Development of a two-site immuno-PCR assay for hepatitis B surface antigen.

Hepatitis B virus (HBV) gene transcription may occur at very low levels resulting in HBsAg concentrations in serum and liver below the limit of detection by currently available immunoassays. An assay has been developed that combines the specificity of two high affinity anti-HBs monoclonal antibodies (MAb) directed against distinct and separate determinants in the 'a' domain of HBsAg with the highly sensitive polymerase chain reaction (PCR) detection method. Following capture of HBsAg present in serum samples, the second anti-HBs MAb, which is biotinylated, is added. Binding of the second antibody allows the subsequent specific binding of streptavidin and a biotinylated linear DNA molecule derived from a bluetongue virus (BTV) gene. Presence of this DNA is then detected by PCR using BTV-specific primers. The PCR product is quantified by a liquid-phase oligonucleotide enzymatic assay, which further increases the sensitivity of the technique. The use of a two-site MAb capture and PCR detection system for HBsAg was shown to greatly enhance the specificity and sensitivity of the assay and detect as little as 0.5 pg of HBsAg in serum samples. It is suggested that the principles of this technique could be applied to measure other low level viral antigens in serum and biological samples.

Endothelin-1 receptor binding assay for high throughput chemical screening.

An endothelin-1 (ET-1) receptor-binding assay, in microtiter format, has been developed for use in high throughput chemical or natural product screening. A rat smooth muscle, clonal cell line derived from embryonic thoracic aorta and designated A10 has been shown to consistently express high-affinity ET-1 receptors. A 96-well microtiter filtration plate, with individual PVDF membranes attached to the bottom of each well, was used for separation. In saturation binding assays, using [125I]Tyr13-ET-1, Scatchard analysis was monophasic, indicating a single high-affinity population of receptors with Kd = 0.12 nM with approximately 40,000 receptors per cell. The Ki values for peptides, known to bind at the ET receptor, were as follows: ET-1, 0.14 nM; ET-2, 0.16 nM; sarafotoxin S6b, 0.6 nM; VIC, 0.2 nM; ET-3, 16 nM; and big human ET, greater than 1 microM. ET receptors on A10 cells were stable for at least 2 months when stored at -20 degrees C. The assay is suitable for automation, because it is stable and reproducible. This method gave a 90% reduction in radioactive waste compared to tissue homogenate assays that use glass fiber filtration and cell harvesters.

Complement C5a receptor assay for high throughput screening.

The complement C5a receptor on U937 cells, a human histiocytic lymphoma cell line, stimulated with dibutyryl-cAMP have been stabilized for at least 3 months at a dilute, ready to use concentration. [125I]-Bolton Hunter labeled C5a, (recombinant, human) has been prepared by reverse phase HPLC to 2200 Ci/mmol. Using a filtration binding assay the Kd from receptor saturation analysis is 10-40 pM and there are 50,000-100,000 receptor sites per cell. These reagents have permitted the development of a reliable, reproducible and convenient drug screening assay, in kit format, for compounds acting at the C5a receptor.

Autoradiographic characterization of (+-)-1-(2,5-dimethoxy-4-[125I] iodophenyl)-2-aminopropane ([125I]DOI) binding to 5-HT2 and 5-HT1c receptors in rat brain.

The 5-HT2 (serotonin) receptor has traditionally been labeled with antagonist radioligands such as [3H]ketanserin and [3H]spiperone, which label both agonist high-affinity (guanyl nucleotide-sensitive) and agonist low-affinity (guanyl nucleotide-insensitive) states of this receptor. The hallucinogen 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) is an agonist which labels the high-affinity guanyl nucleotide-sensitive state of brain 5-HT2 receptors selectively. In the present study, conditions for autoradiographic visualization of (+/-)-[125I]DOI-labeled 5-HT2 receptors were optimized and binding to slide-mounted sections was characterized with respect to pharmacology, guanyl nucleotide sensitivity and anatomical distribution. In slide-mounted rat brain sections (+/-)-[125I]DOI binding was saturable, of high affinity (KD approximately 4 nM) and displayed a pharmacologic profile typical of 5-HT2 receptors. Consistent with coupling of 5-HT2 receptors in the high-affinity state to a guanyl nucleotide regulatory protein, [125I]DOI binding was inhibited by guanyl nucleotides but not by adenosine triphosphate. Patterns of autoradiographic distribution of [125I]DOI binding to 5-HT2 receptors were similar to those seen with [3H]ketanserin- and [125I]-lysergic acid diethylamide-labeled 5-HT2 receptors. However, the density of 5-HT2 receptors labeled by the agonist [125I]DOI was markedly lower (30-50%) than that labeled by the antagonist [3H]ketanserin. High densities of [125I]DOI labeling were present in olfactory bulb, anterior regions of cerebral cortex (layer IV), claustrum, caudate putamen, globus pallidus, ventral pallidum, islands of Calleja, mammillary nuclei and inferior olive. Binding in hippocampus, thalamus and hypothalamus was generally sparse. Of note, choroid plexus, a site rich in 5-HT1c receptors had a high density of [125I]DOI binding sites but [3H]ketanserin binding in this region was low. Studies in which [125I]DOI binding to 5-HT2 receptors was blocked with spiperone revealed persisting robust [125I]DOI binding in choroid plexus, which was guanyl nucleotide-sensitive and displayed a pharmacologic profile consistent with its binding to 5-HT1c receptors. These studies suggest that [125I]DOI may be useful as a radiolabel for visualizing the agonist high-affinity state of 5-HT2 receptors and for visualizing 5-HT1c receptors.

Dissociative binding of alpha- and beta-sulphoxides of biotinylamidoethyl-3-(4-hydroxy-3-[125I]iodophenyl)propionamide to avidin.

Previously we reported the dissociative binding of biotinylamidoethyl-3-(4-hydroxy-3-[125I]iodophenyl)propionamide to avidin [Garlick & Giese (1988) J. Biol. Chem. 263, 210-215]. In the present paper we report the corresponding binding of the alpha- and beta-sulphoxides of this parent compound to avidin. The 1:1 complex (obtained with avidin in excess) of the alpha-sulphoxide derivative with avidin has a dissociation half-life (t1/2) of 25 days, only 1.6 times as fast as the parent compound (t1/2 41 days). However, the corresponding beta-sulphoxide dissociates 446 times faster (t1/2 0.092 day) than the parent compound, this apparently being due to a steric effect. The alpha-sulphoxide is attractive as a tracer reagent to facilitate studies and applications of the avidin-biotin system.

Photoaffinity labeling of dopamine D1 receptors.

A high-affinity radioiodinated D1 receptor photoaffinity probe, (+/-)-7-[125I]iodo-8-hydroxy-3-methyl-1-(4-azidophenyl)-2,3,4,5-tetra hyd ro- 1H-3-benzazepine ([125I]IMAB), has been synthesized and characterized. In the absence of light, [125I]IMAB bound in a saturable and reversible manner to sites in canine brain striatal membranes with high affinity (KD approximately equal to 220 pM). The binding of [125I]IMAB was stereoselectively and competitively inhibited by dopaminergic agonists and antagonists with an appropriate pharmacological specificity for D1 receptors. The ligand binding subunit of the dopamine D1 receptor was visualized by autoradiography following photoaffinity labeling with [125I]IMAB and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Upon photolysis, [125I]IMAB incorporated into a protein of apparent agents in a stereoselective manner with a potency order typical of dopamine D1 receptors. In addition, smaller subunits of apparent Mr 62,000 and 51,000 were also specifically labeled by [125I]IMAB in these species. Photoaffinity labeling in the absence or presence of multiple protease inhibitors did not alter the migration pattern of [125I]IMAB-labeled subunits upon denaturing electrophoresis in both the absence or presence of urea or thiol reducing/oxidizing reagents. [125I]IMAB should prove to be a useful tool for the subsequent molecular characterization of the D1 receptor from various sources and under differing pathophysiological states.

Avidin binding of radiolabeled biotin derivatives.

Three N-acyl derivatives of biotinylethylenediamine were prepared: I, biotinylamidoethyl-3-(3-[125I]iodo-4-hydroxyphenyl)propionamide; II, biotinylamidoethyl-[3H]acetamide; and III, biotinylamidoethyl-3-(3,5-[125I]diiodo-4-hydroxyphenyl)propionamid e. Each compound was combined with a large excess of avidin, yielding 1:1 molar complexes. Aside from a small fraction of each complex that dissociated more rapidly, the dissociation half-lives of these complexes were: I, 41 days; II, 4.4 days; and III, 148 days. The iodo- (mono or di) hydroxyphenylpropionyl moieties of I and III, therefore, contribute significantly to the binding strength of these compounds toward avidin. We also formed 4:1 complexes of I, II, and III with avidin (compound in excess), each of which exhibited biphasic dissociation, with initial half-lives of 4, 3.2, and 24 days, respectively. Thus, I or especially III potentially can be used as a sensitive tracer in quantitative studies with avidin.