Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
1999-05-21
2001-01-09
Beisner, William H. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S288700, C435S286500, C435S287900, C435S004000, C435S013000
Reexamination Certificate
active
06171851
ABSTRACT:
The invention discloses a process and a device for determining the activity of enzymes in liquids, or the concentration and/or activity of inhibitors in liquids.
The determination of enzyme activities in extracts of plants, suspensions of bacteria, homogenates and body fluids such as blood serum, blood plasma, urine, punctate, liquor, cell lines or homogenated tissue has obtained an essential importance for diagnosis, follow up and therapy control.
For example, the discrimination between enzyme proteins and the other proteins in blood serum by chemical means is exceedingly questionable, because the concentrations of the individual enzymes in body fluids are extremely small. The concentration of glutamate-oxaloacetate transaminase in blood serum of a healthy individual is 0,1 &mgr;g/ml, for example. As for comparison, the total protein concentration in blood serum is in the range between 60 to 80 mg/ml, that is, the ratio of these two concentrations is about 1:700.000. Since the determination of the enzyme concentration by chemical means is questionable, the activity of the enzyme is calculated from the rate of its reaction with a suitable substrate.
The so called ELISA assay is known from clinical chemistry. This immunoassay measures the concentration of an enzyme and its corresponding enzym-inhibitor complex present in a tissue or any other sample. Measuring the enzyme activity by this method is impossible, as it measures concentrations regardless to the actual status of the enzyme, active or inhibited.
According to known techniques the activity of an enzyme is measured in such cases by removing at first the enzyme inhibitors corresponding to the enzyme and afterwards determining the activity of the enzyme. The known processes are very laborious, referring to the samples to be measured being tissues as well as to the method, how to remove the enzyme inhibitors from the sample. This method comprises adding to the sample a substance capable of binding the enzyme inhibitors, incubating the so manipulated sample during a definite time to complete the binding of the enzyme inhibitors along with as much homogenous mixing as possible of the sample with the said substance and finally, separating the so manipulated sample from the said substance.
Starting from this point, the purpose of the present invention is to disclose a process and a device for measuring the activity of enzymes in fluids by running the method of measurement largely or completely automatically and also effectively, and for determining the concentration and/or activity of inhibitors in fluids additionally or alternatively.
The inventive process manages the above mentioned problem according to the features of the claims
1
or
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. Accordingly, a process is arranged for measuring enzyme activities in fluids, which comprises withdrawing enzyme inhibitors, that correspond to at least one of the enzymes in the sample, or enzymes, that correspond at least to one inhibitor, adding a substrate to the sample manipulated in this manner, so as to get cleavage products from the substrate by reacting with the enzyme, and detecting the increasing concentration per unit of time of at least one of these cleavage products during an incubation time. The said process is characterized by withdrawing the enzyme inhibitors or enzymes from the sample by means of chromatography.
According to the said invention, it was discovered, that it is possible to remove the enzyme inhibitors from the sample without as much homogenous mixing as possible of the corresponding substance and the sample, and without a following laborious separation process. In connection with this, it was discovered, that one can perform mixing and separation virtually in one step. The said invention has the special advantage, that it enables one now to measure enzyme activities in fluid samples, that is, all kinds of body fluids as well as homogenated tissues, whereby the withdrawing of the enzyme inhibitors by means of chromatography makes it possible, to run the process largely automatically.
For that purpose, the sample is passed in a useful manner through a column filled with a chromatographic carrier, that is treated with a substance capable of binding the enzyme inhibitors. As a result, the enzyme inhibitor is getting concentrated on the column, so as to achieve at the same time an isolation method for these enzyme inhibitors, in a sort of way a side effect of the inventive process.
In order to correlate the results of different measurements, it is necessary to keep to definite experimental conditions. This can be done by diluting the manipulated sample, that is the sample released from the enzyme inhibitors, with a suitable column buffer in a definite manner. Moreover, one may admix a suitable measuring buffer to the sample depending on experimental conditions and the enzyme activities to be measured. In a convenient variant of the inventive process, the assay, that is, the mostly diluted sample together with the test substrate, which reacts with the enzyme to be measured to yield cleavage products, is thermostated during the incubation time.
On principle, there are various possibilities, to detect the concentration increase per unit of time of at least one of the cleavage products of the substrate during the incubation time. It is especially useful, to detect the increase of the concentration by means of fluorescence measurements, as in this way one can observe the concentration increase during the reaction time particulary well.
Moreover, the purpose of the present invention is accomplished by a device in order to measure the activity of enzymes in fluids according to the feature of claim
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. Accordingly, there is provided a column filled with a chromatographic carrier, which is treated with a substance capable of binding enzyme inhibitors corresponding to at least one of the enzymes present in the sample. A sample supply tube is connected in series to one end of the column. To the other end of the column a valve/pump arrangement is connected in series, by which one can fill a substrate as well as at least a portion of the sample into the test tube. Finally, there is provided a detector for measuring the concentration increase per unit of time of at least one of the cleavage products of the substrate.
According to the said invention, it was discovered, that one can run the process of determining enzyme activities and activities and/or concentrations of inhibitors in fluids, in an arrangement consisting of devices connected in series. This process is also characterized in that by means of chromatography enzyme inhibitors corresponding to an enzyme are withdrawn from the sample. In order to do this, the sample is directed successively through the different stations of the arrangement, so that one needs not withdraw the sample at any of these stations in order to manipulate it especially. In particular it is convenient, that the measurements are carried out automatically, as the individual components of the arrangement are steered and controlled automatically.
An effective arrangement of the inventive device allows one to use the column repeatedly; that is, one can measure several samples serially. For that purpose the column contains an excess of the substance capable of binding the enzyme inhibitors in the various samples. Definitely, the capacity of the column is only limited by the amount of excess of this substance.
Moreover, it is convenient, that the column is exchangeable. In this case an used up column can be replaced by a new one. On the other hand, one can prepare the inventive device for different measurements, that is, for measuring activities of different enzymes. For in that case one has to remove different enzyme inhibitors from different samples. Therefore, one has to prepare the column with different substances.
It is convenient with regard to the measuring process running automatically, when the sample supply tube can be fed alternatively from a sample supply, such as a rondel, or from a reservoir containing column buffer. The column buffer i
Schumacher Johannes
Werle Bernd
Beisner William H.
Fitch Even Tabin & Flannery
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