Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1995-12-08
2004-07-27
Saunders, David (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007930, C435S963000, C435S967000, C436S013000, C436S817000, C436S826000
Reexamination Certificate
active
06767708
ABSTRACT:
FIELD OF THE INVENTION
Biologically active steroid compounds susceptible to degradation by oxygen in aqueous protein containing media are given enhanced storage stability by the addition of transition metal chelators.
BACKGROUND OF THE INVENTION
A widely employed method of analyzing the bodily fluids of human beings for the presence or amount of both naturally occurring and synthetic biologically active compounds is by immunoassay. The interaction between the analyte of interest and an antibody which recognizes this analyte is measured. This often provides a relatively fast and inexpensive method of quantitating the amount of a given analyte. The analyte antibody reaction may be measured in a wide variety of techniques. One technique is the competitive assay in which an anti-analyte antibody is immobilized on a solid phase and then reacted with both a known amount of a labeled analyte and a sample suspected of containing analyte. The analyte in the sample then competes with the labeled antibody for binding to the immobilized antibody. The amount of label captured by immobilized antibody is then inversely proportional to the amount of analyte present in the sample.
All analytical techniques require some reference to a standard but such reference is particularly important for immunoassays. The reagents utilized in such assays include biological materials whose reactivity is not exactly reproducible but reproducible only within a given range. In addition the immunological binding between an antibody and its analyte may be influenced by subtle factors which cannot always be controlled. In this regard too rigorous attempts to obtain precise reproducibility are inconsistent with the goal of a fast and inexpensive assay.
Therefore, the practice has developed of providing one or more standards to be included with each run of an immunoassay. For instance the IM
x
® instrument manufactured by Abbott Laboratories can analyze in excess of twenty samples per run. It is typical to include several samples having a known amount of analyte in each run to provide a measure of the variability. Such standard samples are commonly known as controls.
In addition it is also typical to provide a number of samples having known amounts of analyte in order to calibrate and from time to time recalibrate the analyzer. Such standard samples are commonly referred to as calibrators.
For both calibrators and controls it is desirable to use a diluent which displays a behavior in the assay similar to that of the bodily fluid which is to be assayed for analyte. For instance, if human serum is to be analyzed the calibrators and controls may both be diluted in appropriately treated normal human serum. Alternatively an aqueous medium having a protein content similar to serum, for instance a buffered solution of bovine serum albumin (BSA) may be used.
A class of analytes of interest, the steroids, display a tendency to degrade over time in such buffered aqueous protein containing media. This tendency has been observed in both in charcoal stripped human serum and in aqueous solutions of BSA. In this regard, human serum intended for use as a calibrator or control matrix or carrier is charcoal stripped to remove any steroid which might be present. Thus the initial steroid content can be precisely controlled by simply adding a measured amount to the stripped serum.
Thus there is a need for calibrator and control solutions for steroid immunoassays which display extended storage stability under normal field conditions. In particular there is a need for such solutions which do not degrade significantly when stored for extended periods at temperatures between about 2 and 8° C. It is particularly desirable for such solutions to be stable for in excess of six months.
BRIEF DESCRIPTION OF THE INVENTION
Biologically active steroid compounds which are susceptible to degradation by oxidation in dilute aqueous protein in containing solutions which are suitable for the standardization of immunoassays of human bodily fluids are made more storage stable by incorporating a transition metal chelator in the solution. The steroids of interest include the naturally occurring hormonal steroids such as estradiol and progesterone. The chelators of interest include the tetra and higher dentate amino acetic acid chelators such as diethylenetriamine pentaacetic acid (DETP) and ethylenediamine tetraacetic acid. The aqueous media of interest include charcoal stripped normal human serum and aqueous solutions of bovine serum albumin (BSA), especially those buffered to a pH between about 6 and 9. The solutions of particular interest include those with a steroid concentration between about 2.5×10
−11
and 1.0×10
−7
g/mL and a chelator concentration of greater than about 0.1 mM, preferable between about 0.2 and 50 mM.
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Aboleneen Hoda I.
Groskopf William R.
Kuemmerle Steven C.
Williams Gregg T.
Abbott Laboratories
Anderson Regina M.
Saunders David
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