Patent Application
20100055725
The present invention relates generally to enzyme activity assays, and more particularly to an assay system for aminotransferases.
Several heart and liver diseases have been correlated with abnormally high levels of aspartate aminotransferase (AST) present in the blood. Examples of such conditions include pulmonary embolism, viral and toxic hepatitis, acute myocardial infarction, acute pancreatitis, and acute cirrhosis. Similarly, human alanine aminotransferase (ALT) is an enzyme that may be leaked into the blood of a patient suffering from hepatic diseases such as viral hepatitis, hepatocirrhosis, and is used as a key biological marker.
Diagnosis of serum containing ALT and AST provides a good indicator of whether a particular patient is undergoing distress due to a disease. Liver or heart disease may present elevated AST or ALT levels as an indicator. The assays for determining the activity of these enzymes generally involve extracting blood from the patient and immediately employing one of a number of calorimetric or kinetic ultraviolet techniques.
Regardless of the assay format employed to determine aminotransferase activity, it has been common practice to use these assays on venous blood drawn from the patient in a clinical setting. The assays may be performed on serum or plasma separated from the whole blood drawn from the patient. This is due to the fact that hemoglobin content from red blood cells interferes with most measurements. Thus, it is preferable to remove the red blood cells from whole blood in order to avoid excessive light absorption from this protein.
In addition, aminotransferase enzyme activity in the serum is relatively unstable as a function of time, and, for this reason, it has been common practice to analyze serum or plasma relatively quickly once the serum or plasma is separated from whole blood. This practice has meant that serodiagnosis for indications of disorders in which the aminotransferase activities are elevated have been performed in the clinical setting as opposed to a setting distant from the hospital.
Aminotransferases are enzymes that catalyze the transfer of an amino group from a donor co-substrate into an acceptor co-substrate, 2-Oxoglutarate, forming L-glutamate as one of the products of the enzymatic reaction. L-Aspartate is the amino group donor co-substrate for the reaction catalyzed by the Aspartate Aminotransferase (AST) enzyme, and L-Alanine is the amino group donor for the reaction catalyzed by Alanine Aminotransferase (ALT).
Both ALT and AST require the presence of pyridoxal-5′-phosphate (P-5′-P), a protein derived from vitamin B6, as a co-enzyme. This protein attaches to the apoenzyme (i.e., an aminotransferase without this protein) and forms the active site that transfers the amine group from one co-substrate to the other. Blood contains aminotransferases with P-5-P′ and without this coenzyme.
Standard methods for the quantification of aminotransferase activity employ secondary enzymatic reactions that provide an observable change in absorbance. The enzyme-substrate system for the secondary reaction must be abundant enough such that the two-step reaction rate is limited by the first step. More specifically, the most common detection method involves employing enzymes that use nicotinamide-adenine dinucleotide (NADH) as a co-substrate for the enzymatic reduction of the oxo-acid products of the first reaction. The progression of the reaction is monitored as a decrease in absorbance at 339-340 nm created by the consumption of the NADH co-substrate. However, absorption spectroscopy is not as sensitive as fluorometry or luminescence. For example, luminescence measurements are approximately 100 times more sensitive than absorption measurements.
Thus, a need still remains for an assay system for alanine aminotransferase and aspartate aminotransferase that employs fluorescent or bioluminescent labels that are compatible with complex biological fluids. In view of the ever-increasing activity in the biosciences, it is increasingly critical that answers be found to these problems. In view of our aging population having an increase in the occurrence of heart and liver disease, it is increasingly critical that answers be found to these problems. Additionally, the need to save costs, improve efficiencies and performance, and meet competitive pressures, adds an even greater urgency to the critical necessity for finding answers to these problems.
Solutions to these problems have been long sought but prior developments have not taught or suggested any solutions and, thus, solutions to these problems have long eluded those skilled in the art.
The present invention provides an assay system including providing a platform having a contact surface; immobilizing an amino acid group, having an amine side group, on the contact surface; transforming the amine side group, in the amino acid group to a ketone group; and reacting an indicator with the ketone group for displaying a light emission from the indicator.
Certain embodiments of the invention have other aspects in addition to or in place of those mentioned above. The aspects will become apparent to those skilled in the art from a reading of the following detailed description when taken with reference to the accompanying drawings.
The following embodiments are described in sufficient detail to enable those skilled in the art to make and use the invention. It is to be understood that other embodiments would be evident based on the present disclosure, and that process or mechanical changes may be made without departing from the scope of the present invention.
In the following description, numerous specific details are given to provide a thorough understanding of the invention. However, it will be apparent that the invention may be practiced without these specific details. In order to avoid obscuring the present invention, some well-known system configurations and process steps are not disclosed in detail. Likewise, the drawings showing embodiments of the system are semi-diagrammatic and not to scale and, particularly, some of the dimensions are for the clarity of presentation and are shown greatly exaggerated in the drawing FIGs. Where multiple embodiments are disclosed and described, having some features in common, for clarity and ease of illustration, description, and comprehension thereof, similar and like features one to another will ordinarily be described with like reference numerals.
For expository purposes, the term “horizontal” as used herein is defined as a plane parallel to the plane or surface of the substrate, regardless of its orientation. The term “vertical” refers to a direction perpendicular to the horizontal as just defined. Terms, such as “above”, “below”, “bottom”, “top”, “side” (as in “sidewall”), “higher”, “lower”, “upper”, “over”, and “under”, are defined with respect to the horizontal plane. The term “on” means there is direct contact among elements. The term “system” as used herein means and refers to the method and to the apparatus of the present invention in accordance with the context in which the term is used.
Referring now to
In this embodiment, a forward aspartate aminotransferase (AST) catalyzed reaction is measured by first immobilizing the amino acid group 108 on the contact surface 104 using a spacer molecule 114. A surface-linked enzyme substrate 112, having an aspartate molecule or an alinine molecule, includes a spacer molecule 114 and the amino acid group 108. The surface-linked enzyme substrate 112 is then exposed to a co-substrate (2-oxaglutarate) 116 mixed with the specimen containing an aminotransferase enzyme 118, such as aspartate aminotransferase (AST) or an alanine aminotransferase (ALT).
Referring now to
Referring now to
This approach to the system for assay of aminotransferase enables detection of AST and ALT within whole blood, plasma, serum or other biological fluids. Embodiments where the light emission 128 occurs without an external source of illumination are well suited for handheld instrument designs. This approach may be implemented in any format including microscope slides, arrays, well plates, microfluidic channels, filters, porous materials and any combination thereof.
Alternatively the transaminase reactions may be measured by immobilizing L-glutamate on the surface and measuring the transaminase catalyzed conversion of the glutamate into surface-linked 2-oxaglutarate by labeling the ketone groups in this molecule following the approach described in the prior example. The immobilization of one of the co-substrates in the surface enables the implementation of the measurement on spots or array of spots pre-aligned to the appropriate detection optics and detectors.
In one implementation of the assay the hydrazine indicator conjugate 124 absorbs light at wavelengths exceeding 600 nm and emits further in the red. The cyanine dye cy5.5 is an example of such a fluorophore. Other examples of red-emitting fluorescent or luminescent indicators 122 include Alexa Fluor (633 647 660 and 680), allophycocyanin (APC), APC-Cy7, Cy7, Bodipy (630/650-X, 650/665-X, 665/676), Thiadicarbocyanine, TO-PRO-3, TO-PRO-5, TOTO-3, YOYO-3, YO-PRO-3, Q-Dots 650, and others.
Referring now to
An amino acid group 206, such as L-glutamate, is coupled to the luminescent nanocrystal 204. A solution including the aminotransferase enzyme 118, acting to catalyze the transamination, an amine acceptor 208, such as oxaloacetate or pyruvate, and pyridoxal-5′-phosphate (P-5′P), a protein derived from vitamin B6, is required as a co-enzyme for both ALT and AST transamination.
Referring now to
Referring now to
The inventive approach described in this patent may be implemented in any format including microscope slides, arrays, vessels, well plates, microfluidic channels, filters, porous materials and any combination thereof.
Referring now to
In greater detail, a system for assays of aminotransferase according to an embodiment of the present invention, is performed as follows:
It has been discovered that the present invention thus has numerous aspects.
A principle aspect is that the present invention may be implemented in any format including microscope slides, arrays, well plates, microfluidic channels, filters, porous materials and any combination thereof.
Another aspect is the present invention provides a light emission in the red spectrum making the detection and differentiation of bound indicator easier to detect due to the longer wavelength.
Yet another important aspect of the present invention is that it valuably supports and services the historical trend of reducing costs, simplifying systems, and increasing performance.
These and other valuable aspects of the present invention consequently further the state of the technology to at least the next level.
Thus, it has been discovered that the system for assays of aminotransferase of the present invention furnishes important and heretofore unknown and unavailable solutions, capabilities, and functional aspects for detecting and quantifying the amount of aminotransferase in complex biological fluids, such as blood, serum, and plasma. The resulting processes and configurations are straightforward, cost-effective, uncomplicated, highly versatile and effective, can be surprisingly and unobviously implemented by adapting known technologies, and are thus readily suited for efficiently and economically manufacturing assay devices for the detection and quantification of aminotransferase. The resulting processes and configurations are straightforward, cost-effective, uncomplicated, highly versatile, accurate, sensitive, and effective, and can be implemented by adapting known components for ready, efficient, and economical manufacturing, application, and utilization.
While the invention has been described in conjunction with a specific best mode, it is to be understood that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the aforegoing description. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the scope of the included claims. All matters hithertofore set forth herein or shown in the accompanying drawings are to be interpreted in an illustrative and non-limiting sense.
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 60/829,874 filed Oct. 17, 2006 and U.S. Provisional Patent Application Ser. No. 60/829,876 filed Oct. 17, 2006.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2007/081694 | 10/17/2007 | WO | 00 | 3/10/2009 |
| Number | Date | Country | |
|---|---|---|---|
| 60829874 | Oct 2006 | US | |
| 60829876 | Oct 2006 | US |
