The use of immunogold-silver enhancement has its roots in immunohistocytochemistry, in which it has gained widespread use as a specific and sensitive staining tool. More recently this technology has been applied to such other areas as immunoassays. A novel immunoassay- silver enhanced gold-labeled immunosorbent assay (SEGLISA) - is described for the detection of specific analytes in human blood. This non-enzymic, non-isotopic immunoassay has been developed from established microtitration technology incorporating the knowledge and use of immunogold-silver staining. This technique is being developed in several formats, ranging from indirect assays for antibody detection to competitive assays for detection of lipoproteins.We describe a sandwich-type SEGLISA for the measurement of human growth hormone. It has been compared with an equivalent commercially available enzyme-linked immunosorbent assay (ELISA). This novel use of immunogold-silver staining allows an immunoassay that is quick and easy to carry out, is relatively inexpensive, and doesn't suffer from many of the drawbacks associated with immunoassays incorporating enzymic or radioactive labels. The result is permanent and can be read either visually, or quantitatively on a conventional microplate reader.

We describe a decision process for establishing the threshold reliabilities for components of naval major-caliber ammunition. We present a measure of reliability performance, called ef*, which relates directly to the weapons system's performance in a naval gunfire support environment. We use a simulation model to establish this relationship, a regression metamodel to estimate its parameters, and a simple decision process to specify component reliability thresholds which ensure that the ammunition is mission effective. We present this article as an example of the integration of discrete event dynamic system analysis within a decision process.

We describe a new immunoassay for the detection of antibodies to the human immunodeficiency virus. The method is based on a silver enhanced gold-labelled immunosorbent assay (SEGLISA). Test sera are incubated in microtitre wells on which antigens have been coated. If present in the test sera, antibodies to the human immunodeficiency virus bind to the solid-phase antigens. Bound antibodies are quantitated with anti-human immunoglobulin labelled with gold. Positive specimens produce a faint pink deposit which is better visualised by silver enhancement which gives an intense black colour. The intensity of the colour is proportional to the bound antibody concentration. All the reagents are stable and the silver enhancement takes place under ambient light conditions. The assay has many of the advantages of micro enzyme-linked immunosorbent assays but does not suffer from the drawbacks associated with the use of an enzyme label. It requires fewer manipulations and is quicker to carry out than an equivalent enzyme-linked test. As the silver layer is permanent dried wells may be stored for future reading or checking.

A silver-enhanced gold-labelled immunosorbent assay (SEGLISA) for the detection of antibodies to the immunodeficiency virus (HIV) in whole-blood samples is described. This new non-isotopic, non-enzymic immunoassay incorporates use of solid phase viral antigens which bind any HIV antibodies present in the test sample. The antigen/antibody complex is then detected by gold-labelled anti human immunoglobulin G (IgG) followed by silver amplification. We found that whole blood samples give false positives when using a horseradish peroxidase label, whereas the SEGLISA correctly identified 50 HIV antibody positive samples and 50 HIV antibody negative samples when using whole blood. The use of whole blood collected on filter paper is also described. The SEGLISA has good precision (CV = 7.5%) and sensitivity.

Attempts to increase the sensitivity of fluorescein-based fluorescence immunoassays by using multiple labelling have generally been unsuccessful because of concentration quenching. We have labelled antibodies to human immunoglobulin G with multiple fluorescein fluorophores attached by means of a disulphide linkage: this linkage can be rapidly and easily broken by treatment with dithiothreitol, allowing fluorescein to be released from the antibody and measured in free solution. Application of this technique to a fluorescence labelled immunosorbent assay for antibodies to the human immunodeficiency virus gave an approximately 20-fold increase in signal compared with an equivalent assay using fluorescein isothiocyanate.

Human immunoglobulin G, human serum albumin and testosterone were labelled with the 4-aminosalicylic acid derivative of diethylenetriaminepentaacetic acid complexed with terbium ions. An exceptionally large amount of label, of the order of a few hundred moles of complex per mole of analyte, could be conjugated to the compounds tested by the use of poly-L-lysine. Self-quenching appears to be minimal, even with this high local concentration of fluorophores. The tracers were stable at 4 degrees C, and gave competitive calibration graphs at physiological concentrations.

A series of aminoaromatic derivatives of diethylenetriaminepentaacetic acid (DTPA) has been prepared, in a search for terbium chelates suitable for use in fluorescence immunoassay. Most of the derivatives contained heterocyclic rings with at least one nitrogen atom. The fluorescence properties of the terbium chelate of each compound were examined. Although none of the products proved suitable for use in immunoassays, the terbium chelate formed from the product of the reaction between DTPA anhydride and cytosine (4-amino-2-hydroxypyrimidine) was particularly fluorescent and had a long fluorescence lifetime. It was unstable, however, in aqueous solution below pH 9. The fluorescence properties of some europium complexes were also examined.

The development and assessment of a fluorescence-labelled immunosorbent assay for the detection of antibodies to the human immunodeficiency virus is described. Test serum is incubated in microtitre wells on which antigens have been coated. If present in the test serum, antibodies to the human immunodeficiency virus bind to the solid-phase antigens. In turn the antibodies are quantified with anti-human immunoglobulin labelled with fluorescein. Positive samples produce an intense fluorescence which is measured in a spectrofluorimeter. When used to test a panel consisting of normal serum and antibody-positive serum from infected patients the assay proved to be 100% specific and to have a sensitivity of 100%. The assay has many of the advantages of micro enzyme-linked immunosorbent assays, but does not suffer from the drawbacks associated with the use of an enzyme label. It requires fewer manipulations and is quicker to carry out than an equivalent enzyme-linked test.

A rapid single-reagent spectrofluorimetric assay for salicylate in human blood plasma is reported. The method is based on ternary complex formation with terbium and EDTA in alkaline solution; it requires only 10 μl of plasma, and protein precipitation is not necessary. Within-assay relative standard deviations were better than 2.5%, and correlation with the Trinder method was excellent. The procedure is particularly suited for emergency use in cases of suspected aspirin poisoning.

The principles and use of fluorescent labels in immunoassay are reviewed. Comparison is made with radio- and chemiluminescent immunoassays. Possible fluorescent labels are listed. The following types of assay are described: separation fluoroimmunoassay, immunofluorometric assay, fluorescence enhancement and fluorescence quenching assay, fluorescence polarization immunoassay, fluorescence energy transfer immunoassay, fluorescence protection immunoassay, alternative binding immunoassay, release fluoroimmunoassay and time-resolved fluorimetry and phosphorimetry.

Non-separation fluoroimmunoassays are well suited to automation. The potential sensitivity of such assays has not been realised because the most commonly used label (fluorescein) has absorption and emission spectra which coincide with those of blood constituents and because it has a very small Stokes shift. Lucifer yellow VS has a larger Stokes shift than fluorescein and an emission maximum at longer wavelength. We describe an energy transfer fluoroimmunoassay for plasma albumin in which Lucifer yellow VS is used to label albumin and rhodamine B isothiocyanate is used to label anti-albumin antibodies. The assay shows good correlation with a dye-binding method for albumin and has sensitivity and precision which compare favourably with similar assays using a fluorescein label.

The use of a new label for fluoroimmunoassay is described. Lucifer yellow VS is a highly fluorescent vinyl sulphone dye which, under mild conditions, forms covalent bonds with amino and sulphydryl groups but is extremely stable in water. A large Stokes shift (110 nm) and an emission maximum at 540 nm give Lucifer yellow further advantages over the more commonly used labels. The use of the dye as a label has been demonstrated by developing a heterogeneous fluoroimmunoassay for human serum albumin. The fluoroimmunoassay gave comparable results to those obtained using a less specific colorimetric dye-binding assay (r = 0.97, n = 20). The advantages, limitations, and other potential uses of Lucifer yellow are discussed.