Methodological considerations for the human platelet 5-HT2A receptor binding kinetic assay

Analysis of an extensive database of human platelet 5-HT2A receptor binding assays has been conducted in order to identify factors that may affect the assay results. Despite anecdotal reports that storage of frozen platelet pellets may affect 5-HT2A binding affinity and capacity, no quantitative study has been reported in the literature. Analysis of binding data for 373 frozen samples with a storage time up to three years is presented in this paper. It is shown that prolonged storage significantly decreases binding. The loss of binding capacity begins in the first six month of storage. Bmax declines by half after 17 month. The impact of storage time on the binding affinity is much smaller. There is only about 20% increase in the value of affinity K(D) during the half-life of Bmax. Differences in sample storage time may partly explain discrepancies in results between different research groups. Nonspecific binding due to binding to filter material diminishes accuracy and reliability of the binding assays as a result of a decrease in the ratio of specific to nonspecific ratio. A data analysis based on our suggested mathematical model shows that this effect depends on tissue concentration in test tube and becomes pronounced when the concentration is below 0.1 mg protein/ml (at 0.2 nM of ligand). Above 0.1 mg protein/ml, percentage of specific to total binding exceeds 65%, which is an acceptable level for the ratio. The majority of the binding studies reported in the literature employed a tissue concentration more than 0.5 mg/ml, well above the minimal limit sufficient for a reliable assay. However, development of microassays to conserve precious tissue must take the limit into consideration.     

A ligand-receptor binding assay by receptor immobilization

Using transferrin-transferrin receptor binding as a model of ligand-receptor binding, we have developed a new and simple binding assay for the solubilized receptor. Solubilized membrane proteins containing transferrin receptor were immobilized by covalent binding to beads having chemical reactive epoxide groups, and then 125I-labeled transferrin was added to the beads. Dose-dependent, ligand-specific, and saturable binding of 125I-labeled transferrin to the immobilized membrane proteins were demonstrated and a Scatchard analysis derived affinity of Kd = 1.8 X 10(-9) M was obtained. These results indicate that the immobilization of receptors onto beads may be useful in a simple binding assay of the solubilized receptor.     

A high-throughput fluorescent polarization assay for nuclear receptor binding utilizing crude receptor extract.

A homogenous high-throughput assay has been developed to measure the binding between nuclear receptors and test compounds. This assay applies a fluorescence polarization (FP) detection method using human glucocorticoid receptor (GR) as a model system. Crude receptor extract, which requires no additional purification, is used in the assay. The binding conditions (i.e., DMSO tolerance, temperature, stability, and variability) have been investigated and validated. At the optimized conditions, a signal-to-background ratio of 2:1 and a Z'-factor of 0.7 was achieved in a 384-well format. Several known strong and weak GR ligands have been evaluated in this system. Possible interference of fluorescent compounds and methods to identify false positives are also discussed. This FP-based assay system can potentially be used for many soluble nuclear receptors in high-throughput binding assays.     

A dual radioimmunoassay and cytosol receptor binding assay for the measurement of estrogenic compounds applied to urine,fecal and plasma samples

A technique for co-chromatography using tritiated steroids, radioimmunoassay (RIA), and a cytosol receptor binding assay was developed to examine estrogen components following elution from a reverse phase high pressure liquid chromatography (HPLC) system. Urine, fecal or plasma samples from several species of animals were assayed by this method and the results demonstrated compounds previously unreported. Two of these, one labelled E_x in primates, and one labelled E_w in birds are shown to have both cytosol receptor binding activity as well as immunoreactivity.     

A high-performance liquid chromatography assay with ultraviolet detection for olanzapine in human plasma and urine

Olanzapine is a commonly used atypical antipsychotic medication for which therapeutic drug monitoring has been proposed as clinically useful. A sensitive method was developed for the determination of olanzapine concentrations in plasma and urine by high-performance liquid chromatography with low-wavelength ultraviolet absorption detection (214 nm). A single-step liquid–liquid extraction procedure using heptane-iso-amyl alcohol (97.5:2.5 v/v) was employed to recover olanzapine and the internal standard (a 2-ethylated olanzapine derivative) from the biological matrices which were adjusted to pH 10 with 1 M carbonate buffer. Detector response was linear from 1–5000 ng (r²>0.98). The limit of detection of the assay (signal:noise=3:1) and the lower limit of quantitation were 0.75 ng and 1 ng/ml of olanzapine, respectively. Interday variation for olanzapine 50 ng/ml in plasma and urine was 5.2% and 7.1% (n=5), respectively, and 9.5 and 12.3% at 1 ng/ml (n=5). Intraday variation for olanzapine 50 ng/ml in plasma and urine was 8.1% and 9.6% (n=15), respectively, and 14.2 and 17.1% at 1 ng/ml (n=15). The recoveries of olanzapine (50 ng/ml) and the internal standard were 83±6 and 92±6% in plasma, respectively, and 79±7 and 89±7% in urine, respectively. Accuracy was 96% and 93% at 50 and 1 ng/ml, respectively. The applicability of the assay was demonstrated by determining plasma concentrations of olanzapine in a healthy male volunteer for 48 h following a single oral dose of 5 mg olanzapine. This method is suitable for studying olanzapine disposition in single or multiple-dose pharmacokinetic studies.     

A binding assay for serine hydroxymethyltransferase

A sensitive assay for measuring serine hydroxymethyltransferase activity has been developed, based on the binding of N5,N10-[14C]methylene tetrahydrofolate (THF) to DEAE-cellulose paper. The complete assay requires THF, pyridoxal 5'-phosphate, [14C]serine, and enzyme. The reaction is stopped by streaking an aliquot of the reaction mixture onto a square of DEAE-cellulose paper, washing the paper with water to remove unreacted serine, drying the paper, and counting the bound N5,N10-[14C]methylene-THF. To determine that the labeled product was N5,N10-methylene-THF, unlabeled formaldehyde, which exchanges with the labeled methylene carbon, was added after the product had accumulated; 2 min after the addition of formaldehyde the amount of labeled product was reduced by 50%, and by 85% after 10 min. In addition, glycine, which reverses the reaction, and hydroxylamine, which reacts with the methylene carbon, reduced the number of counts bound to the paper. Binding of product to the filter is proportional to both enzyme concentration and assay time. No counts were retained on phosphocellulose filters. This assay represents a new and simple method for measuring serine hydroxymethyltransferase activity, which can be used to measure enzyme activity in tissue homogenates and for screening large numbers of samples.     

A protein binding assay for hyaluronate

A relatively quick and simple assay for hyaluronate was developed using the specific binding protein, hyaluronectin. The hyaluronection was obtained by homogenizing the brains of Sprague-Dawley rats, and then centrifuging the homogenate. The resulting supernatant was used as a source of crude hyaluronectin. In the binding assay, the hyaluronectin was mixed with [3H]hyaluronate, followed by an equal volume of saturated (NH4)2SO4, which precipitated the hyaluronectin and any [3H]hyaluronate associated with it, but left free [3H]hyaluronate in solution. The mixture was then centrifuged, and the amount of bound [3H]hyaluronate in the precipitate was determined. Using this assay, we found that hyaluronectin specifically bound hyaluronate, since other glycosaminoglycans failed to compete for the binding protein. In addition, the interaction between hyaluronectin and hyaluronate was of relatively high affinity (Kd = 5.7 X 10(-10) M), and the size of the hyaluronate did not appear to substantially alter the amount of binding. To determine the amount of hyaluronate in an unknown sample, we used a competition assay in which the binding of a set amount of [3H]hyaluronate was blocked by the addition of unlabeled hyaluronate. By comparing the degree of competition of the unknown samples with that of known amounts of hyaluronate, it was possible to determine the amount of hyaluronate in the unknowns. We have found that this method is sensitive to 1 microgram or less of hyaluronate, and is unaffected by the presence of proteins.     

Determination of the LDL receptor binding capacity of human lymphocytes by immunocytofluorimetric assay

The determination of the LDL receptor binding capacity of human blood lymphocytes was assessed by indirect immunocytofluorimetric assay. To produce the maximal synthesis of the LDL receptor, the cholesterol efflux was enhanced by incubation of lymphocytes with HDL3 subfractions. The binding capacity of the LDL receptor was measured by incubation at 4 degrees C either with LDL and rabbit anti-LDL immunoglobulins or with peptide receptor antibody (ARP-Ig) raised against the NH2-terminal sequence of the LDL receptor. Thereafter complexes were incubated with fluorescein-labelled anti-rabbit immunoglobulin (FITC-Ig). Fluorescence flow cytometry was used to quantify the number of fluorescent lymphocytes and results were expressed as the percentage of lymphocytes with a fluorescent intensity above the threshold. Using preimmune rabbit immunoglobulin and then FITC-Ig, only 5-10% of cells were fluorescent. Neither LDL nor ARP-Ig could bind to homozygous familial hypercholesterolemia (FH) lymphocytes. Normal lymphocytes preincubated with HDL3 could bind LDL or ARP-Ig, the number of fluorescent cells being 59 and 39.2% respectively. Subjects with confirmed or suspected heterozygous FH demonstrated cell fluorescence at about half the normal level.     

Development of a fluorescence polarization binding assay for asialoglycoprotein receptor

Asialoglycoprotein receptor (ASGP-R) has been actively investigated for targeted delivery of therapeutic agents into hepatocytes because this receptor is selectively and highly expressed in liver and has a high internalization rate. Synthetic cluster glycopeptides (e.g., triGalNAc) bind with high affinity to ASGP-R and, when conjugated to a therapeutic agent, can drive receptor-mediated uptake in liver. We developed a novel fluorescent polarization (FP) ASGP-R binding assay to determine the binding affinities of ASGP-R-targeted molecules. The assay was performed in 96-well microplates using membrane preparations from rat liver as a source of ASGP-R and Cy5 fluorophore-labeled triGalNAc synthetic ligand as a tracer. This high-throughput homogeneous assay demonstrates advantages over existing multistep methods in that it minimizes both time and resources spent in determining binding affinities to ASGP-R. At the optimized conditions, a Z' factor of 0.73 was achieved in a 96-well format.     

A microplate binding assay for the somatostatin type-2 receptor (SSTR2)

The clinical importance of somatostatin type-2 receptors (SSTR2) and the study of novel analogues of somatostatin such as OctreoScan or [Tyr3]-octreotide containing DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) as metal chelator led us to develop a methodology to monitor the expression of SSTR2 on tumours of pancreatic origin (e.g. rat AR4-2J cancer cells). Usual binding assay protocols using the commercial [125I][Tyr1]-somatostatin radioligand failed, even in the presence of a cocktail of protease inhibitors with a broad spectrum of activity, possibly due to the high susceptibility of this tracer to proteases expressed in pancreatic cells. We prepared our own radioligand [125I][Tyr2]-octreotide which was shown to be much more resistant to degradation after incubation with AR4-2J plasma membranes. As expected, the increased stability of [125I][Tyr3]-octreotide was associated with good binding to SSTR2. Addition of appropriate protease inhibitors further increased the specific binding of [125I][Tyr3]-octreotide to AR4-2J plasma membranes without affecting the stability of the tracer, suggesting that the protease inhibitors also protect the integrity of SSTR2. Optimal conditions (time, temperature, medium) were developed for a binding assay in 96-well plates using AR4-2J plasma membranes in order to make the assay suitable for high-throughput analysis. This protocol was the basis for studying the in vivo regulation of SSTR2 expression in AR4-2J cells implanted into scid mice after exposure to different compounds.     

A receptor binding assay for paralytic shellfish poisoning toxins: recent advances and applications

We recently described a high throughput receptor binding assay for paralytic shellfish poisoning (PSP) toxins, the use of the assay for detecting toxic activity in shellfish and algal extracts, and the validation of 11-[3H]-tetrodotoxin as an alternative radioligand to the [3H]-saxitoxin conventionally employed in the assay. Here, we report a dramatic increase in assay efficiency through application of microplate scintillation technology, resulting in an assay turn around time of 4 h. Efforts are now focused on demonstrating the range of applications for which this receptor assay can provide data comparable to the more time consuming, technically demanding HPLC analysis of PSP toxins, currently the method of choice for researchers. To date, we have compared the results of both methods for a variety of sample types, including different genera of PSP toxin producing dinoflagellates (e.g. Alexandrium lusitanicum, r2 = 0.9834, n = 12), size-fractioned field samples of Alexandrium spp. (20-64 microm; r2 = 0.9997, n = 10) as well as its associated zooplankton grazer community (200-500 microm: r2 = 0.6169, n = 10; >500 microm: r2 = 0.5063, n = 10), and contaminated human fluids (r2 = 0.9661, n = 7) from a PSP outbreak. Receptor-based STX equivalent values for all but the zooplankton samples were highly correlated and exhibited close quantitative agreement with those produced by HPLC. While the PSP receptor binding assay does not provide information on toxin composition obtainable by HPLC, it does represent a robust and reliable means of rapidly assessing PSP-like toxicity in laboratory and field samples. Moreover, this assay should be effective as a screening tool for use by public health officials in responding to suspected cases of PSP intoxication.     

A microtiter well assay for quantitative measurement of estrogen receptor binding to estrogen-responsive elements

Reproducible, rapid measurement of estrogen receptor (ER) binding to DNA was accomplished in microtiter wells treated so that ER-DNA complexes or DNA bound in preference to free ER. Mixtures of 35S-labeled DNA and [3H]estrogen-charged ER ([3H]ER), incubated to equilibrium in microfuge tubes, were transferred to microtiter wells previously treated with histone followed by gelatin. After binding of the DNA or ER-DNA complex to the treated wells, free ER was removed by washing. Radioactivity retained in each well was measured by placing individual wells from snap-apart microtiter plates directly in scintillation fluid. Binding of DNA was saturable, and ER-DNA complex binding was complete within 2 h at 4 C. The use of 35S-labeled DNA and [3H]ER allowed stoichiometric determination of ER bound to DNA. The amount of ER specifically bound to a consensus estrogen-responsive element (ERE) containing the inverted repeat GGTCAgagTGACC was determined by comparing ER bound to plasmid containing or lacking the ERE. At saturating concentrations of ER, plasmids bearing one, two, and four EREs in tandem bound approximately one, two, and four dimeric ER molecules, respectively. Scatchard analysis of saturation binding data revealed a Kd of 0.15 nM for specific ER binding to a single ERE site. Thus, the assay detects ER retaining both DNA-binding and estrogen-binding functions. ER complexed with DNA in the well was also detected using a monoclonal antibody specific for the receptor. Simple modifications of this method would allow study of other DNA-protein interactions.     

A homogeneous 384-well high-throughput binding assay for a TNF receptor using alphascreen technology

To take advantage of the growing knowledge of cellular signaling pathways, modern-day drug discovery faces an increasing challenge to develop assays to screen for compounds that modulate protein-protein interactions. One bottleneck in achieving this goal is a lack of suitable and robust assay technologies amenable to a high-throughput format. In this report, we describe how we utilized Alphascreen trade mark technology to develop a high-throughput assay to monitor ligand binding to a member of the tumor necrosis factor receptor superfamily. We expressed a fusion protein consisting of the extracellular domain of the OX40 receptor with the constant domains of human IgG. In the presence of OX40 ligand, we determined a binding affinity constant consistent with reported values and optimized the protocol to develop a simple, homogeneous, and sensitive binding assay in a 384-well format. Finally, we assessed if this system could identify small peptides capable of inhibiting the OX40 receptor and ligand interaction. The results showed that the assay was able to detect such peptides and could be used to launch a high-throughput screening campaign for small molecules able to prevent OX40 receptor activation.