A colorimetric immunoassay based on an enzyme inhibitor method.

A competitive binding immunoassay was developed using an enzyme inhibitor for labeling the analyte. Thyroxine was labeled by covalent coupling of the alpha-amino group to the gamma-carboxyl of the glutamyl residue of methotrexate. This thyroxine-methotrexate conjugate was a potent inhibitor of dihydrofolate reductase. When antibody was bound to the thyroxine moiety, the inhibitor was inactivated. Thus, a competitive binding immunoassay for thyroxine was demonstrated based on colorimetric measurement of dihydrofolate reductase activity.

Immunoassay for serum thyroxine monitored by chemiluminescence.

An immunoassay for thyroxine (T4) monitored by chemiluminescence was evaluated with clinical serums. A thyroxine-label conjugate (T4-L) and serum samples were applied sequentially to alkaline Sephadex G-25 columns which adsorbed the thyroxine species. Other serum components and potential interferents were washed from the column with barbital buffer. Subsequently addition of antibody initiated the binding reaction. After 1 h incubation, the antibody was eluted from the column with a barbital buffer wash. The bound T4-L in the eluate was oxidized in a chemiluminescent detection reaction. The peak light intensity, attained in about 1 sec, was related to T4 concentration by means of standards. The intra-assay precision of the chemiluminescence immunoassay was +/-5% (C.V.). Statistical comparison of T4 levels determined for 28 serums by this method and a reference assay was acceptable (y = 0.95x + 5.9, r = 0.98, Sy square root of y . 100 = 13.1%).

Homogeneous substrate-labeled fluorescent immunoassay for IgG in human serum.

A homogeneous substrate-labeled fluorescent immunoassay for IgG has been developed. Purified IgG was covalently labeled with 6-(7-beta-galactosylcoumarin-3-carboxamido)-hexylamine to form a stable conjugate, GU-IgG. The galactosyl residue was hydrolyzed from GU-IgG by beta-galactosidase and the progress of the hydrolysis was monitored by the increase in fluorescence emission at 450 nm with excitation at 400 nm. Antibody to IgG diminished the activity of GU-IgG as a substrate for beta-galactosidase. Competitive binding immunoassays were conducted by allowing added IgG and GU-IgG to compete for a limited number of antibody binding sites. Hence, the fluorescence produced by enzymic hydrolysis increased with the level of added IgG. This method provides a simple and reliable immunoassay for IgG and is applicable to other proteins.

Maternal serum unconjugated estriol and urine estriol concentrations in normal and high-risk pregnancy.

Serum unconjugated estriol levels and urinary estriol levels of concurrent specimens were compared for 6 normal and 6 high-risk subjects throughout the last trimester of pregnancy. Free estriol values in serum exhibited a close correlation with urinary estriol values for both the normal (r = 0.91) and high-risk (r = 0.92) groups. The results of this study indicate that the assessment of high-risk pregnancies now accomplished principally by urinary estriol assays may be performed by more convenient and rapid radioimmunoassays developed for quantification of free estriol in serum.

A column radioassay for the quantification of vitamin B-12.

A rapid and convenient column radioassay for the quantification of vitamin B-12 in clinical specimens has been developed. A mixture of the specimen and radiolabeled B-12 was applied to a column containing immobilized intrinsic factor and allowed to incubate for two hours. A buffer wash then separated bound and free B-12. The average intra- and inter-assay coefficients of variation over the range of interest was about 30 pg/ml in the sample and recovery and parallelism studies gave satisfactory results. The values obtained for clinical specimens by the column procedure correlated well with those obtained by reference method.

The combined radioimmunoassay of triiodothyronine and thyroxine.

An RIA method for the concurrent determination of serum triiodothyronine (T3) and thyroxine (T4) is described. The extraction of the hormones from serum and competitive binding reactions using specific T3 and T4 antisera were conducted in sequence in a single Sephadex column. Precision studies resulted in average interassay relative standard deviations for T3 and T4 of 17 and 9.5%, respectively. The assay also exhibited satisfactory recovery and correlation with reference procedures (T3: y = 0.84x -- 36, r = 0.98; T4: y = 0.96x + 0.06, r = 0.98, where y is the value obtained by the combined method and x is the value obtained by the reference method.

Analytical methods for therapeutic drug monitoring.

The availability of new immunoassay and chromatographic methods has led to the revolution in therapeutic drug monitoring. The immunochemical and chromatographic methods both meet the analytical requirements of sensitivity, precision, and accuracy needed for most TDM applications. The technique chosen for any laboratory will depend upon numerous factors such as the availability of personnel and equipment, the time required to perform the assay, the speed with which the clinician needs the results, and the cost and medical benefit to the patient. It is reasonable to assume that the rapid development of new equipment and methodologies will continue to keep therapeutic drug monitoring one of the most interesting and fastest growing areas in clinical medicine.

Isolation of substances from urine by affinity chromatography.

The applications of affinity chromatography for the isolation and purification of physiological substances from urine have been reviewed. The use of affinity supports for the detection and measurement of various urinary components has also been briefly examined. These examples illustrate the usefulness of urine as a source of fine reagents and the versatility, simplicity and practicality of affinity isolation procedures.

Direct radioimmunoassay for unconjugated estriol in pregnancy serum, with use of a radioiodinated derivative of estriol.

We describe a rapid and direct 125I-based radioimmunoassay for quantification of unconjugated (free) estriol in pregnancy serum. Estriol in serum is adsorbed onto a small column of Sephadex, thereby allowing its separation from proteins and interfering materials. A radioiodinated derivative of estriol (6-ketoestriol-6(o-carboxymethyl)oxime-[125I]tyrosine methyl ester) is added to the column, followed by a limiting amount of antiserum. After incubation, the antibody-bound hormone is separated by a buffer wash. The assay exhibits satisfactory recovery and parallelism, and the intra- and inter-assay coefficients of variation are less than 10%. The values obtained by using the assay correlate well (r = 0.97) with those from a comparison method in which solvent extraction and chromatographic purification are used.

Substrate-labeled fluorescent immunoassay for phenytoin in human serum.

A homogeneous substrate-labeled fluorescent immunoassay has been applied to the measurement of phenytoin concentrations in human serum. We coupled a fluorogenic enzyme substrate, galactosyl-umbelliferone, covalently to a derivative of phenytoin. Under assay conditions, this drug-substrate conjugate was nonfluorescent but became fluorescent upon hydrolysis catalyzed by bacterial beta-galactosidase. When antibody to phenytoin is bound to the drug-substrate conjugate, it is inactive as an enzyme substrate. Addition of phenytoin to competitive-binding reactions relieves the inactivation, and the resulting fluorescence is proportional to the phenytoin concentration. We validated the fluorescent immunoassay by comparing values for phenytoin obtained with this technique to those obtained by gas chromatography and by enzyme immunoassay (EMIT). All three methods correlated well. The major metabolite of phenytoin, 5-(p-hydroxyphenyl)-5-phenylhydantoin, and other drugs at concentrations expected in serum had no effect on the assay. The fluorescent immunoassay is rapid and simple to perform and requires only 2 microL of serum sample per test.

Homogeneous reactant-labeled fluorescent immunoassay for therapeutic drugs exemplified by gentamicin determination in human serum.

We applied a homogeneous reactant-labeled fluorescent immunoassay to the measurement of therapeutic drug concentrations in human serum, exemplified here by gentamicin. A derivative of umbelliferyl-beta-galactoside was coupled covalently to the drug and this conjugate was found to be nonfluorescent under assay conditions. The drug/dye conjugate was a substrate for bacterial beta-galactosidase and yielded a fluorescent product. When the drug/dye conjugate was bound to anti-gentamicin antibody it was inactive as an enzymatic substrate. This inactivation was relieved by the presence of gentamicin in competitive binding reactions. Hence, the rate of production of fluorescence was proportional to the gentamicin concentration. The fluorescent assay yielded values which compared favorably to a radioimmunoassay for gentamicin in clinical serum samples (r=0.94, standard error of estimate=0.66 mg/liter). The fluorescent assay requires only 1 microliter of serum and offers several advantages over existing techniques: sensitivity, specificity, simplicity, and the obviation of radioisotopes.

Evaluation of a new urease reagent strip for detection of Helicobacter pylori in gastric biopsy specimens.

OBJECTIVES: Determine sensitivity and specificity of a new urease reagent strip (URS) test for detection of Helicobacter pylori in gastric biopsy specimens. METHODS: Six paired biopsy specimens were obtained from the greater curvature of the distal antrum, the lesser curvature near the incisura, and the corpus along the greater curvature during 66 procedures on 59 patients with endoscopic findings of gastric antral mucosal erythema or erosions, or gastric or duodenal ulcers. One biopsy from each site was tested with the URS. The second was evaluated with histology. A final antral biopsy was evaluated with a urea/gel test. RESULTS: Twenty-eight of the 66 cases were histologically positive, with H. pylori observed in at least one of the three biopsy sites. The URS test correctly identified all 28. Of 193 individual biopsy specimens, 78 were positive for H. pylori. The URS correctly identified 77. Sensitivity was 0.99, specificity 0.95, positive predictive value 0.93, negative predictive value 0.99, and kappa 0.92. Average time to positive was 20 min. Twenty-seven antral biopsies were histologically positive for H. pylori. The URS test correctly identified all 27, whereas the urea/gel test correctly identified 21. For antral sites, URS sensitivity was 1.00 and specificity 0.95 versus urea/gel test sensitivity of 0.75 and specificity of 1.00. CONCLUSIONS: In this sample, the URS test is as accurate as histology in diagnosing H. pylori infections, and it provides results in less time and at a lower cost than histology.