Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
1998-10-30
2003-10-28
Smith, Lynette R. F. (Department: 1645)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S008000, C435S069100, C435S069600, C435S069700
Reexamination Certificate
active
06638752
ABSTRACT:
I. FIELD OF THE INVENTION
The present invention relates to biodetectors for detecting and quantifying molecules in liquid, gas, or on solid matrices. More specifically, the present invention relates to biodetectors comprising a molecular switching mechanism to express a reporter gene upon interaction with target substances. The invention further relates to methods using such biodetectors for detecting and quantifying selected substances with high specificity and sensitivity.
II. BACKGROUND OF THE INVENTION
The detection of low-levels of biological and inorganic materials in biological samples, in the body or in the environment is frequently difficult. Assays for this type of detection involve multiple steps which can include binding of a primary antibody, several wash steps, binding of a second antibody, additional wash steps, and depending on the detection system, additional enzymatic and washing steps. Such assays further suffer from lack of sensitivity and are subject to inaccuracies. For instance, traditional immunoassays have false negative results of up to 30% when detecting infections.
Molecular probe assays, although sensitive, require highly skilled personnel and knowledge of the nucleic acid sequence of the organism. Both the use of nucleic acid probes and assays based on the polymerase chain reaction (PCR) can only detect nucleic acid which require complicated extraction procedures and may or may not be the primary indicator of a disease state or contaminant. Both types of assay formats are limited in their repertoire in cases where little information is available for the entity to be detected.
Current noninvasive methods to measure a patient's physical parameters, such as CAT or MRI, are expensive and are often inaccessible. Thus, the monitoring of many medical problems still requires tests, which can be slow and expensive. The time between the actual test and the confirmation of the condition may be very important. For example, in the case of sepsis, many patients succumb before infection is confirmed and the infecting organism identified, thus treatment tends to be empirical and less effective. Another example is in screening the blood supply for pathogens.
Verification of a pathogen free blood supply requires a number of labor intensive assays. In the case of HIV-1, the virus that causes AIDS, the current assays screen for anti-HIV antibodies and not the virus itself. There is a window lasting up to many weeks after exposure to the virus in which antibodies are not detectable, and yet the blood contains large amounts of infectious virus particles. Clark et al., 1994,
J. Infect. Dis.
170:194-197; Piatak et al., 1993,
Aids Suppl.
2:S65-71. Thus, screening of the blood supply is not only time-consuming and slow, it may also be inaccurate.
Similarly, the ability to detect substances in the environment, such as airborne and waterbome contaminants is of great importance. For example, it would be desirable to monitor groundwater, to control industrial processes, food processing and handling in real-time using an inexpensive versatile assay. However, current methods are not suited for such “on-line” monitoring.
There are several reasons why current methods are limited. First, access to sufficient amounts of the material to be detected may be difficult. For example, the detection of biological materials can be difficult as the biological materials of interest are often sequestered inside a body, and large quantities can be difficult to obtain for ex vivo monitoring. Therefore, sensitive assays for use on small amounts of material are necessary. This indicates that a method of amplifying the signal is required. Amplification methods have been established for detection of nucleic acids but this is not the case for antigen detection methods.
A second problem is that sensing may be difficult in real-time because the target materials may be present in such small quantities that detection of their presence requires time-consuming, expensive and technically-involved processes. For example, in the case of bacterial infections in the blood, sepsis, there may be only 1-2 bacteria in a 1-10 ml blood sample. Current methods require that the bacteria are grown first in order to be detected. Askin, 1995
, J. Obstet. Gynecol. Neonatal. Nurs.
24:635-643. This time-lag may be detrimental as delaying treatment or mistreating diseases may mean the difference between life and death.
Others have attempted to avoid these limitations by using radioactive or fluorescent tags in combination with antibodies (Harlow et al., (1988),
Antibodies. A Laboratory Manual
(Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press). Antibody-based assays typically involve binding of a primary antibody to the target molecule, followed by a series of washing steps to remove all unbound antibodies. Specific binding is typically detected using an identifier molecule, such a labeled secondary antibody directed against the primary antibody. This step is also followed by multiple wash steps. Alternatively, the primary antibody may be directly attached to a detectable label. Suitable labels have included radioactive tracers, fluorescent tags, and chemiluminescent detection systems. Harlow, et al., 1988
, Antibodies. A Laboratory Manual
(Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press).
The series of steps required using such antibody-based assays to generate a specific signal are time consuming and labor intensive. Furthermore, these type of assays are limited to the detection of antigens fixed to some type of matrix. Examples of this type of detection system include Western blots, immunohistochemistry, and ELISA. The highest sensitivity is currently achieved using radioisotopic and chemiluminescent tags. However, sensitivity, i.e., specific signal over background, of these detection systems frequently remains a limiting factor.
Similarly, background radiation places limits on the sensitivity of radioactive immunoassay techniques. In addition, these techniques are time-consuming and expensive. Finally, radioactive approaches are hostile to the environment, as they present significant waste disposal problems.
Another approach to monitoring substances involves the use of light. Light has the advantage that it is easily measurable, noninvasive and quantitative. Von Bally et al., (1982),
Optics in Biomedical Sciences: Proceedings of the International Conference
(Berlin, N.Y.: Springer-Verlag).
Traditional spectroscopy involves shining light into substances and calculating concentration based upon the absorbance or scattering of light. Von Bally et al., (1982),
Optics in Biomedical Sciences: Proceedings of the International Conference
(Berlin, N.Y.: Springer-Verlag). Optical techniques detect variations in the concentration of light-absorbing or light scattering materials. Von Bally et al., (1982),
Optics in Biomedical Sciences: Proceedings of the International Conference
(Berlin, N.Y.: Springer-Verlag). Near-infrared spectroscopy has proved to be a relatively safe form of radiation that functions well as a medical probe, since it can penetrate into tissues. Further, it is well-tolerated in large dosages. For example, light is now used to calculate the concentration of oxygen in the blood (Nellcor) or in the body (Benaron image), or even to monitor glucose in the body (Sandia). Benaron and Stevenson, 1993,
Science
259:1463-1466; Benaron et al., 1993, in:
Medical Optical Tomography: Functional Imaging and Monitoring,
G. Muller, B. Chance, R. Alfano and e. al., eds. (Bellingham, Wash. USA: SPIE Press), pp. 3-9; Benaron and Stevenson, 1994
, Adv. Exp. Med. Biol.
361:609-617. However, current techniques are limited in that many substances do not have unique spectroscopic signals which can be optically assessed easily and quantitatively. Von Bally et al., (1982),
Optics in Biomedical Sciences: Proceedings of the International Conference
(Berlin, N.Y.: Springer-Verlag). Furthermore, the detection of substances at low concentration is frequently hampered by high background signals,
Benaron David A.
Contag Christopher H.
Contag Pamela R.
Robins & Pasternak LLP
Smith Lynette R. F.
Xenogen Corporation
Zeman Robert A.
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