Simultaneous determination of total IgE and allergen-specific IgE in serum by the MAST chemiluminescent assay system.

We have developed a chemiluminescent immunoenzymometric system. The first commercial application of this chemiluminescent assay (CLA) is the measurement of total IgE and allergen-specific IgE in human serum. The CLA system is a second-generation adaptation of the MAST RIA allergy profiling system. The MAST CLA system assay protocol consists of three steps: overnight incubation of serum, a 4-h incubation with enzyme-labeled antibody, and a 30-min chemiluminescent reaction, which produces a visible image (immunograph) on high-speed Polaroid instant film. The densities of the bands produced on the film are quantified with an inexpensive microprocessor-controlled infrared transmittance densitometer. The novel luminogenic substrates used yield a constant light output for over 2 h with an intensity at least 10-fold greater than that of commercial chemiluminescent reagents. The MAST CLA system exhibits sensitivity, specificity, and precision equal to that of the MAST RIA system (r = 0.96 for 40 serum samples analyzed with 25 allergens). As many as 35 different allergens per sample can be quantified in a single assay. The MAST CLA system requires no standard curve or volume-dependent pipetting steps, incorporates both positive and negative controls for each sample, and quantifies allergen-specific IgE at picomolar concentrations.

Application of the MAST Immunodiagnostic System to the determination of allergen-specific IgE.

The MAST Immunodiagnostic Test System was developed to provide a comprehensive, simple means for the in vitro measurement of multiple antigens or antibodies. The first commercial application of the MAST system incorporates several novel features for cost-effective diagnosis of IgE-mediated allergy in a clinical laboratory or a physician's office. The basis of the MAST system is a unique analytical test chamber, which contains cellulose thread as the solid-phase matrix and allows multiple test results from a single assay. This test chamber incorporates both positive and negative controls and requires no volume-dependent pipetting steps. Immunographic exposure onto high-speed Polaroid instant film allows for quantifying results with an automatic recording infrared-transmittance densitometer. Test results are easily interpreted by using a patient test record provided with the system. The MAST system greatly simplifies testing for allergen-specific IgE, while retaining specificity and sensitivity. Currently, with the MAST system one can simultaneously measure picomoles of allergen-specific IgE in up to 35 different allergen classes. In addition to allergy testing, the MAST technology is applicable to other immunodiagnostic profiles.

Production and characterization of monoclonal antibody to gentamicin.

Splenocytes from BALB/c mice immunized with a gentamicin-hemocyanin conjugate were fused with X63-Ag8.653 murine myeloma cells to produce hybridomas that secreted monoclonal antibody to gentamicin. Sixteen positive clones were obtained. The monoclonal antibody chosen for a gentamicin immunoassay has been characterized with respect to class, subclass, type of light chain, electrophoretic homogeneity, and binding affinity. Gentamicin monoclonal antibody purified from mouse ascites fluid was analyzed by immunoelectrophoresis, double immunodiffusion, enzyme-linked immunosorbent assay (ELISA), and polyacrylamide gel electrophoresis (PAGE). The results show that the antibody is an IgG2a (kappa). Two bands were detected when the purified antibody was electrophoresed on polyacrylamide gels: a major band and a less mobile minor component (5.6% of the major band). Both were IgG2a (kappa). The major band contained antibody which bound 2.1 moles of the substrate-labeled gentamicin derivative, beta-galactosyl-umbelliferone sisomicin, per mole of IgG, whereas one mole of the minor band bound only 0.095 moles of the substrate-labeled conjugate. The antibody has an affinity constant of 1.22 X 10(10) M-1.

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.

Simultaneous determination of phenytoin and phenobarbital in serum or plasma by substrate-labeled fluorescent immunoassay.

This assay system for simultaneously determining phenytoin and phenobarbital in serum and plasma is based on the substrate-labeled fluorescent immunoassay technique. A beta-galactosylcoumarin derivative of phenobarbital and a 4-methylcoumarin phosphodiester derivative of phenytoin are used as substrate labels for Escherichia coli beta-galactosidase and Crotalus atrox phosphodiesterase I, respectively. The smallest measurable concentrations are about 1.6 mg/L for phenytoin, 2.7 mg/L for phenobarbital. Within-run coefficients of variation are about 5% for phenytoin and 2% for phenobarbital, about 6% for both between-runs. Results for phenytoin and phenobarbital in serum and plasma correlate well with those determined by the Ames TDA (r = 0.944 and 0.986, respectively) and Syva's EMIT (r = 0.977 and 0.969, respectively) assays.

Homogeneous substrate-labeled fluorescent immunoassay for carbamazepine.

Carbamazepine is an anticonvulsant drug useful in the management of epilepsy. Because of the narrow therapeutic range, serum carbamazepine monitoring is useful for ensuring adequate drug therapy without toxicity. We report the development of a homogeneous substrate-labeled fluorescent immunoassay for carbamazepine in human serum. A carbamazepine fluorogenic reagent (FR) has been synthesized. Upon hydrolysis by beta-galactosidase, the nonfluorescent FR produces a fluorescent product. This enzymic hydrolysis sin inhibited when the FR binds with antibody against carbamazepine. The inhibition is relieved when carbamazepine competes with FR for available antibody binding sites. Thus, increasing levels of carbamazepine result in increasing levels of fluorescence that can be conveniently monitored with any conventional fluorometer. For low, medium, and high control sera (4, 12, and 16 micrograms carbamazepine/ml), the within-run coefficient of variation for the assay is 5.5%, 1.6%, and 2.9%, respectively, while the respective between-run coefficients of variation are 3.5%, 1.9%, and 2.3%. Fifty-three clinical serum samples were assayed by the SLFIA, gas chromatography (GC), high pressure liquid chromatography (HPLC), and an enzyme immunoassay method. The SLFIA method compares favorably with the HPLC technique (r - 0.97, slope = 1.10, y-intercept = 1.21), the enzyme immunoassay (r = 0.98, slope = 1.07, y-intercept = 0.82), and the GC method (r = 0.95, slope = 1.01, y-intercept = -0.03).

Homogeneous substrate-labeled fluorescent immunoassay for theophylline in serum.

A substrate-labeled fluorescent immunoassay for theophylline in serum is described. 8-(3-Aminopropyl)-theophylline is covalently attached to a fluorogenic enzyme substrate, 7-beta-galactosylcoumarin-3-carboxylic acid. Hydrolysis of this theophylline-labeled substrate by beta-galactosidase yields a fluorescent product. When antibody to theophylline interacts with this substrate, the resulting complex is inactive as an enzyme substrate. For measuring theophylline, competitive protein-binding reactions are set up, with the theophylline in the sample competing with the substrate for the antibody-binding sites. The substrate not bound to antibody is hydrolyzed by beta-galactosidase, producing fluorescence that is proportional to the theophylline concentration. Results for theophylline determined by this method in clinical samples of serum correlated well (r > 0.96) with results obtained by gas-chromatographic or enzyme immunoassay procedures. The within-run CV for three control samples ranged from 1.1 to 2.8%, the between-run CB from 2.3 to 4.5%.

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.

Substrate-labeled fluorescent immunoassay for amikacin in human serum.

A homogeneous substrate-labeled fluorescent immunoassay has been developed to measure amikacin levels in human serum. Amikacin is covalently labeled with the fluorogenic enzyme substrate beta-galactosyl-umbelliferone. This beta-galactosyl-umbelliferone-amikacin conjugate is nonfluorescent under assay conditions until it is hydrolyzed by beta-galactosidase to yield a fluorescent product. When antiserum to amikacin binds the substrate-labeled drug, the antibody complex formation inhibits hydrolysis of the fluorogenic substrate. Reaction mixtures containing a constant level of substrate-labeled amikacin and a limiting amount of antiserum enable labeled and unlabeled amikacin to compete for the antibody-binding sites. Unbound substrate-labeled drug is hydrolyzed by the enzyme to release a fluorescent product that is proportional to the unlabeled amikacin concentration. The amikacin levels found in clinical serum samples with this method were comparable (r = 0.987) to those obtained by radioimmunoassay. The fluorescent immunoassay is rapid and simple to perform and requires only 2 microliters of serum.

A comparison of serum phenytoin determination by the substrate-labeled fluorescent immunoassay with gas chromatography, liquid chromatography, radioimmunoassay and "EMIT".

Patients' sera were analyzed for phenytoin by the Substrate-Labeled Fluorescent Immunoassay (SLFIA), gas chromatography (GC), liquid chromatography (LC), Radioimmunoassay (RIA) and EMIT and the results were compared. The EMIT assay was performed using a "PACER Analyzer". All assay results compared favorably. The correlation coefficients were: SLFIA vs GC, 0.995 (n = 45); SLFIA vs. LC, 0.993 (n = 37); SLFIA vs. EMIT, 0.965 (n = 67); SLFIA vs. RIA, 0.977 (n = 34). These results show that phenytoin levels determined by the SLFIA compare well with those obtained by the other four assay techniques.

Substrate-labeled fluorescent immunoassay for phenobarbital.

An assay for the anticonvulsant drug phenobarbital (PB) has been developed that is based on the principles of the substrate-labeled fluorescent immunoassay. A fluorogenic enzyme substrate, galactosyl umbelliferone, was covalently linked to a derivative of PB. The labeled drug, galactosyl umbelliferone-PB (GUPB), is nonfluorescent under conditions of the assay; however, hydrolysis of the galactosyl moiety by bacterial beta-galactosidase yields a fluorescent product. When GUPB is bound by antibody to PB, it is not a substrate for enzymatic hydrolysis. Thus, only GUPB not bound to antibody is hydrolyzed. In competitive binding reactions, using a fixed concentration of GUPB and a limiting amount of antibody, the PB in serum and the GUPB compete for antibody-binding sites. The fluorescence produced upon enzymatic hydrolysis of unbound GUPB is directly proportional to the concentration of PB. Unknown serum levels of PB are determined from a standard curve of fluorescent intensity versus standard PB concentrations. The assay is specific, sensitive, and easy to perform. It is carried out by adding the equivalent of 2 microliters of serum standard or unknown directly to a cuvette containing 3 ml of a buffered solution of antibody and enzyme. One-hundred microliters of GUPB is added, and the fluorescence intensity is measured after a fixed time (any time from 5 to 90 min). Using clinical specimens, our assay correlated well with a commercial enzyme immunoassay (correlation coefficient = 0.97) and had an interassay precision of less than 7%.

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.

High resolution proton nuclear magnetic resonance investigation of the structural and dynamic properties of d(C15A15)-d(T15G15).

The high resolution proton nuclear magnetic resonance (NMR) spectra of the synthetic DNA block polymer d(C15A15)-d(T15G15) were studied in order to more completely understand telestability in DNA, and to provide fundamental NMR data on DNA helices and random coils. Spectra were measured in the spectral region from 0 to 15 ppm downfield from the usual standard, sodium 4,4-dimethyl-4-silapentane-1-sulfonate(DSS), at various temperatures (24-98 degrees C) in solution containing either moderate or high ionic strength. The effect of actinomycin binding to the block polymer also was studied. The major conclusions derived from this study are as follows: (1) The majority of base pairs in the AT helix of the block polymer have the same conformation as in d(A)n-d(T)25 and d(A)21-d(T)21. (2) The conformation of the GC helix in the block polymer is different from the AT helix and this perturbs the conformation of three or four A-T base pairs at the junction of the AT-GC helix. (3) The conformation of the AT helix is unaffected by salt over the range examined (approximately 0.04 - approximately 2 M), but the conformation of the GC helix changes. (4) There are subtle changes in the conformation of the AT helix as the temperature is increased and resonances characteristic of the random coil and the double-helical state can be simultaneously observed. (5) Binding of actinomycin, which is specific for the GC helix, induces quite large (over 1 ppm) upfield shifts of the resonances from the GC base pairs. This is consistent with an intercalation model in which actinomycin D (Am) is assymetrically sandwiched between two GC base pairs in such a manner that overlap with the guanosine residues is greater than with the neighboring cytidines. (6) The presence of the drug may also perturb A-T base pairs located near the AT-GC junction, but it has no effect on the majority of the AT pairs. However, as expected, Am elevated the Tm of the AT helix, even though it binds to the other end of the DNA.