Title of Invention

A METHOD FOR THE IDENTIFICATION AND/OR THE QUANTIFICATION OF A TARGET COMPOUND

Abstract

An in-vitro method for the identification and/or the quantification of a target compound of the kind such as herein described obtained from a sample, preferably 5 a biological sample of the kind such as herein described, comprising the steps of: - putting into contact said target compound with a capture molecule of the kind such as herein described in order to allow a specific binding between said target compound 10 with said capture molecule, said capture molecule being fixed upon a surface of a solid support of the kind such as herein described according to an array comprising a density of at least 20 discrete regions per cm2 each of said discrete regions being fixed with one species of 15 capture molecules, - performing a chemical or biochemical catalytic reaction leading to the formation of precipitate formed at the location of said binding and obtained by the precipitate of a metallic compound upon the bounded target compound, 20 - determining the possible presence of said precipitate(s) in discrete region(s), and - correlating the presence of said precipitate(s) at the discrete region(s) with the identification and/or a quantification of said target compound.

Full Text

FORM 2 THE PATENTS ACT, 1970 (39 of 1970) THE PATENTS RULES, 2003 COMPLETE SPECIFICATION (See Section 10 and Rule 13) 1. Title of the Invention:- "An in-vitro method for the identification and/or the quantification of a target compound" 2. Applicant(s):- (a) Name : EPPENDORF ARRAY TECHNOLOGIES (b) Nationality : A Belgian Company (c) Address : Rue du Seminaire 20A, B-5000 Namur, BELGIUM 3. Preamble to the Description:-Complete Specification: The following specification particularly describes the Invention and the manner in which it is to be performed. 5 10 Field of the invention The present invention is related to an in-vitro method for the identification and/or the quantification of a target compound obtained from a 15 biological sample by binding to a capture molecule fixed upon chips. The present invention is also related to an identification and/or quantification apparatus based upon said method, that allows the identification and/or the 20 quantification of positive locations of bounded target compounds upon said chips. Background on the invention and state of the art Biological assays are mainly based upon 25 interaction specificity between two biological molecules such two strands of nucleic acid molecules, an antigen and an antibody or a ligand and its receptor. The present challenge of biological assays is to perform simultaneously the multiple detection of molecules present in a sample. 30 Miniaturisation and development of arrays upon the surface of "biochips" are tools that allow multiplex reactions in a microscopic format, said detection being made with a limited volume of sample for the screening and/or the 2 colorimetric labelling (US 5,270,167, US 4,731,325, EP-A-0301141) or the result of an enzymatic activity (EP-A-0393868, WO 86/02733, EP-A-0063810). However, said methods are either characterized by a low sensitivity or 5 are not adequate for the detection of a target compound upon "hybridisation chips", because the precipitate will occur at a certain distance of the reaction binding and its location can not be easily correlated with a specific bounded target compound. In addition, the density of the 10 precipitate of such enzymatic reactions is not enough opaque for allowing a detection by light absorption. It has also been proposed to improve the detection by fixing a soluble product obtained from the enzymatic reaction with a metal before its precipitation. 15 However, as the result of said enzymatic reaction is a soluble product, there is no correlation between the location of the precipitate and the detection of a specific bounded target compound. Aims of the invention 20 The present invention aims to provide a new in-vitro identification and/or quantification method of one or more target compounds present (possibly simultaneously) in a biological sample and that will not present the drawbacks of the state of the art. 25 The present invention aims to provide such a method that is simple and not expensive, that allows the detection of said target compounds by using fixed capture molecules upon arrays of the surface of a solid support. A last aim of the present invention is to 30 provide also a simple and non-expensive apparatus based upon said method, that improves the identification and/or the quantification of bounded target compounds upon "hybridisation chips". 4 Summary of the invention The present invention is related to an in- vitro method for identification and/or quantification of at least one target compound present in a biological sample by 5 its binding upon a capture molecule fixed upon arrays of a solid support (hereafter called "hybridisation chips"), the binding of said target compound upon its corresponding capture molecule resulting in the formation of a metal precipitate at the location of said capture molecule. 10 Advantageously, said in-vitro method comprises the steps of: - putting into contact the target compound with a capture molecule in order to allow a specific binding between said target compound with a (corresponding) capture 15 molecule, said capture molecule being fixed upon a surface of a solid support according to an array comprising at least a density of 20 discrete regions per cm2, each of said discrete regions being fixed with one species of capture molecules, 20 - performing a reaction, preferably a (chemical or biochemical) catalytic reaction, leading to a formation of a precipitate at the location of said binding, - determining the possible presence of a precipitate in a discrete region preferably by the use of a scanner, and 25 - correlating the presence of the precipitate (s) at the discrete region(s) (precipitate pattern) with the identification and/or a quantification of said target compound in the biological sample. The "hybridisation chips" according to the 30 invention are any kind of solid support that allow the formation of arrays of capture molecules (specific pattern) upon one or more of its surfaces. Said solid support can be 5 made of glasses, filters, electronic device, polymeric or metallic materials, etc. Preferably, said arrays contain specific locations (advantageously presented according to a specific pattern), each of them containing normally only 5 one species of capture molecule. The fixation of DNA strands on proteins thereafter specifically attached to sites specific locations on a substrate, is described in the document US-5,561,071. It is also known that capture chemicals can 10 be linked to microtubes that are then spatially arranged in order to produce an array, as described in the document GB-3 319 838, or to obtain the direct synthesis of oligonucleotides on specific surfaces by using photolithographic techniques as described in the documents 15 EP-0476014, US-5,510,270, US-5,445,934, W097/29212, US-5,605,662, US-5,632,957 and W094/22889. All these methods for the fixation of capture molecules on the surface of a solid support in order to obtain the above-described arrays are compatible with the 20 present invention. The target compound is preferably isolated purified, cleaved, copied and/or amplified if necessary by known methods by the person skilled in the art before their detection and/or quantification upon the "hybridisation 25 chips" which is present in a biological compound, or possibly in non-biological compound. Preferably, the formation of a precipitate at the location of the binding is obtained with the fixation of a metallic compound upon the bounded target compound or 30 by the result of a reduction of a metal in the presence of an enzyme. Advantageously, a reduction of silver in the 6 The array according to this invention is used in a diagnostic kit, in a diagnostic and/or quantification apparatus which allows automatic lecture, possibly after a previous treatment, such as purification, cleaving, copying 5 and/or genetic amplification. Preferably, the detection and/or quantification apparatus according to the invention is a system that combines multiple steps or substeps within an integrated system as an automatic nucleic acid diagnostic 10 system (the steps of purification of the nucleic acid sequences in a sample, of amplification (through known genetic amplification methods), the diagnostic and possibly the quantification). The present invention is not intended to 15 claim a process for treatment of human beings or animals to render them free of disease; or a process for treatment of human beings or animals to increase their economical value or that of their products. The biological sample does not include [or 20 it excludes] samples obtained from human beings or animals for diagnostic treatment of human beings or animals. Preferred embodiments of the present invention will be described in the following non-limiting examples in reference to the figures. 25 Short description of the drawings The figure 1 compares the detection of target molecules obtained on arrays composed of DNA capture nucleotide sequences covalently fixed on glass and used to 30 detect 3 concentrations of biotinylated target DNA either in fluorescence or after silver concentration. The figures 2 to 7 represent the spatail arrangement of some elements in various embodiments of the apparatus for performing the detection and/or the quantification method according to the invention. 5 Example 1 Detection of DNA on biochips In this experiment, target DNA labelled is detected by direct hybridisation on capture nucleotide 10 sequences bound to the array. Capture nucleotide sequences were covalently bound on glass and direct hybridisation 15 20 25 30 WE CLAIM:- 1. An in-vitro method for the identification and/or the quantification of a target compound of the kind such as herein described obtained from a sample, preferably 5 a biological sample of the kind such as herein described, comprising the steps of: - putting into contact said target compound with a capture molecule of the kind such as herein described in order to allow a specific binding between said target compound 10 with said capture molecule, said capture molecule being fixed upon a surface of a solid support of the kind such as herein described according to an array comprising a density of at least 20 discrete regions per cm2 each of said discrete regions being fixed with one species of 15 capture molecules, - performing a chemical or biochemical catalytic reaction leading to the formation of precipitate formed at the location of said binding and obtained by the precipitate of a metallic compound upon the bounded target compound, 20 - determining the possible presence of said precipitate(s) in discrete region(s), and - correlating the presence of said precipitate(s) at the discrete region(s) with the identification and/or a quantification of said target compound. 25 2. An in-vitro method as claimed in claim 1, wherein said metallic compound is a magnetic metallic compound. 3. An in-vitro method as claimed in any one of the preceding claims, wherein the reaction leading to 30 the formation of the precipitate is a reduction of a metal in the presence of an enzyme. 4. An in-vitro method as claimed in claim 1 or 2, wherein the reaction leading to the formation of the 25 precipitate is a chemical reduction of silver in the presence of colloidal gold particles coupled to the bounded target compound. 5. An in-vitro method as claimed in any one 5 of the preceding claims, wherein the specific binding between said target compound and its corresponding capture molecule is an hybridisation between two nucleotide sequences. 6. An in-vitro method as claimed in any one 10 of the claims 1 to 4, wherein the binding between said target compound and its corresponding capture molecule is a reaction between an antigenic structure and its corresponding antibody or a hypervariable portion thereof. 7. An in-vitro method as claimed in any one 15 of the preceding claims, wherein the binding between said target compound and its corresponding capture molecule is a reaction between a receptor and its corresponding ligand. 8. An in-vitro method as claimed in any one of the preceding claims, wherein the possible presence of 20 said precipitate is obtained by reflection, absorption or diffusion of a light beam, preferably a laser beam, upon said precipitate. 9. An in-vitro method as claimed in any one of the preceding claims, wherein the presence of said 25 precipitate in a discrete region is obtained by variation of an electromagnetic field or the conductance of an electric current. 10. An in-vitro method as claimed in any of the preceding claims 1 to 9, wherein volume of one or more 30 said precipitate(s) is quantified upon a defined surface of said solid support, wherein images of said defined surface containing one or more precipitate(s) and corresponding to different views, said images containing analogue information, are taken by one or more camera(s) upon 26 illumination by one or more illuminant source(s), spatially arranged relatively to each other according to a predetermined pattern and wherein the corresponding image analogue information of said defined surface containing 5 said precipitate (s) are transformed and converted into digital form or set of digital forms and compared to a first and to a second reference standards to determine the volume of said precipitate(s) to be quantified. 11. An in-vitro method as claimed in claim 10 11, wherein the first reference standard corresponds to a digital form or a set of digital forms obtained from analogue information contained in images taken on the surface of said solid support (1) without precipitate. 12. An in-vitro method as claimed in the 15 claim 11, wherein the second reference standard corresponds to a digital form or a set of digital forms obtained from analogue informations contained in images taken on the surface of said solid support (1) containing said precipitate(s) of known volume. 20 13. An in-vitro method for the identification and/or quantification of a target compound substantially as herein described with reference to forgoing examples and as illustrated with reference to the accompanying drawings. 25 Dated this 21st day of April, 2005. Dr. Ramesh Kr. Mehta] Patent Attorney for the Applicants 30 of Mehta & Mehta Associates

Documents:

327-mumnp-2005-cancelled pages(27-11-2006).pdf

327-mumnp-2005-claims(complete)-(25-4-2005).pdf

327-mumnp-2005-claims(granted)-(19-4-2007).pdf

327-mumnp-2005-claims(granted)-(27-11-2006).doc

327-mumnp-2005-claims(granted)-(27-11-2006).pdf

327-mumnp-2005-correspondence(25-4-2005).pdf

327-mumnp-2005-correspondence(ipo)-(12-03-2007).pdf

327-mumnp-2005-correspondence(ipo)-(27-6-2007).pdf

327-mumnp-2005-correspondence1(24-10-2005).pdf

327-mumnp-2005-correspondence2(12-01-2007).pdf

327-mumnp-2005-description(complete)-(25-4-2005).pdf

327-mumnp-2005-description(granted)-(19-4-2007).pdf

327-mumnp-2005-drawing(complete)-(25-4-2005).pdf

327-mumnp-2005-drawing(granted)-(19-4-2007).pdf

327-mumnp-2005-form 1(12-1-2007).pdf

327-mumnp-2005-form 1(27-11-2006).pdf

327-mumnp-2005-form 1(31-10-2006).pdf

327-mumnp-2005-form 1(6-7-2006).pdf

327-mumnp-2005-form 13(25-4-2005).pdf

327-mumnp-2005-form 13(31-10-2006).pdf

327-mumnp-2005-form 13(6-7-2006).pdf

327-mumnp-2005-form 18(25-10-2005).pdf

327-mumnp-2005-form 2(complete)-(25-4-2005).pdf

327-mumnp-2005-form 2(granted)-(19-4-2007).pdf

327-mumnp-2005-form 2(granted)-(27-11-2006).doc

327-mumnp-2005-form 2(granted)-(27-11-2006).pdf

327-mumnp-2005-form 2(title page)-(complete)-(25-4-2005).pdf

327-mumnp-2005-form 2(title page)-(granted)-(19-4-2007).pdf

327-mumnp-2005-form 26(31-10-2006).pdf

327-mumnp-2005-form 26(6-7-2006).pdf

327-mumnp-2005-form 3(25-4-2005).pdf

327-mumnp-2005-form 5(25-4-2005).pdf

327-mumnp-2005-marked copy(27-11-2006).pdf

327-mumnp-2005-petition under rule 138(11-12-2006).pdf

327-mumnp-2005-petition under rule 138(25-4-2005).pdf

327-mumnp-2005-specification (amanded)-(27-11-2006).pdf

327-mumnp-2005-specification(amanded)-(27-11-2006).pdf

327-mumnp-2005-specification(amanded)-(31-10-2006).pdf

327-mumnp-2005-wo international publication report(25-4-2005).pdf