ELISA

The assay is highly efficient in sample antigen detection and quantification. The assay is suitable for antigens that contain at least two antigenic epitopes capable of binding to antibody. Both monoclonal and polyclonal antibodies are widely used as capturing and detection antibodies in Sandwich ELISA systems. The quantification of antigens happens between two layers of antibodies and therefore the method is termed as sandwich ELISA.

The assay is more sensitive and specific than direct or Indirect ELISA i.e. up to 2 to 5 times. The difficulty in sandwich ELISA lies in optimization and selecting tested pair antibodies. This ensures antibodies chosen are detecting different epitopes on the target protein without interfering with other antibody binding. As a high precision assay, the sample need not be purified before analysis.

 

Simple Procedure
  1. The wells of the microtiter plates are coated with the capture antibody at 1–10 μg/mL concentration in carbonate/bicarbonate buffer. In case unpurified antibodies are used, the concentration of the protein sample should be 10 μg/mL) to compensate for the lower concentration of specific antibody.
  2. The plate is covered with adhesive plastic and incubated at 4°C for 12 hours.
  3. The coating solution is discarded and the plate is washed twice by 200 μL PBS. The PBS is also removed completely by flicking the plate over a sink and the by patting the plate on a paper towel.
  4. The remaining protein binding sites in the wells are blocked by 200 μL blocking buffer (5% non-fat dry milk/PBS) per well.
  5. The plate is covered with the adhesive plastic and incubate for at least 2 hours at room temperature or overnight at 4°C.
  6. The plate is washed twice with 200 µL PBS.
  7. The diluted samples of 100 μL is added to each well. The signals exhibited are compared with a standard curve. Both standards and blank with each plate are incubated for 90 minutes at room temperature.
  8. The concentration range is optimized to obtain a standard curve. This ensures the standards spans the detection range of antibody binding.
  9. the samples are removed and the plate is washed twice with 200 μL PBS.
  10. 100 μL of diluted detection antibody is added to each well. It is important to ensure detection antibody recognizes a different epitope on the target protein to the capture antibody to prevent interference with antibody binding.
  11. The plate should be covered with adhesive plastic and incubated for 2 hours at room temperature.
  12. The plates are washed with PBS four times and 100 μL of conjugated secondary antibody diluted in blocking buffer is added.
  13. The plate is covered with adhesive plastic and incubated for an hour at room temperature.
  14. The plate is washed four times with PBS.
  15. Detection involves enzymes such as Horse radish peroxidase (HRP) and alkaline phosphatase (ALP). Blocking treatment with levamisole or 0.3% H2O2 in methanol. This helps in hindering nonspecific signal.

Competitive ELISA

The assay is the most precise of all ELISA types and the assay is based on competitive binding.

 

Simple Procedure
  1. Unlabeled antibody is incubated with the sample antigen.
  2. The incubated complex is added to the 96-well plates coated with the same antigen in the complex
  3. The unbound antibody is washed away from the plate.
  4. More antigens in the sample leads to lesser antibodies binding to the antigens in the well, hence the procedure is termed competitive.
  5. Secondary antibody conjugated with the enzyme is added to the well.
  6. A suitable substrate is added and enzymes elicit a chromogenic or fluorescent response.

Selection and Coating

  1. Optimizing plate condition for antigen or capture antibody is of paramount importance in developing assay for a specific antigen.
  2. Microplate with minimum protein binding capacity of 400 ng/cm² are usually chosen for running ELISA. The coefficient of variation of protein binding should be less than 5%.
  3. The plate colors are largely determined based on signal detection.
  4. Optimizing plate condition for antigen or capture antibody is of paramount importance in developing assay for a specific antigen.
  5. Usually, polystyrene bottom plates and opaque plates are used for signal detection. While Polystyrene plates are used for colorimetric signals, opaque plates are used for fluorescent and chemiluminescent signals.
  6. It is important to inspect the plates before use as scratches and imperfections would lead to deviations in the data obtained from the assay.
  7. The coating of the plate is achieved by adsorption of the protein to the plastic of microplate i.e. through Hydrophobic interactions between the plastic and non-polar protein.
  8. The plates are coated by adding 2–10 μg/mL solution of protein dissolved in an alkaline buffer. After incubating the plates for at least 12 hours at less than 37° after removing coating solution, blocking buffer is added to ensure binding surfaces of the plastic well are covered.

Buffers

Buffers help in adsorbing antibodies and antigens that are not binding to the surfaces of the coated plate. Largely, the sensitivity of the assay depends on the blocking buffer as it reduces background signal and enhances the signal to noise ratio. An ideal buffer is an agent that eliminates background completely without impacting the epitope for antibody binding.

Detection Strategies

As a biochemical technique, ELISA is mainly used to detect the presence of an antigen or antibody in a sample. Therefore, detection occupies an important role in ELISA. In simple terms, the final step in ELISA always involves adding a signaling agent to the enzyme to produce a visible signal i.e. color change.

Based on detection strategies, ELISAs are classified into three groups: Chromogenic assays, Chemifluorescent assays and chemiluminescent assays.

Some enzymes on addition to the substrate produces a reaction that fluoresces when light particles are emitted at a specific wavelength. When these enzymes find utility in ELISAs, those assays are called Fluorescent immunoassays. Fluorescence units detected are directly proportional to the quantity of analyte in sample.

Chromogenic assays result in colored reaction product that absorbs in light in the visible spectrum and therefore visible to naked eyes. The antigen-antibody complex reacts with the substrate of choice giving rise to colored reaction product. Like Chemifluorescent assays, the color changes are directly proportional to the amount of the analyte.