Lanthanide-based time-resolved luminescence immunoassays

The sensitive and specific detection of analytes such as proteins in biological samples is critical for a variety of applications, for example disease diagnosis. In immunoassays a signal in response to the concentration of analyte present is generated by use of antibodies labeled with radioisotopes, luminophores, or enzymes. All immunoassays suffer to some extent from the problem of the background signal observed in the absence of analyte, which limits the sensitivity and dynamic range that can be achieved. This is especially the case for homogeneous immunoassays and surface measurements on tissue sections and membranes, which typically have a high background because of sample autofluorescence. One way of minimizing background in immunoassays involves the use of lanthanide chelate labels. Luminescent lanthanide complexes have exceedingly long-lived luminescence in comparison with conventional fluorophores, enabling the short-lived background interferences to be removed via time-gated acquisition and delivering greater assay sensitivity and a broader dynamic range. This review highlights the potential of using lanthanide luminescence to design sensitive and specific immunoassays. Techniques for labeling biomolecules with lanthanide chelate tags are discussed, with aspects of chelate design. Microtitre plate-based heterogeneous and homogeneous assays are reviewed and compared in terms of sensitivity, dynamic range, and convenience. The great potential of surface-based time-resolved imaging techniques for biomolecules on gels, membranes, and tissue sections using lanthanide tracers in proteomics applications is also emphasized

Microdetermination of mercury by its inhibition of 3α-hydroxysteroid dehydrogenase

A fluorimetric enzymatic method is described for the determination of mercury(II) which inhibits the activity of the enzyme 3α-hydroxysteroid dehydrogenase (3α-HSD). The method is based upon the oxidation of deoxycholic acid by β-NAD+ in the presence of the enzyme 3α-HSD. The generated NADH is monitored fluorimetrically. Mercury was determined in the range 5-25 × 10-8 M with an accuracy of about 5%. © 1985

Enzymic fluorimetric determination of sulphated and non-sulphated primary bile acids in urine using a rapid solvolysis technique

A simple and rapid enzymic fluorimetric method for the determination of sulphated and non-sulphated primary bile acids in urine has been developed. Octadecylsilane-bonded silica cartridges (Sep-Pak C18) are used for the solid-phase extraction of bile acids (BA) from urine samples. Sulphated BA are solvolysed before measurement with an improved rapid solvolysis procedure. The measurement is based on the reaction of 7 -hydroxylated BA with β-nicotinamide adenine dinucleotide (β-NAD+) in the presence of the enzyme 7a-hydroxysteroid dehydrogenase (7α-HSD). The NADH generated is monitored fluorimetrically. The mean relative standard deviation of the method was 8% (n - 32), the detection limit 0.4 μM and the mean recovery of added BA 99%. The solvolysis step in this procedure takes about 4 h and is faster than the conventional procedures which are usually carried out in an 18-h protocol. Sulphated and non-sulphated primary BA were determined in urine from 16 healthy persons and 67 hospitalised patients suffering from various hepatobiliary diseases. The reference range for 7α-hydroxylated urinary BA was in agreement with previously published results. The method is simple and suitable for routine clinical use. About 30 urine samples can be analysed in one working day

Continuous-flow potentiometric determination of α-amylase activity in serum and urine

An automated saccharogenic potentiometric method for serum or urinary α-amylase is described. Amylase is allowed to act on a buffered at pH 6.9 starch solution under controlled continuous-flow conditions and the reducing sugars produced are left to react with periodate. The consumption of periodate is monitored continuously with a newly constructed periodate-sensitive flow-through electrode. The endogenous reducing substances of serum or urine are measured with the same system by incubation of the sample with the same starch solution, at pH 4.7, in the presence of sodium fluoride as an amylase inhibitor. The difference in the reducing power (as glucose) is used to calculate the amylase activity of the samples. Values obtained with this assay correlate fairly well with those obtained with an amyloclastic method (r = 0.90-0.96). © 1985