An overview of various labeled assays used in medical laboratory diagnosis. Immune and non-immune assays.

In this review, some light is thrown on various labeled immunoassays that depend on antigen-antibody (Ag-Ab) reactions, including immunofluorescence, radioimmunoassay, and enzyme immunoassay (EIA or ELISA). Their definitions, principles, and applications are described, then they are discussed chronologically to show their stepwise development that led finally to full automation. Enzyme labeled immunoblot assays (Western blot, blot spot, and recombinant immunoblot assay), and luminescence (bioluminescence and chemiluminescence) are also discussed chronologically. Labeled assays, that do not involve Ag-Ab reaction but rather, utilizing biotin-streptavidin (BS) interaction and probe-target DNA interaction, are described, together with their applications for DNA/RNA detection and genotyping. Finally, included in the discussion were some luminescent labeled techniques that utilize the immune Ag-Ab reaction together with non-immune BS reaction, such as the luminescent oxygen channeling immunoassay, and its commercialized AlphaLISA, both eliminate the washing steps without sacrificing high sensitivity, or wide dynamic range.

Origin and utility of the reverse dot-blot.

Reverse allele specific oligonucleotide assays provide a robust method for the molecular characterization of high-mutation spectrum disorders. Commercial test have been developed for human leukocyte antigens class I and class II regions of human chromosome 6, the cystic fibrosis transmembrane conductance regulator at 7q31 and strains of human Hepatitis B and C virus. In their most developed form, these assays rely upon highly multiplexed PCR reactions containing biotinylated primers providing a substrate for nonradioactive detection systems. Sophisticated reverse dot-blot technology involves mechanized covalent attachment of activated primary amine-conjugated oligonucleotides to carboxylated nylon membranes or bovine serum albumin. Subsequent to line or dot printing, membranes are stored or sold dry in preparation for hybridization. Circular spots or lines are visualized colorimetrically after hybridization through the use of streptavidin horseradish peroxidase incubation followed by development using tetramethylbenzidine and hydrogen peroxide, or via chemiluminescence after incubation with avidin alkaline phosphatase conjugate and a luminous substrate susceptible to enzyme activation, such as CSPD, followed by exposure to x-ray film. The entire procedure from blood specimen receipt to result usually requires less than 1 day. Because of the simplicity, speed, and generally high sensitivity and specificity, large numbers of individuals can be rapidly screened using this technology. Rapid turnaround is often required in prenatal diagnosis of cystic fibrosis, beta-thalassemia and hemoglobinopathies, giving this technology has special applicability in those genetic diseases. Commercial instruments are available which automate the hybridization and color development. In addition, scanning software can capture the probe reactivity pattern and interpret it in terms of a genotype.