A review of fluoroimmunoassay and immunofluorometric assay.
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Fluorescence↗
Biomedical subjects
J Landon
Publications and source records attributed to J Landon.
Cortisol directly determined in serum by fluoroimmunoassay with magnetizable solid phase.
We developed a direct fluoroimmunoassay for cortisol in serum. This method involves cortisol labeled at the 3 position with fluorescein, and antibodies to cortisol coupled to magnetizable cellulose/iron oxide particles. Sodium salicylate is used as a blocking agent to prevent interference from endogenous binding proteins in serum. Serum sample and labeled cortisol are incubated with the antibody-coupled solid phase for 30 min. The solid phase is then separated and washed to remove free labeled cortisol and endogenous fluorophores of the sample. Finally we elute the antibody-bound fraction of the labeled cortisol into an alkaline methanolic medium and measure its fluorescence. The separation, wash, and elution steps are facilitated by magnetic sedimentation. The assay is sufficiently sensitive, specific, and reliable for routine use and correlates acceptably (r = 0.92) with an established radioimmunoassay. Precision (CV) ranged from 4 to 10% in experiments on three pooled sera; analytical recovery for sera supplemented with as much as 360 microgram of cortisol per liter was 91 to 109%.
Cross Reactions↗
Magnetizable solid-phase fluoroimmunoassay of phenytoin in disposable test tubes.
We developed a fluoroimmunoassay for phenytoin in serum or plasma, based on the magnetic separation technique. The method involves sheep anti-phenytoin serum coupled to magnetizable cellulose/iron oxide particles, with a fluorescein-labeled phenytoin analog as tracer. After magnetic sedimentation of the solid phase from assay mixtures, the free fraction of the tracer is aspirated, removing endogenous fluorophores and other interfering components of the sample. The antibody-bound tracer is then eluted from the solid phase into a methanolic buffer medium and quantitated fluorometrically. The entire procedure, including fluorometry, is performed within disposable polystyrene test tubes. The assay involves only simple reagents and equipment, and correlates closely with established radioimmunoassay (r = 0.97) and gas-liquid chromatographic (r = 0.98) techniques.
Animals↗
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.
Fluoresceins↗
A fully automated, continuous-flow radioimmunoassay for methotrexate.
We describe a fully automated continuous-flow radioimmunoassay for methotrexate. [125I]Histamine-labeled methotrexate was used as tracer. Anti-methotrexate serum was coupled to a magnetizable solid-phase and the bound and free fractions were separated with an electromagnetic field. The assay is precise (CV less than 2.5%) and rapid (30 samples per hour), incubation volume is small (about 160 micro L), and incubation brief (10 min). The accurate timing inherent in the system obviates the need to attain equilibrium, so that assay of each sample takes only 15 min. The assay is sensitive (1--100 microgram/L). There is no significant carryover between samples of high and low concentration. Results by the automated method correlated well with those by both a manual assay in which the same reagents and separation technique are used (r - 0.99) and a competitive protein-binding assay (r = 0.96).
Autoanalysis↗
Multi-fluorescein-substituted polymers as potential labels in fluoroimmunoassay: a system for improved detection sensitivity.
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Fluoresceins↗
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.
Animals↗
Improved 125I-ligands for gentamicin radioimmunoassay.
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Animals↗
A look at the future with regard to immunoassay.
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Antibodies↗
Novel 125I-labeled nortriptyline derivatives and their use in liquid-phase or magnetizable solid-phase second-antibody radioimmunoassays.
Nortriptyline derivatives prepared by reaction with fluorescein isothiocyanate or conjugation to N-acetyl-L-histidine were radioiodinated and the products purified with Sephadex LH-20 columns to obtain two novel nortriptyline radioligands. Antisera were raised in rabbits by immunization with nortriptyline conjugated to succinylated ovine albumin. By use of the iodinated fluorescein derivative we developed a liquid-phase second-antibody radioimmunoassay that gives results correlating closely (r = 0.98) with those by an established radioimmunoassay of similar specificity in the assay of apparent total amitriptyline and its metabolite nortriptyline in serum or plasma from patients being treated with these drugs. With the iodinated N-acetyl-L-histidine derivative we developed a magnetizable solid-phase second-antibody radioimmunoassay. The cross reactivities of amitriptyline and nortriptyline could be made equal by performing the assay at pH 9.0, which makes it possible to measure true total active drug concentrations in patients receiving amitriptyline.
Amitriptyline↗
Evaluation and application of magnetizable charcoal for separation in radioimmunoassays.
We compared conventional charcoal separation of antibody-bound and free antigen with a novel method involving magnetizable particles containing charcoal. The magnetizable charcoal separation method was as effective as the conventional method for a wide range of radioimmunoassays, including those for several steroids and small polypeptide hormones. In a detailed comparison of conventional vs. magnetizable charcoal for radioimmunoassay of aldosterone, the magnetizable charcoal had several advantages: it did not require prior coating with dextran or protein, varied less with time and temperature, required no centrifugation, and took less time for separation. The correlation of results obtained with magnetizable and conventional charcoal separation was excellent (n = 89, r = 0.98, p less than 0.001), and the coefficient of variation for the separation with magnetizable charcoal was 0.5%.
Aldosterone↗
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.
Acrolein↗
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.
Albumins↗
Separation fluoroimmunoassay methods for phenytoin in serum.
A separation fluoroimmunoassay system for phenytoin was established based on the use of a specific rabbit antiserum, a fluorescein-labelled ligand, and precipitation of the antibody-bound fraction of the labelled ligand with sodium sulphate. Simple measures were taken to obviate non-specific binding and matrix effects. Either the free fraction (in the supernatant) or thebound fraction of the labelled ligand was quantitated fluorimetrically. Assays of patient serum samples by either method correlated well with established gas-liquid chromatographic and radioimmunoassay techniques. Advantages of a separation based procedure as compared with previously described non-separation hapten fluoroimmunoassay techniques are that only simple instrumentation and assay reagents are required, and that the separation step may enable the removal of any interfering intrinsic fluorescence of serum samples.
Fluorometry↗
Automated tests for the assessment of thyroid function.
Fully automated methods have been developed for the determination of thyroxine and triiodothyronine levels, antibodies to thyroglobulin and the assessment of thyroid hormone binding proteins in serum, using a continuous flow radioimmunoassay system. In addition the feasibility of a partially automated assay for thyrotrophin levels has been demonstrated. These employ Auto Analyzer modules and antibodies covalently linked to a magnetisable solid phase support. Separation of bound and free antigen is achieved by applying an external magnetic field. The system currently operates at a rate of 30 samples/h and requires only 10 minutes incubation since it is not necessary to reach equilibrium. The results are similar to those obtained by conventional manual techniques, however the precision is improved and operator error eliminated.
Female↗
Anti-drug sera raised by immunisation with a readily available drug-enzyme conjugate.
A phenytoin-enzyme conjugate, readily available as an enzyme-immunoassay reagent, is shown to be a convenient immunogen for production of anti-phenytoin sera for immunoassay purposes.
Animals↗
Polarisation fluoroimmunoassay of phenytoin.
A polarisation fluoroimmunoassay for the determination of phenytoin levels in serum is described. Fluoresceinthiocarbamyl alpha,alpha-diphenylglycine (FTC-DPG) is used as an easily prepared fluorescent-labelled analogue of phenytoin. Interference from the non-specific binding of FTC-DPG by serum proteins is eliminated by proteolytic degradation of samples prior to assay. The method employs stable, non-radioactive reagents, requires no separation procedure, and involves only 1.25 microliter of serum. Analyses of serum samples from patients receiving phenytoin correlate well (r = 0.96) with an established gas-liquid chromatographic method.
Chromatography, Gas↗
The relationship between circadian variations in circulating thyrotrophin, thyroid hormones and prolactin.
Half-hourly blood samples were taken from six clinically euthyroid men over a continuous period of 24 h. Their concentrations of total thyroxine (T4), total triiodothyronine (T3), thyrotrophin (TSH) and prolactin (PRL) were assessed together with the degree of unsaturation of thyroid hormone binding proteins as determined by the thyroid hormone uptake test (THUT). Both T3 and T4 were also measured in urine samples collected serially during the same 24 h period. Significant circadian changes in serum TSH, THUT, serum and urine T4 and serum PRL were demonstrated in all subjects. TSH showed a reciprocal pattern to serum T4, with higher levels during the evening and at night than the daytime. This TSH pattern did not coincide with PRL secretion. Further studies on the same subjects did not show any significant effect of posture, corticosteroid or T4 administration upon circadian changes in TSH. There appeared to be no consistent circadian changes in serum or urinary T3. It seems likely that the TSH circadian rhythm is centrally determined and that free T3 levels are maintained more or less constant by variation in peripheral conversion from T4.
Adult↗