Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1984-11-09
1988-05-10
Nucker, Christine M.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 4, 435 7, 435 68, 43524027, 4351722, 435810, 435948, 436501, 436547, 436548, 436808, 530387, 530388, 530808, 530810, 536 27, C12Q 168, G01N 3353, G01N 33577, C08G 7704
Patent
active
047435399
DESCRIPTION:
BRIEF SUMMARY
The invention relates to a specific antibody to the native form of 2'5'-oligonucleotides; the method for preparing immunogens by coupling oligonucleotides with 2',5' phosphodiester linkages, instead of the common 3'5', and carrying 5' terminal triphosphate to an immunogenic substance in such a way that antibodies of extremely high affinity and specificity can be obtained; and their use as part of highly sensitive immuno-assays for 2'5'-oligo(A) in its native 5'-triphosphorylated form or for 5'-terminal mono- or diphosphorylated (2'-5')adenyl-adenosine oligonucleotides, provided the antibody, has an affinity of 1/100 or more of the affinity to 5'-(phospho).sub.3 (adenylyl 2'-5').sub.2 adenosine; or for depleting biological systems of these substances.
Prior Art: The oligomeric series of polynucleotides, known collectively as 5'-triphospho-(adenylyl 2'-5')adenosine, short name 2'5'-oligo(A), have the structure shown below. ##STR1## The trimer is shown, and the repeat unit is enclosed in dotted lines.
These compounds have been recently discovered and have aroused a great deal of interest because of their association with interferon, and because they represent an entirely novel class of messenger molecules. The literature concerning this subject has been recently reviewed ("The Interferon Renaissance: Molecular Aspects of Induction and Action". Microbiological Reviews, volume 45, pages 244-266 (1981), by M. Minks and J. Gordon). Most of the inferences concerning the biological action of 2'5'-oligo(A) have been indirect, and relied on the measurements of the enzyme 2'5'-oligo(A) synthetase, which is induced in interferon treated cells. The enzyme can readily be measured as it can be made to function very efficiently in cell extracts in the presence of its activator, double stranded RNA. Such measurements are of potential diagnostic usefulness, since elevated levels are found in a variety of diseases, as well as in response to interferon treatment (Lancet ii, 8745, pages 497-499, 1981 by A. Schattner, G. Merlin, S. Levin, D. Wallach, T. Hahn and M. Revel and Journal of Interferon Research, volume 1, 1981, pages 587-594, by A. Schattner, G. Merlin, D. Wallach, H. Rosenberg, T. Bino, T. Hahn, S. Levin and M. Revel, respectively). Furthermore, a patent application has been filed protecting the use of such enzyme assays (German application DE 30 15 462, by M. Revel, A. Kimchi, L. Schulman and D. Wallach). The usefulness of the assay for 2'5'-oligo(A) synthetase is limited by the fact that it is indirect, and the biological active species is 2'5'-oligo(A) itself. The synthetase does not always faithfully reflect the cellular content of 2'5'-oligo(A), as can be seen from example number 6 of this application.
Some attempts have been made to deal with this problem by developing assays for 2,5'-oligo(A) itself. This can be done by measurement, in extracts of cells, of the ability of the 2'5'-oligo(A) to inhibit protein synthesis or activate an endonuclease (see Methods in Enzymology, volume 79, 1981, pages 199-208, by R. G. Williams, R. E. Brown, C. S. Gilbert, R. R. Golgher, D. H. Wreschner, W. K. Roberts, R. H. Silverman and I. M. Kerr). However, these methods are indirect, cumbersome, not suitable for routine analysis, and involve the use of unstable reagents. The Group of Kerr therefore developed improvements including a radio-immune and a radio-binding assay (See Methods in Enzymology, volume 79, 1981, pages 216-227, by M. Knight, D. H. Wreschner, R. H. Silverman and I. M. Kerr). Their radio-immune assay was dependent on the use of an antibody which was insensitive to the presence of the terminal 5'triphosphate essential for biological activity. Their radio-binding assay was based on the binding of 2'5'-oligo(A) to the above nuclease. This assay has been commercialized by Amersham International, using rabbit reticulocyte lysates as the source of the binding protein. While this assay has a sufficient degree of specificity, it is of limited usefulness because of the use of biochemical preparations which are unstable a
REFERENCES:
Chemical Abstracts, vol. 99(25), Abstract No. 209018a, 1982.
Chemical Abstracts, vol. 98, Abstract No. 51665b, 1983.
Munns et al., Biochemistry, vol. 21, 2929-2936 (1982).
Cailla et al., Proc. Natl. Acad. Sci. USA, vol. 79, 4742-4746 (1982).
Gordon Julian
Minks Michael A.
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