Monoclonal anti-ornithine decarboxylase antibody and method of producing same

Monoclonal anti-ornithine decarboxylase antibody is produced by cell hybrids between hypoxanithine phosphoriboxyltransferase deficient myeloma cells and spleen cells derived from an animal previously immunized with ornithine decarboxylase. By means of immunoaffinity chromatography using cyanogen bromide-activated agarose coupled with the antibody, ornithine decarboxylase in animal cell extracts can be highly purified in high yield.

SUMMARY OF THE INVENTION 
This invention relates to monoclonal anti-ornithine decarboxylase antibody, 
to novel hybridoma cells which express such antibody, and to a method for 
producing such hybrid cells and anti-ornithine decarboxylase antibody. 
BACKGROUND OF THE INVENTION 
Ornithine decarboxylase (ODC) is the initial step in the mammalian 
polyamine biosynthetic pathway (Pegg and Williams-Ashman, Polyamines in 
Biology and Medicine, pp. 3-42, Marcel Dekker, New York (1981); Pegg and 
McCann, Am. J. Physiol. 243: C212-C221 (1982). This enzyme exhibits rapid 
and many fold changes in activity in response to a wide variety of stimuli 
and there is evidence that stimulation may be linked to cell growth and 
tumor promotion (Boutwell et al., Adv. Enzyme Reg. 17: 89-112 (1979)); 
Russell, Pharmacology 20: 117-129 (1980); Pegg and McCann, (1982 supra.). 
Detailed studies of the underlying biochemical mechanism of the regulation 
of ornithine decarboxylase have been hampered by the small amounts of this 
protein present in mammalian tissues (Pritchard et al., Biochem. Biophys. 
Res. Commun. 100: 1597-1603 (1981)); Seely et al., Biochem. J. 206: 
311-318 (1982) and the consequent difficulty in obtaining the purified 
protein. Recently, several groups have described the purification of 
ornithine decarboxylase to homogeneity from rat liver (Kameji et al., 
Biochem. Biophys. Acta 717: 111-117 (1982)); Kitani, et al, J. Bio. Chem. 
258: 235-239 (1983)) or mouse kidney (Persson, Acta Chem Scand. 35: 
737-738 (1981); Seely et al., Biochemistry 21: 3394-3399 (1982) and 
specific antisera have been raised to purified enzyme in rabbits (Persson, 
Acta Chem. Scand 36: 685-688 (1982)); Seely, et al J. Bio Chem. 258 
2496-2500 (1983). However, even from these relatively rich sources of the 
enzyme, only small amounts of it were obtained and improved methods of 
isolating the enzyme from crude extracts were needed to study the enzyme 
in other cell extracts. 
Fusion between myeloma cells and spleen cells from immunized donors has 
been shown to be a successful method of deriving homogenous antibodies. 
Thus, continuous cell lines of genetically stable hybridoma cells capable 
of producing large amounts of monoclonal antibodies against maligant 
tumors and specific viruses and their antigenic determinants have been 
developed. More particularly, according to U.S. Pat. No. 4,172,124 to 
Koprowski et al, antibodies demonstrating a specificity for malignant 
tumors can be produced by somatic cell hybrids between hypoxanthine 
phosphoriboxyltransferase deficient myeloma cells and spleen or lymph 
cells derived from an animal previously primed with tumor cells. Also, 
according to U.S. Pat. No. 4,196,265 to Koprowski et al., continuous cell 
lines of genetically stable fused cell hybrids capable of producing large 
amounts of monoclonal antibodies against specific viruses and their 
antigenic determinants have been developed. 
It would be desirable if such cell fusion techniques could be employed to 
provide a reliable and standard supply of anti-enzyme antibodies, e.g. 
anti-ODC antibodies, which in turn could be used to react with mammalian 
ODC, as for example in an immunoaffinity column, from which the active 
enzyme could then be eluted, recovered and used for the study of polyamine 
synthesis and its regulation during cell growth. 
BRIEF DESCRIPTION OF THE INVENTION 
This invention contemplates a novel continuous hybridoma cell line which 
expresses monoclonal anti-ODC antibody, to the use of such cell line in 
production of such antibody, and to a method for producing such cell line. 
The invention also contemplates a method for obtaining large amounts of 
ODC for use in the study of polyamine synthesis and cell growth. 
DETAILED DESCRIPTION OF THE INVENTION 
According to the present invention a novel continuous hybridoma cell line 
which expresses anti-ODC antibody is obtained by immunizing an animal with 
ODC, preferably purified ODC, forming fused hybrid cells between 
antibody-producing cells from the immunized animal and myeloma cells, 
cloning the hybrids and selecting clones which express anti-ODC antibody. 
More specifically, a mouse or other animal is injected with purified ODC 
and the antibody producing cells of the animal's spleen are then fused 
with a cancerous type of mouse cell or myeloma cell. The hybrid cell so 
formed produces the anti-ODC antibody molecule of its spleen cell parent 
and continually grows and divides like its parent myeloma cell. The clone 
of cells producing such antibody are selected and grown as a continuous 
cell line from which large amounts of anti-ODC antibody is harvested. 
In the alternative the clonal hybrid cells may be injected into a 
histocompatable animal where they proliferate, producing high levels of 
anti-ODC antibody which can be recovered from the animal's ascites fluid. 
Thus, the present invention makes available on a relatively large scale a 
reliable and standard supply of anti-ODC antibody for use in the 
immunolocalization of antigen in normal, premalignant and malignant 
tissues; purification of ODC from difficult sources via immunoaffinity 
chromatography; as an aid in the synthesis of cDNA from ODC mRNA and 
cloning of the gene for ODC, and immunodiagnosis of tumors.

The following is a typical procedure for preparing a hybrid cell line which 
produces anti-ODC antibodies, and the procedural steps are generally 
known. While this procedure refers to fusing myeloma cells of a BALB/c 
mouse with the spleen cells of BALB/c mice primed with ODC, the procedure 
is applicable using myeloma cells and anti-ODC antibody producing cells 
from another source. 
A. PREATION OF SPLEEN CELLS FOR FUSION 
The enzyme ornithine decarboxylase was purified from kidneys of 
androgen-treated mice through pyridoxamine-Sepharose affinity 
chromotography using the technique as described by Seely et al in 
Biochemistry 21: 3394-3399 (1982). The enzyme was about 10-20% pure, based 
on the comparison of its specific activity with that of the homogeneous 
enzyme Seely et al, Biochemistry, supra. The enzyme was used to immunize a 
BALB/c female mouse by subcutaneous admistration of about 5 .mu.g 
emulsified in Freund's complete adjuvant. The mouse was reimmunized 4 
weeks later with a further 5 .mu.g of the enzyme in incomplete adjuvant 
given intraperitoneally. After an additional 4 weeks, 5 .mu.g of the 
enzyme were administered intravenously, and 4 days later the mouse was 
sacrified and a spleen cell suspension was prepared in the manner taught 
by Gerhard et al., Eur. J. Immunol. 5: 720-725 (1975). Red blood cells 
were lysed by incubation of 15 minutes at 4.degree. C. in NH.sub.4 Cl 
(0.83%). The resulting cell suspension was washed by one centrifugation 
(800.times.g) through heat-inactivated calf serum and one centrifugation 
in protein-free medium (RRMI 1640, buffered with 7.5 mM HEPES, pH 7.2). 
B. PREATION OF MYELOMA CELLS FOR FUSION 
BALB/c (P3.times.63 Ag8-variant 653) myeloma cells derived from the MOPC-21 
line and deficient in HPRT (E.C.2.4.2.8) as described by Kearney et al J. 
Immunol 123: 1548-1550 (1979), were maintained in Eagle's minimum 
essential medium (MEM) containing 10% fetal calf and 10% horse serum. The 
growth of P3.times.63 Ag8 myeloma cells is inhibited by selective 
hypoxanthine-aminopterin-thymidine (HAT) medium. 
C. PRODUCTION OF HYBRIDS 
Production of hybrids was accomplished by mixing 10.sup.7 BALB/c 
(P3.times.63 Ag8) myeloma cells with 10.sup.8 spleen cells obtained from 
the ODC immunized BALB/c mouse. The cell mixture was centrifuged at 
800.times.g and the cells were resuspended for fusion in a 50% solution 
(w/v) of polyethylene glycol (PEG 4000) diluted in minimum essential 
medium (MEM) without serum following the procedure described by Koprowski 
et al., Proc. Natl. Acad. Sci. USA 74: 2985-2988 (1977), and by Herlyn et 
al., Proc. Natl. Acad. Sci. USA 76: 1438-1442 (1979). The resulting 
hybridoma cells designated B 11 were cloned in 
hypoxanthine-aminopterin-thymidine (HAT) medium by limiting dilution as 
described by Galfre and Milstein Meth. Enzymol. 73: 3-46 (1975). 
D. KARYOLOGICAL ANALYSIS 
The P3.times.63 Ag8 parental cells contained an average of 63 chromsomes 
and BALB/c spleen cells an average of 40 chromosomes. 
The Hybridoma cell line designated B 11 was deposited with The Wistar 
Institute of Anatomy and Biology, Philadelphia, Pa., U.S.A. on Oct. 12, 
1982 and with the American Type Culture Collection (ATCC), Rockville, Md., 
U.S.A., Deposit Accession No. ATCC HB 8372, on Oct. 5, 1983. The deposits 
are available pursuant to the patent laws and regulations of the United 
States and of those countries foreign to the United States in which 
counterparts of this application are filed. The availability of a deposit 
does not constitute a license to practice the invention of this 
application in derogation of any patent issued thereon or on any division 
or continuation of this application. 
E. TESTING OF THE CLONES FOR PRODUCTION OF ANTI-ODC ANTIBODY 
Polyvinyl chloride microtiter 96 well plates (Dynatech, Alexandria, VA) 
were coated with 50-100 .mu.g second antibody (goat anti-mouse IgG) by 
addition of a solution of 0.05M borate buffer, pH 8 which was allowed to 
dry overnight at room temperature. After washing the wells three times 
with RIA buffer (phosphate buffered saline (PBS), 0.12M NaCl, 0.012M 
Na.sub.2 PO.sub.4, 0.0015M KH.sub.2 PO.sub.4, containing 10% agamma 
globulin horse serum and 0.08% sodium azide), 0.05 ml of the culture 
supernatants were added and incubated at 4.degree. C. overnight. The 
supernatant was removed after washing three times with RIA buffer and 0.05 
ml of a solution containing about 10,000 cpm [5-.sup.3 
H].alpha.-difluoromethyl ornithine (DFMO)-labled ornithine decarboxylase. 
This labeled protein was prepared by reacting the partially purified mouse 
kidney enzyme ODC with [5-.sup.3 H]DFMO (15 Ci/mmol) as described by Seely 
et al., J. Biol. Chem. 258: 2496-2500 (1983). Incubation was continued for 
at least 1 hour at room temperature or overnight at 4.degree. C. The 
solution was then removed, the wells washed 3 times with RIA buffer and 
their contents solubilized in 0.5N NaOH and counted. Controls for 
non-specific binding were included by omitting either the second antibody 
or the culture supernatant. Less than 20-30 cpm was bound under these 
conditions. One clone B 11 which gave a strong positive response binding 
about 500 cpm of the labeled antigen was re-cloned several times and grown 
up as described above. The immunoglobulin produced by this clone and 
designated MK-1 was found to be of the IgM type by Ouchterlony 
immunodiffusion analysis using monospecific antisera purchased from 
Bionetics, Bethesda, MD. The commercial goat anti-mouse IgG preparation 
used in the initial screening has significant ability to bind mouse IgMs 
also, and this accounts for the initial detection of the MK-1 antibody. 
The hybridoma cells B 11 were grown as an ascites form by intraperitoneal 
injection into pristane-treated mice (Galfre and Milstein, Meth. Enzymol. 
73 (part B) 1-46 (1981)), and the resulting ascites fluid was used as a 
source of the monoclonal antibody MK-1. 
F. PRECIPITATION OF ORNITHINE DECARBOXYLASE AND [5-.sup.3 H]DFMO-ORNITHINE 
DECARBOXYLASE 
Mouse kidney ornithine decarboxylase (about 30 units in 0.1 ml of buffer A 
which consists of 25 mM Tris-HCl, pH 7.5, 0.1 mM EDTA and 2.5 mM 
dithiothreitol) was incubated with 0.06 ml of various dilutions of ascites 
fluid containing monoclonal antibody MK-1 (diluted into 150 mM Tris-HCl, 
pH 7.5, 1 mM EDTA, 0.02% Brij 35; buffer A) for 2 hours at 
0.degree.-4.degree. C. A further 0.06 ml of a solution (3 mg/ml) of goat 
anti-mouse IgM was then added and the samples shaken gently overnight at 
0.degree.-6.degree. C. 0.08 ml of a 10% suspension of protein A bacterial 
adsorbent (Miles Laboratories, Elkhart, IN) was then added and the samples 
shaken for 2 hours at 0.degree.-6.degree. C. After centrifugation at 
15,000.times.g for 1 minute, 0.1 ml aliquots of the supernatant were 
assayed for ornithine decarboxylase activity. The precipitation of 
[5-.sup. 3 H]DFMO-ornithine decarboxylase was carried out in the same way 
starting with a solution containing 2952 cpm in 0.1 ml. The 
immunoprecipitate was washed twice in 0.5 ml of buffer A, resuspended in 
0.2 ml water and counted as previously described (Seely et al, 1983 
supra). Control experiments using another ascites fluid containing 
monoclonal IgM (designated 1116NS10) which was prepared against a human 
adenocarcinoma cell line (Brockhaus et al., J. Biol. Chem. 256: 
13,223-12,225 (1981)) indicated no loss of orithine decarboxylase activity 
or precipitation of the [5-.sup.3 H]DMFO-ornithine decarboxylase under 
these conditions. A positive control was provided by the use of rabbit 
antiserum against orithine decarboxylase. This was used in the same way, 
but the goat anti-mouse IgM was omitted. 
A major advantage of the hybridoma technique for production of anti-ODC 
antibody is that a homogeneous antigen is not required for immunization, 
and in the experiments herein described only partially purified ornithine 
decarboxylase protein was used. However, it is necessary to have either 
the purified protein of interest or a specific way to identify its binding 
to the antibodies in order to screen potential clones of 
antibody-secreting cells. Such identification was achieved by using 
[5-.sup.3 H]DFMO to specifically label ornithine decarboxylase. DFMO is an 
enzyme-activated irreversible inhibitor of ODC and forms a covalent bond 
with the enzyme. This interaction is extremely specific and even in crude 
cell extracts ornithine decarboxylase is the only protein which becomes 
labeled (Seely et al., (1982) supra). The use of [5-.sup.3 H]DMFO-labeled 
ornithine decarboxylase to identify positive clones helped in the 
isolation of anti-ODC antibody designated MK-1, which recognized the 
native enzyme since the solution antigen was used for detection rather 
than insolubilized protein used in many ELISA techniques. However, it is 
unlikely that antibodies directed at the active site of ornithine 
decarboxylase would be found using this screening method since DFMO binds 
at the active site. When added to solutions containing either native 
ornithine decarboxylase from rat or mouse tissues or [5-.sup.3 
H-DFMO]-labeled ornithine decarboxylase, the monoclonal antibody, MK-1, 
did not lead to rapid loss of the activity or precipitation of the 
radioactivity. However, precipitation was achieved by addition of a second 
antibody (IgG) specific for mouse IgM followed by protein A (see Table 1, 
below). 
TABLE 1 
______________________________________ 
Precipitation of Ornithine Decarboxylase by Monoclonal Antibody 
______________________________________ 
MK-1 
Example A. Precipitation of Native Enzyme 
Ornithine 
Antibody added Decarboxylase Percent 
(0.06 ml of dilution 
Activity Remaining 
Precipi- 
shown) (units) tated 
______________________________________ 
None 27.1 0 
Rabbit antiserum (1:50) 
0.1 99 
Monoclonal antibody (1:50) 
8.7 68 
Monoclonal antibody (1:100) 
8.9 67 
Monoclonal antibody (1:500) 
8.8 68 
Monoclonal antibody (1:2500) 
22.7 16 
______________________________________ 
Example B. Precipitation of Enzyme which has been Labeled and 
Inactivated by Reaction with [5-.sup.3 H]DFMO 
[5-.sup.3 H]DFMO- 
Labeled 
Ornithine Percent 
Antibody added Decarboxylase Precipi- 
(0.06 ml of dilution 
Precipitated (cmp)* 
tated 
______________________________________ 
None 0 0 
Rabbit antiserum (1:50) 
2756 93 
Monoclonal antibody (1:50) 
2133 72 
Monoclonal antibody (1:500) 
1153 39 
Monoclonal antibody (1:2500) 
250 8 
______________________________________ 
*A total of 2952 cpm was added. 
Referring to Table 1, it will be noted that not all of the ornithine 
decarboxylase was precipitated under the latter described conditions and 
the amount precipitated being somewhat variable from experiment to 
experiment. In Example A of Table 1 using mouse kidney extracts which 
contained about 25 ng of ornithine decarboxylase in 0.1 ml, approximately 
70% of the activity was precipitated by about 1:500 fold dilutions of the 
ascites fluid. Increasing the antibody concentration by 10-fold did not 
increase the extent of precipitation, but all of the enzyme could be 
precipitated by polyclonal rabbit antiserum under the same conditions. In 
the experiment, the monoclonal antibody, MK-1, was incubated with enzyme 
for 2 hours at 4.degree. C., but increasing the time or temperature did 
not increase the percentage precipitated. 
Similiarly, the monoclonal antibody MK-1 was able to precipitate about 70% 
of the radioactivity present in [5-.sup.3 H]DFMO-labeled ornithine 
decarboxylase in the presence of the second antibody and protein A, 
although rabbit antiserum could precipitate more than 90% of this material 
(Example B, Table 1). Larger amounts of the monoclonal antibody were 
needed to bring about precipitation of the inactivated labeled antigen 
than of the native enzyme (see Table 1). 
G. IMMUNOAFFINITY CHROMATOGRAPHY TESTS 
The MK-1 antibody was purified from ascities fluid by ammonium sulfate 
precipitation and gel filtration on Sephacryl S-300 (Hudson, et al., 
Practical Immunology, Blackwell Scientific Publications, Oxford pp. 
221-222 (1980)). Fractions eluted from this column containing pure 
antibody (determined by polyacrylamide gel electrophoresis under 
denaturing conditions) were used to prepare immunoaffinity columns. 
Cyanogen bromide-activiated Sepharose 4B was reconstituted and washed 
according to the manufacturer's instructions. It was then coupled with the 
MK-1 antibody by overnight incubation and the antibody (2-5 mg protein/ml) 
in PBS, pH 8.0. Determination of the A.sub.280 of the solution before and 
after coupling indicated that about 80-95% of the antibody was coupled to 
the Sepharose. Remaining sites on the gel were blocked by reaction with 
0.5M ethanolamine at room temperature for several hours or overnight at 
4.degree. C. Columns containing about 2 ml of the gel were prepared, 
washed and buffer A containing 0.3 mM L-ornithine and 20M pyridoxal 
phosphate and stored at 4.degree. C. until use. 
Experiments using immunoaffinity columns containing bound MK-1 antibody 
prepared as described above showed that both active ornithine 
decarboxylase and the [5-.sup.3 H]DFMO-labeled derivative were retained on 
these columns. Several different strategies for eluting the material from 
these columns were tested. Low ionic strength buffer of either high (11.5) 
or low (2.5) pH eluted the protein, but were not compatible with recovery 
of enzyme activity in good yield. However, as shown in FIG. 1, a stepwise 
gradient of the chaotropic agent NaSCN eluted the enzyme with excellent 
retention of activity (measured after dialysis to remove the thiocyanate). 
About 82% of the enzyme activity applied was recovered with 91.5% of this 
amount (75% of the total) in the NaSCN fractions. 
In obtaining the data plotted in FIG. 1, partially purified mouse kidney 
ornithine decarboxylase (through DEAE cellulose chromatography) was 
applied to an immunoadsorbant column prepared as described above. The 
column was eluted with 2 ml fractions of buffer A containing 0.3 mM 
L-ornithine and 20 .mu.M pyridoxal phospate (loading buffer) containing 
the concentration of NaSCN shown in FIG. 1, and the ornithine 
decarboxylase (ODC) activity eluted determined after overnight dialysis 
against 60 volumes of buffer A to remove NaSCN. 
The immunoaffinity chromatography columns can also be used to purify crude 
preparations of ornithine decarboxylase (see FIG. 2). After application of 
a crude mouse kidney extract, virtually all of the protein was eluted in 
the first 4 fractions (curve with open circles), while 93% of the 
recovered ornithine decarboxylase activity was eluted as a sharp peak when 
1M NaSCN in loading buffer (see arrow) was applied (curve with solid 
dots). Even with the crude extracts which had been fractionated only by 
ammonium sulfate precipitation (Seely et al., Biochemistry 21: 3394-3399 
(1982)) at least 60-fold purification was achieved by a single pass 
through the immunoaffinity column (FIG. 2). When the extracts were 
partially purified by DEAE-cellulose chromatography (Seely, et al., (1982 
supra)) prior to immunoaffinity chromatography at least 2000-fold 
purification was achieved. 
Referring to FIG. 3, the same procedure as used in obtaining the data for 
FIG. 2 was employed except [5-.sup.3 H]DFMO-ornithine decarboxylase was 
chromatographed and elution was carried out with 0.4M NaSCN (arrow). 
Approximately 84% of the applied radioactivity was recovered from the 
column and 26.5% in the peak after 0.4M NaSCN. However, a lesser fraction 
of the labeled ornithine decarboxylase was retained by the column. This 
result is consistent with the results of Table 1 and other data suggesting 
that the monoclonal antibody MK-1 has a higher affinity for unmodified 
ornithine decarboxylase than for the DFMO-inactivated protein. 
The immunoaffinity columns prepared as above could be reused several times 
without significant loss of acitvity. Immunoaffinity columns prepared in 
the same way with a mouse monoclonal IgM (1116NS10) directed against the 
Le.sup.b blood group antigen (Brockhause et al., (1981 supra)) did not 
retain native or DFMO-labeled ornithine decarboxylase (FIG. 4) indicating 
that the results are not due to a non-specific adherence of ornithine 
decarboxylase to the column adsorbent. 
In FIG. 4 the large graph shows results with [5-.sup.3 H]DFMO-labeled 
ornithine decarboxylase which was applied in loading buffer and eluted 
first with this buffer and then with 50 mM diethylamine, pH 11.5 and by 50 
mM acetate, pH 2.6 (treatments known to release the labeled enzyme from 
MK-1 antibody columns). The recovery of radioactivity was about 85%. The 
inset in FIG. 4 shows chromatography of partially purified ornithine 
decarboxylase in the same manner as FIG. 2, except that fewer fractions 
were collected. The recovery of ornithine decarboxylase activity was 96% 
of that applied. 
H. IMMUNOPRECIPITATION OF LABELED ORNITHINE DECARBOXYLASE FROM MICE TREATED 
WITH [.sup.35 S]METHIONINE 
Female BALB/c mice were untreated or treated with androgens to induce 
ornithine carboxylase as previously described (Seely and Pegg, (1983), 
supra)). The mice were then given 500 .mu.Ci of [.sup.35 S]methionine 
(1284 Ci/mmol) by intraperitoneal injection of a 0.2 ml solution in PBS. 
The mice were sacrificed 30 minutes later and the kidneys removed, 
homogenized in 3 vol. of 25 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 2.5 mM 
dithiothreitol and centrifuged at 100,000.times.g for 45 min. Aliquots of 
0.05 ml of the supernatant were incubated with 0.15 ml of a 1:50 dilution 
of the MK-1 ascites fluid (diluted in buffer A) for 3 hours at 
0.degree.-4.degree. C. Thirty .mu.l of a solution (30 mg/ml) of goat 
anti-mouse IgM was then added and incubation continued overnight at 
0.degree.-4.degree. C. After 16 hours, bacterial protein A adsorbent (0.3 
ml of 10% solution) was added and the sample incubated with shaking for an 
additional 2 hours at room temperature. The mixture was diluted by additon 
of 0.75 ml of buffer A and centrifuged at 15,000.times.g for 30 seconds. 
The pellet was washed 4 times in buffer A and finally resuspended in 0.1 
ml of a solubilizing buffer containing 2% sodium dodecyl sulfate, 5% 
2-mercaptoethanol, 10% glycerol and 62.5 mM Tris-HCl, pH 6.8. After 
heating in a boiling water bath for 5 minutes the sample was centrifuged 
at 15,000.times.g for 1 minute and the supernatant used for polyacrylamide 
gel electrophoresis on a 10% discontinuous buffer system of Laemmli 
(1970). The gel was fixed overnight in 10% trichloroacetic acid, 10% 
glacial acetic acid, 30% methanol, impregnated with EN.sup.3 HANCE dried 
and exposed to Kodak XAR-5 X-ray film at -70.degree. C. for 3 days. 
Control samples using either a monoclonal antibody not directed against 
ornithine decarboxylase or a polyclonal rabbit antiserum to ornithine 
decarboxylase were prepared in the same way. 
The foregoing experiment showed that the monoclonal antibody in conjunction 
with protein A could be used to demonstrate the rapid synthesis of 
ornithine decarboxylase in the kidneys of androgen-treated female mice. As 
shown in FIG. 5, a band of labeled protein having a M.W. of about 55,000 
could be precipitated from extracts of kidneys of such mice given [.sup.35 
S]methionine, 30 minutes before death (Lanes 2 and 7). As shown in Lane 3, 
this band was not present in similarly treated extracts from female mice 
not treated with androgens which have a 400-fold lower content of 
ornithine decarboxylase (Seely and Pegg, (1983), supra)). The band 
corresponded exactly to a marker of [5-.sup.3 H]DFMO-labeled ornithine 
decarboxylase (Lane 5) and to a band precipitated by polyclonal rabbit 
antiserum to ornithine decarboxylase (Lane 6). This band was not found 
when another monoclonal antibody (1116NSID) not directed against ornithine 
decarboxylase was used as a control (Lanes 1 and 4). The band precipitated 
by the rabbit antiserum (Lane 6) was more intense than that precipitated 
by MK-1 (2 and 7) which is consistent with the more complete precipitation 
of ornithine decarboxylase by the rabbit antiserum. However, the bands had 
identical mobility. A vancant lane was left between lanes 7 and 8 to allow 
for some contamination by the very heavily labeled total extract sample 
placed in Lane 8. 
The immunoaffinity purification described in this specification may be used 
in the isolation of ornithine decarboxylase from mammalian cell extracts 
in which this enzyme represents only a very small percentage of the total 
protein. Although the species specificity of the interaction of the 
monoclonal antibody MK-1 has not been investigated in detail, it does 
interact with the enzyme ODC from mouse, rat and hamster tissues, and 
cells from these species could be used as sources of enzyme. Although the 
degree of precipitation of the enzyme by the monoclonal antibody was 
somewhat variable, the experiments provided no evidence in favor of 
multiple forms of the enzyme. The labeled band of ornithine decarboxylase 
precipitated by the monoclonal antibody from kidney extract of mice given 
[.sup.35 S]methionine coincided exactly with the band precipitated by the 
polyclonal rabbit antiserum and with the [5-.sup.3 H]DFMO labeled marker 
of ornithine decarboxylase. The substantial incorporation of [.sup.35 
S]methionine into this band provides direct experimental evidence showing 
that ornithine decarboxylase has a rapid rate of synthesis and 
degradation. Ornithine decarboxylase represents only 1 part in 5-10,000 of 
the soluble protein in the mouse kidney even after androgen stimulation, 
but the radioactivity incorporated into the ornithine decarboxylase band 
amounts to about 1% of the total incorporation into protein. This 
indicates that the enzyme protein must turn over much more rapidly than 
the average kidney protein. These results, therefore, confirm previous 
reports based on indirect evidence that ornithine decarboxylase protein 
has a very short half life. In vitro studies of the mechanism by whch the 
rapid degradation of ornithine decarboxylase is brought about should be 
aided by the availability of the radioactively labeled protein. This 
substrate can now be prepared by administration of [.sup.35 S]methionine 
to androgen-treated mice followed by immunoaffinity chromatography of the 
kidney extracts. 
MATERIALS 
L-[1-.sup.14 C]Ornithine (57 Ci/mol), DL-[5-.sup.3 
H].alpha.-difluoromethylornithine (15 Ci/mmol) and L-[.sup.35 S]methionine 
(1284 Ci/mmol) and EN.sup.3 HANCE were obtained from NEN, Boston, Mass. 
Bacterial protein A absorbent was obtained from Miles Laboratories, 
Elkhart, IN. Cyanogen bromide-activated Sepharose was purchased from 
Pharmacia Fine Chemicals, Piscataway, N.J. Other reagents came from Sigma 
Chemical Co., St. Louis, MO.