J. Landon

A detailed investigation of circulating IgE levels in a normal population.

Total circulating IgE levels were measured in a carefully selected normal population, using a highly sensitive double antibody assay. The mean level of 36.3 u/ml found in this group is much lower than that reported in all previous studies. No circadian rhythm was evident, but IgE levels varied slightly from day‐to‐day. IgE levels in 100 serum samples were compared using the double antibody assay and the Phadebas lest kit. based on a solid phase technique. A poor correlation was obtained with values below 100 u/ml, however, there was an excellent correlation for samples with IgE levels above this figure.

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. Marks 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 Crohns disease, twenty-two ulcerative colitis, and forty-one functional bowel disorders. All the patients with Crohns 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.

Polarisation fluoroimmunoassay of gentamicin.

A polarisation fluoroimmunoassay has been developed for the routine determination of gentamicin levels in serum. The method employs fluorescein-labelled gentamicin which is easily, reproducibly and economically prepared and has excellent shelf-life. The assay is fast (minimum incubation time 2 min) and requires 1.25 mul of serum. There is no separation step prior to readout of results. Recovery experiments reveal no serum effects. Intra- and inter-assay coefficients of variation are of the order of 10% or less. Estimations of gentamicin levels in patient serum samples correlate closely with those obtained by bioassay (r = 0.93) and radioimmunoassy (r = 0.97).

The measurement of urinary digoxin and dihydrodigoxin by radioimmunoassay and by mass spectroscopy

A radioimmunoassay for urinary digoxin is described which includes an initial solvent extraction to remove factors in urine which cause non-specific interference in the assay. The recoveries obtained using different solvents are compared and the non-specific factors influencing the assay investigated further. These effects were overcome by the use of a small urine volume (10 mul) in a direct, unextracted, urine assay and the results obtained correlated closely with those from the assay using prior extraction (r=0.99). No false positive results were obtained with unextracted urine samples from hospitalised patients not receiving digoxin. The specificity was also determined with regard to the natural steroids, spironolactone and the metabolites of digoxin including dihydrodigoxin. The metabolite dihydrodigoxin, with a saturated lactone ring, was not detected whereas the mono-, and bis-digitoxo-sides and digoxigenin metabolites did cross react in the assay. It was not possible to separate dihydrodigoxin and digoxin by thin-layer chromatography or solvent extraction due to their similar structures, however, mass spectroscopy was successful in this respect and was employed to obtain the ratio of dihydrodigoxin to digoxin in extracted urine samples. Levels of urinary digoxin excreted by patients maintained on different oral doses of the drug were measured. The percentage excreted in the urine as digoxin correlated closely with the oral dose (r = 0.96) but was found to be lower than that reported in most previous studies. Mass spectroscopy measurements showed that an average of 16.4% (range 12.2-19.7%) of the total oral dose was excreted as dihydrodigoxin in the urine of nine patients investigated.

Solid-phase, magnetic particle radioimmunoassay

A solid-phase radioimmunoassay system has been developed based on the use of antibodies covalently linked to polymer-coated iron oxide (EnzacrylR). An electro-magnet is employed both to mix the particles during incubation (by switching the field on and off) and to separate the antibody-bound and free fractions. This obviates the need for vertical rotation and for the time-consuming, multiple centrifugations required with conventional solid phase procedures. The system is universally applicable and methods have been established for the assay of thyroxine, human placental lactogen and digoxin. The thyroxine assay was employed as a model and it was shown that the results obtained for serum samples correlated closely with those using a routine liquid-phase radioimmunoassay. The applicability of employing a second antibody linked to the iron oxide particles was also studied.

Estimation of serum gentamicin by quenching fluoroimmunoassay.

A new type of non-isotopic immunoassay, applied to the determination of serum gentamicin, is reported. The method is based on partial quenching of fluorescence observed when fluorescein-labelled gentamicin is bound by anti-gentamicin serum. The fluorescence intensity of the labelled gentamicin in an unseparated immunoassay incubation mixture therefore serves to indicate the extent of binding, which is related to the amount of competing unlabelled gentamicin present. Precision and accuracy are shown to be similar to those of the best existing methods for gentamicin, while the new assay is more rapid and technically simpler, and avoids the use of expensive radio-chemicals with their attendant health hazard. Assays of patient samples correlate with established bioassay and polarisation fluoroimmunoassay methods.

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.

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.

A simple competitive protein binding assay for plasma cortisol

Abstract The availability of cortisol labelled with 75 Se has permitted the development of a simple and rapid competitive protein binding assay for cortisol. Plasma samples are diluted with water and heated at 70° to destroy endogenous transcortin, and an aliquot is then incubated with 75 Se-labelled cortisol and charcoal-treated rabbit serum. Separation of the bound and free fractions is achieved by a Sephadex equilibration technique. Results compare closely with those obtained using a specific, automated fluorimetric assay. The assay is more specific and technically simpler than manual fluorimetric methods.

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.