Homogeneous immunoassays

5.6 Homogeneous immunoassays

The degree of antigen–antibody interaction is determined with separation of bound and free components and has the advantage that equilibrium established in the assay is not disturbed. Many techniques have been used, including fluorescence polarization and fluorescence quench- ing, but the technique is now becoming more common with the availability of sophisticated sur- face plasmon resonance equipment that can very sensitively detect the interaction of antibody with antigen.

5.6.1 Plasmon resonance

This technique utilizes a surface sensitive detector which consists of: (a) a ligand immobilized to the detector; and (b) the detector which can measure the absorption of an analyte on the same spot and at the time

when it occurs. Biosensors allow real-time biomolecular interactions to be monitored without having to use a

label. They can analyse interactions between proteins, nucleic acids, carbohydrates, lipids and low molecular weight molecules, such as signalling substances and drugs. The main advantage is that molecules do not need to be purified, but can be studied in crude extracts, lipid vesicles, viruses and bacteria as well as eukaryotic cells.

The first system to be commercially available was the BIAcore ® (Pharmacia Biosensor, Uppsala, Sweden). This system combines a detector, a gold sensor chip, and integrated liquid- handling fluidic components for transport of samples and reagents to the site of interaction. The ligand is immobilized to a surface, such as dextran coated on the gold sensor chip. When the ana- lyte is injected into the instrument, it passes over the immobilized ligand by means of the fluidic system. Adsorption of the analyte from the continuous flow of sample is measured as a function of time. After the pulse has passed the chip surface, the analyte dissociates and this can be monitored. The surface optical detection technique of BIAcore ® uses surface plasmon resonance ( Jonsson et al. 1991). Polarized light illuminates the sensor and is reflected into the detector array. Through surface plasmon resonance, light is displaced at one angle of incidence, and this is measured as a dark spot on the detector. This angle for extinction of light is changed when analyte associates or dissociates from the ligand.

An alternative to the BIAcore ® is the IAsys ® (Affinity Sensors, Cambridge, UK) which has a dif- ferent optical detection system using a resonance mirror, and uses a stirred cuvette instead of a fluidic system.

Bisensors are used to monitor biomolecular interactions, such as molecular recognition, affinity, kinetics and multimolecular interactions. Kinetics and affinity measurements are deter- mined from the curves generated in response to the association or dissociation of analyte from ligand (Altschuh et al. 1992). The association phase is a function of the kinetic properties of the reaction as long as the mass transport of analyte to the immobilized ligand is not a limiting factor

in the reaction. The association constant (k a ) is obtained from a series of analyte concentrations and the detection range extended from 10 3 to 10 6 /M/s. The dissociation rate constant (k d ) is ascer- tained by measuring the dissociation of bound analyte in buffer flow after the sample has passed the chip surface. The association equilibrium constant (K a ), or affinity constant, is calculated

C H A P T E R 5: Immunoassay C H A P T E R 5: Immunoassay