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Biomedical subjects

J Landon

Publications and source records attributed to J Landon.

At least 55 records · Page 3Linked to original sources
PubMed1988

Production of polyclonal antibodies in ascitic fluid of mice: technique and applications.

Balb/c mice immunised intraperitoneally with insulin developed significantly higher antibody concentrations in their serum than mice immunised subcutaneously. The antibody response was dose related, 50 ug amounts being more effective than either 20 ug or 5 ug amounts. In contrast, the antibody titres in the ascitic fluid of mice immunised with growth hormone were significantly higher after 5 ug when compared to 25 and 100 ug amounts. Repeated intraperitoneal injections of an emulsion of sheep IgG or human growth hormone in complete Freund's adjuvant, together with a single intraperitoneal injection of Pristane, induced ascites formation in most mice within 5 weeks. Up to 90 mL could be obtained from a single mouse by abdominal tapping, and the antibodies derived from the ascitic fluid proved suitable for application in a radioimmunoassay.

Animals↗
PubMed1988

Development of separation fluoroimmunoassays for bendazac and 5-hydroxy bendazac.

Two distinct separation fluoroimmunoassays were developed to determine serum levels of bendazac and its main metabolite 5-hydroxy bendazac for use in early pharmacokinetic studies and for long-term therapeutic drug monitoring should this prove necessary. In both assays fluorescein-labeled analyte was used as tracer and the antibodies were coupled to magnetizable particles. The use of a magnetizable solid phase enabled a simple and rapid separation step and thus a high sample throughput. There was no crossreactivity between the drug and its metabolite in either assay, indicating that highly specific antibodies had been produced. The results from both assays correlated well with serum levels determined by a high performance liquid chromatography method.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1988

Design and development of a single-reagent polarization fluoroimmunoassay for methamphetamine.

The design and development of reagents suitable for use in single-reagent polarization fluoroimmunoassay of methamphetamine is described. The single-reagent method requires that a substantial fraction of antibodies exhibit fast dissociation of bound tracer. Antiserum obtained early in the course of immunization of a sheep with a methamphetamine immunogen had suitable dissociation characteristics. Tracers with bridges of different length between methamphetamine and the fluorescein label group were prepared and evaluated. A single-reagent assay with sensitivity and specificity suitable for urine screening application was demonstrated.

Animals↗
PubMed1988

Development of a direct fluoroimmunoassay for serum levels of 17-hydroxyprogesterone.

A direct, rapid and highly specific fluoroimmunoassay for determining serum levels of 17-hydroxyprogesterone has been developed. It is based on the use of a sheep antiserum covalently coupled to magnetisable particles and fluorescein-labelled steroid. Sodium salicylate is employed to eliminate interference from endogenous binding proteins in serum. The sensitivity of 0.5 nmol/L is adequate for clinical purposes. Analytical recovery, linearity and precision are satisfactory and the results obtained correlate closely with those of an established radioimmuno-assay using 3H-labelled steroid and the same antiserum after initial sample extraction and chromatography. The values found for serum from normal adult subjects ranged from 1.0 to 12.6 nmol/L while those from treated and untreated patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency were 1.5 to 19.0 and 28.0 to 655 nmol/L, respectively.

17-alpha-Hydroxyprogesterone↗
PubMed1988

Design of the immunogen and label for use in a fluoroimmunoassay for paracetamol.

The design of immunogens for raising antisera containing antibodies specifically directed against paracetamol is discussed. A derivative of the drug with minimal structural alteration was synthesised, coupled to the carrier protein key-hole limpet haemocyanin, and used to immunise sheep. The evaluation of the resulting antisera is described. Preparation of a fluorescent label with reduced bridge-binding then allowed the development of a polarisation fluoroimmunoassay for paracetamol in buffer, suitable for conversion to a direct serum assay.

Acetaminophen↗
PubMed1988

Development of a specific polarisation fluoroimmunoassay for paracetamol in serum.

A specific polarisation fluoroimmunoassay for the measurement of paracetamol levels in serum has been developed for use in emergency toxicological screening. It is based on the use of a fluorescein-labelled analogue and a sheep antiserum and exploits the rapid dissociation kinetics of the hapten-antibody complex to enable the label and antiserum to be combined as a single reagent. Paracetamol levels are determined by adding 5 microL of serum to 1.5 mL of the single reagent, incubation at ambient temperature for a few minutes and measurement of fluorescence polarisation. In addition to its speed and simplicity, the assay is both accurate and precise and the results obtained correlate closely with those from the commonly used chemical and enzymatic techniques.

Acetaminophen↗
PubMed1988

Direct single-reagent fluorescence polarisation immunoassay for valproic acid in serum.

A fluorescence polarisation immunoassay for quantitating serum concentrations of valproic acid was developed and validated. Its low molecular weight and lack of structural features caused difficulties in producing suitable antibodies. However, success was achieved using 2-propyl-6-aminohexanoic acid to make the fluorescein-labelled drug and two immunogens, the first using glutaraldehyde to link the drug derivative to keyhole limpet haemocyanin, and the second by carbodiimide activation of cellulose hydroxyl groups and coupling them to the drug derivative and killed Mycobacteria. It was found that both immunogens produced a good antibody response in sheep. The antibodies were highly specific and the assay results correlated well with an in-house gas-liquid chromatographic method.

Chromatography, Gas↗
PubMed1988

Development of a direct [125I]radioimmunoassay for serum levels of 17-hydroxyprogesterone.

We describe a direct, rapid, sensitive and highly specific radioimmunoassay for determining serum levels of 17-hydroxyprogesterone. It is based on the use of highly specific sheep antiserum, 125I-labelled tracer, dextran-coated charcoal to separate the antibody bound and free fractions, and sodium salicylate to eliminate interference from endogenous binding proteins in serum. Intra- and interassay coefficients of variation are less than 8% and recovery is satisfactory. Sensitivity is 3.5 fmol per assay tube (0.14 nmol/l). Results correlate closely with those of an established technique using 3H-labelled steroid after initial solvent extraction and column chromatography of samples (y = 1.02x - 0.16; r = 0.998). The values found for serum from normal adult subjects ranged from 1.0 to 12.1 nmol/l while those from treated and untreated patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency were 1.4-18.4 and 27.3-650 nmol/l, respectively.

17-alpha-Hydroxyprogesterone↗
PubMed1987

The production and characterisation of antisera to 17-hydroxyprogesterone.

Sheep were immunised with 11-deoxycortisol-21-hemisuccinate-bovine serum albumin (11-deoxycortisol-21-HS-BSA) or with 17-hydroxyprogesterone-7 alpha-carboxyethyl thioether-keyhole limpet haemocyanin (17-OHP-7 alpha-CETE-KLH) or with 17-OHP-3-(O-carboxymethyl)oxime-KLH (17-OHP-3-CMO-KLH). The resultant antisera were assessed using [3H]17-OHP and dextran-coated charcoal to separate the antibody bound and free fractions. All sheep produced antisera with an apparent affinity constant of from 1.4 to 6.6 X 10(9) 1/mol. Those raised against 11-deoxycortisol-21-HS-BSA had titres ranging from 1:12,000 to 1:78,000 but showed significant cross-reactivity with many of the steroids tested. Sheep immunised with 17-OHP-7 alpha-CETE-KLH had antisera titres of from 1:102,000 to 1:180,000 and only 17-hydroxypregnenolone cross-reacted significantly (10-20%). The best antisera were raised in sheep immunised with 17-OHP-3-CMO-KLH. Titres ranged from 1:168,000 to 1:390,000 and there were about 8 g/l of specific antibodies which cross-reacted 5.7% or less with 17-hydroxypregnenolone, and less than 0.5% with progesterone, 11-deoxycortisol and the other steroids studied. The antisera to 17-OHP-3-CMO-KLH were further assessed using [125I]17-OHP; titres ranged from 1:5,700,000 to 1:18,000,000 with affinity constants of from 1.67 to 2.5 X 10(10) 1/mol. They showed minimal or no cross-reactivity with the steroids studied. Reimmunisation after an 8-month interval produced antisera with a higher affinity constant and even lower cross-reactivity with other steroids.

17-alpha-Hydroxyprogesterone↗
PubMed1987

Radioimmunoassay of salivary digoxin by simple adaptation of a kit method for serum digoxin: saliva/serum ratio and correlation.

A robust commercial radioimmunoassay kit for serum digoxin was adapted to salivary assay by simple procedural modifications and shown to have satisfactory sensitivity, precision, accuracy, and specificity. The digoxin saliva/serum concentration ratio in 50 hospital patients was 0.67 +/- 0.10 (mean +/- SD), and the levels were well correlated (r = 0.96). Similar saliva/serum ratios were found in two normal subjects who took digoxin orally, and the free fraction in serum, determined by equilibrium dialysis, was found to equate with the salivary drug level. Digoxin appeared in saliva at its maximum level within 1 min of intravenous injection. Although previous studies of salivary digoxin have given widely discrepant results, the present findings are entirely consistent with the simple expectation that digoxin should appear in saliva by passive diffusion. Salivary sampling is suitable for the monitoring of digoxin therapy and pharmacokinetics, and the modified radioimmunoassay should be of value to laboratories wishing to introduce this technique.

Digoxin↗
PubMed1987

Development of fluoroimmunoassays for flecainide.

Antibodies raised in sheep against a flecainide:protein conjugate and fluorescein-labeled drug were used to develop simple fluoroimmunoassays for the measurement of flecainide acetate in serum or plasma. A rapid, nonseparation assay, based on fluorescence polarization was optimized for therapeutic drug monitoring. A separation fluoroimmunoassay, using antibodies covalently linked to magnetizable particles to avoid the need for centrifugation, was also optimized and validated for monitoring flecainide therapy. It is applicable to lipemic, hemolyzed or icteric samples unsuitable for the nonseparation approach and for laboratories with access only to a simple fluorimeter. Finally, the separation fluoroimmunoassay was modified slightly to improve markedly sensitivity for use in pharmacokinetic studies.

Flecainide↗
PubMed1987

The raising and characterisation of antibodies to salicylate.

Polarisation fluorescence was used to assess the antibody response in sheep immunised with 4-aminosalicylic acid coupled to keyhole limpet haemocyanin and to develop a polarisation fluoroimmunoassay for subsequent clinical use. The fluorescent label was prepared by coupling 4-aminosalicylic acid to fluorescein isothiocyanate. All immunised sheep produced antibodies and a simple assay was developed using salicylic acid as the standard. The simple fluorescence polarisation approach enabled the rapid and direct study of the immuno-reactivity of a large number of pure chemical analogues of salicylic acid. Each was assessed for its ability to compete with the fluorophore-labelled analyte for antibody-binding sites, relative to salicylate, thereby enabling investigation of the steric and chemical characteristics important in the recognition and binding of a hapten to antibodies.

Aminosalicylic Acid↗
PubMed1987

Single-reagent polarization fluoroimmunoassay of methamphetamine in urine.

We developed a polarization fluoroimmunoassay for the specific detection of methamphetamine in urine. Sheep antiserum raised against methamphetamine was premixed with a fluorescein-labeled amphetamine tracer in a single reagent. The use of labeled amphetamine gave a more sensitive and more specific assay than did the use of a labeled methamphetamine tracer. The assay system had equal sensitivity for the d- and l-isomers of methamphetamine while the cross reactivity of amphetamine and other structurally related compounds was 3% or less. With a positive/negative cutoff concentration set at 1 mg/L, the polarization fluoroimmunoassay reliably identified human urines that had been reported positive for methamphetamine by an established gas-liquid chromatographic method.

Animals↗
PubMed1987

Polarisation fluoroimmunoassay for quinine in serum and urine.

The development and validation of a polarisation fluoroimmunoassay for the antimalarial drug quinine is described. The assay is performed either by sequential addition of the reagents or by a single-reagent technique whereby the tracer and antibodies are premixed. Serum samples require pepsin digestion prior to assay while urine specimens are assayed directly. The reliability criteria of the assay are satisfactory and no cross-reaction is detected with quinidine (the optical isomer of quinine) or with common antimalarial drugs. The assay was applied to the measurement of quinine in urine specimens obtained from a single-dose pharmacokinetic study and the results correlated with those of the benzene extraction fluorescence method for quinine measurement.

Adult↗
PubMed1986

Single-reagent polarization fluoroimmunoassay for theophylline in serum.

A polarization fluoroimmunoassay for theophylline was developed employing fluorescein-labeled drug and antiserum precombined in a single reagent. Assay was performed simply by addition of sample to an aliquot of the single reagent, incubation, and determination of fluorescence polarization. Because of the relatively rapid dissociation kinetics of the labeled drug from antibody binding, added unlabeled theophylline caused displacement within a practical time period. The precision, accuracy, and specificity of the simplified single-reagent assay were similar to those obtained by a conventional immunoassay procedure using the same reagents. Results for the assay of patients' serum specimens correlated well with those by an established enzymoimmunoassay.

Animals↗
PubMed1986

Single-reagent polarisation fluoroimmunoassay for the cocaine metabolite, benzoylecgonine, in urine.

A polarisation fluoroimmunoassay is described for the detection in urine of benzoylecgonine, the major metabolite of cocaine. The method uses a single reagent comprising sheep anti-benzoylecgonine serum pre-mixed with a fluorescein-labelled benzoylecgonine derivative as tracer. Under the assay conditions the antiserum has equal cross-reactivity with cocaine and the metabolite. The test is performed by the addition of 10 microL of urine to an aliquot of the single reagent, incubation for a few minutes and measurement of fluorescence polarisation. The assay has a positive/negative cut-off level of 1 mg/L benzoylecgonine and results compare favourably with the EMIT-d.a.u.TM (Syva) system.

Antibody Specificity↗