Increasing sensitivity

Enzyme-based immunoassays, blotting techniques and immunohistochemistry have become the norm for the detection of antibodies and for characterization of their specificity. These systems have been developed in several ways to increase their sensitivity and ease of application. Biotin, a small molecule (244 Da) which binds with very high affinity to the proteins avidin or streptavidin, can itself be bound covalently to immunoglobulins without the loss of activity. A single biotinylated reagent can then be used in any test system with suitably labeled avidins. Since avidin can bind up to four biotin molecules, this gives the possibility of multilayered systems which offer increased sensitivity.

Alternative approaches to the detection of antibody utilize protein A, a bacterial surface protein which binds specifically to certain classes of IgG. Protein A in its natural or recombinant form or protein G, an IgG-binding protein from streptococci, are widely available. Protein G has broader specificity for subclasses of IgG and binds better to sheep and goat IgG than protein A. Second antibody systems do, however, remain the method of choice, particularly with the availability of good monoclonal antibodies specific for individual immunoglobulin classes or subclasses.

Although conventional detection systems are usually for sufficient sensitivity for the detection of antibodies, the detection of trace antigens such as growth factors or their receptors can require the necessity for an amplified detection system which will quantitate the binding of a primary antibody. Increased sensitivity is possible by the use of sandwich techniques involving several layers of antibody, enzyme-anti-enzyme complexes, biotin-avidin complexes or by newly developed specific amplification systems. One example of an enhancement system uses alkaline phosphatase conjugated to the detecting antibody to generate NAD from NADP. The NAD is then used in a pair of cycling redox reactions to reduce a colorless precursor to a colored formazan dye. The reaction product can be detected at 492 nm by conventional readers. The amplification step results in an increase in sensitivity of over 100-fold and is the basis of several rapid dipstick-type tests.

Chemiluminescence-based techniques have replaced colorimetric ELISAs and immunoblotting techniques in many laboratories because of their much greater sensitivity. A number of substrates which yield chemiluminescent products upon cleavage by peroxidase (luminol) or alkaline phosphatase (dioxetane phosphate esters) are available, usually in the form of kits containing all necessary reagents. For ELISA-based assays 96-well microplate lumino-meters are used. In chemiluminescence-based blotting techniques the signal can be detected using photographic film or specially designed plate detectors.

Time-resolved fluorescence techniques such as the DELFIA (Pharmacia) system, have been developed to replace the radioimmunoassay for the measurement of hormones etc. in large diagnostic laboratories. These assays are equally applicable to detection of antibodies, for example, in the diagnosis of infectious diseases. DELFIA utilizes Europium'+ bound to proteins through a covalently-linked F.DTA derivative. Excess of the chelators naphthoyltrifluoroacetone and trioctylphosphine are then used to form a fluorescent complex in solution at low pH.

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