Ouabain immunoassay and kit and ouabain labeled with 125I or...

Details Ouabain immunoassay and kit and ouabain labeled with 125I or... Ouabain immunoassay and kit and ouabain labeled with 125I or...

1999-01-20

2001-02-20

Le, Long V. (Department: 1641)

Chemistry: analytical and immunological testing

Optical result

With fluorescence or luminescence

C436S093000, C436S519000, C436S815000, C436S822000, C436S501000, C436S820000, C436S811000, C436S817000, C436S526000, C436S518000, C436S524000, C436S537000, C435S007210, C435S007240, C435S007900, C435S007920, C435S007930, C435S007250, C435S004000, C435S005000, C435S007800, C435S007100, C435S007200, C435S007940, C435S007950, C530S388240, C530S388250, C530S389200, C530S389300, C530S389800, C530S388900, C536S024310

Reexamination Certificate

active

06190921

ABSTRACT:

This application is based on International Application Serial No. PCT/FI98/00167, filed Feb. 25, 1998, which claims priority to Finland Application Serial No. FI 970801, filed Feb. 26, 1997.
The present invention deals with the use of a reactive compound in immunological analyses as a tracer, which compound comprises ouabain or an analog thereof attached to a compound labeled by a detectable radioiodine or fluorogen. The invention covers also the methods in which the reactive compounds are used in the measurements of the concentrations of ouabain, ouabain isomers, ouabagenin or ouabagenin isomers in biological fluids, especially in plasma for the diagnoses of cardiovascular and endocrine diseases and other harmful conditions.
Sodium-potassium-adenosine triphosphatase (Na—K—ATPase) is a cell membrane enzyme, the main function of which is to transport the sodium ion to the extracellular space. Since it is known that cardiac glycosides inhibit Na—K—ATPase (Goto et al., Pharmacol. Rev. 44;377-399, 1992), it has been assumed that the compounds resembling glycosides act as physiological natriuretic hormones. The search for natriuretic hormones has led to the purification and chemical characterization of a cardiac glycoside ouabain from human plasma (Hamlyn et al Hypertension 10, [Suppl I]; I-71-I-77, 1987; Hamlyn et al. Proc. Natl. Acad. Sci. USA 88:6259-6263, 1991; Ludens et al., Hypertension 17:923-929, 1991). Ouabain or a structurally closely resembling compound has also been isolated from the mammalian adrenal cortex (Shaikh et al. J. Biol. Chem. 266: 13672-13678, 1991) and from hypothalamus (Alaghband et al., J. Endocrinol. 98:21-226,1983). High levels of ouabain have been found in the adrenals, and adrenalectomy or changes in the intake of electrolytes lead to changes in plasma ouabain levels (Ludens et al., Hypertension 19:721-724,1992; Hamilton et al., Current Opinion in Endocrin. and Diabetes 1:123-131, 1994; Laredo et al., Endocrin. 153:794-797, 1994). However. the measurement methods used in the above mentioned studies have not been reliable. Ouabain isolated from human plasma or bovine hypothalamus may differ slightly from plant-derived ouabain, although they have similar characteristics in liquid chromatography and mass spectroscopy (Zhao et al., Biochemistry 34:9893-9896, 1995).
Plant-derived ouabain has been used as a drug named G-strophantin for the treatment of heart failure. Since there is ouabain also in the human body, it was assumed that the secretion of ouabain could be elevated in cardiovascular diseases, for example. Now it is clear that the plasma levels of ouabain are very low and their reliable measurement is problematic. It is possible to reach the necessary sensitivity with radioimmunological analyses, but high sensitivity leads to unspecific reactions. The first radioimmunoassay of ouabain was published in 1986 (Masugi et al., Biochem. Biophys. Res. Commun. 135:41-45, 1986). The method used a polyclonal antiserum and tritiated ouabain as the tracer. Later several researchers have developed immunoanalyses for the measurements of ouabain in body fluids (Hamlyn et al. Proc Natl Acad.Sci.USA 88;6259-6263; Naruse et al., Hypertension 23, [Suppl I]; I-102-I-105, 1994; Harris et al., Hypertension 17:936-943,1991) However, the plasma ouabain levels reported have varied considerably, from below 5 (Lewis et al., Hypertension 24:549-555, 1994) to 440-530 pmol/l (Gottlieb et al., Circulation 96:420-425, 1992; Rossi et al., J. Hypertension 13(10):1181-1191, 1995), when the samples are extracted by a solid phase system. Thc high variability shows clearly that there is a need for a sensitive and reliable measurement method of ouabain for clinical and physiological studies and for diagnostic purposes.
Zhao et al., (Biochemistry 34:9993-9896, 1995, recently showed that naphtolyated ouabain-like compounds isolated from human plasma or from bovine hypothalamus differ from autenthic ouabain in a reverse phase HPLC. The naphtolyated compounds had a different CD spectrum from naphtolyated authentic ouabain. They concluded that the previously isolated compound is a new isomer of the plant-derived ouabain, in which there is a different attachment for hydroxyl groups and/or a different stereochemistry in the steroid moiety. It is notable that the native isomer does not differ from the plant-derived ouabain in mass spectroscopy or in chromatographical and immunological characteristics, whereby immunoanalysis of ouabain can be used for measurement of an endogenic compound.
When ouabain was isolated as a candidate for an endogenous natriuretic hormone, plasma levels of ouabain were measured by an ELISA method (Hamlyn et al., reference above). By using this method human plasma samples extracted by a resin were found to contain 138 pmol/l of ouabain immunoreactivity. Later even higher plasma ouabain levels have been reported, but the nature of the immunoreactivity was not studied (Gottlieb et al., Rossi et al. references above). The immunoreactive material extracted from large plasma volumes behaved identically with the authentic ouabain in two different reverse phase HPLC systems (Harris et al., reference above). Four other subsequent studies were however not able to confirm this result The presence of ouabain could not be shown when plasma samples were extracted by solid phase methods and measured by two different ELISA methods with a sensitivity of 30-60 pmol/l (Gomez-Sanches et al., Am J. Hypertension 7:647-650,1994) or by a radioimmunoassay with a sensitivity of 75 pmol/l (Doris et al. Hypertension 23(5):632-638,1994). Nor did HPLC analyses with larger plasma volumes give any evidence for the presence of ouabain in human plasma (Lewis et al., Doris et al., see above). Very low plasma ouabain levels have also been documented, but the serial dilutions of plasma extracts were not comparable with those obtained with ouabain (Worgall et al. J Hypertension 14(5):623-629, 1996) and with HPLC analyses detectable amounts of immunoreactivity could not be shown when test eluated with authentic ouabain (Worgall et al., Gomez-Sanches et al., see above). Based on the facts presented above it appears clear that the low sensitivity of the immunoassays using tritiated ouabain or of the ELISA methods used so far is the main reason why the results of the above mentioned studies have led to the conclusion that there is no ouabain in human plasma.
The purpose of the present invention is to achieve a sensitive and reliable measurement method for plasma ouabain or for its analogs to be used in the immunoanalyses of ouabain in the diagnoses of e.g. diseases related to heart failure. This purpose has been fulfilled according to the invention by using polyclonal antisera of high affinity and radioiodinated or fluorogen labeled ouabain compounds of high specific activity and reverse phase separation with C
18
resins to achieve a very sensitive immunoanalysis, which can be used for routine measurements of endogenous ouabain or its analog.
The essential features of the invention have been presented in the enclosed patent
In the course of the invention it was found, somewhat surprisingly, that the use of radioiodine, especially of
125
I, or a fluorogen, especially of a lanthanide such as Europium instead of
3
H dramatically improves the sensitivity of the ouabain radioimmunoassay. Other isotopes of iodine such as
131
I can also be used, sine the isotopes are chemically similar. The ouabain compound labeled by
125
I or by Eu-chelate had clearly better sensitivity—about 100-fold higher than the previously used ELISA methods or radioimmunoassays using
3
H tracers. In place of the Eu chelate Samarium or Terbium chelates could be used, since their chelate residues are similar to that in the Eu chelate. The radioimmunoassay presented in the invention is able to detect ouabain levels of 0.5 fmol/assay tube. The previously described most sensitive methods detect 10-20 fmol/assay tube or well (Harris et al., see above). The method presented in the invention combined with C
18
reverse phas

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