Time-Resolved Fluorescence Receptor Assay for Benzodiazepines

Abstract

A novel non-isotopic receptor binding technique for the detection of benzodiazepines is described. A benzodiazepine labeled with europium chelate was prepared and employed as a labeled ligand, and time-resolved measurements of the long lifetime fluorescence of europium chelate allowed avoidance of interference due to proteins in the receptor preparation. Experimental results demonstrate a sigmoid inhibition curve, with binding of the labeled ligand inhibited by comparable concentrations of the unlabeled drug. The proposed assay may provide a simple procedure for the measurement of benzodiazepines in biological systems and a useful tool in the screening of natural substances for new classes of benzodiazepine-like compounds.     

Non-isotopic receptor assay for benzodiazepines using a biotin-labeled ligand and biotin-immobilized microtiter plate.

A non-isotopic receptor assay for benzodiazepine drugs was developed using a biotin-labeled ligand, biotin-1012S. Biotinylated bovine serum albumin (biotin-BSA) was immobilized onto the wall of microtiter plate wells by simple adsorption. Avidin peroxidase conjugate could be extracted from solution owing to its strong interaction with biotin. The amount of avidin peroxidase taken up on the wall was then determined by measuring the enzyme activity. The competition between immobilized biotin on the wall and free biotin for avidin provided the basis for a solid-phase avidin-biotin binding assay. By this binding assay, not only biotin but also biotin-1012S could be measured sensitively. Because 1012S is a ligand with high affinity to benzodiazepine receptors, biotin-1012S could be utilized as a probe ligand for a non-isotopic receptor assay. Based upon the competition between biotin-1012S and various benzodiazepine drugs for the receptor binding sites, a non-isotopic receptor assay was demonstrated.     

Determination of Toxins Using Enzyme-Amplified Receptor Assay

Abstract

A new receptor based assay is described for the determination of toxins which have high affinities for the acetylcholine receptor. The method is based upon the hindrance of the normal binding of a synthetic enzyme-drug conjugate with a high affinity for the acetylcholine receptor protein by the presence of toxins acting as antagonists. The activity of the enzyme marker system, glucose-6-phosphate dehydrogenase covalently conjugated to desipramine, is monitored by colorimetric detection of the rate for NADH formation at 340 nm. The procedure proposed is designed to provide a simple toxin screen which can be done in a minimally equipped laboratory while achieving the required sensitivity. The technique is illustrated for snake venoms from Bungarus multicintus, Naja naja, and the alkaloid tubocurarine. Aspecific binding responses are shown to have minimal effect on the assay.     

A Simple,But Reliable Assay of Sex Hormone Binding Globulin

Abstract

The likelihood of erroneous results is very high when a single dose of ligand (e.g. dihydrotestosterone) is employed in the assay of sex hormone binding globulin (SHBG) using precipitation with ammonium sulfate. However, correct results will be obtained if several ligand doses are used and the calculations are based on a Scatchard plot from which non-specific binding has been eliminated. The reliability of such a multiple-dose SHBG assay was tested. As established by an analysis of variance, the results of the measurements were independent of the volume assayed. Furthermore, by assaying 25 plasma sample in duplicate, an average within-assay coefficient of variation of 5.5% was obtained. The assay of the same samples on different occasions gave an estimate of between-assay variation ranging from 3.5% to 8.9% for various types of plasma. Moreover, the results of the assay of 170 plasma samples were well correlated (r = 0.8) with those obtained by a steady state electrophoresis. Thus the multiple-dose precipitation assay gives reliable results and is suitable for routine measurements of SHBG.     

Preparation of Monomeric Affinity-Purified Fab'-ß-D-Galactosidase Conjugate for Immunoenzymometric Assay

Abstract

A Simpler method for the preparation of monomeric affinity-purified Fab'-ß-D-galactosidase conjugate is described. Rabbit (anti-human IgG) serum was subjected to successive processes of pepsin digestion to convert IgG to F(ab')_2′ reduction with 2-mercaptoethy on a column of human IgG-Sepharose 4B. The affinity-purified Fab' thus obtained without using gel filtration was reacted with excess of maleimide groups introduced into ß-D-galactosidase from Escherichia coli. The monomeric Fab'-ß-D-galactosidase conjugate formed was separated from unconjugated Fab' by gel filtration and from unconjugated ß-D-galactosidase by affinity chromatography on a column of goat (anti-rabbit IgG) IgG-Sepharose 4B. By immunoenzymometric assay technique for human IgG, the monomeric conjugate was compared with a monomeric conjegate prepared by a previously reported complexmethod and non-monomeric conjugate which contained 3.7 Fab' molecules per ß-D-galactosidase molecule. The present monomeric conjugate provided as sensitive a dose-response curve as the previously reported monomeric conjugate and a more sensitive dose-response curve than the non-monomeric conjugate.     

Fully Automated Synthesis of Novel TSPO PET Imaging Ligand [18F]Fluoroethyltemazepam

Introduction: Benzodiazepines, including temazepam are described as TSPO antagonists. In fact, TSPO was initially described as a peripheral benzodiazepine receptor (PBR) with a secondary binding site for diazepam. TSPO is a potential imaging target of neuroinflammation because there is an amplification of the expression of this receptor. Objectives: Herein, we developed a novel fluorinated benzodiazepine ligand, [¹⁸F]Fluoroethyltemazepam ([¹⁸F]F-FETEM), for positron emission tomography (PET) imaging of translocator protein (18 kDa). Methods: [¹⁸F]F-FETEM was radiolabelled with an automated synthesizer via a one-pot procedure. We conducted a [¹⁸F]F-aliphatic nucleophilic substitution of a tosylated precursor followed by purification on C18 and Alumina N SPE cartridges. Quality control tests was also carried out. Results: We obtained 2.0–3.0% decay-uncorrected radiochemical activity yield (3.7% decay-corrected) within the whole synthesis time about 33 min. The radiochemical purity of [¹⁸F]F-FETEM was over 90% by TLC analysis. Conclusions: This automated procedure may be used as basis for future production of [¹⁸F]F-FETEM for preclinical PET imaging studies.     

Competitive Protein Binding Assay of Camp1 Using Thyroid Cytosol

Abstract

We describe here a competitive protein-binding assay (CPBA) of cAMP which employs a crude thyroid cytosol preparation as the ligand-binding reagent. The affinity constant (Ka) of the binding of cAMP to the thyroid receptor varied between 1 and 4 × 10⁹ M^?1 in the assay. ATP, ADP and AMP did not interfere in the assay. Cross -reaction of cGMP, 1.8%, and that of cIMP, 36%, were comparable to those observed with previously described CPBAs of cAMP. The range of the assay standard curve was 0.5—20.0 pmoles per tube. Coefficient of variation was 8.3% within an assay and 8.7% between assays. The assay was applied to measurement of cAMP in biologic fluids and tissues. The results were comparable to those obtained with previous methods.     

Synthesis of 3-aminopropyl β-glycoside of sialyl-3′-lactose and derived neoglycoconjugates as a tumor vaccine prototype and artificial antigens for the control of immune response

Starting from the biotechnologically available trisaccharide sialyl-3′-lactose, representing the carbohydrate portion of the tumor-associated ganglioside GM3, the corresponding 3-aminopropyl β-glycoside (1) and 3-(4-maleimidobutanoylamino)propyl glycoside were synthesized. The reaction of the latter with a thiolated derivative of the Megathura crenulata hemocyanine (KLH) afforded a carbohydrate—protein conjugate, a tumor vaccine prototype containing about 330 trisaccharide ligands attached to KLH. N-Stearoylation of ligand 1 gave the model neoglycolipid for comparative study of the activity of mono-and polyvalent immunogens and the natural ganglioside GM3. A monovalent conjugate, in which ligand 1 is linked to biotin through an oligo(ethylene glycol) spacer and a polyvalent conjugate with a polyacrylamide carrier were also prepared. These conjugates are meant as covering antigens to assess the specificity and efficiency of the immune response in the ELISA assay. Dedicated to Academician O. M. Nefedov on the occasion of his 75th birthday. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 11, pp. 2016–2023, November, 2006.     

Detection of Aflatoxins in Capsicum Spice Using an Electrochemical Immunosensor

Abstract

Electrochemical immunosensor based on immobilized aflatoxin B1 (AFB)–albumin conjugate and polyclonal antibody against AFB1 was performed for a competitive assay of AFB1 in capsicum spice. Spiked samples were used for construction of calibration curve. The proved limit of detection was 2.4 ppb. Immunosensor long-term stability was also estimated. Decrease of immunosensor sensitivity was less than 10% when stored in a refrigerator and approximately 22% for the immunosensor preserved at laboratory temperature for two weeks. Consequent performance of immunosensors for assay of real capsicum spice samples with proven aflatoxins presence produced good correlation with the data from valid method (high-performance liquid chromatography with fluorescence detector).     

Pre-Incubation and Low Temperatures in Quantitative Radioreceptor Assays

Abstract

The detection limits of drugs in quantitative RRA are primarily determined by their affinities towards the receptor. Yet, the concentration of radiolabeled ligand, necessary for quantification of receptor-bound drug, increases the theoretical detection limit. Therefore the influences of low temperatures and pre-incubation on the detection limit was studied.     

A Simplified Analytical Procedure for the Determination of Diazepam in Intravenous Admixtures

Abstract

A Simplified gas-Chromatographic technique was developed for the determinatilon of diazepam in intravenous admixtures. Aliquots of a methanolic solution of diazepam were further diluted in various aqueous vehicles: double-distilled water, 5% dextrose in water, 0.9% sodium chloride injection, Lactated Ringer's injection, and Ringer's injection. The assay procedure consisted of a simple extraction technique from the aqueous system into an nbutyl acetate phase using prazepam as the internal standard, peak-height ratios of diazepam:prazepam from the chromatograms were then plotted against the chromatograms were then plotted agaisnt theoretical aqueous diazepam concentra tions. The y-intercept and correlation coefficient of the data were also determined.

Results of the study show that the diazepam:prazepam peakheight ratios exhibited consistent correlation coefficients in the five fluids studied. The retention time for diazepam was about 7.5 minutes, while prazepam had a retention time of 12 minutes.     

Enzyme-Amplified Receptor Assay Predictive Model and Analytical Limits

Abstract

A theoretical basis for enzyme amplified receptor assay (ERA) is presented and criteria are established for the general case of any receptor system. Analytical criteria necessary to perform this assay are identified based upon the relevant affinity constants, the concentrations of the enzyme-labelled ligand and receptor, and the analyte.     

A Screening Assay for the Tetrachlorodibenzo-p-dioxin Receptor using the [125I]iodovaleramide Derivative of Trichlorodibenzo-p-dioxin as the Binding Ligand

Abstract

A relatively simple assay method for the putative cytosolic ‘receptor’ that binds 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds is described. The assay is based on specific binding of [¹²⁵I]dioxin to cytosol ‘receptor’ protein. Saturation is ensured by competition experiments in which unlabeled TCDD and other competitors displace the radiolabeled ligand from specific binding sites. This assay has been applied to estimation of levels of ‘receptor’ in cytosol.     

Characterization of Anti-Chloramphenicol Antibodies by Enzyme-Linked Immunosorbent Assay

Polyclonal antibodies against conjugates of chloramphenicol succinate and chloramphenicol base with proteins were obtained and characterized in direct ELISA. Antiserum against a conjugate of chloramphenicol (CAP) base with BSA (direct coupling) was very specific and showed cross-reactivity only with CAP succinate (11.3%) and CAP base (4.6%); whereas, antisera against a conjugate of CAP succinate with a protein recognized CAP succinate strongly as an initial compound. In direct ELISA, antisera against a conjugate of CAP succinate with KLH (homologous assay) and CAP base with BSA (heterologous assay) showed similar sensitivity: IC₅₀ were 1.3 and 1.5 ng mL^?1, respectively. Applicability of the immunoreagents obtained was shown in the analysis of CAP residues in milk (3.5% fat content). Detection limit of 0.3 ng mL^?1 was obtained for milk diluted 5 times.     

A Piezoelectric Quartz Crystal Biosensor as a Direct Affinity Sensor

Abstract

A microprocessor controlled piezoelectric detector as sensor was employed to monitor in real time protein adsorption and immunoreactions using piezoelectric quartz crystals (AT-cut) with a basic resonant frequency of 10 MHz. The adsorbed protein was an immunoglobulin (h-lg G); in the immunosensing a covalent immobilized molecule (the pesticide 2,4-D) formed the receptor for the immobilized ligand sample (Mab anti-2,4-D) in a competitive assay.     

Radioreceptor Assay of Opioid Peptides in Selected Canine Brain Regions

Abstract

A radioreceptor assay using the opioid delta receptor-preferring ligand D- ²ala, D- ⁵leu leucine enkephalin (³H-DADL) and the broader-specificity ligand ³H-etorphine was used to measure five HPLC-purified neuropeptide fractions derived from the peptide-rich fraction of tissue homogenates of nine anatomical regions of the canine brain. The receptoractive peptides studied were methionine enkephalin, alpha-neo-endorphin, dynorphin 1-8, methionine enkephalin-Arg-Phe, and leucine enkephalin. These peptides derive from two larger precursors: proenkephalin A, which contains methionine enkephalin, leucine enkephalin, methionine enkephalin-Arg-Phe; and proenkephalin B, which contains alpha-neo-endorphin and dynorphin 1-8. Receptoractive peptides were measured in the peptide-rich fraction derived from homogenates of canine hypothalamus, pituitary, caudate nucleus, amygdala, hippocampus, mid-brain, thalamus, pons-medulla, and cortex.     

Pretreatment-free immunochromatographic assay for the detection of streptomycin and its application to the control of milk and dairy products

A rapid pretreatment-free immunochromatographic assay was developed for the control of the streptomycin (STR) content in milk and dairy products. The assay is based on the competition between an immobilized STR–protein conjugate and STR in a sample to be tested for the binding to monoclonal anti-STR antibodies conjugated to colloidal gold during the flow of the sample along a membrane strip with immobilized reactants. It is possible to improve the cut-off level of positive and negative samples distinguished by a change in the molar STR to protein ratio in the immobilized conjugate. The cut-off level (500 ng mL⁻¹) thus achieved corresponds to the stated MRL of STR in milk and dairy products. For STR concentrations in the range of 16–250 ng mL⁻¹ its content can be quantitatively measured based on the degree of binding of a colloidal gold label in the test strip zone with the immobilized STR–protein conjugate. The duration of the assay is 10 min. The selected sizes of membrane pores and colloidal gold particles allow the assay to be carried out at room temperature without additional reactants and pretreatment. The applicability of the assay for milk, whole milk, sour clotted milk, and kefir with different fat content (from 0.5% to 6%) was confirmed. The results of quantitative immunochromatographic assay show good correlation with traditional ELISA (r was equal to 0.935 and 0.940 for the series tested).     

Amperometric Enzyme-Amplified Receptor Assay For Tricyclic Antidepressants

Abstract

Enzyme-amplified receptor assay performed by competitive binding of amitriptyline drug and enzyme labeled drug for acetylcholine receptor binding sites is described. the change in enzyme activity is followed by the rate of NADH oxidation at a platinum electrode with polarizing potential of 700 mV. the response obtained using amperometric technique compares well with results from spectrophotometric studies carried out for comparison purposes.     

A novel microplate reader-based high-throughput assay for estrogen receptor binding

Coumestrol is a well-known ligand for the estrogen receptor (ER). The compound itself is fluorescent, and its fluorescence intensity at 408?nm increases upon binding to the ER. Here we describe a novel binding assay in 96-well plate format for estrogenic compounds, based on the competition between fluorescent coumestrol and estrogenic compounds for binding to the ligand binding domain (LBD) of the ER-alpha. Displacement of coumestrol was measured as a decrease in fluorescence intensity using a Victor² 1420 multilabel reader. Competitive binding curves for the well-known estrogenic compounds, 17β-estradiol (E₂), ethinylestradiol, 4-nonylphenol, 4-octylphenol, genistein, bisphenol A, tamoxifen and diethylstilbestrol were constructed by using 7–10 different concentrations of the compounds and a fixed concentration of ER-α-LBD (14?nmol) and coumestrol (100?nmol). IC₅₀ values and relative potencies (compared to E₂) of the estrogenic compounds were determined. The assay was validated by comparing the relative potencies to those from standard radioligand binding assays in the literature. Within day and between day variations were determined and the performance of the assay was assessed by determining the coefficients of variation and Z′ values. The present fluorescent binding assay has proven to be fast and easy, and allows accurately quantifying the binding affinity of estrogenic ligands. The method is also suitable as a high-throughput screening assay for ER ligands.     

A Simultaneous Enzyme-Linked Immunoassay for Human Thyroglobulin

Abstract

A “simultaneous” enzyme-linked immunoassay for the measurement of thyroglobulin in human serum was established using polyclonal rabbit antibody, in which rabbit anti-human thyroglobulin IgG-coated silicone rubber rod, rabbit anti-human thyroglobulin monovalent fragment (Fab')-β-D-galactosidase conjugate and serum sample were mixed at the same time. In order to improve the assay performance, an appropriate amount of Fab'-β-D-galactosidase conjugate was carefully chosen.

The present assay was simple, rapid, highly sensitive and excellently reproducible. The sensitivity of the assay was approximately 1.5 amol/tube corresponding to 0.5 ng/ml using 2 μ1 of serum, which was higher than that in the two-step sandwich enzyme-linked immunoassay previously reported. The coefficients of variation for different levels of serum thyroglobulin determined by the present assay ranged from 3.4–7.2% and 2.5–6.1 % for within and between run, respectively.