Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2000-12-01
2003-02-25
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S005000, C435S810000
Reexamination Certificate
active
06524809
ABSTRACT:
The present invention relates to a method for detecting materials, notably to a method in which two or more viral components are attached to the material which endow the material with a detectable property by which the presence of that material may be detected and it presence in a sample verified.
BACKGROUND TO THE INVENTION
Many techniques exist for analysing samples to detect the presence of a material in that sample. For example, chemical analysis, chromatographic techniques and spectroscopic techniques can be used to identify individual chemical molecules. However, such techniques cannot readily be used to identify organisms present in a sample.
Nucleic acid methods, such as deoxyribonucleic acid (DNA) hybridisation can be used to detect DNA or ribonucleic acid (RNA). However, these methods lack sensitivity. Amplification methods, such as the polymerase chain reaction (PCR), can be used to amplify nucleic acid targets which can then be detected. However, such approaches are complex and expensive and are specific for the detection of nucleic acids.
Immuno methods such as Western blotting or enzyme immunoassay (EIA) or enzyme labelled immuno-absorbent assay (ELISA) can be used to detect a given molecule, for example a protein. However, these methods have detection limits in the picamol (pmol) to sub-picamol per liter range and thus are not suitable where very low levels of the material to be detected are present.
In order to increase the sensitivity of such detection methods, it has been proposed to label the detecting antibody with radioactive materials, enzymes, gold particles or bacteriophage. However, these proposals are often non-specific due to the non-specificity of the attachment of the label to the target or other materials present in the sample being assessed.
It has been proposed in for example PCT Application No WO92/02633 to identify bacteria in a sample by treating the sample in a first cultivation stage with a virus, or phage, which is specific to and infects the bacterium expected to be present in the sample. The infected sample is then treated to kill or remove any exogenous phage particles, that is those which have not infected the bacterium cells and are thus protected within the cell. The resultant bacterial cells are then cultured in a second cultivation stage to cause the phage particles protected within the infected bacterium cells to replicate and lyse. This releases a new generation of phage particles into the culture medium. These can be detected by carrying out the second cultivation stage in the presence of further of the first bacterium cells and detecting the dead bacterium cells formed with each replication of viral particles within an infected cell. Alternatively, the proteins released by the lysis of the viral particles can be detected by protein staining or other methods.
If the expected bacterium is present in the initial sample, viral particles will be present in the infected bacterium cells from the first cultivation of the sample and will be detected in the second cultivation stage. However, if the sample did not contain the expected bacterium, no infection of the sample cells would occur in the first cultivation stage, so that no viral particles would be carried over to replicate and lyse in the second cultivation stage.
This technique is known as the phage amplification technique.
In a modification to the phage amplification technique proposed in PCT Application No WO97/22713, the second cultivation stage is carried out in the presence of a susceptible bacterium which has a replication rate greater than that used in the first cultivation stage. In this way slow growing bacteria, for example Mycobacterium tuberculosis, can be detected rapidly due to the acceleration effect of the second cultivation stage.
However, both variations of the phage amplification technique require that a virus which is specific to and can infect the expected bacterium be used, which severely limits the range of materials which can be identified and the range of viral particles which can be used.
Not only would it be desirable to detect specific organisms in a sample, but it is also desirable to detect a wide range of other materials in samples. For example, it is often desired to detect heavy metals and other contaminants in water or in waste products, to determine whether two or more materials interact with one another in a manner which could affect a user or the environment. Specifically, it would be desirable to provide a simple but effective means for determining the effect of a medicament or pharmaceutical on an organism or medical condition which it is desired to treat with the medicament or pharmaceutical. In vitro homogeneous immunoassay techniques, such as scintillation proximity and fluorescence polarisation/fluorescence quenching are used to report on molecular interactions in screening assays.
These assays are fast and simple to carry out, but generally have a low sensitivity. The yeast two hybrid system is used as an in vivo assay system, where interacting proteins act as a promoter of gene transcription, thereby indicating molecular interactions within the cell. However, this protein/protein interaction can be disrupted by the medicament under assessment. The yeast two hybrid system also requires that the components of the assay be synthesised within the yeast cells. This limits the range of molecular interactions which can be studied.
I have now devised a method which can be applied to the detection of a wide range of materials and the products of the interaction of such materials, so as to provide an assay technique which has widespread application. The method of the invention can use a wide range of marker materials to identify the materials to be detected giving the user flexibility in the materials which are used.
In the method of the invention two different viral particles or active components of a virus are attached directly or indirectly as tags to the material to be detected, hereinafter denoted as the target material. These tags endow the target material with characteristic properties which distinguish the tagged material from the properties of either of the viral particles individually. The tagged material is then examined for the presence or otherwise of these distinctive properties by cultivating the tagged material in the presence of a bacterium which can be infected by the two viruses.
The term virus is used herein to denote true viruses and organisms which infect bacteria in manner similar to a true virus. Thus, the term virus includes:
a. Components of a virus which have the characteristics of the virus from which they are derived;
b. Packaged phagemids, which are crosses between plasmids and viruses and can grow as plasmids in bacterial hosts, but which can be packaged and secreted as if they were viral particles in the presence of a helper virus although they cannot independently produce viral progeny;
c. Viruses which are lysogenic for bacteria and can grow, replicate and produce progeny in the bacteria without lysis of the bacteria which can continue to grow and replicate.
If viral infection of bacterial cells is carried out using an excess of bacteria over that required to achieve parity between the infecting viruses and the infectable bacterial cells, a given bacterium cell is unlikely to be infected by more than one virus particle. The amount at which such dual infection becomes sufficiently unlikely that it will not distort the results of the assay method of the invention can be calculated statistically and is denoted herein as the statistical amount. Such a statistical calculation can be confirmed by simple trial and error tests.
In the method of the invention, two viral particles are physically linked together through the target material and can thus each infect the same bacterial cell so as to endow that cell with both of the characteristic properties of the infecting viruses. A bacterial cell infected by both viruses and possessing the sum of the two characteristic properties can readily be distinguished from cells w
Bourque & Associates PA
Microsens Biophage Limited
Park Hankyel T.
LandOfFree
Analytical method using multiple virus labelling does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Analytical method using multiple virus labelling, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Analytical method using multiple virus labelling will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3161383