Immunoassays. Classification and applications

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Lesson 5. Immunoassays

Classification. Antibody-Antigen Complex Formation. Assay Formats. Homogeneous and heterogeneous immunoassays. Labels in immunoassays: radioisotopes, fluorescence, chemiluminescent, enzymes. Enzyme Immunoassays: ELISA.     Immunoassay curve fitting.

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PODCAST 1. LISTEN ME FIRST! GENERAL INTRODUCTION FOR THE LECTURES OF 9/12 AND 10/12 IN ONLY ONE POST

PART 1. SUMMARY OF THE TECHNIQUE

ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for detecting and quantifying soluble substances such as peptides, proteins, antibodies, and hormones. Other names, such as enzyme immunoassay (EIA), are also used to describe the same technology. In an ELISA, the antigen (target macromolecule) is immobilized on a solid surface (microplate) and then complexed with an antibody that is linked to a reporter enzyme. Detection is accomplished by measuring the activity of the reporter enzyme via incubation with the appropriate substrate to produce a measurable product. The most crucial element of an ELISA is a highly specific antibody-antigen interaction.

The enzyme linked immunosorbent assay (ELISA) is a powerful method for detecting and quantifying a specific protein in a complex mixture. Originally described by Engvall and Perlmann (1971), the method enables analysis of protein samples immobilized in microplate wells using specific antibodies. ELISAs are typically performed in 96-well or 384-well polystyrene plates, which passively bind antibodies and proteins. It is this binding and immobilization of reagents that makes ELISAs easy to design and perform. Having the reactants of the ELISA immobilized to the microplate surface makes it easy to separate bound from non-bound material during the assay. This ability to use high-affinity antibodies and wash away non-specific bound materials makes ELISA a powerful tool for measuring specific analytes within a crude preparation.

Although many variants of ELISA have been developed and used in different situations, they all depend on the same basic elements:

  1. Coating/capture–direct or indirect immobilization of antigens to the surface of polystyrene microplate wells.
  2. Plate blocking–addition of irrelevant protein or other molecule to cover all unsaturated surface-binding sites of the microplate wells.
  3. Probing/detection–incubation with antigen-specific antibodies that affinity-bind to the antigens.
  4. Signal measurement–detection of the signal generated via the direct or secondary tag on the specific antibody.

The most commonly used enzyme labels are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Other enzymes have been used as well; these include β-galactosidase, acetylcholinesterase, and catalase. A large selection of substrates is available commercially for performing ELISA with an HRP or AP conjugate. The choice of substrate depends upon the required assay sensitivity and the instrumentation available for signal-detection (spectrophotometer, fluorometer, or luminometer).

PODCAST 2. INTRODUCTION TO THE DIAGNOSIS OF INFECTION DISEASE. ELISA FORMATS

PART 2. HOW DOES ELISAs WORKS? INSIDE A MICROWELL

Inside a microplate. ELISA sandwich for the detection of a virus. Step by Step.

Protocol 1. ELISA sandwich for the detection of a virus. Example of protocol, step-by-step

Warning! in this summary, the format is a sandwich with indirect labelling, in which the primary antibody (blue) is no labelled and requires a secondary labelled antibody!

View sandwich ELISA protocol summary diagram.

Introduction

A sandwich ELISA measures antigen between two layers of antibodies (capture and detection antibody). The target antigen must contain at least two antigenic sites capable of binding to antibodies.

Monoclonal or polyclonal antibodies can be used as the capture and detection antibodies in sandwich ELISA systems. Monoclonal antibodies recognize a single epitope that allows quantification of small differences in antigen. A polyclonal is often used as the capture antibody to pull down as much of the antigen as possible.

Sandwich ELISAs remove the sample purification step before analysis and enhance sensitivity (2–5 times more sensitive than direct or indirect).

Coating with capture antibody

  • Coat the wells of a PVC microtiter plate with the capture antibody at 1–10 μg/mL concentration in carbonate/bicarbonate buffer (pH 9.6).
    Unpurified antibodies (eg ascites fluid or antiserum) may require increased concentration of the sample protein (try 10 μg/mL) to compensate for the lower concentration of specific antibody.
  • Cover the plate with adhesive plastic and incubate overnight at 4°C.
  • Remove the coating solution and wash the plate twice by filling the wells with 200  μL PBS. The solutions or washes are removed by flicking the plate over a sink. The remaining drops are removed by patting the plate on a paper towel.

Blocking and adding samples

  • Block the remaining protein-binding sites in the coated wells by adding 200 μL blocking buffer (5% non-fat dry milk/PBS) per well.
  • Cover the plate with adhesive plastic and incubate for at least 1–2 h at room temperature or overnight at 4°C.
  • Wash the plate twice with 200 µL PBS.
  • Add 100 μL of diluted samples to each well. Always compare signal of unknown samples against those of a standard curve. Run standards (duplicates or triplicates) and blank with each plate. Incubate for 90 min at 37°C.
    Ensure concentration of standards spans the most dynamic detection range of antibody binding. You may need to optimize the concentration range to obtain a suitable standard curve. Always run samples and standards in duplicate or triplicate.
  • Remove samples and wash the plate twice with 200 μL PBS.

Incubation with detection and secondary antibody

  • Add 100 μL of diluted detection antibody to each well.
    Check that the detection antibody recognizes a different epitope on the target protein to the capture antibody. This prevents interference with antibody binding. Use a tested matched pair whenever possible.
  • Cover the plate with adhesive plastic and incubate for 2 h at room temperature.
  • Wash the plate four times with PBS.
  • Add 100 μL of conjugated secondary antibody, diluted in blocking buffer immediately before use.
  • Cover the plate with adhesive plastic and incubate for 1–2 h at room temperature.
  • Wash the plate four times with PBS.

Detection

Horse radish peroxidase (HRP) and alkaline phosphatase (ALP) are the two most widely used enzymes for detection in ELISA assays.

Consider that some biological materials have high levels of endogenous enzyme activity (such as high ALP in alveolar cells, high peroxidase in red blood cells) that may result in nonspecific signal. If necessary, perform an additional blocking treatment with levamisol (for ALP) or 0.3% H2O2 in methanol (for peroxidase).

ALP substrate
P-Nitrophenyl-phosphate (pNPP) is the most widely used substrate for most applications. Measure the yellow color of nitrophenol at 405 nm after 15–30 min incubation at room temperature and stop the reaction by adding equal volume of 0.75 M NaOH.

HRP chromogenes
The substrate for HRP is hydrogen peroxide. Cleavage of hydrogen peroxide is coupled to oxidation of a hydrogen donor which changes color during reaction.

TMB (3,3’,5,5’-tetramethylbenzidine)
Add TMB solution to each well, incubate for 15–30 min, add equal volume of stopping solution (2 M H2SO4) and read the optical density at 450 nm.

OPD (o-phenylenediamine dihydrochloride)
The end product is measured at 492 nm. Keep and store the substrate it in the dark as it is light sensitive.

ABTS (2,2’-azino-di-[3-ethyl-benzothiazoline-6 sulfonic acid] diammonium salt)
The end product is green and the optical density can be measured at 416 nm.

Always handle with care and wear gloves as some enzyme substrates are considered hazardous (potential carcinogens).

Data analysis

Prepare a standard curve from the serial dilutions data with concentration on the x axis (log scale) vs absorbance on the Y axis (linear). Interpolate the concentration of the sample from this standard curve.

Buffers and reagents used

Bicarbonate/carbonate coating buffer (100 mM)
Antigen or antibody should be diluted in coating buffer to immobilize them to the wells: 3.03 g Na2CO3,6.0 g NaHCO3 in 1000 ml distilled water, pH 9.6

PBS
1.16 g Na2HPO4, 0.1 g KCl, 0.1 g K3PO4, 4.0 g NaCl (500 ml distilled water) pH 7.4.

Blocking solution:
Commonly used blocking agents are 1% BSA , serum, non-fat dry milk, casein, gelatin in PBS.

Wash solution:
Usually PBS or Tris-buffered saline (pH 7.4) with detergent such as 0.05% (v/v) Tween20 (TBST).

Antibody dilution buffer:
Primary and secondary antibody should be diluted in 1x blocking solution to reduce non specific binding.


General note

  • For accurate quantitative results, always compare signal of unknown samples against those of a standard curve. Standards (duplicates or triplicates) and blank must be run with each plate to ensure accuracy.
  • Sandwich ELISA procedures can be difficult to optimize and tested match-paired antibodies should be used. This ensures the antibodies are detecting different epitopes on the target protein and do not interfere with the other antibody binding. We are unable to guarantee our antibodies in sandwich ELISA unless they have been specifically tested.

Inside a microplate. ELISA sandwich for the detection of a seroprevalence (IgG or IgM) against the virus. Step by Step.

Protocol 2. ELISA sandwich for the detection of a seroprevalence (IgG/IgM) against the virus. Step by Step.

Warning! in this summary, the primary antibody (blue) is the antibody found in the sample (the analyte) which can be IgG (as depicted here) or IgM, which are produced by the host to neutralized the virus, indicating current or past infection (or vaccination)

View sandwich ELISA protocol summary diagram.

Coating antigen to microplate

  • Dilute the antigen to a final concentration of 20 µg/ml in PBS or other carbonate buffer. Coat the wells of a PVC microtiter plate with the antigen by pipetting 50 µl of the antigen dilution in the top wells of the plate. Dilute down the plate as required.
    Test samples containing pure antigen are usually pipetted onto the plate at less than 2 µg/ml. Pure solutions are not essential, but as a guideline, over 3% of the protein in the test sample should be the target protein (antigen). Antigen protein concentration should not be over 20 µg/ml as this will saturate most of the available sites on the microtiter plate.
    Ensure the samples contain the antigen at a concentration that is within the detection range of the antibody.
  • Cover the plate with an adhesive plastic and incubate for 2 hr at room temperature, or 4°C overnight. The coating incubation time may require some optimization.
  • Remove the coating solution and wash the plate three times by filling the wells with 200 µl PBS. The solutions or washes are removed by flicking the plate over a sink. The remaining drops are removed by patting the plate on a paper towel.


Blocking

  • ​Block the remaining protein-binding sites in the coated wells by adding 200 µl blocking buffer, 5% non-fat dry milk or 5% serum in PBS, per well. Alternative blocking reagents include PEG or BSA.
  • Cover the plate with an adhesive plastic and incubate for at least 2 hr at room temperature or, if more convenient, overnight at 4°C.
  • Wash the plate twice with PBS.


Incubation with primary and secondary antibody

  • Add 100 µl of diluted primary antibody (serum) to each well.
  • Cover the plate with an adhesive plastic and incubate for 2 hr at room temperature.
    This incubation time may require optimization. Although 2 hr is usually enough to obtain a strong signal, if a weak signal is obtained, stronger staining will often be observed when incubated overnight at 4°C.
  • Wash the plate four times with PBS.
  • Add 100 µl of conjugated secondary antibody, diluted at the optimal concentration (according to the manufacturer) in blocking buffer immediately before use.
  • Cover the plate with an adhesive plastic and incubate for 1-2 hr at room temperature.
  • Wash the plate four times with PBS.


Detection and Data analysis (as above)

PART 3. CLASSIFICATION. DIRECT AND INDIRECT

​ELISA formats–direct, indirect, and sandwich ELISA

There are several formats used for ELISAs. These fall into either direct, indirect, or sandwich capture and detection methods. The key step is immobilization of the antigen of interest, accomplished by either direct adsorption to the assay plate or indirectly via a capture antibody that has been attached to the plate. The antigen is then detected either directly (labeled primary antibody) or indirectly (such as labeled secondary antibody). The most widely used ELISA assay format is the sandwich ELISA assay, which indirectly immobilizes and indirectly detects the presence of the target antigen. This type of capture assay is called a “sandwich” assay because the analyte to be measured is bound between two primary antibodies, each detecting a different epitope of the antigen–the capture antibody and the detection antibody. The sandwich ELISA format is highly used because of its sensitivity and specificity.

Diagram of common ELISA formats including direct ELISA, indirect ELISA, and sandwich ELISA method

Diagram of common ELISA formats (direct vs. sandwich assays). In the assay, the antigen of interest is immobilized by direct adsorption to the assay plate or by first attaching a capture antibody to the plate surface. Detection of the antigen can then be performed using an enzyme-conjugated primary antibody (direct detection) or a matched set of unlabeled primary and conjugated secondary antibodies (indirect detection).

Direct versus indirect ELISA detection strategies

Among the standard assay formats discussed and illustrated above, where differences in both capture and detection were the concern, it is important to differentiate between the particular strategies that exist specifically for the detection step. Irrespective of the method by which an antigen is captured on the plate (by direct adsorption to the surface or through a pre-coated “capture” antibody, as in a sandwich ELISA), it is the detection step (as either direct or indirect detection) that largely determines the sensitivity of an ELISA.

The direct detection method uses a primary antibody labeled with a reporter enzyme or a tag that reacts directly with the antigen. Direct detection can be performed with an antigen that is directly immobilized on the assay plate or with the capture assay format. Direct detection, while not widely used in ELISA, is quite common for immunohistochemical staining of tissues and cells.

The indirect detection method uses a labeled secondary antibody or a biotin-streptavidin complex for amplification and is the most popular format for ELISA. The secondary antibody has specificity for the primary antibody. In a sandwich ELISA, it is critical that the secondary antibody is specific for the detection of the primary antibody only (and not the capture antibody) or the assay will not be specific for the antigen. Generally, this is achieved by using capture and primary antibodies from different host species (e.g., mouse IgG and rabbit IgG, respectively). For sandwich assays, it is beneficial to use secondary antibodies that have been cross-adsorbed to remove any secondary antibodies that might have affinity for the capture antibody.

Comparison of direct, indirect, and sandwich ELISA detection methods

Competitive ELISA

Besides the standard direct and sandwich formats described above, several other styles of ELISA exist:

Competitive ELISA is a strategy that is commonly used when the antigen is small and has only one epitope or antibody binding site. One variation of this method consists of labeling purified antigen instead of the antibody. Unlabeled antigen from samples and the labeled antigen compete for binding to the capture antibody. A decrease in signal from the purified antigen indicates the presence of the antigen in samples when compared to assay wells with labeled antigen alone.


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