Chemistry: analytical and immunological testing – Composition for standardization – calibration – simulation,...
Reexamination Certificate
2001-10-27
2003-10-21
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Composition for standardization, calibration, simulation,...
C435S005000, C435S007920, C435S007900, C435S007100, C436S518000, C436S018000, C436S017000, C436S016000
Reexamination Certificate
active
06635485
ABSTRACT:
FIELD OF THE INVENTION
The present invention concerns calibrators or calibration solutions which are based on a human serum matrix and which are used in a method for detecting cytokeratin, a process for producing them, a method for stabilizing cytokeratin in human serum and an immunological method for detecting cytokeratin in a sample.
BACKGROUND OF THE INVENTION
Immunological detection methods have become very important in diagnostics. They are characterized by a high specificity and sensitivity and hence they are also very suitable for detecting low concentrations of analytes in biological fluids. Immunological detection methods are of major importance particularly in the fields of infectious diseases, fertility and thyroid diagnostics, for metabolic diseases and for diagnosing tumour diseases. At present carcino-embryonic antigen (CEA), alpha-fetoprotein (AFP), prostate-specific antigen (PSA) and cytokeratins (CK) are for example among the most significant tumour markers.
Human cytokeratins are building blocks of the intermediary filaments which are the major components of the cytoskeleton of epithelial cells. More than 19 cytokeratins are known of which cytokeratins 1 to 8 are referred to as basic cytokeratins and cytokeratins 9 to 19 are referred to as acidic cytokeratins. The cytokeratins can aggregate in the cell to form tetramers. A tetramer consists of two basic and two acidic cytokeratin molecules in each case. Linear aggregation of tetramers results in the formation of filaments. Intact cytokeratin molecules are water-insoluble as integral components of the intermediary filaments of epithelial cells. The complexity and composition of the cytokeratins differs in the various epithelial tissues i.e. epithelial cells have cytokeratin compositions that are typical for the respective tissue. The soluble fragments of cytokeratin 19 (CK 19) which are also referred to as CYFRA 21-1 are particularly relevant for tumour diagnostics. A concentration of CYFRA 21-1 that is increased in comparison to healthy persons indicates the presence of a tumour disease. Increased values have previously been found in the following tumours: bronchial carcinoma, ovarial cancer, cervix carcinoma, bladder carcinoma and in tumours of the head and neck. The main indication for CYFRA 21-1 is to monitor non-small cell bronchial carcinomas.
A method for detecting CK 19 by a sandwich ELISA technique is described for example in EP-A-0 460 190. In this method the body fluid sample to be examined is incubated with at least two receptors R1 and R2, R1 and R2 being monoclonal antibodies which each detect different epitopes of CK 19. One of the antibodies is labelled with biotin and the other carries a different label. The sandwich complex comprising R1, CK 19 and R2 binds to a solid phase coated with streptavidin. After separating the solid from the liquid phase, the label is measured in one of the two phases and preferably in the solid phase. The tumour marker CK 19 can be detected in the sample on the basis of the signal that is obtained.
When carrying out such a test it is important that the measured value that is obtained can be at least classified qualitatively as positive (tumour marker is present) or as negative (the tumour marker is not present). This applies especially to the classification of measurement data that are obtained by means of automated systems. It is often also desirable or necessary to quantify the concentrations of tumour marker. Hence the test system must be calibrated with reagents that contain a defined concentration of analyte before carrying out the measurements. These defined reagents are referred to in the following as calibrators. The terms calibration solution, calibration standard, standard solution or control are used synonymously for the term calibrator.
An important requirement for a calibrator is high stability. On the one hand it is necessary to ensure the accuracy of the test, an essentially 100% recovery of the analyte in the calibrator and a good signal to noise ratio. On the other hand a reliable reproducibility of the result of the determination must be guaranteed over a long time period. Hence calibrators must be insensitive to their environmental conditions over a time period of several weeks i.e. to temperature, direct solar radiation on the laboratory bench, pH value, buffer conditions etc. If the conditions are unfavourable there is a risk of hydrolysis, proteolysis or denaturation of the calibrator. The use of a calibrator that is no longer intact would lead to erroneous measurements.
Many tests are carried out in serum samples. In order to ensure comparability of the measurements, the calibrator should therefore also be based on a serum matrix. The more sensitive the measuring system, the larger the differences in measurement will become due to the jump in the matrix between sample and calibrator. It has been shown that cytokeratin is unstable in a serum matrix. When a calibrator containing cytokeratin is stored in a liquid state, the cytokeratin is destroyed and denatured and hence such a calibrator cannot be used for a long time period.
Hence in the past the serum matrix was replaced by an artificial matrix the composition of which mimics that of serum. The artificial matrix is generally based on a buffer to which various salts and proteins (for example bovine serum albumin) are added in order to simulate as closely as possible the natural human serum environment with regard to salt and protein concentration as well as pH value. Although this enabled the analyte cytokeratin to be stabilized in the calibrator, a disadvantage of this procedure is that comparability is not optimal especially with sensitive measuring systems since the underlying matrix of the samples is human serum. There is therefore a jump in the matrix. This can lead to measuring errors with samples in the low measuring range near to the cut-off value. In extreme cases this could lead to a disparity between the value determined for a positive cytokeratin signal using the calibrator based on an artificial matrix and the value determined for a human serum sample even when the cytokeratin analyte is present in each case at a comparable concentration.
DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS
Hence the object was to provide a calibrator for methods for detecting cytokeratin which is based on a serum matrix and preferably a human serum matrix and which is stable for a period of at least several weeks even under unfavourable ambient conditions.
The object is achieved by a calibrator whose essential components are serum, preferably human serum, cytokeratin and aprotinin. It surprisingly turned out that the addition of the protease inhibitor aprotinin can effectively stabilize cytokeratin. Consequently it is possible to provide a calibrator for cytokeratin detection methods which is manufactured on a natural serum matrix basis and in particular is based on a human serum matrix. This avoids a jump in the matrix when measuring the actual serum samples.
Furthermore it turned out that the calibrator according to the invention has a long shelf life and temperature stability. Thus the recovery rate for the cytokeratin analyte dissolved in the calibrator, preferably CK 19, is almost 100% in a sandwich immunoassay after stressing the lyophilised calibrator for three weeks at 30 to 40° C. Thus the cytokeratin is not destroyed and is still immunologically recognized by the antibodies used in the test even after the time-temperature stress. The measured concentrations of cytokeratin in the various calibrators measured at the start of the stability test essentially correspond to the concentrations after the stress test. Hence the stability and precision of the calibrator according to the invention is comparable with the stability of a corresponding calibrator based on an artificial matrix. Even when the calibrator is stored at 2 to 8° C. in a liquid or reconstituted form which corresponds to the usual storage temperature of reagents in a refrigerator, a cytokeratin recovery of almost 100%
Bodenmueller Heinz
Franken Norbert
Kyriatsoulis Apostolos
Moessner Ellen
Pappert Gunter
Davis Deborah A.
Le Long V.
Roche Diagnostics GmbH
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