Note: Descriptions are shown in the official language in which they were submitted.
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PROCESS FOR PRODUCINC ~IUMAN INTERFERON
AND METIIOD FO~ ASSAYING THE INTERFERON PRODUCTIVITY OF BLOOD
Field of the Invention
The present invention relates to a process for producing human-
specific interferon (abbreviated as " HuIFN" hereinafter) using whole human
blood and to a method for assaying the HuIFN productivity of human blood.
Description of the Prior Art
In recent years, clinical tests whereby the level of a blood en-
zyme or its metabolite is determined chemically have been in a wide use.
Since HuIFN, a blood component, exhibits antiviral- and antitumor
activities, it has been proposed to include the level of serum HuIFN in the
items of clinical test. Such proposal, however, has not been realized
because serum HuIFN is minute.
Blood consists of a fluid, plasma, in which are suspended formed
elements such as erythrocyte, leukocyte and platelet. One mm3 blood
generally contains, in addition to 7 . 4X103 leukocytes and 3X105 platelets in
adult, 5,4X106 erythrocytes in man or 4.8X106 in woman.
It is well known that HuIFN is produced by human leukocyte.
In conventional processes to produce HuIFN, viable leukocytes
separated from human blood are used. For example, as is evident from
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Hans Strander and Kari Cantell, Ann. Med._ex~. Fenn., Vol.
44, pp.265 273 (1966), and the specifications of Japan Patent
Kokai Nos.6,111~74 and 94,008/78, leukocytes are separated
from other element3 present in whole blood, and then allowed
to produce HuIFN.
Detailed studie~ on these conventional processes
confirmed that they, however, give a low recovery yield of
leukocytes ~rom blood, i.e. 30-50~, and damage leukocytes
during the separation to lower the viability to 40-60~ and,
eventually, the overall recovery yield to 10~30%, as well as
that the separated leukocytes give an inconsistent HuIFN
production; these facts render the estimation of the HuIFN
productivity of blood very difficult, and cause an obstacle
in mass-production of HuIYN.
Summary of the Invention
According to an a~pect of tha invention there is
provided a clinical assay for detacting human cancer, comprising:
collecting whole blood from a subject; incubating in vitro
a predetermined amount of the whole blood in the presence of
an effective amount of an anticoagulant and an effective amount
of a virus under conditions appropriate for the production
of interferon; and determining the interferon level in the
resultant culture, whereby the determination of an interferon
level below a predetermined amount is indicative of the possible
presence of human cancer~
According to a further aspect of the invention there
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is provided a clinical a~say for detecting human cancer,
comprising: collecting whole blood from a subject; removing
the pla~ma from the donated whole blood; suspending the residual
solid in a member selected from thle group con~isting of saline,
.isotonic buffer, and nutrient culture medium; incubating in
vitro a predetermined amount of thle resultant cell suspension
in the presence of an effective amount of an ant~coagulant
and an effective amount of a virus under conditions appropriate
for the production of interferon; and determining the interferon
level in the resultant culture, whereby the determination of
an interferon level below a predetermined amount is indicative
of the possible presence of human cancer.
Detailed Descri~ion of the Invention
.
As the results of my researches for the ~ass-production
of HuIFN using preciou~ human blood, as well as for the assay
of the HuIFN productivity by use of a donated blood, the present
inventor eventually found that a large amount of HuIFN can
be produced with an ea~e by incubating a whole blood in a veqsel
while exposing the whole blood to an anticoagulant and a viru~.
The present inventor also found that the HuIFN productivity
of a whole blood can be determined with an ease and a high
reproducibility by incubating a whole blood in a vessel while
exposing the whole blood to an anticoagulant and a virus, and
titrating the accumulated HuIFN.
Detailed studies confirmed that the exposure to an
intact or inactivated virus of an amount of 20-200,000 HA/ml
whole blood is favor-
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able . " I IA " represents the unit of the haemagglutination titer of a virus .
The wording of "whole blood" means fresh blood preparations
collected from donors, and also suspensions which are obtained by removing
plasma liquid from such blood preparations and suspending the residual
formed elements in an suitable non-plasma liquid, e . g . physiological saline,
buffer solution or nutrient culture medium.
Any anticoagulant capable of preventing the coagulation of such
whole blood does not affect HuIFN production is usable in the invention.
For example, heparin, acid citrate-dextrose (ACD) and citrate-phosphate-
dextrose ( CPD ) are favorable .
The viruses usable in the invention are those which are capable
of inducing HuIFN production in the whole blood. For example, Sendai
virus or Newcastle disease virus may be used intact or after inactivation.
The inactivated viruses usable in the invention include those whose repro-
ductivities are partially or completely suppressed, for example, by uv-
irradiation, heating or treatment at an extreme pH. An appropriate range
for inoculum of the virus is 20-200,000 HA/ml whole blood.
The step of incubating the whole blood in a vessel while exposing
the whole blood to the anticoagulant and virus is carried out in such a
manner that the whole blood is exposed in the vessel to the anticoagulant
and virus to produce HuIFN. For example, to the prescribed amounts of
the anticoagulant and virus in the vessel is added an appropriate amount of
the whole blood, and the mixture is incubated therein. Alternatively, a
mixture of the anticoagulant and whole blood is placed in a vessel, added
with the virus, and incubated. In this incubation step, a suitable medium,
e. g . physiological saline, isotonic buffer solution or nutrient culture me-
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dium, may be used adclitionally.
Tank, jar, flask, test tube, ampul and micro plate well of anyshape and volume may be used as the vessel in the invention.
The incubation conditions are those under which HuIFN is pro-
ducible: for example, temperature range of 30-~0C; and incubation time,
5-50 hours. In this case, priming or superinduction may be carried out if
necessary .
After incubation to produce HuIFN and an optional dilution with
physiological saline or isotonic buffer solution, the whole blood is then
separated with suitable procedure(s), such as centrifugation or filtration,
to remove formed elements such as blood cells, and the resultant supernat-
ant or filtrate containing HuIFN is subjected to purification or titration.
The HuIFN can be purified to obtain an HuIFN preparation having
a possible highest purity by combination of conventional procedures, e. g .
salting-out, dialysis, filtration, concentration, adsorption and desorption by
ion exchange, gel filtration, affinity chromatography using a suitable ligand
such as antibody, isoelectric point fractionation and electrophoresis.
The obtained HuIFN is advantageously feasible as injection or
drug for an external or internal use in the prevention and treatment of
human diseases alone or in combination with one or more substances.
The HuIFN productivity of human whole blood can be determined
according to the invention by titrating the HulFN level in the above de-
scribed supernatant or filtrate. For the purpose of such titration, any
assay can be used as long as the HuIFN production by the whole blood is
titrated therewith; e.g. bioassay, radioimmunoassay and enzyme-linked
immunosorbent assay.
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In recent years, enzyme-linked immunosorbent assay ha~ been
developed as a highly safe, convenient and speedy assay. Any enzyme-
linked immunosorbent assay capable of titrating IFN as the antigen is em-
ployable in the invention. For example, double antibody sandwich tech-
nique and modified double antibody sandwich technique are favorable.
It was confirmed that the HuIFN productivities determined in this
way are very useful for clinically testing the individual donor.
The method according to the invention confirmed that the blood
collected from a cancer patient is much lower in blood HuIFN productivity
than those collected from healthy volunteers.
The following experiments further explain the present invention.
Experiment 1
Effect of pretreatment on the HuIFN productivit~of blood
The effect of pretreating blood on the productivity of HuIFN was
studied. In this Experiment, fresh blood samples from three healthy volun-
teers were used after heparini~ation.
The treated bloods used in this Experiment were as follows: a
plasma-free suspension, obtained by centrifuging blood to remove plasma
liquid and suspending the residual formed elements in RPMI 1640 medium to
give the same element density as that in blood, and an ammonium chloride-
treated suspension, obtained by treating blood with Tris-HCl buffer (pH
7.2) containing 0.7596 ammonium chloride in usual way to effect the haemoly-
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sis of the erythrocytes, centrifuging the mixture and suspending the re-
sultarlt erythrocyte-free formed elements in RPMI 1640 medium to give the
same element density as that in blood.
One ml aliquots of the heparini~ed or treated blood were placed in
different plastic test tubes which were then added with 0.1 ml aliquots of
physiological saline containing Sendai virus in respective amount of 0, 100,
or 1,000 HA, followed by 16-hour incubation at 37C. The incubated
mixtures were uv-irradiated to completely inactivate the Sendai virus, and
centrifuged to obtain supernatants which were then assayed for HulFN
titers per ml whole blood.
The HuIFN titer was determined by the dye uptake assay report-
ed in Anne L . R . Pidot, Applied Microbiology, Vol .22, No.4, pp .671-677
(1971). The haemagglutination titer (HA) was determined by the method as
reported by J.E. Salk, The Journal of Immunology, Vol.49, pp.87-98 (1944)
with slight modification.
The results are given in Table 1.
As is evident from these results, the whole blood and plasma-free
suspension containing the whole formed elements of blood are favorable for
assaying the HuIFN productivity because of its high and consistent HuIFN
productivity. It was also confirmed that the ammonium chloride-treated
suspension wherein the erythrocytes were haemoly~ed and removed gives a
low and inconsistent HuIFN productivity.
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Table 1
Healthy volunteer
TreatmentSenclai virus _ _
A B C
. _ _
0 30 60 10
No treatment 100 3,800 2,500 4,200
1,000 3,700 2,600 ~,500
~ . . _ , _ . _ _
suspension1 ~ ooooo 4 16000 22 84oo 4 200
_ . _ . . _ _ _
treated suspension 1, 000 0 3000 1070
. _ . . _
Experiment 2
Effect of virus_inoculum on the productivity of HuIFN
The effect of virus inoculum on the productivity of HuIFN was
studied. Fresh blood samples from three healthy volunteers and two cancer
patients were used after heparini~ation.
According to the method as described in Experiment 1, 1 ml
aliquots of either heparinized blood sample were placed in different test
tubes, added with O.1 ml aliquots of physiological saline containing Sendai
virus in respe~tive amount of O, 2, 20, 200, 2,000, 20,000, or 200,000 HA,
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incubated, and assayed for HuIFN titers per ml b:lood.
A series of experiments using 2,000,000 HA Sendai virus per ml
blood was scheduled, but not clone because preparation of such a high-titer
Sendai virus was unsuccessful.
The results are given in Table 2.
T able 2
Healthy volunteer Cancer patient
Sendai virus
(HA)
D E F G H
0 S0 80 20 10 0
2 130 70 30 10 0
2,600 1,~00 5,400 10 30
200 2,~00 2,400 5,800 20 70
2,000 4,100 2,100 6,300 20 190
20,000 3,500 2,300 8,800 10 140
200,0004,400 2,200 7,300 . 10 180
2,000,000 ND ND . ND ND ND
Note: ND means not done.
As ;s evident from these results, virus inocula in the range of
20-200,000 ~lA/ml blood are favorable.
It was confirmed that the blood collectetl from a cancer patient is
much lower in blood HuIFN productivity than those collected from healthy
volunteers. This suggests that the assay of blood HuIFN productivity is
helpful for the detection of cancer in its early stage.
Several embodiments of the present invention are disclosed here-
inafter .
Production of HuIFN
Example 1
One ml of a heparinized fresh blood from a healthy volunteer was
placed in a plastic test tube, added with 1,000 HA of Sendai virus, incu-
bated at 37C for 20 hours, and uv-irradiated to completely inactivate the
virus. After centrifuging the mixture, the resultant supernatant was
assayed for HuIFN titer.
The HuIFN production was about 3, 600 units per ml blood .
Example 2
One ml of a hepar;nized fresh blood from a healthy volunteer was
added with 2, 000 HA of Newcastle disease virus wherein 90~ of the repro-
ductivity had been inactivated. After incubating at 37C for 15 hours, the
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mixture was assayed for HuIFN titer similarly as in Example 1.
The l-luIFN production was about 2, 800 units per ml blood .
Example 3
A heparinized fresh blood from healthy volunteers was centrifuged
to remove plasma. The formed elements so obtained were then centrifugally
washed in physiological saline, and suspended in RPMI 1640 medium to give
the same element density as that in blood.
The resultant suspension was placed in a mini jar, and added
with 500 HA of Sendai virus per ml suspension. After incubating at 37C
for 16 hours, the mixture was treated and assayed for HuIFN titer similarly
as in Example 1.
The HuIFN production was about 3, 000 units per ml blood .
Example 4
A suspension containing the formed blood elements was prepared
similarly as in Example 3.
The suspension was placed in a mini jar, added with 300 units of
HuIFN per ml suspension, and incubated at 37C for 6 hours. Thereafter,
the suspension was further added with 1, 000 HA of Sendai virus per ml
suspension, and incubated at 37C for additional 16 hours. The resultant
was treated and assayed for HuIFN similarly as in Example 1.
The HuIFN production was about 27, 000 units per ml blood .
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Assay of blood HuIFN productivity
Example 5
One ml of a heparinized fresh blood from a healthy volunteer,
28-year old man, was treated similarly as in Example 1, and subjected to
bioassay for HuIFN titration.
The HuIFN productivity was about 3, 600 units per ml blood O
Example 6
One ml of a heparinized fresh blood from a healthy volunteer,
33-year old woman, was treated similarly as in Example 2, and assayed for
HuIFN titration similarly as in Example 5.
The HuIFN productivity was about 2, 800 units per ml blood .
Example 7
A heparinized fresh blood from a healthy volunteer, 61-year old
man, was treated similarly as in Example 3 to obtain a suspension containing
the formed blood elements.
One ml of the suspension was placed in .a plastic test tube, and
added with 1,000 HA of Sendai virus. After incubating at 37C for 16
hours, the mixture was subjected to double antibody sandwich technique,
an enzyme-linked immun~sorbent assay, for HuIFN titration.
The HuIFN productivity was about 3, 400 units per ml blood .
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This value was consistent with that obtained by bioassay.
Example 8
A heparinized fresh blood from a cancer patient, 68-year old man,
was treated similarly as in Example 5 to obtain an HuIFN productivity of
about 140 units per ml blood.
Example 9
A heparinized fresh blood from a cancer patient, 55-year old
woman, was treated similarly as in Example 5 to obtain an HuIFN productiv-
ity of about 70 units per ml blood.
It will be obvious to those skilled in the art that various changes
and alterations may be made without departing from the scope of the inven-
tion and the invention is not to be considered limited to what is described
in the specification.
