Quantitation of estrogen receptor mRNA in breast carcinoma by branched DNA assay.

A quantitative nucleic acid hybridization assay for determination of estrogen receptor (ER) mRNA in breast carcinoma is described. The assay, which is based on the branched DNA (bDNA) technology, requires 20 mg of tissue, is simple, highly specific, and reproducible, and correlates reasonably well with an established methodology (r = 0.87). The assay has a dynamic range of 3 x 10(3)-6 x 10(7) copies of ER mRNA per well. ER message as high as 2.5 x 10(6) copies per well could be detected in normal breast tissues. Thus a sensitivity of 3 x 10(3) ER copies per well was sufficient to analyze clinical specimens. In the present studies, accurate measurement of tissue weight enabled direct reporting of the ER mRNA values as the end point results. The bDNA assay provides a useful tool for the detection and quantitation of ER mRNA in research and routine clinical laboratories.

Quantitation of progesterone receptor mRNA in breast carcinoma by branched DNA assay.

Expression of progesterone receptor (PR) mRNA is indicative of a normal gene regulation mechanism mediated by functional estrogen receptor (ER). A simple assay which can reliably detect and quantitate PR mRNA levels in a small amount of tissue will be of value for studying functional status of ER. We have developed a quantitative nucleic acid hybridization assay for PR mRNA in breast carcinoma. The assay, which is based on the branched DNA (bDNA) technology, is simple, highly specific, and reproducible, requires 20 mg of tissue, and correlates reasonably well (r = 0.86) with an established methodology. The assay has a dynamic range of 3 x 10(3)-6 x 10(7) copies of PR mRNA per well. PR message as high as 3.9 x 10(5) copies per well could be detected in normal breast tissues. Thus a sensitivity of 3 x 10(3) PR copies per well was sufficient for testing clinical samples. In the present studies, accurate measurement of tissue weight enabled direct reporting of the PR mRNA values as the end point results. The bDNA assay provides a useful tool for the detection and quantitation of PR mRNA in research and routine clinical laboratories.

Immunoassays for serum C-reactive protein employing fluorophore-labelled reactants.

Two simple rapid and precise fluorescence assays for determining serum levels of C-reactive protein (CRP) are described which employ sheep antibodies to CRP covalently linked to magnetisable cellulose/iron oxide particles. The first (a fluoroimmunoassay) is based on competitive binding of CRP in the sample or standard with fluorescein-labelled CRP, a 30 min incubation time, simple separation with a magnet followed by elution of the bound fraction into alkaline methanol and fluorescence quantitation. In the second (a 'sandwich' immunofluorometric assay) an excess of solid-phase linked antiserum is incubated with sample and fluorescein-labelled purified sheep anti-CRP immunoglobulin followed by separation, elution and quantitation of the bound fraction. The assays cover the ranges 3-400 mg/l and 3-70 mg/l respectively and the results correlate well with those obtained by radial immunodiffusion and radioimmunoassay.

Fluoroimmunoassay of digoxin in serum.

A heterogeneous (solid-phase separation) fluoroimmunoassay for digoxin in serum was developed employing antibodies coupled to magnetisable cellulose/iron oxide particles and a fluorescein-labelled digoxin derivative as tracer. Intrinsic fluorophores and other potentially interfering components of serum samples were reliably and completely removed at the separation and wash steps of the assay procedure which were facilitated by magnetic sedimentation. In order to attain adequate sensitivity (detection limit 0.2 micrograms/l (0.26 nmol/l) serum digoxin), a sample volume of 500 microliters was necessary. Assay results for patients' specimens correlated well with those obtained using established charcoal--separation (r = 0.96) and magnetisable solid-phase (r = 0.95) radioimmunoassays. The feasibility of a "stat" adaptation of the fluoroimmunoassay that involved only two standards (0.5 and 4 micrograms/l digoxin) was demonstrated. The stat method would be suitable for the assay of urgent or single specimens.

Composite polyacrolein-coated cellulose magnetisable particles: an "autoreactive" magnetisable solid-phase with superior buoyancy properties.

A composite solid-phase was prepared that combined the autoreactive properties of polyacrolein with the superior buoyancy of cellulose-based particles. The advantage of using such particles in immunoassays is demonstrated in a fluoroimmunoassay for human immunoglobulin G.

Magnetisable solid-phase fluoroimmunoassay of human immunoglobulin G in serum.

A simple fluoroimmunoassay is described for the determination of immunoglobulin G (IgG) in serum. It employs IgG labelled with fluorescein and magnetisable polyacrolein/iron oxide particles covalently linked to goat anti-human IgG serum. Diluted patient sample or standard and fluorescein-labelled IgG are incubated for 30 min with solid-phase antibody, the antibody-bound and free antigen separated simply by means of a magnet and the fluorescence in the supernate (free fraction) determined, which directly reflects the IgG concentration of the standard or sample. Correlation studies with two other immunological methods showed good agreement.

Magnetizable solid-phase fluoroimmunoassay of thyroxine by a sequential addition technique.

We describe a simple fluoroimmunoassay for the determination of thyroxine concentrations in serum. The method, "sequential addition, separation fluoroimmunoassay," involves both thyroxine labeled with fluorescein and magnetizable cellulose/iron oxide particles to which antibodies to thyroxine have been covalently linked. Serum sample or standard is incubated with an excess of the solid-phase antibody; the particles, which now carry most of the antigen in the sample, are sedimented onto a magnet and the supernate, which contains endogenous fluorophores and other interfering factors, is removed and discarded. Excess labeled thyroxine is then added, and, after incubation, the fluorescence in the supernate (free fraction), which is related directly to the amount of thyroxine in the sample is measured. For the whole procedure, including fluorometry, each sample is treated entirely within disposable polystyrene test tubes. Correlation studies with two different radioimmunoassays showed good agreement.

Use of antibodies against the label in non-separation non-isotopic immunoassay: 'indirect quenching' fluoroimmunoassay of proteins.

Antibodies against the label are introduced as a potentially useful reagent in nonisotopic immunoassay. They may permit end point determination without the need for a separation step, provided (i) that steric hindrance selectively prevents their binding to the antibody-bound fraction of the labelled antigen in an immunoassay mixture, and (ii) that their binding to the label in the free fraction results in a change in its signal. This 'indirect' approach was investigated in systems employing the fluorescein label and antibodies to fluorescein which quenched the fluorescence of free labelled antigen. 'Indirect quenching' fluoroimmunoassays for human serum albumin, human immunoglobulin G and human placental lactogen were demonstrated. These assays for proteins may be contrasted with conventional non-separation techniques, which are usually best suited to the determination of haptens.

Solid-phase fluoroimmunoassay of human albumin in biological fluids.

A simple fluoroimmunoassay for the determination of albumin levels in serum, urine and cerebrospinal fluid is described. It employs magnetisable particles to which antibodies to human serum albumin are covalently linked, and albumin labelled with fluorescein. Equilibrium is reached within 30 min, when separation of the bound and free fractions of the labelled albumin is performed by precipitation of the particles either with a magnet or by centrifugation. Measurement of the fluorescence in the supernatant (the free fraction) reflects the albumin concentration of the standards or samples. Correlation studies with an automated immunoprecipitation technique show good agreement.

Non-separation fluoroimmunoassay of human albumin in biological fluids.

Determination of albumin levels in biological fluids by a simple, rapid, non-separation fluoroimmunoassay is described. The method ("indirect quenching" fluoroimmunoassay) employs albumin labelled with fluorescein and antibodies against both albumin and fluorescein. Anti-fluorescein serum is added to the conventional immunoassay mixture of sample (or standard), labelled albumin and anti-albumin serum. The fluorescein groups of the free fraction of the labelled albumin become bound by anti-fluorescein antibody and their fluorescence is quenched, whereas the bound fraction is sterically protected from similar quenching. The extent of quenching thus reflects the relative amounts of the free and bound fractions in the unseparated assay mixture. Correlation studies with an automated immunoprecipitation technique show good agreement.