Specific antibody to the native form of 2'5'-oligonucleotides, the method of preparation and the use as reagents in immunoassays or for binding 2'5-oligonucleotides in biological systems

A new specific antibody to 5'-terminal mono-, di- or triphosphorylated (2'-5')adenyl-adenosine oligonucleotides and a method of producing it have been found. The antibody can be used for the quantitative analysis of the oligonucleotides mentioned above in any one of the well known methods of immunological analysis.

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 and need careful handling, because of the 
presence of degradative enzymes which will preferentially break down the 
analyte, and because of the finite affinity of the binding protein, which 
places a limit on the attainable sensitivity. All of the above 
disadvantages are eliminated in the present invention. 
In addition to the antibody of the Kerr group mentioned above, monoclonal 
antibodies have been prepared ("Monoclonal antibodies to 
5'-triphospho-(2'5')adenyl adenosine oligonucleotides" in Proceedings of 
the National Academy of Sciences of the United States, volume 79, pages 
4742-4746, 1982, by H. Cailla, C. LeBorgne de Kauol, D. Roux, M. Delaage 
and J. Marti), which are also specific for the 2'5' linkage, but not for 
the presence of the terminal 5'-triphosphate, in spite of the promise of 
the title. The Kerr group used as immunogen (A2'p).sub.2 A, oxidized with 
periodate to generate a dialdehyde from the 2' terminal ribose, and cross 
linked this is bovine serum albumin with the aid of cyanoborohydride. 
Cailla et al prepared di-succinyl 2'5' ApA and cross linked this to human 
serum albumin with the use of carbodiimide. 
Since it is clearly preferable to obtain antibodies specific for the 
biologically active form of the molecule, it can be concluded that either 
these groups had earlier attempted this and failed, or else they did not 
attempt to use the native form of 2'5'-oligo(A) with terminal 5' 
triphosphate because they anticipated that it would be degraded in the 
immunization procedure. 
According to the invention the new antibody may be prepared by parenterally 
administering to a vertebrate living animal an immunogen consisting of an 
immunogenic substance coupled with a fully or partially 3'-ribose and 
2'-terminal ribose acylated 2'5'-oligo(A), at time intervals suitable to 
induce immunization of said animal, gathering blood which contains the 
resulting specific antibody, and obtaining antiserum from said blood by 
clotting and optionally centrifugation, or plasma from said blood by 
addition of anticoagulant and centrifugation. 
It is surprising that the antibody preparations obtained contain only a 
small fraction of molecules with the limited specificity reported in the 
above two works. 
According to the state of the art, nucleotides and oligonucleotides can be 
protected from phosphodiesterase attack by reagents which form O-ribose 
adducts. This is because of the need of the phosphodiesterase to form a 
cyclic structure through a 2' hydroxyl (in the case of the common 3'-5' 
linked oligonucleotides) as part of its mechanism, as is shown below: 
##STR2## 
See, for example, "Biochemistry. The chemical reactions of living cells", 
by D. E. Metzler. Academic Press, New York, 1977, pages 381-382.

It is surprising that O'ribose modification, which comprises the attachment 
of the 2'5'-oligo(A) via a linker which promotes its accessibility to the 
immune system of the animal being immunized, also protects the 5'-terminal 
triphosphate and permits us to raise antibodies of the specificity 
revealed in this invention. 
The ribose hydroxyls can be acylated with any suitable acidic anhydride, as 
is well known in the state of the art. See for example the patent of I. 
Yamamoto (U.S. Pat. No. 4,350,761, 1982) for the preparation of adducts 
with cyclic AMP. Examples of acidic anhydrides which are suitable for this 
purpose are e.g. dicarboxylic acid anhydrides. 
The preferred form is succinic anhydride, and formula of the resulting e.g. 
fully succinylated 2'5'-oligo(A) is as shown: 
##STR3## 
Partially succinylated molecules may also be used. 
The acyl 2'5'-oligo(A) derivative is then coupled to a suitable immunogenic 
substance with a carbodiimide reagent. Suitable immunogenic substances 
with e.g. proteins, peptides, carbohydrates and phospholipids. The 
carbodiimide reagent can be 1-ethyl-3-(3'-dimethylamino 
propyl)carbodiimide, 1-ethyl-3-(3-diethylaminopropyl)-carbodiimide, 
1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide, 
1-cyclohexyl-3-(4-diethylaminocyclohexyl)-carbodiimide or 
N.methyl-N,N'-di-tert-butylcarbodiimidium tetrafluoroborate or other 
suitable reagent. 
After the coupling reaction e.g. with succinic anhydride, the molecule may 
have the structure below, where R represents e.g. an immunogenic protein 
coupled via an available amino group: 
##STR4## 
The position of the protein is not defined by the reaction conditions, and 
in principle, any of the succinyl groups could be reacted, or even 
combinations, when sterically possible. The exact number and positions of 
the succinyl groups are also not exactly defined by the reaction 
conditions, and great heterogeneity is therefore possible. In spite of 
this, antibodies of great homogenity and specificity are obtained by the 
conditions of this invention. 
The immunogenic protein may be any suitable protein, such as bovine serum 
albumin, immunoglobulin of the same or other species as used for the 
immunization, or keyhole limpet hemocyanin. The latter is to be preferred. 
Any suitable animal can be used for the immunization, such as sheep, goats, 
horses, rabbits, rats, mice, chickens, as is commonly used. Rats yield the 
best results. The titer of the antibody in the serum is optimally assayed 
by the nitrocellulose binding assay described in the examples, using 
either [.sup.3 H]-2'5'-oligo(A) prepared according to a previously 
published procedure ("Synthesis of 2'5'-oligo(A) in extracts of 
interferon-treated Hela cells", Journal of Biological Chemistry, volume 
254, 1979, pages 5058-5064, by M. A. Minks, S. Benvin, P. A. Maroney and 
C. Baglioni) or [.sup.32 P]-pCp-2'5'-oligo(A), labelled by means of the T4 
polynucleotide kinase reaction, and commercially available from Amersham 
International. The maximum titer is reached after two booster injections. 
The anti-serum so obtained is active at high dilutions (&gt;50,000-fold), is 
stable to freezing, thawing and lyophilization, and can thus be packaged, 
shipped or stored with no difficulty. The serum is also totally free of 
any 2'5'-oligo(A) degrading activity, especially at the high dilutions 
used in the assays. This is in contrast to the cell extracts used in the 
radio-binding assay mentioned above. 
The 2'5'-oligo(A) can be extracted from biological material by any of the 
commonly used extraction procedures for making cell-free extracts, 
provided care is taken to avoid either degradation or re-synthesis under 
the extraction conditions. Homogenization with buffered saline may be 
used, but acid extraction is to be preferred. Optimally, cold 
trichloroacetic acid extraction is used, preferably in the range 5-20%. 
The trichloroacetic acid can then be removed by ether extraction, or 
preferably by freon-octylamine extraction according to a published 
procedure ("Extraction procedures for use prior to HPLC nucleotide 
analysis using microparticle chemically bonded packings", Journal of 
Chromatographic Science, volume 15, 1977, pages 218-221, by S-C. Chen, P. 
R. Brown and D. M. Rosie). This procedure is excellent for all kinds of 
biological materials, including cultured cells, organs, tissues or any 
biological fluids such as blood, urine, cerebrospinal fluid, etc. The 
extract may be lyophilized to reduce the volume and remove extraneous 
volatile material. If desired, an additional step of chromatography (High 
Performance Liquid Chromatography or conventional column chromatography) 
may be used. Stepwize chromatography on diethylaminoethyl cellulose is 
especially simple and useful for removal of the cellular ATP, which may be 
at concentrations .gtoreq.10.sup.6 -fold the analyte concentration. 
According to the invention the resulting specific antibody is used for the 
assay of 5'-terminal mono-, di- or tri-phosphorylated (adenylyl 
2'-5').sub.n adenosine in biological material by any commonly used methods 
of immunological analysis, namely by determination of free or bound ligand 
or determination of free or bound antibody or determination of 
antibody-ligand interaction by means of a signal which is modulated by the 
antibody-antigen reaction. For instance, this can be done either 
determining the reduction of binding of a known highly radioactive ligand, 
such as [.sup.32 P]-pCp-labelled 2'5'-oligo(A), after binding the 
antibody-ligand complex to a solid support, such as nitrocellulose. The 
ligand can be labelled with any suitable radioactive isotope. It is also 
possible to label the ligand with a fluorescent, enzymatic or luminescent 
moiety. The assay can also be of the sandwich type, where antibody of one 
specificity is bound to a solid support, used to bind the unknown analyte, 
and detected with an antibody of second specificity. Of this pair forming 
the sandwich, one may be the specific antibody defined by the present 
invention, and the other may be of the type described by the groups of 
Kerr or Cailla described above, or directed against the adenosine, as 
described by Erlanger and Beiser (Proceedings of the National Academy of 
the United States, volume 52, 1964, pages 68-74). The label need not be in 
the ligand for any of the above assays, but may be in one of the 
antibodies, or in a second antibody directed against one of the primary 
antibodies, as is well-known in the art. 
The preferred form of assay for detection of fmoles of 5'-terminal mono-, 
di- or tri-phosphorylated (adenylyl-2'-5').sub.n adenosine in extracts 
from biological material is the competition binding assay already 
mentioned, where the unknown is mixed with a known amount of known 
radioactive ligand, incubated with a dilution of the antibody, mixed with 
suitable buffer, poured over nitrocellulose in a filtration apparatus, and 
the excess unbound ligand removed by washing, and counting in a 
scintillation counter. The assay is fast and simple, and the reaction is 
complete in a few minutes at reduced temperature. The assay by reduction 
of binding of the labelled 2'5'-oligo(A) is as shown in FIG. 1. 
Even more sensitive assays can be constructed with the use of labelled 
antibodies. 
The main application of such assay at present lie in the field of research 
into the biochemistry of interferon action, and in the biochemistry of 
action of hormones, lymphokines, growth factors etc. The wide importance 
of 2'5'-oligo(A) is evidenced by the fact that 2'5'-oligo(A) synthetase is 
induced in a variety of systems other than interferon such as in the 
differentiating chick oviduct following estrogen withdrawal, as described 
("2-5A synthetase: assay, distribution and variation with growth hormone 
status", Nature, volume 278, 1979, pages 471-473, by G. R. Stark, W. J. 
Dower, R. T. Schimke, R. E. Brown and I. M. Kerr), or in Friend 
Erythroleukemia transformed mouse erythroblasts, triggered into 
differentiation by various reagents ("Increased levels of 
interferon-induced (2'-5') oligo-isoadenylate synthetase in mature 
lymphocytes and in differentiated Friend erythroleukemic cells", Journal 
of Interferon Research, volume 1, 1981, pages 559-569, by A. Kimchi). 
Since the real action of the synthetase depends on its induction and on 
the simultaneous presence of its activator double stranded RNA, such 
information is of limited usefulness. In the examples which follow, we 
demonstrate using the methods of this invention, that indeed, in 
interferon treated HeLa cells, with a high level of 
2'5'-oligo(A)-synthetase, the 2'5'-oligo(A) is unaltered from the basal 
level, while in the differentiating Friend Erythroleukemia cells, there is 
a parallel increase in synthetase and 2'5'-oligo(A). 
An additional use for the antibody made according to the methods of the 
present invention is as a 5'-terminal mono-, di- or tri-phosphorylated 
(adenylyl 2'-5').sub.n adenosine chelating agent. It can be added to 
cell-free protein synthesizing systems, and rescue them from the 
inhibition by added or endogenous 2'5'-oligo(A). It can also be used 
therapeutically, where it is desired to reduce the cellular levels of 
2'5'-oligo(A). 
Since 2'5'-oligo(A) is such an important newly discovered signal molecule, 
it is anticipated that the methods of this invention will lead to the 
discovery of multitudes of novel uses in research, diagnosis and therapy. 
Diagnostic applications of the 2'5'-oligo(A) synthetase for viral 
infection, leukemias, auto-immune and connective tissue diseases have 
already been explored by the group of Revel in the publication in Lancet 
(see above) and for monitoring interferon treatment, in the Journal of 
Interferon Research (also mentioned above). Wide applications of assays 
for the biologically active molecule itself, which yield more direct and 
relevant information by simpler methodology, can be anticipated in all of 
the above circumstances. 
The more sensitive assays for 2'5'-oligo(A) provided by this invention also 
permits more sensitive assays for the enzyme 2'5'-oligo(A) synthetase. 
Further, since the enzyme requires double standed RNA for activity, this 
also provides an extraordinarily sensitive assay for double stranded RNA. 
With this method, it is therefore also possible to determine the small 
amounts of double stranded RNA in biological material which would be 
necessary for the activation of the 2'5'-oligo(A)-synthetase. 
In order to explain more fully the invention, the following examples are 
set forth, it being understood that these examples are not intended to 
limit the scope of the invention. 
EXAMPLE 1 
Preparation of the specific antibody to the native form of 
2'5'-oligonucleotides 
The 2'5'-oligo(A) itself is prepared in quantity using interferon treated 
Hela cell extracts themselves prepared by published methodology (Minks et 
al., J. Biol. Chem., 254, 1979, pp 5058-5064). The 500 ml reaction mixture 
contains 5 mM ATP, 3 mM fructose-1,6-biphosphate, 100 mM KOAc, 25 mM 
Mg(OAc).sub.2, 10 mM HEPES/KOH, pH 7.4, 1 mM dithiothreitol, 10 .mu.g/ml 
poly(rI).poly(rC) and 1 mg/ml of post-mitochondrial supernatant from 
interferon treated Hela cells. The mixture is incubated for 15 hr at 
30.degree. C., at which stage conversion of ATP to 2'5'-oligo(A) is &gt;30%. 
The reaction is terminated by boiling for 3 min and denatured proteins are 
removed by centrifugation or filtration. 
The 2'5'-oligo(A) is separated from ATP and other mononucleotides by ion 
exchange chromatography on diethylaminoethylcellulose (DEAE cellulose). 
The clarified boiled extract is diluted with an equal volume of distilled 
water, and loaded on to a DEAE cellulose column equilibrated with 50 mM 
NaCl or KCl in 10 mM Tris HCl, pH 7.6, or with 50 mM NH.sub.4 HCO.sub.3 or 
triethylamine bicarbonate pH adjusted to 7.6. The column is loaded with 
100 A.sub.260 units of the extract per ml of packed column volume. 
Typically, a 400 ml column is used. The oligonucleotides are eluted with a 
salt gradient from 50 to 400 mM of the selected salt, using 4 l of 
gradient. The ATP elutes first, followed by the 2'5'-oligo(A) oligomers in 
order of increasing polymeric size. This can be determined from the 
ultraviolet absorption profile of the column effluent. The desired 
fractions are pooled, and the 2'5'-oligo(A) desalted by precipitation with 
5 volumes of acetone, or by lyophilization if the volatile buffer is used. 
The 2'5'-oligo(A) is succinylated in a reaction mixture containing 10% 
(v/v) triethylamine, 50 mg/ml succinic anhydride and 5 mM 2'5'-oligo(A). 
Typically, 5 ml reaction volume is used. Incubation is at room temperature 
for 10 min. The nucleotides are precipitated with 5 volumes of acetone, 
followed by de-salting on a column of Sephadex G25. 
The succinylated 2'5'-oligo(A) is coupled to keyhole limpet hemocyanin 
(from Calbiochem) as follows. The hemocyanin is dissolved at 10 mg/ml in a 
10 mg/ml solution of succinyl 2'5'-oligo(A), and 
1-ethyl-3(3'-dimethylaminopropyl)carbodiimide. HCl is added gradually 
while readjusting the pH to 5.5 with KOH. The condensation reaction is 
allowed to proceed overnight at room temperature in the dark. The 
conjugate is freed from reactants by dialysis against 0.15M NaCl, 0.01M 
potsassium phosphate buffer, pH 6.8 (PBS) for 2 days at 4.degree. C. with 
at least 4 buffer changes. The degree of coupling is verified by the 
spectral alteration before and after coupling; using the A.sub.260 value 
as a measure of the 2'5'-oligo(A) content; or by radioactivity, by 
inclusion of a small portion of radioactive 2'5'-oligo(A) in the original 
coupling reaction. The degree of substitution obtained is in the range 
80-100 moles of oligonucleotide per mole of protein. 
Successful immunization of rats, mice or rabbits is obtained with the 
following procedures. Primary injections are with succinyl-2'5'-oligo(A) 
conjugate in an equal volume of Complete Freund's Adjuvant and boosters 
are in an equal volume incomplete Freund's Adjuvant. The conjugate is at 1 
mg/ml in PBS. Rats are injected intraperitoneally with 0.5 mg of conjugate 
and boosted with the same amount at two weekly intervals. Mice are 
immunized similarly, but with 100 .mu.g per injection. Rabbits are 
immunized by subdermal injection of 1 mg of conjugate at five sites on the 
back, and boosted with 0.75 mg at three weekly intervals. Rats and mice 
are bled by the tail vein and rabbits by the ear vein 10 days after the 
previous injection. Rats are preferably 150-200 g females, mice 30 g and 
rabbits 3 kg. 
EXAMPLE 2 
Titer of specific antisera 
The titers of the antisera are determined from the reciprocal of the 
dilution which gives 50% retention of the radioactivity to a 
nitrocellulose filter. The antiserum dilution is incubated in 25 .mu.l of 
20 mM KOAc, 10 mM HEPES-KOH, pH 7.4, 1.5 mM Mg(OAc).sub.2 containing 1 
mg/ml bovine serum albumin (BSA) and 5-6000 cpm of [.sup.32 P] terminally 
labelled 2'5'A tetramer pCp (Amersham International: specific activity, 
approximately 3000 Ci/mmole). After incubation at 0.degree. for 1 hr, the 
mixtures are diluted with 3 ml of ice cold 90 mM KCl, 20 mM Tris-HCl, pH 
7.6 and passed through 25 mm discs of nitrocellulose (Millipore, type 
HAWP, 0.45.mu.) with a filtration apparatus. The filters are washed with a 
further 2 portions of the same buffer, and retained radioactivity counted 
by Cerenkov radiation in a scintillation counter. Maximal titers of 
50,000, 5,000 and 1,000 are usually obtained for rats, mice and rabbits, 
respectively, and after the second booster injection. 
EXAMPLE 3 
Competitive binding assays 
In the simple radio-immune assay described in Example 2, the unknown is 
determined by dilution of the radioactivity bound from the constant amount 
of known radioactive probe. Other related substances will bind to the 
antibody more or less well, depending on their relative affinities, and 
may thus also give a reduction in the bound radioactivity. This inhibtion 
is a measure of the affinity of the related compound for the antibody, and 
will also indicate to what extent such compounds might interfere with the 
assay, if present in the material being analysed. The concentration of the 
related substance giving 50% inhibition of the binding of the radioactive 
probe is used as a measure of its relative affinity. A collection of 
2'5'-adenylates and related compounds were prepared as follows. The dimer, 
trimer, tetramer and pentamer were obtained from the DEAE-cellulose 
fractionation of example 1. The tetramer was partially digested with 
bacterial alkaline phosphatase and the products separated by high 
performance liquid chromatography on a Partisil SAX column (Whatman), with 
10 mM potassium phosphate buffer, pH 6.8, 20% ethanol and a gradient from 
10 to 500 mM KCl. Acetyl and succinyl derivatives of the tetramer were 
obtained by reaction with acetic and succinic anhydride, according to the 
method of Example 1. Other compounds were from commercial sources. The 
amounts of the various compounds which gave 50% inhibition of the maximal 
binding of the [.sup.-32 P]-labelled probe (see Example 2) are listed 
below. 
______________________________________ 
fmoles 
______________________________________ 
pppA(2'p5'A).sub.4 2.5 
pppA(2'p5'A).sub.3 2.5 
pppA(2'p5'A).sub.2 2.5 
pppA(2'p5'A) 850 
2-O--succinyl pppA(2'p5'A).sub.3 
2,0 
2-O--acetyl pppA(2'p5'A).sub.3 
2.5 
ppA(2'p5'A).sub.3 12 
pA(2'p5'A).sub.3 25 
A(2'p5'A).sub.3 500 
A(2'p5'A).sub.2 500 
A(3'p5'A).sub.2 2,500,000 
pA(3'p5'A).sub.3 250,000 
AMP, ADP, ATP, GTP, 
&gt;3,000,000 
CTP, UTP, NADH, pCp 
______________________________________ 
The assay is therefore able to detect fmole amounts of oligonucleotides 
with appropriate specificity. The affinity of the antibody is so high that 
the same results will be obtained with any reaction volume between the 25 
.mu.l specified in the preceeding example and 1 ml. The affinity of the 
antibody is .about.10.sup.12 M.sup.-1. The affinity of the antibody for 
the 5'-phosphorylated forms of 2'5'-oligo(A) is 20-200 times that for the 
de-phosphorylated core. This supports the concept that the antibody is 
specific for 5'-phosphorylated 2'5'-oligo(A). 
EXAMPLE 4 
Preparation of extracts for 2'5'oligo(A) radioimmunoassays 
This is an example for cells growing in culture, but is universally 
applicable to any biological material: cell cultures, organs, tissues, 
bodily fluids such as blood, urine, cerebrospinal fluids, milk or any 
other suitably available material. Cells in a petri dish are rapidly 
washed with buffered saline (this step is dispensed with in the case of 
bodily fluids), then enough ice cold 5% trichloracetic acid (TCA) to cover 
the layer of cells (3 ml for a 7 cm diameter petri dish) and the dish is 
kept on ice for 5 min. The TCA and detached cells are removed and 
centrifuged to remove the insoluble material, and the dish and pellet are 
re-extracted with a similar volume of 5% TCA and the supernatants pooled. 
Alternatively, in the case of a cell suspension culture, the cells are 
pelleted by centrifugation at room temperature, the cells resuspended in a 
volume of buffered saline equal to the volume of the pellet, and 
approximately 10 volumes of 5% TCA are added. The insoluble material is 
removed by centrifugation and the pellet extracted with 1/2 the previous 
volume of 5% TCA. The supernatants are pooled. All operations with TCA are 
performed between 0.degree. C. and 5.degree. C. The pooled supernatants 
are freed of TCA by extraction with an equal volume of 30% (v/v) 
tri-N-octylamine in Freon 113 (Dow Chemicals). The upper aqueous phase is 
collected and the Freon phase re-extracted with 1/2 the preceeding volume 
of distilled water. Since biological material may contain ATP in the mM 
range, and it is desirable to measure 2'5'-oligo(A) in the nM range, it 
may be desirable to remove such 10.sup.6 -fold excess of ATP, which might 
interfere with the assay. The ATP and other nucleotides which might 
interfere with the assay may then be removed chromatographically with DEAE 
cellulose (Whatman DE-52) equilibrated with 125 mM NH.sub.4 HCO.sub.3 (pH 
7.8). The loading ratio is the same as for the preparative columns of 
example 1, but microcolumns of 200 .mu.l are optimal for this analytical 
scale. The column is washed with 15 ml of the same buffer to remove the 
ATP and other unbound material. The 2'5'-oligo(A) is eluted with 2 ml of 
0.5M NH.sub.4 HCO.sub.3, and the samples are lyophilized after addition of 
1/10th volume of ethanol. The samples may be stored indefinitely in 
anhydrous form and re-dissolved with suitable volumes of solvent for the 
radio-immuno assay as described in example 3. 
EXAMPLE 5 
Determination of 2'5'oligo(A) levels in differentiating cells 
Friend mouse erythroleukemia cells can be induced to differentiate and 
become erythropoietic under the influence of a variety of reagents, 
especially dimethylsulfoxide. Such cells are also known to produce the 
enzyme 2'5'-oligo(A) synthetase. Before the method of the present 
invention, it was not possible to determine the 2'5'oligo(A) directly in 
these cells. The determination is described in this example. The Friend 
cells (clone FBU or N46AP) are grown in Dulbecco's modified Eagle's Medium 
containing 10% fetal calf serum, 2% (v/v) dimethylsulfoxide for FBU or 
1.4% (v/v) DMSO for N46AP and 1 mM glutamine. The cells are diluted with 
the same medium daily to maintain a density of 5.10.sup.5 cells per ml. At 
various times, 30 ml cultures are collected and processed as described in 
the preceeding example. The lyophilized material is dissolved in the 
buffer corresponding to the reaction mixture for the assay, as in example 
2, but containing 0.1 mg/ml of bovine serum albumin. The 2'5'-oligo(A) 
content is determined from the reduction of the binding of the [.sup.32 P] 
labelled probe as in example 2 and 3. The following results are obtained: 
______________________________________ 
2'5'-oligo(A) 
Cell line Days post-DMSO 
(fmoles/10.sup.6 cells) 
______________________________________ 
FBU 0 2.6 
1 28 
2 28 
4 20 
N46AP 0 7.5 
1 7.8 
2 4.3 
3 17.5 
5 63 
6 53 
______________________________________ 
In the case of the N46AP cell line, there is a delay between the first 
appearance of the 2'5'-oligo(A) synthetase and the 2'5'-oligo(A) itself, 
while in the FBU line the two go in parallel. 
This example demonstrates for the first time the possibility of 
determination of 2'5'-oligo(A) in cells not treated with interferon. 
EXAMPLE 6 
Determination of 2'5'-oligo(A) in interferon treated Hela cells 
Cultures of Hela cells respond very well to interferon in the induction of 
2'5'-oligo(A) synthetase (see example 1). The basal 2'5'-oligo(A) itself 
was too low to be measured by other methodology. This example demonstrates 
such measurement. Hela cells of the S3 line are cultivated in the presence 
of 20 units/ml of interferon .alpha.2 for 24 hrs. The cells are 
centrifuged and processed according to example 4. The 2'5'-oligo(A) 
synthetase is assayed by methods well described in the literature in 
parallel cultures. The synthetase increases 10-fold over the basal level, 
while the 2'5'-oligo(A) itself remains at the basal level of 0.45.+-.0.15 
nM. This shows that 2'5'-oligo(A) synthetase levels are not invariably 
indicative of the biologically active species, 2'5'-oligo(A). 
EXAMPLE 7 
Enhanced sensitivity assay for 2'5'-oligo(A) synthetase 
Because of the extremely efficient reaction condition of cell-free systems, 
it is possible to assay low levels of 2'5'-oligo(A) synthetase. However, 
it is sometimes desirable to measure low levels from extremely small 
amounts of precious starting material. By coupling the extremely sensitive 
measurement of 2'5'-oligo(A) according to the methods of the present 
invention, with the enzymatic assay for 2'5'-oligo(A) polymerase, the 
enzyme can be determined with 10.sup.5 -fold higher sensitivity than was 
previously possible. In the example, Swiss mouse 3T3 fibroblast cells are 
stimulated into a cell cycle by a variety of hormones and factors (for 
literature on this subject see the article "Criteria for the establishment 
of the biological significance of ribosomal protein phosphorylation" in 
Current Topics in Cellular Regulation, volume 21, 1982, pages 89-99, by J. 
Gordon, P. J. Nielsen, K. L. Manchester, H. Towbin, L. Jimenez de Asua and 
G. Thomas). Quiescent 3T3 cells in Dulbecco's modified Eagle's minimal 
medium and 10% fetal calf serum, in 10 cm Petri dishes are treated 
overnight with various reagents. On the following day the monolayers are 
washed with phosphate buffered physiological saline and the cells 
suspended in 2 ml per dish of the same. The cells are centrifuged at 800 g 
for 5 min and the packed cells treated with twice the pellet volume of 10 
mM HEPES-KOH, pH 7.4, 20 mM KOAc, 1.5 mM Mg(OAc).sub.2, 2 mM DTT, 0.1% 
(v/v) triton X-100, and 10% glycerol, for 10 min at 0.degree.. The cell 
debris are removed by centrifugation at 30,000 g for 10 min and the 
supernatant is used as the source of 2'5'-oligo(A) polymerase. The 
supernatant (40 .mu.l, 30 A.sub.260) is mixed with 100 .mu.l of 
poly(rI).poly(rC)-agarose beads (P-L. Biochemicals) and 100 .mu.l of 20 mM 
Tris-HCl pH 7.6, 225 mM KOAc, 12 mM Mg(OAc).sub.2, 1 mM dithiothreitol, 10 
.mu.M phenylmethylsulfonylchloride and 10% glycerol. The non-adsorbed 
material is removed after centrifugation to pellet the beads and the 
agarose is washed three times with 200 .mu.l aliquots of the latter 
buffer. The 2'5'-oligo(A) synthesis is allowed to proceed overnight in the 
presence of 50 .mu.l of the latter buffer supplemented with 10 mM ATP 
containing 500,000 cpm of [.sup.3 H]-ATP. The reaction is terminated by 
addition of 150 .mu.l of 90 mM KCl, 20 mM Tris-HCl pH 7.6. The beads are 
removed by centrifugation and the supernatant divided into 2 portions, one 
for a conventional assay and the second for the radio-immune assay of the 
reaction product. One portion (198 .mu.l) is analysed for conversion of 
the ATP to 2'5'-oligo(A) from the retention of radioactivity on an 
analytical DEAE cellulose column (see example 4). The other portion (2 
.mu.l) is diluted in the range 250 to 10,000-fold and the 2'5'-oligo(A) 
determined by the radioimmune assay (see example 3). The following results 
are obtained. 
______________________________________ 
[.sup.3 H]--ATP .fwdarw. 
Radio-immune assay 
[.sup.3 H]--2'5'-oligo(A) 
for 2'5'-oligo(A), 
Treatment cpm/assay pmoles/assay 
______________________________________ 
None 556 50 
+10 .mu.M prosta- 
632 100 
glandin F2.alpha. 
+100 u/ml inter- 
4109 2000 
feron 
Background of assay 
(197) 0 
______________________________________ 
With radioactive ATP, more than 10.sup.7 cells must be extracted to obtain 
a significant signal. The sensitivity is limited by the high Km of the 
enzyme (c. 1 mM) and the background of the assays itself. In general, it 
is difficult to discriminate lower than 1/10.sup.4 of the input ATP. The 
antibody assay improves both the sensitivity and the selectivity. The 
discrimination factor between 2'5'-oligo(A) and ATP is ca. 10.sup.6 -fold, 
and this can be enhanced to 10.sup.8 -fold with a DEAE cellulose column 
step. The minimal amount of 2'5'-oligo(A) to give a signal is in the fmole 
range, considerably lower than that needed to detect the 2'5'-oligo(A) in 
the above table. The enhanced sensitivity of the radio-immune assay is 
thus 10.sup.5 -fold. It thus permits the use of smaller amounts of 
biological material, biological material with very low activity, or the 
measurement of smaller amounts of material which activitates the 
synthetase, than would otherwise be possible. 
EXAMPLE 8 
Non-isotopic immuno-assays for 2'5'-oligo(A) 
Great developments have been made in recent years in easier and safer 
alternatives to radio-immunoassay techniques. For example, such methods 
have been developed which are of great simplicity and sensitivity, based 
on nitrocellulose as a solid support and peroxidase-conjugated antibodies 
as label (see "A dot-immunobinding assay for monoclonal and other 
antibodies", Analytical Biochemistry, volume 119, 1982, pages 142-147, by 
R. Hawkes, E. Niday and J. Gordon and U.K. Patent Application No. 2099578 
"New Kits for Immunological Analysis", 1981, by J. Gordon, R. Hawkes, E. 
Niday and H. Towbin). Conjugated 2'5'-oligo(A) is prepared with bovine 
serum albumin instead of hemocyanin according to example (1). This is 
diluted to 0.2 mg/ml in TBS (0.01M Tris-Cl, pH 7.6, 0.15M NaCl), 1 mg/ml 
BSA. Rat anti-serum against 2'5'-oligo(A) prepared according to example 
(2) is diluted 1:10,000 in TBS, 1 mg/ml BSA, 0.01% Nonidet P40 (Shell 
Chemicals). Standard and unknown samples are diluted into 1 ml portions of 
the diluted anti-serum, and incubated for 1 hr at room temperature. These 
are then added to the nitrocellulose and incubated for a further 2 hrs at 
room temperature, with gentle agitation. The strips are then washed with 
TBS and incubated a further 2 hrs in 1 ml of peroxidase conjugated rabbit 
anti-rat immunoglobulins (DAKO, Copenhagen), diluted 1:500 in TBS, 1 mg/ml 
BSA, 0.01% Nonidet P40. The strips are then washed again with TBS and 
incubated in 1 ml 0.6 mg/ml chlornaphthol and 0.01% H.sub.2 O.sub.2 in TBS 
for 15 min. The inhibition of the color reaction is then taken as a 
measure of the amount of unknown 2'5'-oligo(A). A reduction of 50% is 
obtained with 100 fmoles of 2'5'-oligo(A). 
EXAMPLE 9 
Kits for assay of 2'5'-oligo(A) 
Such kits may consist of two parts: (a) for the extraction and (b) for the 
performance of the assay itself. These are provided either together or 
separately. 
(a) comprises materials for carrying out the procedure of example (4) in a 
stable, packaged form. Trichloracetic acid is provided in packs containing 
5 g, to be made up to 100 ml with distilled water. Physiological saline pH 
6.8 is provided in packs containing 0.88 g NaCl, 0.14 g KH.sub.2 PO.sub.4 
and 0.23 g K.sub.2 HPO.sub.4.3H.sub.2 O, to be made up to 100 ml with 
distilled water. Freon 113 and tri-N-octylamine are provided in ampoules 
of 35 and 15 ml, respectively, which, when mixed, provide the non-aqueous 
phase for the extraction of the trichloracetic acid. The provision of the 
latter two ingredients as part of a kit is especially useful as they are 
not generally available in biochemical or clinical laboratories. The 
latter volumes provide enough for approximately 50 assays. Disposable 
microcolumns are made by packing 0.2 ml of DEAE cellulose (Whatman DE52) 
equilibrated with 125 mM NH.sub.4 HCO.sub.3 and 0.05% NaN.sub.3 as 
preservative, in disposable polypropylene 2 ml Econocolumns (from Biorad 
Corporation). These are packed in the kit with the top sealed with the 
stopped provided and the bottom with a luer fitting. NH.sub.4 HCO.sub.3 is 
provided in packs of 9.88 g, which can be made up to 1 liter for the 
column wash buffer or to 250 ml for the elution buffer. 
(b) For a radio-immune assay, the kit contains 2'5'A tetramer triphosphate 
[.sup.32 P]Cp 3' end labelled, 25 .mu.Ci, as supplied by Amersham 
International, rat antiserum against 2'5'-oligo(A) prepared as in example 
(1). The antiserum is diluted 50,000 fold in 20 mM KOAc, 10 mM HEPES-KOH, 
pH 7.4, 1.5 mM Mg(OAc).sub.2, 1 mg/ml bovine serum albumin, divided into 
1.25 ml portions and lyophilized. Each portion provides material for 50 
assays following reconstitution. Washing buffer is made from portions of 
0.67 g KCl, 0.24 g Tris and 1.9 ml 1N HCl. The mixture is lyophilized and 
packed. Each pack provides 100 ml of wash buffer when reconstituted with 
distilled water. Alternatively, the probe may be made by conjugation of 
succinyl 2'5'-oligo(A) with tyrosine by methods known for cyclic AMP (see 
Cailla, H. L. & Delaage, M.A. in Analytical Biochemistry 48 (1972), page 
62), and labelled with [.sup.125 I] by the chloramine T method. 
The kit may also be assembled for enzyme-based immuno-assay. In its 
simplest form, multi-well dishes are provided with 3 mm.times.3 mm squares 
of nitrocellulose (Millipore type HAWG) in each well. The squares are 
prepared with 0.5 .mu.l of 1:50 dilute antiserum and saturated with 10% 
horse serum as in example (8). The remaining components of the kit are as 
described in U.K. Patent Application No. 2099758.