A method for diagnosing mammalian breast cancer by detecting in
the physiological fluid of said mammal an antigen (ACRT) having
immune cross-reactivity with human reverse transcriptase, or detecting
antibodies against said ACRT or detecting antibody-ACRT complexes,
wherein the reverse transcriptase is substantially purified, has
a molecular of about 70,000 and a sedimentation coefficient on a
glycerol gradient of between 5 and 5.5 S. A process for the purification
of reverse transcriptase from human milk.
1. A method for diagnosing mammalian breast cancer by detecting,
in a physiological fluid of said mammal, antibodies having immuno-reactivity
towards human reverse transcriptase, which comprises:
mixing a sample of said fluid containing said antibodies with an
excess of reverse transcriptase substantially purified having a
molecular weight of about 70,000 and a sedimentation coefficient
on a glycerol gradient of between 5 and 5.5 S, and being capable
of binding to said antibody and of forming a complex therewith,
detecting said complex.
2. The method of claim 1, which further comprises separating said
complex from said excess reverse transcriptase.
3. The method of claim 1, wherein said substantially purified reverse
transcriptase is detectably labeled.
4. The method of claim 3, wherein said label is selected from the
group consisting of a chromophore, a radiolabel, and an enzyme label.
5. The method of claim 4, wherein said radiolabel is .sup.125 I.
6. The method of claim 5, wherein said detection is carried out
by scintillation counting.
7. The method of claim 1, wherein said detection is carried out
by a colorimetric assay.
8. The method of claim 1, wherein said detection is carried out
adding to said mixture a second antibody raised against said first
antibodies in said fluid.
9. The method of claim 8, wherein said second antibody is detectably
10. The method of claim 9, wherein said label is an enzyme.
11. The method of claims 9 or 10, wherein the reverse transcriptase
is bound on a water insoluble support.
12. The method of claim 11, wherein said support is a strip of
plastic or cellulose.
13. A method of diagnosing mammalian breast cancer by detecting,
in a physiological fluid of said mammal, a complex of an antigen
(ACRT) having cross-reactivity towards human reverse transcriptase
and an antibody therefor, wherein said reverse transcriptase is
substantially purified, has a molecular weight of about 70,000 and
a sedimentation coefficient on a glycerol gradient of between 5
and 5.5 S.
14. The method of claim 13, which further comprises
separating said complex into its individual components and then
detecting the individual components.
15. The method of claim 14, wherein said separation is carried
out by acidifying a solution containing said complex in the presence
of polyethyleneglycol of molecular weight 6000.
16. The method of claim 15, which further comprises electrophoresing
said mixture of components of ACRT, antibody and polyethylene-glycol
on an agarose-containing material.
17. The method of claim 14, wherein said component being detected
is an antibody.
18. The method of claim 14, wherein said component being detected
19. In a method of diagnosing mammalian breast cancer by immunoassay,
the improvement which comprises detecting in a physiological fluid
of said mammal a material selected from the group consisting of
a complex of an antigen (ACRT) having immune cross-reactivity with
human reverse transcriptase enzyme with an antibody therefor, and
an antibody against ACRT.
20. The method of claim 19 wherein said reverse transcriptase enzyme
is from human milk.
21. The method of claim 8 wherein said second antibody is obtained
from a hybridoma from human or nonhuman animal sources.
22. The method of claim 19 wherein said material being detected
is an antibody against ACRT.
23. The method of claim 19 wherein said material being detected
is a complex of ACRT with an antibody therefor.
24. The method of any of claims 1, 13 or 19 wherein said human
reverse transcriptase enzyme is substantially purified from human
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a reverse transcriptase enzyme
from human milk, and its use in the detection of breast cancer.
2. Description of the Prior Art
The diagnosis of human cancer has been carried out in the prior
art by detecting the presence of a variety of polypeptides in human
Thus, for example, Bjorklund, U.S. Pat. No. 4,160,019 describes
the isolation of a cancer associated polypeptide antigen (CAPA)
showing monospecificity and being present in a wide variety of human
cancers of different localizations. The material possessing CAPA
activity is isolated by homogenizing malignant tissues from autopsies,
carcinoma tissues of various types and sites being collected to
make a tumor pool. The purified CAPA is a polypeptide based on the
single peptide chain, soluble at a pH within the range of about
1-3.5, and denaturing irreversibly at pH's exceeding about 4.5.
The antigen is hygroscopic, turns yellow and becomes inactivated
and smeary when it takes up moisture. The authors established that
CAPA originates from cancer cell walls. The polypeptide has a molecular
weight within the range of 20,000-27,000 and does not contain any
carbohydrate or nucleic acid. The presence of CAPA has been detected
in cancers as varied as those originated in the intestines, colon,
rectum, pancreas, breast, ovaries, prostate, testes, and the like.
Freedman et al, U.S. Pat. No. 3,663,684 describe a carcinoembryonic
antigen showing so-called "CEA-activity". CEA is used
for detection of cancer of the colon and is extracted from the antigen-containing
tissues by a glycoprotein solvent. Its molecular weight is about
200,000, although it may be as low as 70,000. It is a glycoprotein.
Davidson et al, U.S. Pat. No. 4,146,603 also describe a class of
glycoproteins found to be produced by human cancer cells and present
in the sera of cancer patients. This glycoprotein is called "TSGP"
(tumor specific glycoprotein). TSGP is generated by tumor cells
regardless of the tumor concerned. It appears in the circulatory
system of humans suffering from lung, mammary, colon, uterine, or
gastric carcinomas, melanomas, and the like. It is used for the
early diagnosis of individuals with any kind of malignant disease.
None of the aforementioned diagnostic tests is specific for breast
The presence of a reverse transcriptase enzyme in human breast
cancer tissue has been established by Gerard, G. F. et al, Nature,
256: 140-143 (1975). In 1977, Ohno et al, Proceedings of the National
Academy of Sciences, USA, 74: 764-768 (1977), described the properties
of a reverse transcriptase purified from human breast cancer particles.
This enzyme is a DNA polymerase with reverse transcriptase properties,
sedimenting between 5 and 6 S and having a molecular weight estimated
about 70,000. The enzyme was purified by fractionating breast tumor
for particulate material of density 1.16-1.18 g/cm.sup.3, solubilizing
this sample and applying it to a column of polyacrylamide agarose
gel. Fractions containing the reverse transcriptase were then pooled
and loaded onto a phosphocellulose column. The reverse transcriptase
was then isolated by a fractionation on this column. Non-malignant
samples yield no enzyme activity with the properties of the reverse
transcriptase of Ohno et al. The reverse transcriptase from the
Ohno et al preparation has been characterized immunologically and
has been determined to be related to the reverse transcriptase of
the Mason Pfizer monkey virus (Ohno et al, Proceedings of the National
Academy of Sciences, USA, 74: 2144 (1977)).
Other studies (Schlom et al, Science, 175: 542 (1972), Dion, A.
S. et al, Cancer Research 34: 3509 (1974), Feldman et al, Proceedings
of the National Academy of Sciences, USA, 70: 1976 (1973)) have
shown that some human milk samples contain RNA-dependent DNA nucleotidyl
transferase activity (reverse transcriptase). The activity from
human milk has, however, never been isolated, purified or immunologically
A need continues to exist for a rapid and efficient, as well as
highly specific diagnostic test for the detection of human breast
cancer. Such a test could be used for example, to qualify a generalized
diagnosis of cancer (obtainable with the previously described tests)
to a diagnosis of breast cancer.
SUMMARY OF THE INVENTION
It is therefore an object of the invention to provide a selective
diagnostic test for the determination of mammalian breast cancer.
Another object of the invention is to provide a reverse transcriptase
enzyme substantially purified from human milk.
Still another object of the invention is to provide a method for
the purification of a reverse transcriptase enzyme from human milk.
A further object of the invention is to provide a method for detecting
human breast cancer by using substantially purified reverse transcriptase
enzyme from human milk in a detectably labeled form.
Still a further object of the invention is to provide detectably
labeled reverse transcriptase enzyme substantially purified from
human milk, antibodies raised against this enzyme, in their substantially
purified form and/or in a detectably labeled form.
These and other objects of the invention which will hereinafter
become more readily apparent have been attained by providing:
A method for diagnosing breast cancer in mammals by detecting,
in a physiological fluid of said mammal an antigen (ACRT) having
immunocross-reactivity with a human reverse transcriptase enzyme,
such as that deriveable from human milk wherein said enzyme has
a molecular weight of 70,000 and a sedimentation coefficient on
a glycerol gradient, of 5-5.5 S.
Another object has been attained by providing a method as described
above, wherein either ACRT-immune antibodies are detected or antibody-ACRT
complexes are detected.
Another object has been attained by providing a reverse transcriptase
enzyme substantially purified from human milk, having a MW of 70,000
and a sedimentation coefficient on glycerol gradient of 5-5.5 S.
Another object has been attained by providing a reverse transcriptase
as described above, in detectably labeled form.
Another object has been attained by providing a method of purifying
a reverse transcriptase from human milk containing particles of
density 1.15-1.25 and which particles comprise a lipid-rich outer
layer coating a core containing said reverse transcriptase in admixture
with nucleic acid and other enzymes, which comprises:
separating said particles from the main body of said human milk;
removing said lipid-rich outer alyer by treating said particles
with a phospholipase enzyme;
separating said nucleic acid from said reverse transcriptase enzyme
and said other enzymes from said core;
purifying said reverse transcriptase from said remaining admixture
of enzymes of said core.
BRIEF DESCRIPTION OF THE DRAWINGS
A more complete appreciation of the invention and many of the attendant
advantages thereof will be readily obtained as the same becomes
better understood by reference to the following detailed description
when considered in connection with the accompanying drawings, wherein:
FIG. 1 shows the chromatographic elution profile of human milk
DNA polymerase (reverse transcriptase) from a diethylaminoethyl-cellulose
column (DEAE.sup.R -cellulose).
FIG. 2 shows the chromatographic elution profile of human milk
DNA polymerase (reverse transcriptase) from a poly(C)agarose column.
FIG. 3 shows the chromatographic elution profile of human milk
DNA polymerase (reverse transcriptase) from a Sephadex.RTM. (bead-formed
dextran gel cross-linked with epichlorohydrin).
FIG. 4 shows the comparative inhibition of the human milk reverse
transcriptase and polymerase .alpha.,.beta. and .gamma. by IgG directed
against the human milk reverse transcriptase. Enzyme activity in
the presence of immune IgG is expressed as a percentage of the activity
in the presence of an identical amount of control IgG. Symbols:
: polymerase .alpha.; .quadrature.: polymerase .beta.; .DELTA.:
polymerase .gamma., and : reverse transcriptase.
FIG. 5 shows the titration of DNA polymerases from other human
sources against IgG to human milk reverse transcriptase. Symbols:
: polymerase .alpha. from NC-37 cells; .DELTA.: polymerase .gamma.
from NC-37 cells; .quadrature.: polymerase .gamma. from HeLa cells;
and : reverse transcriptase from human milk.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention has succeeded in providing a highly sensitive,
fast and selective method for the detection of mammalian breast
cancer. The present inventors have discovered that a macromolecular
antigen having cross-reactivity with human milk reverse transcriptase
is present in the physiological fluids, especially the serum, of
a mammal (e.g., a human) with breast cancer. The antigen will be
called hereinafter ACRT (Antigen having Cross-Reactivity with Reverse
The essence of the invention relates to the isolation, purification
and characterization of a reverse transcriptase enzyme from the
milk of normal lactating humans. This enzyme is capable of being
used in a binding assay for the detection of breast cancer. The
enzyme resembles reverse transcriptase of RNA tumor viruses in its
primer template specificities and cation requirement (Gallagher,
R. E., et al, Proceedings of the National Academy of Sciences, USA,
71: 1309 (1974), and Steel, L. K., et al, Cancer Letters, 2: 291
(1977)). The purified enzyme is devoid of terminal transferase activity
and is not immunologically related to human DNA polymerases .alpha.,.beta.
In order to prepare the enzyme, high-density particles (.rho.=1.15-1.25,
preferably p>1.2) are obtained from defatted human milk (Feldman,
S. P., et al, Proceedings of the National Academy of Sciences, USA,
70: 1976 (1973)). Samples of human milk are pooled, defatted and
centrifuged. The skim milk is filtered through cheesecloth, mixed
with glycerol and centrifuged at 45,000-50,000 rev/min for 1-2 hours.
After centrifugation, the sediment is separated and constitutes
the milk concentrate. Particles with a density greater than about
1.2 g/cm.sup.3 are prepared from the milk concentrate according
to the method of Feldman, S. P. et al (Proceedings of the National
Academy of Sciences, USA, 70: 1976 (1973)).
The particles contain a lipid rich membrane coat surrounding a
central "core" containing nucleic acid plus the reverse
transcriptase plus other enzymes. The preparation of the enzyme
thus comprises removal of the lipid rich membrane followed by breaking
up of the core, separation of nucleic acid therefrom and final purification
of the enzyme.
In order to remove the membrane, dithiothreitol (DDT) or any other
sulfhydryl-containing reducing agent is added together with a phospholipase
enzyme (for example, phospholipase C, available commercially) to
the milk concentrate. The solution is incubated for a few minutes,
preferably 2 minutes, at about 37.degree. C. and then for about
15-30 minutes, preferably 20 minutes, at about room temperature,
preferably 25.degree. C. The resulting solution is cooled to 0.degree.-10.degree.
C., and a water immiscible organic solvent, such as for example,
anhydrous ether, is added thereto to form an emulsion by gentle
mixing. After centrifugation of the resulting emulsion, the lower
aqueous phase is removed and the aqueous solution is then layered
over a discontinuous metrazamide gradient (Sanner, Cancer Research,
36: 405 (1976)). Metrazamide is available commercially; the gradient
consists of 1.5 volume parts of 25% metrazamide (.rho.>1.18),
1.0 volume parts of 18% metrazamide (.rho.>1.10) in a buffer
containing an alkali metal halide salt such as sodium chloride,
and a metal chelating agent, such as EDTA. This buffer is at a pH
between 7.5 and 9.0, preferably at a pH of 8.0. An appropriate buffer
is 10 mM tris-HCl (pH 8.0), 150 mM NaCl, and 2 mM EDTA. The solution
which has been layered over the discontinuous metrazamide gradient
is centrifuged for about 90 minutes at 45,000-50,000 rev/min at
4.degree.-10.degree. C. The high-density particles are resuspended
in 10 mM buffer at pH 8.0, containing a detergent (1-5%) capable
of breaking up the core. Non-ionic detergents, well-known to those
skilled in the art are preferably used. For example, 1% N-P.sub.40,
0.5 M KCl and particles, are gently stirred for about 1 hour at
about 40.degree. C. The sample is then desalted by standard desalting
techniques such as for example, use of coarse Sephadex G-25.RTM..
The reverse transcriptase activity is purified from these high-density
particles by a sequential chromatography on two types of materials.
The first chromatographic step rids the enzyme from contaminant
nucleic acid. Any material having adsorption properties for nucleic
acids can be used; for example, diethylaminoethyl-cellulose (DEAE-52
cellulose.RTM.). Thus, desalted material containing broken up high-density
milk particles is applied to DEAE-cellulose equilibrated with a
buffer capable of maintaining a pH between 7.5 and 8.5. An appropriate
buffer is for example, 10 mM tris-HCl (pH 7.5), 20% glycerol, 0.2%
detergent and 2 mM sulfhydryl reducing agent, such as DTT. After
application of the sample, the chromatographic column is washed
with the buffer and the reverse transcriptase activity is batch-eluted
with the buffer to which at least 0.3 M KCl have been added. Fractions
having polymerase activity with (dG).sub.12-18.(C).sub.n (a hybrid
of polycytidylate and deoxyguanylate that is 12 to 18 nucleotides
long) as described below, are collected.
The fractions having polymerase activity are pooled, desalted and
applied to the second chromatographic material, a poly(C) agarose
column equilibrated in the same or different buffer.
Other chromatographic materials capable of separating reverse transcriptase
from other enzymes will work. Thus, agarose coupled to synthetic
polyguanylate, or synthetic polythymidilate, or synthetic polyadenylate
can be used. These materials work in a "pseudo-affinity chromatography"
fashion. Still other columns could be loaded with immobilized antireverse
transcriptase antibody, and the like.
When using poly(C) agarose, the column is washed with buffer and
the DNA polymerase activity is eluted with a linear gradient of
0 to 0.8 M KCl in the same buffer. The peak of enzyme activity elutes
at about 0.20 M KCl. The fractions containing the main peak of DNA
polymerase activity are pooled and ovalbumin is added to about 200
.mu.g/ml to stabilize the enzyme. Other stabilizing materials can
be bovine serum albumin, gelatin, etc. The enzyme is stable, can
be kept at 40.degree. C., and can be used for characterization studies.
The molecular weight of the DNA polymerase was determined by three
methods: (i) SDS (sodium dodecyl sulfate)-polyacrylamide gel electrophoresis,
(ii) Sephadex G-200.RTM. chromatography, and (iii) velocity sedimentation
analysis. Molecular weights can be determined by using standard
protein markers such as phosphorylase A (94,000), bovine serum albumin
(68,000), and ovalbumin (43,000). The peak coming off the last column
material, with a 200-fold purification over the high-density particles,
contains a major polypeptide band corresponding to a molecular weight
of about 70,000. Molecular sieving of the material through Sephadex
G-200.RTM., in the presence of 0.5 M KCl indicates that the human
milk reverse transcriptase has a molecular weight of 70,000. The
enzyme is sedimented through a linear glycerol gradient (10-30%
by volume) and the enzyme active fractions are located by assaying
with the primer template (dG).sub.12-18.(C).sub.n. The enzyme activity
sediments between 5 S and 5.5 S, slightly faster than the bovine
serum albumin marker.
In order to demonstrate that the DNA polymerase isolated from human
milk has reverse transcriptase activity, the following synthetic
primer-template specificities were determined. In general, the viral
reverse transcriptase show a preference for (dT).sub.12-18.(A).sub.n
(a hybrid of polyadenylate and deoxythymidilate that is 12 to 18
nucleotides long) and not for (dT).sub.12-18.(dA).sub.n (a hybrid
of poly(deoxyadenylate) and deoxythymidilate which is 12 to 18 nucleotides
long (Spiegelman et al, Nature (London) 228: 430 (1970)). Reverse
transcriptase will also use (dG).sub.12-18.(C).sub.n (a hybrid of
poly(citydylate) and deoxyguanylate that is 12 to 18 residues long)
and (dG).sub.12-18.(Cm).sub.n (a hybrid of poly(2'-O-methylcitydylate)
and deoxyguanylate which is 12 to 18 residues long) as templates
for the synthesis of polydeoxyguanylate (Sarngadharan, M. G., et
al, Nature (London) New Biology, 240: 67 (1972); Gerard, G. F.,
Biochem. Biophys. Res. Commun., 63: 706 (1975)). The cellular DNA
polymerases, specifically polymerase .gamma., will inefficiently
transcribe (dG).sub.12-18.(C).sub.n at low salt concentrations,
that is, 0.05 M KH.sub.2 PO.sub.4 or no added KCl (Sarngadharan,
M. G., et al, supra, Gerard, G. F., supra and Knopf, K. W., et al,
Biochemistry 15: 4540 (1976)), and will not utilize poly(2'-O-methylcitydylate).oligodeoxyguanylate
for the synthesis of poly(dG).(Knopf, K. W., et al, supra).
The response of human milk DNA polymerase to synthetic primer templates
appears in Table 1. Assays are described below in the section on
TABLE 1 ______________________________________ [.sup.3 H]dNMP,
polymerized Template [.sup.3 H]dNTP Divalent cation (pmole) ______________________________________
(A).sub.n .multidot. (dT).sub.12-18 TTP Mn.sup.2+ 0.16 (A).sub.n
.multidot. (dT).sub.12-18 TTP Mg.sup.2+ 0.70 (dA).sub.n .multidot.
(dT).sub.12-18 TTP Mn.sup.2+ 0.10 (dA).sub.n .multidot. (dT).sub.12-18
TTP Mg.sup.2+ 0.08 (C).sub.n .multidot. (dG).sub.12-18 dGTP Mn.sup.2+
0.05 (C).sub.n .multidot. (dG).sub.12-18 dGTP Mg.sup.2+ 0.50 (Cm).sub.n
.multidot. (dG).sub.12- 18 dGTP Mn.sup.2+ 0.25 (Cm).sub.n .multidot.
(dG).sub.12-18 dGTP Mg.sup.2+ 0.50 Primer alone (dT).sub.12-18 TTP
Mg.sup.2+ * <0.01 (dT).sub.12-18 dGTP Mg.sup.2+ * <0.01 (dG).sub.12-18
TTP Mg.sup.2+ * <0.01 (dG).sub.12-18 dGTP Mg.sup.2+ * <0.01
______________________________________ *Same results with Mn.sup.2+.
TTP: thymidine triphosphate dGTP: deoxyguanosine triphosphate dNTP:
deoxynucleoside triphosphate dNMP: deoxynucleoside monophosphate
The response shows that the enzyme can synthesyze poly(dT) in the
presence of (dT).sub.12-18.(A).sub.n and Mg.sup.2+ (10 mM). It will
also utilize templates (dG).sub.12-18.(C).sub.n and (dG).sub.12-18.(Cm).sub.n
when synthesizing polydeoxyguanylate. Primer alone with either Mg.sup.2+
or Mn.sup.2+ gives not detectable activity. This pattern of primer
template utilization by the human milk DNA polymerase excludes the
possibility that it is a terminal deoxynucleotidyl transferase.
The utilization of (dG).sub.12-18.(Cm).sub.n in the presence of
Mg.sup.2+ shows that this enzyme is not a cellular polymerase found
in human milk. The results show a pattern of activities that are
consistent with those obtained with reverse transcriptase from animal
viruses (Sarngadharan, M. G., et al, supra; Gerard, G. F., supra).
Further characterization of the DNA polymerase with reverse transcriptase
activity can be carried out by immunochemical methods. An antibody
is prepared against the purified DNA polymerase from human milk
and the IgG fraction is obtained by standard methods. One or two
micrograms of the immune IgG inhibit the DNA polymerase 70% but
do not inhibit polymerase .alpha.,.beta. or .gamma. from human milk.
A number of enzyme neutralization studies can be carried out with
purified polymerases .alpha. and .gamma. from other human sources.
Neither DNA polymerase .alpha. from human lymphoid cells nor DNA
polymerase .gamma. from NC-37 cells or from HeLa cells are inhibited
by the antibody to human milk DNA polymerase. An antibody binding
to polymerase .gamma. (IgG fraction) can be obtained, which binds
to the enzyme to form an antigen-antibody complex but does not neutralize
activity (Robert-Guroff, M. and Gallo, R. C., Biochemistry 16: 2874
(1977)). Binding is assayed with the antibody (IgG) to polymerase
.gamma. and the human milk DNA polymerase. Complex formation can
be determined by velocity sedimentation through linear glycerol
gradients. The fractions are assayed for DNA polymerase activity
and the shape and position of the curve can be compared to control
gradients. When this test is carried out, the DNA polymerase having
reverse transcriptase activity from human milk does not bind to
the antibody to polymerase .gamma.. The .gamma. polymerase from
HeLa cells and NC-37 cells do not bind to the antibody to human
milk DNA polymerase. This indicates that this last polymerase is
not immunologically related to polymerases .alpha., .beta., or .gamma.
from human sources.
When the human milk reverse transcriptase is compared immunologically
to the reverse transcriptases of some RNA viruses, enzyme neutralization
studies with antibody to reverse transcriptase against simium sarcoma
virus, baboon endogoneous virus and RD-114, are all negative.
All of these results indicate that the reverse transcriptase isolated
from high-density particles of human milk is immunologically distinct
from DNA polymerases .alpha., .beta., or .gamma.. The enzyme has
no terminal transferase activity, as indicated by its inability
to incorporate either .sup.3 H-labeled deoxyguanosinemonophosphate
or .sup.3 H-labeled thymidinemonophosphate when oligodeoxyguanylate
or oligodeoxythymidilate is used as a primer.
The present inventors have discovered that patients with breast
cancer exhibit in their serum an antigen (ACRT) which is immunologically
cross-reactive with the reverse transcriptase from human milk described
above. Furthermore, in patients with breast cancer, the antigen
may be present in the serum by itself, or in the form of an antibody-antigen
complex. In addition, freely circulating antibody against ACRT may
also be present in cancer patients. This indicates that the detection
of ACRT in mammalian serum, by itself of in complexation with antibodies,
or detection of the antibodies themselves, serve as a selective
diagnostic test for breast cancer. Such a diagnostic test can be
carried out by standard well known binding assay methodology.
When ACRT is detected in mammalian serum, it is possible to incubate
a sample of mammalian serum containing the ACRT with a dissociatable
complex of a binding macromolecule and a detectably labeled reverse
transcriptase purified from human milk as described above. Among
the detectable labels useable in the present invention are radiolabels,
enzyme labels, chromophoric labels, or other labels.
Radiolabels for example, can be divided into two types: those with
an internal label and those with an external label. With an internal
label, an existing atom in the reverse transcriptase enzyme is replaced
by a radioactive isotope of that atom (e.g.: C.sup.14 for C.sup.12,
H.sup.3 for H.sup.1). With an external label, an atom or atoms of
a radioactive isotope (e.g., I.sup.131 or I.sup.125) are substituted
for an existing atom on the reverse transcriptase; to achieve stability,
a covalent link is established between the enzyme and the label.
The enzyme with an external label such as I.sup.125 is not identical
with the unlabeled enzyme, but its behavior is practically indistinguishable
from the latter. Both the internal or external label methodology
can be used in the present invention. The most preferred method
is that of using an external label, most preferably radioactive
iodine. It is known that iodine can be substituted into the aromatic
side-chain of tyrosine residues, as well as other amino acids such
as histidine. Many procedures have been described for iodination,
and they can all be used in the present invention. The most common
technique is the chloramine T technique (Greenwood, F. C. et al,
Biochemical Journal, 89: 114 (1963)). This procedure is simple since
all that is required is mixing a solution of the substantially purified
reverse transcriptase, sodium iodide having radioactive iodine and
chloramine T; the reaction is terminated by the addition of a reducing
agent, preferably sodium metabisulfite.
For a general description of labeling techniques, see Chard: "An
Introduction to Radioimmunoassay and Related Techniques", North
Holland Publishing Co., Amsterdam, N.Y., Oxford, 1st Edition, 1978.
Labels other than radioactive labels can, of course, be used since
they are well known in generalized binding assay techniques. Thus,
for example, alternatives to isotopic labels may be (a) chromophoric
labels: such as fluorescent, ultraviolet absorbing or visible light-absorbing
labels. These are advantageous because of their long shelf life
and absence of radiation. (b) Enzyme labels: since specific enzymes
can be coupled to other molecules by covalent links, a highly specific
enzyme may be covalently reacted with the substantially purified
reverse transcriptase (see for example, Engvall et al, "Enzyme-Linked
Immunoabsorbent Assay.Elisa", the Journal of Immunology, vol.
109: 120 (1972)). (c) Other tracers: such as free radical labels
or bacterial labels can also be used in the present invention.
In order to carry out a competitive binding assay, such as a colorimetric
assay or a radioimmunoassay, it is necessary to provide a binding
macromolecule which has reversible affinity for the detectably labeled-containing
reverse transcriptase. Such a binding macromolecule is most generally
an antibody raised against reverse transcriptase substantially purified
from human milk. It is also obvious that the binding macromolecule,
preferably antibody, should as far as possible be directed only
to the reverse transcriptase which the assay is intended to measure,
not to a wide variety of similar materials which would intefere
with specificity. Human antibodies or "hybridoma" antibodies
from human or animal sources could of course also be used.
The preparation of anti-sera in animals is a well known technique
(Chard, supra, pp. 385-396). The choice of animal is usually determined
by a balance between the facilities available, and the likely requirements
in terms of volume of the resulting anti-serum. A large species
such as goat, donkey and horse may be preferred because of the larger
volumes of serum readily obtained; however, it is also possible
to use smaller species such as rabbits or guinea pigs which often
have high titer anti-sera. Usually, subcutaneous injection of substantially
purified reverse transcriptase coupled to guinea pig albumin and
emulsified in Freund's complete adjuvent is sufficient to produce
anti-reverse transcriptase anti-sera. The detection of antibodies
can be carried out by testing the anti-sera with appropriate detectably
labeled reverse transcriptase. Fractions that bind labeled reverse
transcriptase are isolated and further purified if necessary.
It is of course possible to use other than antibodies as the binding
macromolecule. The use of cell receptors specific for reverse transcriptase
or of any circulating binding proteins equally specific for reverse
transcriptase can be used.
The general competitive binding assay techniques useful for the
detection of minute amounts of organic molecules, such as hormones,
proteins, antibodies, and the like are well known in the art (Chard,
supra). Any of these competitive binding assay techniques can be
used for the purposes of the present invention. A small amount of
serum from a patient suspected of having breast cancer is incubated
with a complex of the detectably labeled reverse transcriptase and
the binding macromolecule. If ACRT is present in the sample serum,
it will displace detectably labeled reverse transcriptase from the
binding macromolecule binding sties and "free" the labeled
enzyme. It is then necessary to determine the distribution of labeled
enzyme between the free and the bound form. Usually, but not always,
this requires that the bound fraction be physically separated from
the free fraction; a variety of techniques can be used for that
purpose. All of the techniques exploit physical-chemical differences
between the labeled enzyme and its free and bound form. The general
available methodologies have been described by Yalow (Pharmacol.
Rev. 28: 161 (1973)). These techniques include adsorption of free
antigen to solid phase material, such as cellulose, charcoal, silicates
or ion exchange resins; precipitation of antigen-antibody complexes
by second antibody; salting out techiques or organic solvents; adsorption
or complexing of antibody to solid phase material; electrophoretic
separation on cellulose, starch gel or polyacrylamide gel, and the
The choice of technique depends on the speed, simplicity, applicability
and cost. It is a simple matter of choice for anyone skilled in
the art and therefore, the generalized techniques will not be described
in further detail.
Particularly preferred among the aforementioned techniques are
adsorption methods, double-antibody methods and solid phase systems.
In adsorption methodology, the non-specific adsorption of proteins
to particle surfaces is used as a method for the separation of bound
and free detectably labeled enzyme. The procedure depends on the
fact that only the labeled enzymes and not the binding macromolecules
or bound complexes have the adsorption property. The most preferred
adsorption procedure, which is highly useful in the present case,
is adsorption on charcoal or silicates. The most commonly used of
the available charcoals are the Norit range (Norit SX1) with a maximum
particle size of 63 .mu.m.
"Double" or "second" antibody methods depend
on the precipitation of the bound complex with an antibody directed
to the binding macromolecule. The second antibody is specific to
the .gamma.-globulin of the species in which the first antibody
was raised, for example, if a guinea pig anti-reverse transcriptase
is used in the primary reaction of an assay for ACRT, an antibody
to guinea pig .gamma.-globulin raised in a goat may be used for
the separation step. Although most commonly used in radioimmunoassays,
this concept can be applied to any binding macromolecule for which
an antibody is available. Separation by this technique requires
a relatively large concentration of second antibody and a correspondingly
large amount of the species of .gamma.-globulins of which the first
antibody forms a part must be included; for this purpose, a second
antibody system always involves the addition of carrier protein,
either whole serum or .gamma.-globulin from the species in which
the first antibody was raised. The use of coupling of the second
antibody to an insoluble matrix such as cellulose is more economical
and efficient and has been described by den Hollander et al (Kirkham
et al, "Radioimmunoassay Methods" 419 (1971)).
Solid phase systems in general have been increasingly utilized
in recent years. When the binding macromolecule is covalently coupled
to an insoluble support, both it and the bound complex can readily
be separated from the soluble free fraction. A wide variety of solid
phase supports have been described which include particles of dextran
and cellulose, and continuous surfaces such as polystyrene or polypropylene
discs, or the walls of plastic or glass tubes. Plastic surfaces
exhibit adsorptive properties, and simply exposing such surface
to an appropriate dilution of the immunoglobulin antibody, e.g.,
IgG, will lead to the attachment of a proportion of the antibody
molecules thereon. The bond is probably ionic or hydrophobic and
not covalent. Covalent bonding, however, can be readily obtained
by the incorporation of crosslinking agents such as glutaraldehyde
and other agents in the antibody solution used for the coating.
Coated tube systems offer great convenience in the actual performance
of assays and the technique can be widely used in commercial kits.
In one preferred embodiment, the antibody is covalently attached
to the inside of a test tube and labeled enzyme is also incorporated
in the tube. A single addition of a sample of fluid being tested
is then added to the test tube. After incubation, the contents of
the tube are emptied and the tracer is detected by standard methodology.
The binding macromolecule can also be attached to a particulate
solid phase by any one of a number of techniques designed to yield
a covalent link between the protein and the particles, such as for
example, diazotization or cyanogen bromide activation. The resulting
material is then extensively washed to insure that no free .gamma.-globulin
molecules remain. Alternative approaches include the use of antibody
entrapped in the interstices of a polyacrylamide gel or covalently
bound to magnetic particles (polymer-coated iron oxide). With the
latter system, mixing and separation can be simply achieved by the
application of a magnetic field.
After incubation of the test sample with the binding macromolecule-labeled
enzyme complex, and separation of the labeled enzyme, it is necessary
to detect the label by some physical or chemical means. When the
label is a second enzyme, said second enzyme is assayed by the addition
of a substrate which upon reaction releases a ultraviolet or visible
light-absorbing product. Thus, for example, the second enzyme may
be alkaline phosphatase. To the conjugate reverse transcriptase-alkaline
phosphatase is added a solution of p-nitrophenylphosphate at pH
8.0. p-Nitrophenylphosphate has substantially no absorbance at 400
nm. However, on being acted upon by alkaline phosphatase, the substrate
releases p-nitrophenol which has a large absorption coefficient
at 400 nm and is yellow. In such a simple fashion then, appearance
of yellow coloration is a direct indication of the presence of ACRT
in the serum of the patient.
When the label is radioactive iodine, scintillation counting is
the method of choice. Radioactive iodine is a .gamma.-ray emitter
and therefore intimate contact between the isotope and the scintillator
is unnecessary. The scintillator in these cases usually consists
of a crystal of sodium iodide coated with thallium usually formed
as a well; as the radiation strikes, the molecules making up the
crystal lattice, ionization occurs and results in a light flash
which is then detected by the photomultiplier. If the radioisotope
used for labeling is C.sup.14 or H.sup.3, liquid scintillation is
appropriate to detect .beta.-particles.
Patients having breast cancer may also contain freely circulating
antibody against ACRT. In such case, a sample of serum from the
patient suspected of having breast cancer, is mixed with a large
excess of detectably labeled reverse transcriptase. After an appropriate
incubation time, sufficient to allow formation of antibody enzyme
complexes, bound complexes and antibodies are separated from excess
remaining labeled enzyme. This is carried out by adsorbing excess
labeled enzyme onto an appropriate material having adsorbing capacity
therefor but not adsorbing capacity for complex or free antibody.
The non-adsorbed solution can then be assayed by the appropriate
methodology (radioactivity or colorimetric, or the like) for the
presence of detectably labeled antibody-reverse transcriptase complex.
It is also possible to prepare an immobilized reverse transcriptase
by binding the enzyme to an insoluble support. Incubation of the
resulting insoluble enzyme with a sample of serum will cause antibody
therein to bind to the insoluble support. In order to detect the
bound antibody, a double antibody or second antibody technique is
utilized. The second antibody is raised against reverse transcriptase
immune IgG. The second antibody is then detectably labeled, for
example, with a radioactive label or with an enzyme. Incubation
of the second antibody with the insolubilized antibody enzyme complex
will cause attachment of the second antibody to the insoluble support.
After washing remaining soluble second antibody, the solid insoluble
support is detected by radioactivity measurement or by appropriate
enzyme assay. When an enzyme assay is used, this technique is the
well known Elisa technique (Engvall et al, Journal of Immunology,
vol. 109: 129 (1972)). A preferred embodiment of the system is achieved
by coating a tube with reverse transcriptase and then adding a sample
of serum thereto, followed by an enzyme-labeled preparation of anti-immune
reverse transcriptase IgG. The enzyme activity remaining in the
tubes after washing provides a measurable amount of specific antibodies
in the serum. Coating of polystyrene tubes is a preferred embodiment.
Another preferred embodiment is the attachment of reverse transcriptase
to the surface of a cellulose of plastic strip. Incubation of this
strip with a serum sample containing reverse transcriptase immune
IgG and further incubation of the resulting strip with detectably
labeled anti-reverse transcriptase immune IgG will attach the second
detectably labeled antibody to the strip. Washing of the strip and
counting thereof (if radiolabeled is used) or incubation of the
strip with a drop of appropriate color-generating substrate (if
enzyme labeled is used) will indicate the presence of antibody against
ACRT in the serum of the patient.
A complex of ACRT and complementary antibody has been found to
be present in breast cancer patients. Therefore, methods for the
detection of the antibody enzyme complex are also part of the present
A preferred method is to dissociate the antibody-antigen complex
into its free components and analyze those separately by the methods
discussed previously. For example, a sample of serum from a patient
suspect of having breast cancer is mixed with 3 parts by volume
of a solution of polyethyleneglycol (PEG) in 10% saline buffer,
wherein the PEG is of molecular weight 6000. The final concentration
of PEG should be 1-10%, preferably 2-5% by volume. The addition
of PEG to the serum, precipitates C.sub.lq binding-IgG complexes
which comprise fully formed complement binding complexes (true complexes)
and initial complement binding complexes (precursor complexes).
The precipitate of PEG and complexes is separated from the supernatant
and deposited on top of a 10-50% gradient of PEG, molecular weight
20,000, in saline phosphate buffer pH 7.2, 0.01 M phosphate. The
centrifuge tube is ultra-centrifuged with the aid of a W.sup.2 T
integrator capable of maintaining a constant value of the g rating
at 10,000.+-.2000 xg for 10-30 minutes, preferably about 20 minutes.
The ultra-centrifugation causes the material to develop into a series
of bands. The bands are fractionated from the tube by a standard
fraction recovery system and the band between 20 and 22% contains
the ACRT-containing complex. This band is isolated and the complexes
are separated from the PEG. Standard separation of PEG from the
complexes can be carried out for example, by film electrophoresis.
The contents of the 20-22% band are acidified to a pH 2.5 with an
appropriate buffer. The acidification step separates the complexes
into antigen and antibody components but, the presence of PEG, prevents
the loss of the tertiary structure of these components. The acidified
mixture is then added to a block of agarose (normally 2-5 mm thick,
10 cm long), into a well or channel formed at the center thereof.
Standard agarose useful for high endosmotic flow (for example, sigma
type 3.sup.R) can be used. Electrophoresis for 1 hour in 0.5 M sodium
barbital buffer pH 8.6 is carried out at 1 mamp/cm. The antigen
moves to the anode while the antibody moves to the cathode and the
PEG remains in the well or trough. All that remains is to detect
the antibody or antigen at the cathode or anode respectively, by
any of the previously discussed methods. Thus, for example, the
anode fraction of the agarose is cut and suspended in a beaker with
saline, and further detection of the ACRT can be carried out. Alternatively,
a cellulose acetate membrane can be laid on the surface of the agarose
block after electrophoresis. The cellulose acetate membrane adsorbs
surface proteins and, after appropriate staining (for example, with
Coomassie Blue or other standard stain) serves to pinpoint the presence
of protein in the agarose block. The appropriate sections of the
block are then added to a beaker containing saline and analyses
of the constituents are carried out as above.
Any other method for the specific detection of circulating antibody-antigen
complexes can also be used. Two such techniques have been recently
described in the patent literature. Masson et al, U.S. Pat. No.
4,062,935 describe a method of analyzing a sample for antibody-antigen
complexes which includes a step of adding to the sample a solution
of rheumatoid factor (RF) or Cl.sub.q (a component of complement)
which have the property of combining with complexes but not with
either free antigen or free antibody. Another, more efficient and
general methodology is that given by Soothill et al, U.S. Pat. No.
4,141,965. Soothill et al describe a latex particle agglutination
test useful for the determination of an immune complex which can
readily be applicable to the present invention. A sample comprising
the antibody-ACRT complex is incubated with coated latex particles
and IgM antibodies, the latex particles being coated with milk reverse
transcriptase and the IgM antibodies being low affinity antibodies
raised against milk reverse transcriptase in a non-human animal.
In the absence of antibody-ACRT complex, the latex particles will
agglutinate. When however, antibody-ACRT complex is present in the
serum, an inhibition of agglutination will be observed due to the
interaction of the low affinity IgM with the ACRT-containing complex.
A latex particle agglutination technique can also be generally
used for the detection of single constituents of the complex and
therefore represents another available methodology for the detection
of circulating ACRT or circulating antibody. This technique does
not involve the use of a detectably labeled enzyme having reversible
affinity for antibody but rather the use of the unlabeled enzyme
itself. See for example, Sawai et al, U.S. Pat. No. 4,118,192 or
Hoffmann-LaRoche, British Pat. No. 1,384,399. In these techniques,
antibody raised against reverse transcriptase is supported on an
insoluble carrier particle, usually a latex particle, thus sensitizing
the insoluble carrier particle. The supported transcriptase immune
IgG is then reacted with a sample suspected of containing the ACRT.
The sensitized latex agglutinates to a degree which is proportional
to the amount of ACRT present in the serum. The agglutination is
followed by irradiating the resulting reaction mixture with light
having a wave-length in the range of 0.6-2.4 microns. The determination
of absorbance can be performed with a spectrophotometer similar
to that used in near infrared spectrometry. Polystyrene latexes
or styrene butadiene latexes can readily be used; however, other
particles such as dispersed coccal bacteria, cell membrane fragments,
micro particles of inorganic oxides such as silica, silica alumina,
and alumina or finely pulverized minerals, metals and the like are
also readily useable. These latex agglutination techniques not only
make it possible to determine low concentrations of ACRT but enables
the determination of the antigen in trace amounts and with comparable
specificity to those of the ratioimmunoassay methodology. The amount
of ACRT can be determined by measuring the absorbance as described
above, or alternatively by measuring the rate of reaction, or the
reaction time required for the absorbance to reach a perscribed
value. When latex particles are coated with reverse transcriptase,
agglutination thereof occurs when the serum sample contains antibody.
The Sawai et al methodology is also applicable in the inhibition
of agglutination mode. In this mode, latex particles are coated
with reverse transcriptase. The particles are then incubated with
reverse transcriptase immune IgG. The so-formed complex is mixed
with serum suspected of containing ACRT. If the serum sample contains
ACRT, the latter will compete for the antibody binding sites and
inhibit the agglutination of the enzyme-covered latex particles.
The techniques and materials of the present invention for the detection
of breast cancer can be readily automated. A noteworthy development
in the field of automated radioimmunoassay is the recent patent
of Brooker et al, U.S. Pat. No. 4,022,577.
Among the kits useful in the present invention are those of the
general type described by Szczesniak, U.S. Pat. No. 3,899,298. Such
kits comprise a carrier being compartmented to receive at least
one, or at least two, or at least three or more, containers and
to maintain said containers in close confinement. A first container
may contain detectably labeled reverse transcriptase. Another container
may contain reverse transcriptase immune IgG raised in an appropriate
animal. These materials may be in the freeze-dried state or suspended
in a buffer solution. When in a freeze-dried state, the buffer solution
may be in a third container. Alternatively, the complex of reverse
transcriptase immune IgG and detectably labeled reverse transcriptase
may be present in one container and buffer solution which may be
added at the time of testing may be present in another container.
Alternatively, the first container may be a test tube being coated
at the inner surface thereof with reverse transcriptase immune IgG.
A second container may contain detectably labeled reverse transcriptase
in the presence or absence of buffer. At the time of testing for
ACRT in the serum, the buffer suspension of labeled enzyme is added
to the antibody coated test tube and a drop or two of the test serum
containing the suspected antigen is added to the tube. Alternatively,
the first container may be a test tube coated at its inner surfaces
with reverse transcriptase immune IgG and containing in complexation
therewith, the detectably labeled enzyme. At the time of testing,
the addition of the suspect test sample is then sufficient to carry
out the methodology. Other containers in the carrier may contain
the elements necessary for the separation of bound and free detectably
labeled enzyme. Thus, such containers may contain charcoal, silicates
or second-antibodies useful in the "second antibody technique"
described previously. Another container, for example, may contain
detectably labeled anti-reverse transcriptase immune IgG for use
in the determination of circulating anti-ACRT in the serum. Any
number of variations and permutations consistent with the various
techniques and for use in the detection of either ACRT, antibody
or ACRT-antibody complex can be envisioned for the preparation of
the kit. These are all matters of choice determined by the ease
of handling, rapidity and efficiency of the testing.
The methodology of the present invention can be used to specifically
diagnose the presence of breast cancer. ACRT, its antibodies or
ACRT-antibody complexes are not present in non-lactating patients
without breast cancer. Thre is no evidence that ACRT is present
in patients with any other type of cancer. Since milk from lactating
women contains the enzyme, it should be kept in mind that presence
of the ACRT in serum may denote either breast cancer or lactation.
This however isn't really a significant clinical problem. Less than
1% of women under 35 have breast cancer.
Having now generally described the invention, the same will be
further illustrated by means of specific examples which are presented
herewith for purposes of illustration only and are not intended
to be limiting thereof, unless otherwise specified.
Isolation and Purification of Reverse Transcriptase from Human
Samples (N=10 to 20) of human milk were pooled (1 to 2 liters),
defatted with 0.1 M EDTA (pH 8.3) and centrifuged at 2,500 g for
10 minutes at 40.degree. C. The skim milk was filtered through two
layers of cheesecloth, mixed with glycerol to a final concentration
of 20%, and centrifuged at 48,000 rev/min for 90 minutes at 40.degree.
C. in a Beckman Ti-14 batch rotor. The fluid was aspirated and the
outer wall of the rotor was scraped with a spatula and washed with
3 to 6 ml of 0.01 M tris-HCl (pH 8.0) (milk concentrate). Particles
with a density (.rho.) greater than 1.2 g/cm.sup.3 were prepared
according to a modification of the method described by Feldman et
al (Proceedings of the National Academy of Sciences, USA, 70: 1976
(1973)). One ml of 1 M dithiothreitol was mixed with 1 ml of polyvinyl
sulfate (1 mg/ml) and kept at 40.degree. C.; 3 ml of the milk concentrate
was then added to the tube followed by 100 .mu.l of phospholipase
C type 1 (10 mg/ml in 1% bovine serum albumin). The solution was
incubated for 2 minutes at 37.degree. C. and then for 20 minutes
at 25.degree. C. The solution was then placed in an ice bath, 3
ml of cold anhydrous ether was added, and after being mixed gently,
the resulting emulsion was centrifuged at 1500 g for 10 minutes
at 40.degree. C. The lower aqueous phase was removed, and the ether
was removed from the solution by blowing a gentle stream of filtered
air over the surface for 5 to 10 minutes. The resulting solution
was layered over a discontinuous metrazamide gradient consisting
of 1.5 ml of 25% metrazamide and 1.0 ml of 18% metrazamide in TNE
(10 mM tris-HCl (pH 8.0) at 150 mM NaCl, and 2 mM EDTA) and centrifuged
for 90 minutes at 48,000 rev/min at 40.degree. C. in a Spinco SW
50.1 rotor. The high density particles were resuspended in 0.01
M tris HCl (pH 8.0) containing 1% N-P.sub.40 and 0.5 M KCl and were
gently stirred for 1 hour at 40.degree. C. The sample was desalted
by centrifugation through 30 ml of coarse Sephadex G-25. The desalted
material obtained by centrifugation through Sephadex G-25 was applied
to a 10 ml column of DEAE-cellulose equilibrated with buffer A (10
mM tris HCl (pH 7.5), 20% glycerol, 0.2% N-P.sub.40, and 2 mM DTT).
After the sample was applied, the column was washed with 30 ml of
buffer A and the DNA polymerase activity was batch-eluted with buffer
A to which 0.3 M KCl had been added. Portions (10 .mu.l) of each
fraction from the DEAE-52 column and similar portions of fractions
from subsequent column purification steps were assayed for polymerase
activity with (dG).sub.12-18. (C).sub.n as described below. FIG.
1 shows the profile of the DEAE-cellulose column.
The peak fractions from the DEAE-cellulose column were pooled,
desalted by centrifugation through Sephadex G-25 and applied to
a 3-ml poly(C)agarose column equilibrated in buffer A. The column
was washed with 10 ml buffer A and the DNA polymerase activity was
eluted with a 15 ml linear gradient of 0 to 0.8 M KCl in buffer
A. The peak enzyme activity was eluted at 0.22 M KCl. The fractions
containing the main peak of DNA polymerase activity were pooled
and ovalbumin was added to 200 .mu.g/ml. This material was kept
at 4.degree. C. and was used as the starting material for characterization
studies. FIG. 2 shows the poly(C)agarose column profile.
Enzymatic Assay for the Reverse Transcriptase Enzyme
Enzymatic activity of the human milk DNA polymerase with various
synthetic polynucleotides was determined as follows: assay mixtures
containing 10 .mu.l of enzyme were initiated by adding 40 .mu.l
of a mixture that gave a final concentration of 50 mM tris-HCl (pH
8.0); 60 mM KCl; 1 mM DTT; either 10 mM MgCl.sub.2 or 0.5 mM MnCl.sub.2,
7.6 .mu.M [.sup.3 H] TTP (2,000 to 4,000 counts per minute per picomole,
or 7.6 .mu.M [.sup.3 H] dGTP (2,000 to 4,000 counts per minute per
picomole); and bovine serum albumin (0.5 mg/ml). All reactions were
performed at 37.degree. C. for 30 minutes. Acid insoluble precipitates
were collected on filters and the radioactivity was counted by means
of a liquid scintillation counting system.
Characterization of Reverse Transcriptase from Human Milk (Molecular
The enzyme-active fractions from the poly(C)agarose column from
Example 1 were pooled and applied to a 20-ml Sephadex G-200 column
equilibrated in buffer A containing 0.5 M KCl. The column had been
standardized with ovalbuminum (43,000), bovine serum albumin (68,000),
and phosphorilase A (93,000). Fractions (250 .mu.l) were collected
and 10-.mu.l portions were assayed for DNA polymerase activity with
(dT).sub.12-18.(A).sub.n. The milk DNA polymerase eluted 2 to 3
fractions before bovine serum albumin indicating an apparent molecular
weight of 70,000. The Sephadex G-200 chromatography profile is shown
in FIG. 3.
Comparative Inhibition of Human Milk Reverse Transcriptase and
Human Polymerase .alpha.,.beta. and .gamma. by IgG Directed Against
the Human Milk Reverse Transcriptase
FIGS. 4 and 5 show antibody neutralization studies of partially
purified human milk DNA polymerases with IgG fraction of antibody
to human reverse transcriptase. Enzyme activity in the presence
of immune IgG is expressed as a percentage of the activity in the
presence of an identical amount of control IgG. The results demonstrate
that immune IgG against reverse transcriptase (anti-reverse transcriptase)
inhibited the reverse transcriptase 70% but did not inhibit polymerases
.alpha.,.beta. or .gamma. from human milk (FIG. 4). Neither DNA
polymerase .alpha. from human lymphoid cells nor DNA polymerase
.gamma. from NC-37 cells or from HeLa cells was inhibited by the
antibody to reverse transcriptase (FIG. 5).
Preparation of Reverse Transcriptase Immune IgG
A solution containing 200 .mu.g of human milk DNA polymerase (reverse
transcriptase) obtained from the poly(C)agarose column was divided
into 8 portions of 1 ml each. For the first two immunizations, a
portion of the transcriptase (25 .mu.g) was emulsified with an equal
volume of complete Freund's adjuvant and injected into 6 subcutaneous
sites on the back of a New Zealand rabbit. Subsequent immunizations
were made at weekly intervals and were similarly performed except
that incomplete adjuvant was used. A total of 8 immunizations were
performed and the animal was bled from the ear vein at the end of
the 8th week. Samples (50 ml) of blood were taken, the serum was
recovered, and the IgG fraction was purified by precipitation with
50% ammonium sulfate and by DEAE-52 cellulose chromatography. The
purified IgG fractions were free of ribonuclease and deoxyribonuclease
activity. Normal (nonimmune) IgG was similarly purified from serum
obtained from the same rabbit prior to immunization. The IgG fractions
were dyalized against 0.01 M tris-HCl (pH 8.0) and stored at -70.degree.
C. The protein concentration of IgG was determined by optical absorption
at 280 nm, with an assured extinction coefficient for a 1% solution
and a 1 cm path length of 14.2 (Little, J. R. et al, Methods in
Immunology and Immunochemistry, 2: 343 (1969)).
Having now generally described this invention, it will be apparent
to one of ordinary skill in the art that the same can be carried
out with minor modifications or changes which do not affect the
content or spirit thereof.