Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1996-10-24
2004-04-20
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S006120, C435S007500, C435S969000, C422S082010, C422S098000, C436S517000, C436S518000, C436S161000
Reexamination Certificate
active
06723516
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to chemical methods for detecting preselected analytes in a flowing liquid stream. More specifically, the invention relates to a novel method for continuously monitoring the eluant from a liquid chromatography system so as to enable detection and quantitation of an analyte or analytes contained therein.
BACKGROUND
Processing biological materials often involves the use of liquid chromatography to separate and harvest a cellular product or metabolite of interest from cellular debris or cell media in a fermentation vessel. Typically, extracting the product of interest is accomplished using a series of chromatographic separations. In general, the chromatographic eluant is monitored using an ultraviolet-visible (UV-VIS) light-range spectrophotometer, and fractions which are thought to contain the product of interest are collected and pooled. These pooled fractions are further analyzed using any of a variety of biochemical analyses, such as Western blotting, SDS-PAGE, ELISAS, protein sequencing, or the like, in order to, first, determine if they correspond to the presence of the target analyte and, second, quantitate the analyte identified. The use of such a procedure to harvest a cellular product of interest, using well-known and characterized process steps, is time consuming and costly in terms of labor and production. In addition, the development of new process methodologies for separating and harvesting new compounds is compromised by the relatively slow process of resolving and identifying the specific compounds.
Previous attempts to develop post-column on-line detectors have been directed at analytical liquid chromatography and not preparative chromatography. In general, such detectors consist of two cells. The first cell is an enzyme reactor-detector in which a specific enzyme, for which the substrate is the target analyte, is immobilized on a solid-phase support. An enzyme-catalyzed reaction proceeds within this enzyme reactor cell between the immobilized enzyme and the substrate contained in the chromatography eluant, thereby generating an electroactive species such as hydrogen peroxide. The electroactive species is then detected in a second reactor cell which contains, for example, an electrochemical detector which makes an amperometric determination of the electroactive species. Thus, the immobilized enzyme contained in the first cell may be one or more of any number of specific oxidases which react with sugars, acids or alcohols to generate hydrogen peroxide.
Such a detection system utilizes both the specificity of immobilized enzymes and the high sensitivity of electrochemical detection; however, this system has certain deficiencies. For example, such a configuration requires the eluant to flow into two cells rather than one cell. In addition, the detection of the target analyte is indirect since it is based on the generation of an electroactive species which in turn is a product of the reaction between the target analyte and the enzyme. Non-specific electrochemical signals, i.e., background noise, may be generated by other cellular or media components which are capable of eliciting an amperometric response. The incorporation of an enzyme in the first cell dictates that special buffers must be used.
Moreover, users of the combined enzyme-electrochemical detection system are faced with a limited number and availability of useful enzymes. Such restricted availability of enzymes with desired specificities constrains this mode of detection with respect to the number and type of analytes that can be detected. Furthermore, the enzyme-catalyzed reaction must generate a species that is electroactive. In general, the time required for the analyte to react with the enzyme and then generate the electroactive species is on the order of minutes.
In other systems where enzymes are not utilized, the detection cell often incorporates an immobilized protein which binds to the target compound. However, in order to detect that binding event a secondary label, such as a fluorescently labeled antibody or antigen, is introduced. Such systems are usually not coupled with preparative chromatography systems as the target analyte is chemically altered by the fluorescent tag. In addition, as in the enzyme reactor detectors, these measurements are not made on a continuous basis.
Mass biosensors have been used to measure microquantities of biological materials, and involve the use of a modified surface which selectively binds a particular component. As explained in commonly assigned U.S. Pat. No. 5,130,257 to Baer et al., European Patent Publication No. 416,730 (inventors Tom-Moy et al.), U.S. Pat. No. 5,306,644 to Myerholz et al. and co-pending U.S. patent application Ser. No. 08/167,273, filed Dec. 13, 1993 (entitled “Method and Reagents for Binding Chemical Analytes to a Substrate Surface, and Related Analytical Devices and Diagnostic Techniques,” inventors Tom-Moy et al.), a preferred type of mass biosensor uses a piezoelectric crystal as an acoustic waveguide. These sensors operate on the principle that changes in the amount of mass attached to their surface cause shifts in the resonant frequency. A typical device is constructed on a piezoelectric substrate and has interdigital input and output transducers defined by precise electrode fingers. Selective mass detection with such devices is achieved by coating the surface of the device with a chemically reactive layer that preferentially reacts with the substance to be detected, such that the mass present on the reactive layer changes proportionately, i.e., relative to the amount of the substance to be detected. These devices thus function as chemical sensors that can measure the concentration of analytes in a solution.
For example, and as explained in U.S. Pat. No. 5,306,644 cited above, piezoelectric surface wave devices have been used to measure the concentration of a specific antigen in solution using a conventional assay format, as follows. The mass-sensitive surface of the device is coated with a receptor layer which contains the antibody corresponding to the antigen, thereby forming a sample-sensing device. A reference device is also used which does not contain the antibody in the receptor layer. The devices are then exposed to a sample solution, and antigen present in the solution will bind to the receptor layer of the sample-sensing device, thereby increasing the mass loading of the surface. Radio frequency energy coupled into the device through an input transducer, such as an interdigital transducer (IDT), is converted to a surface acoustic wave confined to within about a few wavelengths of the surface. The velocity of the surface acoustic wave will vary according to the mass loading on the top surface of the device. The surface acoustic wave propagates along the surface of the device until it encounters the output transducer, such as an IDT, which converts the surface acoustic wave back into radio frequency energy. The change in propagation velocity of the surface acoustic wave corresponds to the mass bound to the surface of the crystal. The output frequency is converted to a voltage which is proportional to the phase difference between sample and reference devices. Such acoustic waveguide devices can utilize different wave motions, including surface transverse waves (STWs), Rayleigh waves (SAWs), Lamb waves, and surface-skimming bulk waves (SSBWs), although STW devices are preferred.
The present invention addresses the deficiencies in the art described above by providing a method for detecting a specific product in the eluant of a liquid chromatography system. The method employs a piezoelectric mass biosensor for continuous on-line monitoring of preselected analytes in a flowing liquid stream.
SUMMARY OF THE INVENTION
The present invention provides a method which yields accurate detection and quantitation of an analyte or multiple analytes in a flowing liquid stream, wherein the flowing liquid stream is obtained from any means by which constituents of a mixture are resolved to
Baer Richard L.
Doherty Thomas P.
Spira-Solomon Darlene J.
Tom-Moy May
Achutamurthy Ponnathapu
Agilent Technologie,s Inc.
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