John F. Burd

Specific protein-binding reactions monitored by enzymatic hydrolysis of ligand-fluorescent dye conjugates

Abstract Specific protein-binding reactions were monitored utilizing ligand-fluorescent dye conjugates which were substrates for porcine esterase. Biotin was coupled directly to umbelliferone through an ester bond, while 2,4-dinitrophenyl derivatives were conjugated to fluorescein via an ester linkage. Hydrolysis of these nonfluorescent esters with an esterase yielded fluorescent products, and the reaction rate was related quantitatively to the conjugate concentration. When the conjugated ligands were bound to their specific proteins (avidin or antibody to the dinitrophenyl residue), they were inactive as substrates. This assay allowed the measurement of unbound conjugate without separation from protein-bound conjugate. The inactivation of the conjugated substrates was relieved by the addition of free (unconjugated) ligand in competitive binding reactions. In addition to measuring free ligand levels, this assay was used to detect specific binding proteins, e.g., during the purification of antibody to the dinitrophenyl residue.

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.

A colorimetric assay for serum lactate dehydrogenase.

Abstract A simple, rapid, colorimetric assay for serum lactate dehydrogenase is described. The method requires 0.05 ml serum and a 12-min incubation at 37° and utilizes a tetrazolium salt in conjunction with a stable intermediate electron carrier (Meldola Blue). Good correlation between the colorimetric assay and a reference spectrophotometric assay was observed.

Immunochemical studies on lactate dehydrogenase: Preparation and characterization of anti-M lactate dehydrogenase antiserum: Development of an immunochemical assay for lactate dehydrogenase isoenzymes in human serum

Abstract 1. 1. Inhibitory antibodies against highly purified Rhesus monkey muscle M4 lactate dehydrogenase ( l (+)-lactate:NAD+ oxidoreductase, EC 1.1.1.27.) were prepared in rabbits and goats. The specificity and sensitivity of immunochemical reactions were successfully exploited to develop a simple, specific and reliable assay of lactate dehydrogenase subunits in human serum. 2. 2. The antiserum specifically inhibited the enzymatic activity of purified human lactate dehydrogenase isoenzymes in proportion to their M monomer content, indicating the existence of four active sites per tetramer. 3. 3. The antiserum also specifically inhibited the M4 lactate dehydrogenase activity of partially purified preparations of enzyme from several animal species. The degree of inhibition differed from species to species, but was specific for the M subunit in all enzymes tested.

Substrate-labeled fluorescent immunoassay for phenobarbital.

Summary 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 β-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 μl 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%.

Homogeneous Substrate-Labeled Fluorescent Immunoassay for Carbamazepine

Summary: 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 β‐galactosidase, the nonfluorescent FR produces a fluorescent product. This enzymic hydrolysis is 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 μg 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, v‐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 = ‐0.30).

Serum theophylline determination by fluorescence polarization immunoassay utilizing an umbelliferone derivative as a fluorescent label

Abstract We report the development of a non-radioisotopic homogeneous immunoassay to determine serum theophylline concentrations using fluorescence polarization. Theophylline is a therapeutic drug which is used in the treatment of asthma. Monitoring this drug in serum is necessary to ensure effective dosage and to avoid toxicity. The fluorescence polarization immunoassay is based on labeling the analyte with a fluorescent probe and monitoring the changes in the probes fluorescence polarization occurring in the hapten-antibody reaction. Previous reported fluorescence polarization immunoassays invariably involved the use of fluorescein as a fluorescent label. The existence of polymorphic forms in fluorescein and the presence of contaminants in commercial preparations of fluorescein have led us to use an alternative fluorophore, namely, an umbelliferone derivative, in the polarization immunoassay. This fluoreceent probe is characterized by a high quantum yield, a large extinction coefficient, a large Stokes shift, and the absence of nonspecific interaction with normal human serum. The spectroscopic properties of this label including both the excitation and emission polarization spectra are presented, and the procedure to obtain the optimized fluorescence polarization standard curve is described. Using the polarization immunoassay technique, the theophylline concentrations of 25 clinical serum samples were determined. The values obtained correlated well with values determined by high-pressure liquid chromatography (equation of linear regression analysis y = 0.97 x + 1.33; and correlation coefficient, 0.93).

Enzymic hydrolysis of intramolecular complexes for monitoring theophylline in homogeneous competitive protein-binding reactions

Abstract A novel approach in the design of fluorogenic substrate-analyte conjugates that can be used in a substrate-labeled fluorescent immunoassay (SLFIA) is described. The new SLFIA uses an enzyme substrate molecule that contains a fluorophore component and a quencher component, separated by a chain containing a bond which can be hydrolyzed by an enzyme. The feasibility of using this approach, in the construction of a fluorophore-quencher-analyte conjugate for monitoring analytes in homogeneous competitive protein binding reactions was demonstrated by using flavin-N 6 -(6-aminohexyl-theophylline) adenine dinucleotide (FAD-Theophylline) as the intramolecularly quenched fluorogenic substrate. Hydrolysis of the FAD-theophylline by nucleotide pyrophosphatase yielding FMN and AMP-theophylline restores the fluorescence to the expected level of FMN. Antibody to theophylline, however, inhibits the enzymic hydrolysis, and this inhibition is relieved in competitive binding when theophylline is added.