Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-01-24
2003-10-28
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S006120, C435S007200, C435S007300, C435S007400, C435S075000, C435S007600, C435S007700, C435S007800, C435S007900, C424S001490, C436S504000, C436S536000, C436S538000, C436S542000, C530S324000, C530S344000, C530S350000, C530S415000, C530S812000, C530S816000, C530S827000
Reexamination Certificate
active
06638725
ABSTRACT:
This application claims priority to Japanese Patent Application Nos. 15980/1999, filed Jan. 25, 1999 and 174536/1999, filed Jun. 21, 1999, the contents of which are incorporated by reference.
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for assaying a receptor binding substance in an aqueous sample (e.g., biological material, sea water, river water, ground water and the like), or a method for assaying the receptor binding property of a chemical substance, and an assay reagent to be used for this method.
BACKGROUND OF THE INVENTION
Recent reports have revealed that substances induce various biochemical reactions that occur in a living body. These substances bind with their receptors in the body and cause release of signal transmitters that induce such reactions. The receptors are largely divided into those present on the cell membrane and those present in a cell or nucleus.
The receptors present on the cell membrane are hormone receptors such as adrenergic receptor, luteinizing hormone receptor and the like. Many of these receptors are responsible for intracellular regulation through signal transmission and amplification via tyrosine kinase, adenylate cyclase and the like that are regulated by the interactions of the receptors and their ligands. Those present in a cell or nucleus are exemplified by estrogen receptor, retinoide receptor and the like.
Some receptors act as a carrier in blood, such as transferrin. In particular, the action mechanism of nuclear receptor is considered to involve binding of a nuclear receptor and its ligand, which activates a nucleic acid binding region of the receptor, and binding thereof with the nucleic acid to control transcription and translation from the nucleic acid, which ultimately results in the control of various reactions in the body.
The binding of substance and receptor has been investigated and studied as models representing various reactions in the body. By the receptor is meant a substance which is not an antibody and which shows hormone binding property, biochemical messenger, steroid, drug, drug metabolite, polypeptide, protein, vitamin, alkaloid, monosaccharide, disaccharide, polysaccharide and the like.
There are some methods to evaluate in vitro the receptor binding property of a substance. They include a method using a radioisotope (Obourn, J. D. et al.,
Biochemistry,
32, 6229-6236, 1993), a method using fluorescence depolarization (Bolger, R. et al.,
Environ. Health Perspect.,
106, 551-557, 1998), a method using surface plasmon resonance (Ward, L. D. et al.,
J. Biol. Chem.,
269, 23286-23289, 1994), a method using microcalorimeter for thermodynamic assay (Moore, J. L. et al.,
J. Biol. Chem.,
271, 21273-21278, 1996) and the like.
As a method in vivo, there have been reported and practiced a method using a cultured cell (Soto, A. M. et al., Environ. Health Perspect., 103, 113-122, 1995), a method using recombinant yeast (Arnold, S. F. et al.,
Environ. Health Perspect.,
104, 544-548, 1996) and the like.
The reported method using fluorescence depolarization by Bolger et al comprises competitively reacting a fluorescent-labeled tracer with a receptor and an assay target substance with a receptor, and measuring depolarization of the fluorescence due to the binding of the tracer and the receptor. This method is defective in that the use of the fluorescent-labeled tracer causes lower reactivity and the method is subject to an influence of a contaminating fluorescent substance in the sample and the cloudiness of the sample.
The method using a radioisotope by Obourn et al comprises competitively reacting an RI-labeled tracer with a receptor and an assay target substance with a receptor, and quantitatively assaying the radioisotope bound with the receptor. This method can be used only in a specific facility because it uses a radioisotope that limits facility and workability.
The method using surface plasmon resonance by Ward et al uses an immobilized receptor, and analyzes the direct molecular interaction between the assay target substance and the receptor. However, this method fails to simultaneously process plural samples, and requires an expensive sensor chip and a special apparatus.
The method for thermodynamic assay by Moore et al is superior in that it does not require immobilization of receptor or use of a labeled tracer. However, this method also fails to simultaneously process plural samples and requires a special apparatus.
In recent years, a need has arisen for a broad screening of endocrine disrupting chemical (EDC) (Toyama, C., Clinical Endocrinology, 46, 517-528, 1998), that requires examination of receptor binding property of known or new tens of thousands of chemical substances. The above-mentioned methods cannot process plural samples simultaneously, and therefore, enormous time and labor are needed. There is an urgent demand on an assay method for the interaction between a receptor and a chemical substance, that can achieve a high throughput.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide a method capable of simultaneous processing of plural test samples for the receptor binding property of a chemical substance, which does not require immobilization of the receptor or a special device, and a reagent to be used for this method.
The present invention is based on the finding that a ligand not bound with a receptor can be assayed alone with ease by the steps comprising competitively reacting a ligand and an assay target substance with the receptor in a solution, and, without physically removing the ligand bound with the receptor, further adding an antibody against the ligand and a labeled ligand to allow reaction.
Accordingly, the present invention provides the following.
1. A method for assaying the receptor binding property of an assay target substance, comprising the steps of
(a) competitively reacting a known concentration of a ligand and the assay target substance with a known concentration of the receptor in a solution,
(b) measuring, without physically removing the ligand bound with the receptor prior to the assay, the amount of a free ligand in the solution using one or more antibodies against the ligand, and
(c) determining the receptor binding property of the assay target substance using the amount of the free ligand as an index.
2. The method of 1 above, wherein the step (b) comprises adding one or more antibodies against the ligand and a labeled ligand to the reaction mixture obtained in step (a) to allow competitive reaction of the antibody and the free or labeled ligand, and measuring the amount of the label bound or not bound with the antibody.
3. The method of 1 above, wherein the antibody retains its activity by 60% or more in a 1% organic solvent.
4. The method of 1 above, wherein the ligand is not labeled, bound, processed or denatured.
5. The method of 1 above, wherein the receptor is used in an amount that produces binding of 50% or more of the ligand with the receptor.
6. The method of 1 above, wherein the receptor is selected from the group consisting of receptors of hormone, drug, drug metabolite, polypeptide, protein, saccharides, biochemical messenger and vitamin and ligand binding domains thereof.
7. A reagent for assaying the receptor binding property of a substance, comprising a reagent containing a known concentration of a receptor, a reagent containing a known concentration of a ligand of a known concentration of the receptor, and a reagent for measuring a free ligand, which contains one or more antibodies against the ligand.
8. The reagent of 7 above, further comprising a reagent containing the ligand which has been labeled.
9. The reagent of 7 above, wherein the antibody retains its activity by 60% or more in a 1% organic solvent.
10. The reagent of 7 above, wherein the ligand is not labeled, bound, processed or denatured.
11. The reagent of 7 above, wherein the receptor is used in an amount that produces binding of 50% or more of the ligand with the receptor.
12. The reagent of 7 above, wherein the receptor is s
Ishibashi Takuya
Kawamura Yoshihisa
Matsui Kazuhiro
Nishii Shigeaki
Soya Yoshihiro
Cook Lisa V.
Le Long V.
Morrison & Foerster / LLP
Toyo Boseki Kabushiki Kaisha
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