Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
1999-05-06
2002-10-15
Chin, Christopher L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C436S043000, C436S519000, C436S800000, C436S807000, C436S809000, C435S004000, C435S005000, C435S006120, C435S007800, C435S007210, C435S007220, C435S007250, C435S007920, C435S007930, C435S007940, C435S021000, C435S069400, C435S070100, C435S071200, C435S091500, C435S173300, C435S242000, C435S320100, C435S091500, C435S091500, C435S091500, C435S962000, C435S968000, C530S300000, C530S301000, C530S324000, C530S350000, C530S351000, C530S395000, C530S397000, C530S399000, C530S402000, C530S403000
Reexamination Certificate
active
06465259
ABSTRACT:
BACKGROUND OF THE INVNETION
Receptor binding assays have been commonly employed for studying receptor ligand interactions in e.g. neurochemistry, neurobiology. psychopharmacology and related fields. Receptor assays are also used as an analytical method to measure drug levels in biological matrices. The principle of these methods is based on the competition of a labeled ligand and the analyte for binding to a certain receptor. Up till now, receptor assays have mostly been performed with radioactive ligands. Due to the low density of receptor binding sites in most tissue e.g. 10 to 100 pmol ligand bound per gram of tissue the ligand used must have high affinity and selectivity for the binding sites as well as high specific radioactivity in order to determine low levels of ligand. Radioactive ligands are commercially available that can be selectively measured at very low levels due to their high specific activity. Besides, since most of the radioisotopes are incorporated in the molecule, this type of labeling has no influence on the binding affinity of the ligand towards the receptor. Since the use of radioactivity has several well documented disadvantages such as limited shelf life, problematic handling and care required in the disposal thereof, non-radioactive ligands, such as fluorescent-labeled ligands, have been synthesized for different receptor types. To date however the results have not been such that reliable quantitative data for trace amounts of analytes can be obtained in a simple and satisfactory manner that they can offer an alternative for those types of analysis carried out with radioactive receptor assays (RRAs).
Examples of a non radioactive receptor assay are provided in general for a large number of receptors in WO93/03382 of Tyler McCabe. They describe how numerous attempts to characterize receptors using fluorescent ligands were carried out. The references cited on page 3 of the PCT application are hereby also incorporated by reference. The receptors mentioned are &agr;-adrenergic &bgr;-adrenergic, opioid, adenosine, glucagon, steroid and dopamine receptors. They indicate there were problems with quantification and visualisation by direct fluorescence measurement due to autofluorescence and lack of specificity. They provide a group of fluorescent ligands suitable for determining ligand-receptor interactions intracellularly and extracellularly and to determine the specificity and affinity of uncharacterised compounds. They analyse intracellular versus extracellular events by selecting a fluorescent probe which emits different fluorescent intensities depending on the pH of the environment. They carry out their tests on 100% tissue. The receptors mentioned were opioid, potassium channel, glibenclamide and glycine. The assays actually illustrated are a fluorescein labelled ligand for the opioid receptor, nitrobenz-2-oxa-1,3-diazol-4-yl(=NBD) labelled ligands for the potassium channel and glibenclamide and glycine receptors. They indicate the fluorescent value is corrected for by subtracting the value of auto fluorescence. This means the assay they employ can never be sufficiently sensitive to obtain reliable quantification or detection of trace amounts of analytes.
One of the important receptors for pharmacologists and doctors is the benzodiazepine receptor. Benzodiazepines are extremely widely used drugs and have been primarily used for the treatment of anxiety and insomnia. It is accepted the pharmacological effects are medicated through specific receptors in the central nervous system. This receptor has been found to be extremely difficult to tackle for determining low concentrations of benzodiazepine receptor binding analytes. In vitro receptor radioligand interactions have been used to investigate the mechanism of the pharmacological effects and also to investigate new benzodiazepine drugs.
An alternative method has been the use of non radioactive immunoligand assays. These solve the problems associated with the use of radioactivity. The disadvantages thereof are however numerous. In immunoassays the binding molecule is an antibody that has been generated against the ligand to be determined. In order to generate antibodies the ligand must first be linked to a large carrier for example BSA. The position of the linkage of the BSA to the ligand restricts the selectivity of the antibody. The antibody is not selective for the linkage position. The sensitivity of immunoassays is not correlated to the pharmacological effectivity of the ligand. The affinity between ligand and antibody is based merely on chemical structure not an the chemical structure that determines the pharmacological effectivity. A ligand can have a high affinity for the antibody whet such Ligand is hardly pharmacologically effective and vice versa. For example the Merck label for the fluorescent polarisation immunoassay for benzodiazepines (Vitalab Eclair) hardly has any affinity for the benzodiazepine receptor (Ki=200 nM). With regard to metabolites nothing can be stated with regard to their pharmacological activity. In addition when multiple ligands need to be determined multiple antibodies are required for each separate ligand. In the receptor ligand assays a single ligand can suffice to assay for multiple analytes. The immunoligand technology is for example illustrated in EP-A-0.264.797 of Abbott.
Specifically for the benzodiazepine receptor for example fluorescent-labeled ligands have also been used as non radioactive labeled ligand for the characterization of the benzodiazepine receptor [g-i]. Such characterisation experiments however are not subject to the degree of sensitivity required for analyte detection and quantification at trace limits. The content of the cited articles will however be presented furtheron in order to create a more complete overview of the technology and the specific problems.
Further to the above more recent publications for the benzodiazepine receptor address use of fluorescent-labeled ligands as labeled ligand for a benzodiazepine receptor assay [b (1991),j (1993). c(1995)] as opposed to simple characterisation.
In the fluorescent benzodiazepine receptor assays developed by Takeuchi et al. [b,c], the free fractions of label were quantified after collection of these fractions by centrifugation. Since membrane-bound receptors exhibit background fluorescence [c] the problem of autofluorescent interference was expected to be reduced in these assays. However this was not the case. Additional measures were required and the sensitivity of these assays left a lot to be desired,
Takeuchi and Rechnitz in [b] already in 1991 described quantifying the free fraction of the ligand. They used a HPLC-system in conjunction with AMCA-Ro7-1986 (AMCA-didesethylflurazepam) as ligand in their fluorescence receptor assay. The use of HPLC was required as a pretreatment to eliminate possible interference in the matrix. The supernatant after the bound/free separation by centrifugation was injected directly onto the HPLC column without further cleanup such as filtration. To obtain enough difference in the fluorescence signal between the maximal binding and the non-specific binding, they also had to use a high amount of receptor material, 50 mg/ml. Such a test is not practical for large scale commercial use due to the prohibitive cost of using such high amounts of receptor material. In addition the use of such high amounts of animal tissue is undesirable also from an ethical point of view.
Takeuchi et al. [j and c] later addressed the problem of autofluorescence in an alternative and preferred manner as disclosed in their articles of 1993 and 1995. Specifically they stated in the latter article “Because the commercially available benzodiazepine receptor preparations are only partially purified their supernatants exhibit strong background fluorescence and may interfere with the measurement of fluorophore labeled ligands”. To solve this problem in the cited article they disclose developing a time-resolved fluorometric
De Zeeuw Rokus Arie
Ensing Kornelis
Janssen Maria Johanna
Chin Christopher L.
Cook Lisa R
Merska B.V.
LandOfFree
Non radioactive receptor assay suitable for quantitative and... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Non radioactive receptor assay suitable for quantitative and..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Non radioactive receptor assay suitable for quantitative and... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2994862