Screening for analytes using labeled receptors

Details Screening for analytes using labeled receptors Screening for analytes using labeled receptors

2001-05-09

2004-04-13

Ceperley, Mary E. (Department: 1641)

Chemistry: analytical and immunological testing

Biospecific ligand binding assay

Utilizing isolate of tissue or organ as binding agent

C435S007930, C436S504000, C436S526000, C436S527000, C436S530000, C436S531000, C436S534000, C436S535000, C436S545000, C436S546000, C436S815000, C436S817000, C436S829000

Reexamination Certificate

active

06720192

ABSTRACT:

INTRODUCTION
Receptor binding assays have been commonly used for the assessment of the pharmacological properties of New Chemical Entities (NCE). Due to the introduction of combinatorial chemistry in the pharmaceutical industry, in an attempt to find succesful drug candidates, an enormous increase in NCE requires a concomitant demand for high through-put screening systems. Due to their specific properties receptor assays have been considered valuable analytical tools for the quantitation of highly potent drugs that exert their pharmacological action via a receptor interaction.
The term receptor is exclusively used for proteins which can interact with hormones, neurotransmitters and drugs or poisons yielding or blocking a pharmacological response. Thus therefore antibodies, circulating or membrane bound proteins e.g. enzymes cannot be considered receptors even if they should have ligand binding properties.
The principle of receptor binding assays is based on the competition between a ligand and an analyte for binding to a certain receptor. After incubation of ligand, analyte and receptor followed by separation of the receptor bound and the free fraction of the ligand by either filtration, centrifugation or dialysis, subsequently one or both resulting fractions are quantitated. The subsequently acquired data can be used for assessment of the affinity of a NCE for the receptor or for quantitation of a particular receptor binding analyte.
Up till now, all receptor assays have been construed around the use of a (radio) labeled ligand. Furthermore generally receptors present in animal tissue or cultivated cell lines have been used after having undergone only little purification. Typical receptor densities in commonly used receptor assays range from 10-100 picomole per gram tissue. This subsequently implies that the amount of displaceble labeled ligand in such assays is limited. The use of radioactive ligands in such cases is notwithstanding this attractive because radioactivity can be detected with good sensitivity and limited back-ground signals from such receptor material. Another important reason in favor of use of radioactive labels is that development is easy once a compound has been identified that binds to a particular receptor with high affinity. Replacing 1-6 hydrogen atoms by the same number of tritium atoms yields a product that has a receptor affinity similar to that of the unlabeled ligand. However disadavantages such as the limited shelf-life and the problems encountered with the use of radioactive tracers has stimulated the search for alternatively labeled ligands. Another motivation for such search was also the expectation that alternative labels might improve the sensitivity of the assay with regard to limits of quantitation. Taken into consideration the physical half-life and the counting time of each sample it can be calculated for tritium by way of example that only 1 out of each million labeled molecules is detected.
Almost all approaches with non-radioactive labeled ligands have been with fluorescent labels. The development of fluorescent ligands with a high receptor affinity, if the ligands itself does not have sufficient native fluorescence, is quite difficult and a compromise between affinity and fluorescence properties has always been required. The aforementioned low receptor density implies that the maximal signal is limited and lies close to the limitations of available instrumentation. High amounts of receptor containing material needs to be used per assay in order to sufficiently increase the signal.
Furthermore the currently used receptor containing materials in receptor assays contain large amounts of non-receptor proteins which cause a high fluorescence background.
Time-resolved fluorescence or fluorophores that emit at wavelengths >500 nm could partially solve this problem but provides no real break-through. In patent application PCT/NL96/00418, Janssen et al. have reviewed the existing literature on assays and recognized these problems. The review is incorporated herein by reference. Janssen et al subsequently modified the procedure to quantitate the bound fraction of labeled ligand after dissociation of the labeled ligand from the receptor which took place after the separation of the bound and free fractions of the labeled ligand. Initially they developed a fluorescent labelled benzodiazepine receptor ligand attempting to obtain a homogeneous assay using polarisation fluorescence.
The adapted procedure with the dissociation step opened a new array of detection modes for the ligand. Principally if a high affinity receptor binding compound has unique physico-chemical properties e.g. halogen atoms which can be detected with gas chromatography with electron capture detection, it can be used as a ligand in such a receptor assay.
It can be concluded that the development of non-radioactive labeled ligands is difficult because the bulky groups, required to obtain e.g. highly fluorescent probes that emit at higher wavelengths at the same time reduce the receptor affinity of the product. Furthermore a further disadvantage is that per ligand-receptor interaction only one detectable functionality is present.
Benzodiazepine receptors in particular remain important in clinical pharmacology because of their widespread use. Benzodiazepines are extensively metabolized yielding a range of active and inactive metabolites. The overall effect of a benzodiazepine can therefore not be properly related to the concentration of only the parent drug. Quantitation of the individual compounds, parent drug and metabolites on the other hand is highly impractical and assays that could measure the sum of all active compounds could be beneficial.
To solve such a problem an immuno assay could be used, However the affinity of the individual compounds towards the applied antibody does not correlate with the affinity of these individual compounds towards the receptor. This thus precludes the measured response being a proper reflection of the total pharmacological effect. It is for this reason that the use of the receptor which mediates the pharmacological effect in an assay forms a logical step.
Traditional receptor assays using radioactive or non-radioactive ligands require a separation step enabling quantitation of bound and or free fractions of the labeled ligand. Procedures used for the separation are dialysis, centrifugation and filtration. The selection depends on available instrumentation and equilibrium dissociation constants of the labeled ligand and of the analyte. It is a requirement that the separation step may not alter the amount of receptor bound ligand.
Dialysis is a slow process, up to 24 hours. Automation is cumbersome because collection of the fractions containing the bound plus free and the free fraction of label and analyte is hardly possible. In addition miniaturisation is precluded.
Centrifugation with or without precipitation of the labeled ligand or the soluble or solubilized receptor has limited applicability for receptor assays because the separation process can change the bound fraction of the labeled ligand. Moreover it is difficult to quantitatively collect sedimented receptors. Automation of centrifugation steps also remains problematic. Filtration is the method of choice albeit that due to the amounts of receptor containing tissue per sample minimal filter surface areas are required. This is to avoid clogging of these filters and to warrant accurate quantitation of the bound fraction of the labeled ligand. There are a number of filtration devices (Skatron, Brandell, Millipore) that allow parallel filtration of up to 96 samples. It should be noted however that filtration capacity per filter is limited and that the precision of the methods is strongly dependent on the differences in filtration rate between the individual filters. With the exception of the Millipore system it is not possible to collect either the free fraction of the labeled ligand or the bound fraction of the labeled ligand after dissociation from the collected receptor fraction.
It i

Affiliated with

Also associated with

Rating

Screening for analytes using labeled receptors does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Screening for analytes using labeled receptors, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Screening for analytes using labeled receptors will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3235668