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Biomedical subjects

J Landon

Publications and source records attributed to J Landon.

At least 73 records · Page 4Linked to original sources
PubMed1986

Single-reagent polarisation fluoroimmunoassay for cotinine (a nicotine metabolite) in urine.

A rapid, single-reagent, non-separation, non-isotopic immunoassay was developed for determining levels of the nicotine metabolite, cotinine, in urine. The single reagent was prepared by pre-mixing an appropriate dilution of sheep anti-cotinine serum with a fluorescein-labelled cotinine tracer. All normal reliability criteria were satisfied. The assay was found to be specific for cotinine and there was no cross reactivity with other available nicotine metabolites and structurally-related compounds. The results obtained correlated closely with those of an established radioimmunoassay. The assay was well-suited to application in the discrimination of active smokers from non-smokers (and passive smokers).

Buffers↗
PubMed1986

Direct homogeneous phosphoroimmunoassay for carbamazepine in serum.

In this "phosphoroimmunoassay," a phosphorescent label is used: erythrosin (tetraiodofluorescein). Its long-lived phosphorescence was detected after pulsed excitation in a time-resolved luminescence spectrometer. To achieve convenient open-atmosphere phosphorimetry in liquid solution at room temperature, we used sodium sulfite as an "in situ" chemical deoxygenator, to eliminate oxygen quenching of the excited triplet state. Time-resolved detection allows for complete rejection of short-lived background signals, including those from fluorescent or light-scattering components of biological fluids that can interfere in fluoroimmunoassays. We chose the antiepileptic drug carbamazepine (CBZ) and its active metabolite CBZ-10, 11-epoxide (CBZE) to demonstrate the development and validation of nonseparation assays based on quenching the phosphorescence of erythrosin-labeled drug upon binding to antibody. These two compounds cross reacted equally with the antiserum used; hence, we measured the activity of both in patients' sera. Alternatively, simple pre-treatment of the sample with acid destroys CBZE immunoreactivity and enables specific assay of CBZ.

Carbamazepine↗
PubMed1986

Immunoassays for low concentrations of albumin in urine.

We have developed and validated simple, rapid immunoassays to measure concentrations of albumin in urine ranging from 5 to 200 mg/L. We use antiserum to human albumin, raised in sheep, and we separate the antibody-bound and free fractions of albumin by using a second antiserum, produced in donkeys, against the Fc fragment of sheep IgG. These two antisera can be mixed and added to assay tubes as a single reagent without inhibiting antigen binding. Samples or standards are incubated for at least 30 min with fluorescein-labeled albumin and the premixed antiserum reagent. After brief centrifugation, the supernate is discarded and the precipitate containing the bound fraction is dissolved and its fluorescence measured. Alternatively, 125I-labeled albumin can be used as tracer with all other reagents unchanged; in this case one simply counts the radioactivity of the bound fraction. A reference interval of less than 0.2-2.8 mg/mmol of creatinine was obtained for untimed, daytime, midstream urine specimens from 80 healthy subjects.

Adolescent↗
PubMed1985

C-reactive protein as an aid in the differentiation of functional and inflammatory bowel disorders.

Eighty-two patients were investigated on their first visit to the outpatient department of St. Mark's Hospital, London, for the assessment of abdominal symptoms. In addition to the clinical examination, a rectal biopsy, routine tests and appropriate special investigations, blood was taken from each patient for the determination of erythrocyte sedimentation rate, C-reactive protein and alpha-1-acid glycoprotein. Nineteen patients were finally diagnosed as having Crohn's disease, twenty-two ulcerative colitis, and forty-one functional bowel disorders. All the patients with Crohn's disease had an elevated erythrocyte sedimentation rate and C-reactive protein level as had 11 (50%) of the patients with ulcerative colitis, but none with functional disorders. All cases of ulcerative colitis could be diagnosed by rectal biopsy. Measurement of alpha-1-acid glycoprotein provided no additional diagnostic information. A combination of rectal biopsy, and measurement of the erythrocyte sedimentation rate and C-reactive protein successfully distinguishes between inflammatory disease of the large and small bowel and functional bowel syndrome.

Adult↗
PubMed1985

Dual-label fluoroimmunoassay for simultaneous determination of primidone and phenobarbital.

A dual-label magnetizable solid-phase fluoroimmunoassay for direct determination of serum levels of primidone and its main metabolite, phenobarbital, in a single tube was developed and optimized. Fluorescein isothiocyanate was used to label phenobarbital and a rhodamine 101 derivative (XRITC) to label primidone; these could be independently quantitated with no fluorimetric "cross-talk." The dual-label assay employs mixed immunochemical reagents but is otherwise similar in performance to conventional, single-drug, magnetizable solid-phase fluoroimmunoassays. The ability to measure simultaneously two related analytes, such as a drug and its metabolite, represents a useful extension of immunoassay. This was illustrated in the present work by assay of sera from patients on primidone therapy. Dual-label assay results correlated well with those by a conventional enzymoimmunoassay (for primidone) and by a polarization fluoroimmunoassay (for phenobarbital).

Fluorescent Antibody Technique↗
PubMed1985

The development of a fluoroimmunoassay for beta-2-microglobulin in serum.

A liquid-phase fluoroimmunoassay has been developed for the measurement of beta-2-microglobulin in undiluted serum or plasma. Protein labelled with fluorescein-isothiocyanate at a molar ratio of 1:2 respectively is employed as a tracer together with an antiserum to beta-2-microglobulin raised in a sheep. Separation is achieved by means of rabbit anti-sheep immunoglobulin G and, following precipitation of the bound fraction by centrifugation, the free fraction together with any potentially interfering factors is removed in the supernatant. Finally the precipitate is dissolved and the fluorescence measured. The assay is simple and rapid in that all reactants, including the second antibody, can be added at the start of the assay and equilibrium is attained within 60 min. The assay is reproducible and sufficiently sensitive to allow measurement of normal serum levels and has a wide range avoiding the need for sample dilution. Results correlated well with those of an established radioimmunoassay.

Fluorescein-5-isothiocyanate↗
PubMed1985

Direct determination of theophylline in serum by fluoroimmunoassay using highly specific antibodies.

Described is the development of a fluoroimmunoassay for theophylline using a fluorescein labelled derivative of theophylline as tracer and antibodies coupled to magnetizable solid-phase particles. Three approaches are described for the preparation of antibodies for theophylline, of which one produced highly specific, high titre antibodies. The fluoroimmunoassay using these antibodies required a 10 microL sample, reached equilibrium within 5 min, and the results correlated closely with those of an established enzymoimmunoassay method. Potentially interfering endogenous fluorophores from the serum sample were reliably removed at the separation step of the bound and free fractions. There was no significant cross-reactivity with all other structurally related compounds.

Animals↗
PubMed1985

A radioimmunoassay for retinol-binding protein in serum and urine.

In this radioimmunoassay for human retinol-binding protein (RBP), sample (serum, diluted as much as 3600-fold; or urine, diluted or undiluted) or calibrant (purified RBP in phosphate buffer containing bovine serum albumin) is incubated with 125I-labeled RBP and rabbit anti-human RBP antiserum for 24 h before separation with second antibody/polyethylene glycol. The assay's sensitivity is 5 micrograms/L, its useful working range 10 to 200 micrograms/L. The between-batch CV is 12.5% at 12 micrograms/L, 7% at 120 micrograms/L. Results correlate well (r = 0.99) with those by radial immunodiffusion. The reference interval for RBP in serum of men is 39-75 mg/L (mean 57), significantly (p less than 0.01) broader than for women (29-66 mg/L; mean 48). The 2.5-97.5 centile interval for RBP in urine is from less than 0.5 to 12.2 micrograms per millimole of creatinine (median 5.8). RBP in urine is unstable below pH 5.0 at 37 degrees C.

Animals↗
PubMed1984

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.

C-Reactive Protein↗
PubMed1984

Development of a non-extracted 'two-site' immunoradiometric assay for corticotropin utilizing extreme amino- and carboxy-terminally directed antibodies.

The development of a 'two-site' immunoradiometric assay (i.r.m.a.) for the direct estimation of human corticotropin-(1-39)-peptide in plasma is described. The assay is based on the simultaneous addition of 125I-labelled sheep anti-(N-terminal corticotropin) IgG (immunoglobulin G) antibodies and rabbit anti-(C-terminal corticotropin) antiserum to standards and unknowns (0.5 ml) followed by 18h incubation. The use of solid-phase reagents was avoided in order to minimize non-specific effects and the time required for reactants to reach equilibrium. Instead, the separation of corticotropin-bound from free labelled antibody is achieved by the addition of sheep anti-(rabbit IgG) antiserum, which precipitates bound labelled antibody by complex-formation with rabbit anti-corticotropin antibodies, which are also hormone-bound. Several 125I-labelled sheep anti-(N-terminal corticotropin) IgG preparations were assessed in the i.r.m.a. Although each was derived from antisera raised to a thyroglobulin conjugate of synthetic corticotropin-(1-24)-peptide (Synacthen), purification of immunoglobulins before iodination by selective immunoadsorption resulted in preparations with distinct specificities which demonstrated marked differences in binding to intact human corticotropin-(1-39)-peptide. These preparations are compared in combination with two rabbit anti-(C-terminal corticotropin) antisera. A 'two-site' assay based on the use of 125I-labelled sheep anti-[ corticotropin-(2-16)-peptide] IgG and rabbit anti-[corticotropin-(34-39)-peptide] antiserum was optimized, since steric inhibition of antibody binding was avoided with this combination and because the measurement of only intact human corticotropin-(1-39)-peptide and not fragments was assured by the use of terminal antibodies. This i.r.m.a. is characterized by rapid equilibration of reactants, a wide 'operating range' (the precision of dose estimates was less than 4% over the range 30-2200 pg/ml) and high sensitivity [8 pg of corticotropin/ml (95% confidence interval 3.7-12.0) (4 pg minimal detectable mass) can be detected directly in plasma].

Adrenocorticotropic Hormone↗
PubMed1984

A liquid-phase two-site immunoradiometric assay for human prolactin.

The development of a 'two-site' immunoradiometric assay for human prolactin (hPrl) is described. The assay is based on the addition of radio-iodinated sheep anti-hPrl immunoglobulin G (IgG) and rabbit anti-hPrl serum to standards and unknowns followed by 3 h incubation. The use of solid phase reagents was avoided in order to minimize non-specific effects and the time required for reactants to reach equilibrium. Instead, the separation of hPrl-bound and free labelled antibody is achieved by the addition of sheep anti-(rabbit IgG) serum which precipitates bound labelled antibody by complex formation with rabbit anti-hPrl antibodies which are also hPrl-bound. Varying the order of addition of specific antibodies had a pronounced effect on the 'operating range' and sensitivity of resultant assays. This was attributed to competition between labelled and unlabelled antibodies for binding sites on the hPrl molecule. The immunoradiometric assay employing 'simultaneous addition' of specific antibodies was compared to a 'simultaneous addition' hPrl radioimmunoassay developed using the same sheep antiserum as that used to prepare the radioiodinated sheep anti-hPrl IgG. This immunoradiometric assay is characterized by rapid equilibration of reactants, a wide 'operating range' (the precision of dose estimates was less than 10% over the range 8-10000 mU/l), and high sensitivity (2.6 mU/l, 13 pg). In contrast, the hPrl radioimmunoassay required an incubation of 18 h, demonstrated a much reduced 'operating range' (the precision of dose estimates was less than 10% only over the range 25-1500 mU/l) and reduced sensitivity (9.8 mU/l, 49 pg).

Antibodies↗
PubMed1984

Growth hormone response to clonidine is impaired in patients with central sympathetic degeneration.

Serum growth hormone (GH) levels before and after intravenous clonidine (1.5 mcg/Kg) were measured in normal subjects and patients with chronic idiopathic autonomic failure (AF) due to central sympathetic degeneration. Normal subjects showed a rise in plasma GH from 2 +/- 1 mU/l to 27 +/- 12 mU/l. Basal levels were similar but there was no rise in GH levels in the AF patients. There was no difference in sedation in the two groups, indicating similar central nervous system penetration of the drug. Systolic blood pressure fell in both groups. Diastolic blood pressure fell significantly in normals but not in the AF patients. Clonidine-induced growth hormone release may be a potentially useful neuroendocrine marker indicating derangement of certain components of the central alpha-adrenergic system in man.

Autonomic Nervous System Diseases↗
PubMed1984

Influence of the hapten-fluorophore bridge on binding parameters in a fluoroimmunoassay for carbamazepine.

Raising antibodies to a hapten (drug or steroid) requires that it be coupled to a carrier protein through a bridge such that the hapten has maximum exposure with minimum changes in its configuration. In raising antisera in six sheep against carbamazepine (CBZ) coupled to bovine albumin, we found that some subpopulations of the antibodies recognized the bridge linking the drug to the carrier protein. To study the influence of the bridge on a fluoroimmunoassay for CBZ, we prepared four tracers by linking the carbamyl nitrogen of CBZ to fluorescein via four alkyl bridges of different lengths and structures. We calculated various binding parameters--including antibody affinity, binding capacity, and heterogeneity index--for each tracer and chose for the final fluoroimmunoassay the tracer that gave the best displacement with CBZ. We then optimized and validated the assay for direct measurement of CBZ in serum or plasma. The antibodies are coupled to magnetizable particles, which greatly facilitates separation and ensures removal of endogenous interferents.

Amines↗
PubMed1984

Single-reagent polarization fluoroimmunoassay for barbiturates in urine.

We describe a rapid polarization fluoroimmunoassay for screening for the presence of barbiturates in urine. The single reagent is prepared by pre-mixing antiserum with a fluorescein-labeled barbiturate derivative as tracer. Use of a mixture of two sheep antisera, raised against different barbiturate immunogens, results in an assay with a broad cross-reactivity spectrum for most common barbiturates. One adds urine to the pre-mixed reagent, incubates the solution for a few minutes at room temperature, and measures the fluorescence polarization. The tracer/antiserum reagent is stable for at least six months at 4 degrees C. Results compare favorably with two other commonly used screening techniques for barbiturates, thin-layer chromatography and the EMIT-stTM (Syva) system. Although designed as a qualitative screen, some drugs can be quantified by means of a standard curve of the relevant barbiturate.

Barbiturates↗
PubMed1984

A two-site immunoradiometric assay with magnetizable particles: human liver ferritin as a model.

Using human liver ferritin as a model, we have developed a two-site immunoradiometric assay. Sample (or standard) is incubated with excess rabbit antiserum and 125I-labeled specific sheep antibodies directed against different antigenic determinants until equilibrium is reached. Then excess sheep anti-rabbit immunoglobulin G serum, covalently linked to magnetizable particles, is added. The radioactivity bound to these particles is directly proportional to the amount of ferritin present. The assay is reproducible and precise throughout the clinically important range for ferritin in serum. Late addition of second antibodies coupled to magnetizable particles is a novel, simple, rapid, and universally-applicable separation technique for two-site immunoradiometric assays. By this approach, the first immunological reaction can occur in liquid phase, which shortens the reaction time, maximizes sensitivity, and avoids the need for continuous mixing, as compared with methods in which one of the first antibodies is immobilized on a solid phase.

Adult↗
PubMed1983

Polarisation fluoroimmunoassay of serum and salivary cortisol.

Polarisation fluoroimmunoassays for serum and salivary cortisol were developed using the steroid labelled at the 3-position with fluorescein and antiserum raised in sheep. To avoid interfering factors present in the biological fluids, cortisol was extracted with dichloromethane prior to assay, which was then accomplished without the need for any further separation or blank-correction procedures. The methods were sufficiently sensitive to cover the normal morning ranges for serum and salivary cortisol and correlated acceptably with established fluorimetric and radioimmunoassay techniques. The salivary cortisol assay was more precise than the serum assay, reflecting the greater case with which cortisol may be extracted from saliva. The potential usefulness of salivary cortisol assays was illustrated by a preliminary study showing that the circadian variation in three children was similar to that in an adult.

Adult↗
PubMed1983

Assessment of a latex-agglutination-inhibition card test for serum gentamicin, with a study of the effects of potential interfering factors.

A latex-agglutination-inhibition test for serum gentamicin, based on inhibition by gentamicin of antibody-induced agglutination of gentamicin-coated latex particles, was found to be sufficiently reliable for use in therapeutic monitoring and to correlate well with a variety of established immunoassays. The test (a nonseparation, nonisotopic immunoassay) is performed on cards and has a simple visual end point by inspection for the presence or absence of agglutination. Severely elevated bilirubin or lipid levels, or gross hemolysis (which may cause interference with other nonseparation immunoassays) had no effect on the card test. With raised rheumatoid factor or complement, the cards gave accurate recovery of added gentamicin at 5 and 10 mg/L but low recovery at 2 mg/L. Of 67 patients' sera, three from one individual caused nonspecific agglutination of the latex and could not be assayed. The card test can be recommended for laboratories handling infrequent or small numbers of samples and for those without access to instrumentation.

Fluorescent Antibody Technique↗