5. Enzyme Linked Immunoassays  


ELISA was developed in 1970 and became rapidly accepted. A wide variety of assay principles can be used in ELISA techniques. Currently the most important ones are;-

  1. Competitive methods
  2. Sandwich methods
  3. Antibody capture methods

Competitive methods

One component of the immune reaction is insolubilized and the other one labeled with an enzyme. The analyte can then be quantified by its ability to prevent the formation of the complex between the insolublized and the labelled reagent. Advantages of this approach are that only one incubation step is necessary and that the "prozone effect" at high analyte concentrations cannot occur. Disadvantages are that the concentration range in which the analyte can be quantified without sample dilution is rather narrow and that the antigen or antibody (in cases where either may be present in a sample e.g. hepatitis B) produce the same response, and can therefore cannot be distinguished in a one step assay.

Sandwich (Indirect) methods

  1. The method in which the same component of the immune reaction (e.g. the antibody) is used in the insolubilized and the enzyme labelled form. The other component, the analyte (i.e. the antigen in the sample forms a bridge between the two reagents.)
  2. The method in which one component (usually the antigen) is used in an insolubilized form to bind the analyte from the sample (the antibody),which is subsequently determined by addition of labelled second antibody against the same class of antibody as the analyte antibody or protein A.

In principle, quantification can be achieved over an extremely wide analyte concentration range in such sandwich methods. The "prozone effect" can be avoided in the following ways;- (i) using sequential incubation steps for sample and label, or (ii) by using monoclonal antibodies. Modification of the test in (2) so that antibodies of a specific class such as IgM, can give spurious results if antibodies from other immunoglobulin classes are also present in the sample. Also RF ( rheumatoid factor ) is known to be a potentially interfering factor.

Sandwich inhibition methods

The sample containing the analyte (usually antibody) is pre-incubated with a fixed amount of its binding partner (ie. the antigen ) after which the remaining amount of antigen is determined in a sandwich assay. These methods usually are complicated and have a limited measuring range. The method allows for simultaneous detection of antibody or antigen, if either of these 2 analytes is present in the sample.

Antibody capture methods

These methods used to detect antibodies of specific immunoglobulin subclasses, by first reacting the sample with e.g. insolubilized anti-IgM,and subsequently with either enzyme labelled antigen followed by enzyme linked antibody. Neither antibodies from other immunoglobulin subclasses nor rheumatoid factor interfere significantly in such assays. They are widely used for the diagnosis of acute infections by IgM detection. These assays may be used for detecting IgG and IgA. Considering trends towards simplification of assays and quantification of analytes over a wide concentration range. It must be expected that competitive and sandwich inhibition methods will decrease in importance, and the sandwich and antibody capture methods will be the main assay principles in the future.

Assay Characteristics

The use of monoclonal antibodies has lead to many improvements in ELISA systems.

  1. Higher sensitivity ;- either by selection of antibodies with a extremely high affinity, or by reduction of the height and variability of the background reaction, which makes very low concentrations of analyte more readily detectable.
  2. Higher specificity ;- by avoiding the presence of any antibody in the assay system with specific reactivity against non-analyte epitopes, and by selecting combinations of monoclonal antibodies which may further increase specificity.
  3. Higher practicality ;- e.g. by introducing simultaneous incubation of label, solid phase and sample without risk of "prozone effect".

The enzyme label ;- Most of the assays employ horse-radish peroxidase, alkaline phosphatase, or B-D-galactosidase. The most interesting recent developments has been in new methods to detect these enzymes rather than the use of new enzymes. Fluorimeters were introduced in 1984 for the detection of alkaline phosphatase and B-D-galactosidase. Methods are available to detect horse radish peroxidase by means of chemilumininescence. Fluorimetric and luminometric methods offer higher sensitivity and a wider measuring range than conventional spectrometry. TMB is gradually replacing mutagenic substrates such as OPD, leading to increased sensitivity and safety.


Microplate ELISA: coloured wells indicate reactivity. The darker the colour, the higher the reactivity

Single Radial Haemolysis

Virological Methods Slideset