Note: Descriptions are shown in the official language in which they were submitted.
THE INVENTION
This invention is directed to a novel group of
compounds, herein termed Recognins. Recognins are made by ;~-
treatiny tumor cells or artificial cancer cells and separating
the desired products. The Recognins may be used to prepare
their Chemoreciprocals, i.e., by contacting the Recognins or
the Recognins on a support with body fluids. These Chemo~
reciprocals are useful for diagnostic and therapeutic purposes~
i.e., or diagnosing and treating cancers.
.
One of the Recognins of the present invention is
Astrocytin. Astrocytin is produced from brain tumor tissue,
: . .
preferably brain glioma tumor tissue. Protein fractions con-
taining the Astrocytin precursor are first extracted from the
tissue. A preferred method of accomplishing the extraction
is to treat the tissue with a neutral buffer under conditions
of homogenization or other techniques to disrupt the cells
and tissues in order to solubilize protein fractions which
;~ :
contain the Astrocytin precursor. ~-
At this point, the Astrocytin precursor is still
bound to many large molecular weight substances including
protein, glycoproteins, lipoproteins, nucleic acids, nucleo-
proteins, etc. The solubilized proteins are then separated
from the resultant tissue extract. The extract solution from
.. . .
the tissue is then clarified to remove insoluble particles.
The low molecular weight contaminants are then removed from
the resultant solution, by a perevaporation concentration
- technique. The solution which is obtained is then treated to
cleave Astrocytin precursor from other contaminants in order
to obtain the protein raction having a pK range between 1
~ ,.
and 4. Thus, for example, the solu~ion is placed on a chroma-
tographic column and eluted with increasingly acidic solvents.
All of the fractions which are eluted in the neutral or acid
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range down to pK 4 are discarded and those fractions with pK
range 1-4 are collected. The elua-te is then treated to obtain
a product having a molecular weight of about 8,000. This is
accomplished, for example r by first filtering the material to
remove low molecular weight substances, i.e., those below
l,000 molecular weight, and filtering again to remove those
above 25,000. The fraction having a molecular weight between
1,000 and 25,000 is then furthex treated, i.e., by thin layer
gel (TLG) chromatography, to obtain Astrocytin.
Thus Astrocytin may be produced b~ extracting brain
glioma tumor tissue with a neutral buffer, by repeated homo-
genization and high speed centrifugation, separating from
the resulting extract the fraction having a pK range of from
about 1 to 4, separating from said fraction the substances
having a high molecular weight, i.e., up to about 230,000, and
isclating therefrom the product Astrocytin having a molecular
weight of about 8,000. ~;
The product Astrocytin prepared in accordance with
this process is characterized by forming a single line pre-
cipitate with its specific antibody in quantitative precipit.in
tests and Ouchterlony gel diffusion tests, being soluble in
water and aqueous solutions having an acid or neutral pH,
; and insoluble at an alkaline pH, having a spectrophotometric
absorption peak wave length of 280 m~and having a molecular
weight of about 8,000.
' Astrocytin is also characterized by having a
I very high percentage of residues of glutamic acid and aspartic
acid and a very high ratio of these acids to histidine. A
further analysis of Astrocytin is provided below.
In a manner similar to that described above,
another Recognin, called Malignin, is produced from artificial
cancer cells, i.e., cancer cells grown in vitro. Malignin has
:,' ~: .
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a molecular weight of about 10,000 and similar but distinct
amino acid residue compositi.on to Astrocytin, i.e. high ratios ~ -:
of glutamic acid and aspartic acid and high ratios of these
acids to hist.dine. A further analysis of Malignin is provided `~.
below. : -
: Thus, Malignin can be produced by extracting ~ ;
; artificial cancer cells grown in culture with a neutral buffer ::
hy repeated homogenization and high speed centrifugation,
separating from the resulting extract the fraction having a pK
range of about 1 to 4, separating from said fraction the : :
substances having a high molecular weight, i.e. up to about .
230,000, and isolating therefrom the product having a molecular
weight of about 10,000.
Malignin prepared in accordance with this process ~ :
.. is characterized by forming a single line precipitate with its
specific antibody in quantitative precipitin tests and Ouchter-
lony gel diffusion tests, being soluble in water and aqueous ~.
solutions having an acid or neutral pH, and insoluble at an `.
alkaline pH, having a spectrophotometric absorp~ion peak Wave .
length of 280 m ~ and having a molecular weight of about 10,000. ~.v
: Recognins are further characterized by being
capable of complexing with bromoacetylcellulose to form bromo~
. acetylcellulose-Recognin and producing the specific antibodies
; Anti-Recognin upon injection into mammals, said Anti-Recognin .
being toxic to brain tumor cells in vitro and producing fluor- .
: escence of glioma cells when coupled with fluorescëin, as
described in further detail below.
Recognins, such as Astrocytin, Malignin and
`. similar substances are useful as products which may be intro- ~ :
30 duced into a biological system to reduce foreign reactions,
such as by coating a material with a Recognin. A further
example may be to introduce a Recognin in order to produce
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the Chemoreciprocals in the biological system. They may also
be used nutritionally to encourage the growth of a particular
biological system of which they are a part. A further utility
of Recognin is the production of Target reagents which comprise
the complexes of the Recognin with a carrier to facilitate -~
its applicability in biological systems. Thus, for example,
the complex conveys the physical-chemical characteristics of
the Recognin itself. The carrier should be selected from
those which form a complex with the Recognin and which are
substantially biologically inert.
Any substance known in the art which will form a
stable complex with polypeptides or proteins may be useful for i ;~`
complexing with the Recognin. An example is a cellulose-based
material, such as bromoacetyl-cellulose. In addition to being ;
inert to the biological system, the carrier should be one that
does not alter the specific physical-chemical properties of
the Recognin which are useful for the purposes set forth herein.
The complexes of the Recognin and its carrier are `~
useful for producing, separating and identifying its chemo-
reciproccal in any biological system with which it is brought
into contact. The Recognin~carrier complex is also useul
for stimulating the production of its chemoreciprocal precursor
in any biological system into which it is introduced.
One class o Chemoreciprocals are the anti-
Recognins, i.e. anti-Astrocytin and anti-Malignin. These may
be made by injecting the Recognin into a biological system.
~, An immunologically efective dose of Recognin is brought into
I contact with bodily tissues or fluids in a manner which
1~ induces an antibody response in accordance with techniques
known in the art for producing antibodies. The anti-Recognins
may be used for the delivery of materials such as diagnostic,
nutritional and therapeutic agents to specific cells or sites
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in a biological system which comprises introducing said agent
in complexed form with the anti-Recognin into the biological
system. The anti-Recognins are also useful for diagnosing the
presence of tumor cells in a histology section, by applying
the Anti-Recognin conjugated with a labeling subs-tance such as
dyes and radioactive substances, to said section, whereby
staining or radioactive labeling occurs only with tumor cells.
Yet another use for anti-Recognins is for increasing the
yield of other useful Chemoreciprocal products (such as TAG
described below) from a mammal which comprises injecting an
immunologically effective dose of Recognin into the mammal,
or other biological system.
Another class of Chemoreciprocals i5 Target -~
reagents complexed with their chemoreciprocals. For example,
the Target product of Astrocytin complexed with a carrier such
as bromoacetylcellulose is brought into contact with anti-
Astrocytin. This type of compound may be complexed with and ;~
used for the delivery of diagnostic, nutritional and thera- ~ ~
peutic agents to specific cells or sites in a biological system. ~ `
These compounds may also be used for purification procedures.
For example, Anti-Astrocytin may be made by the decomplexing ~ ;
of Bromoacetylcellulose-Astrocytin-Anti-Astrocytin by hydrolytic
treatment with an acid or proteinase enzyme. Target reagents
are also useful for increasing the amount of TAG products
(described below) in a biological system, such as by bringing
an immunologically effective dose of Target into contact with
bodily tissues or fluids.
Additional Chemoreciprocals are TAG reagents
(e.g. Target-Attaching Globulins). The TAG products are
produced by bringing Target reagents into contact with body
fluids for varying periods of time to form a complex and
cleaving TAG therefrom. Two useful embodiments are S-TAG and
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F-TAG.
A process for producing S~TAG (Slow-Target~
Attaching-Globulin) comprises reacting blood serum or other
body fluid with Target (i.e. Bromoacetylcellulose-Malignin) for -
approximately two hours or more at a low temperature, e.g.
about 4C, and cleaving S-TAG rom the resulting material, e.g~
with dilute acid for approximately two hours at a temperature
of about 37C. The produc~ S-TAG prepared in accordance w.ith
this process is characterized by being soluble in aqueous
buffered solutions, forming a single line precipitate with
its corresponding Recognin in Ouchterlony gel diffusion testsr
being non-dialyzable in cellophane membranes, being retained
by millipore filters which retain molecules over 25,000 ,~
molecular weight, having molecular weights in different states
of aggregation as determined by thin layer gel chromatography
of approxlmately 50,000, and multiples thereof into the macro-
~I globulin range and having a spectrophotometer absorption peak
! ; wave length of 280 m ~.
A process for producing F-TAG (Fast-Target-
0 Attaching-Globulin) comprises reacting blood serum or other
body fluid~with Target (i.e. Bromoacetylcellulose-Malignin)
for approximately 10 minutes at a low temperature, ~e.g. about
4C, and cleaving F-TAG from the resulting material, e.g.,
with dilute acid for approximately two hours at a temperature
::
of about 37C. The product F-TAG prepared in accordance with
, this process is~characterized by being soluble in aqueous
buffered solutions, forming a single line precipitate with
,
its corresponding Recognin in Ouchterlony gel difusion tests,
` being non-dialyzable in cellophane membranes, being retained ``
by millipore filters which retain molecules over 25,000
1~ molecular weight, having molecular weights in different
states of aggregation as determined by thin layer gel chroma-
i, , ,, ~'
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7 t;~
tography of approximately 50,000, and multiples thereof into
the macroglobulin range and having a spectrophotometer
absorption peak wave length of 280 m~.
TAG products are useful for detecting cancer
tumors in living mammals by determining the concentration of
S-TAG and F-TAG produced by a known vclume of the mammal's
blood serum or other body fluid and correlating this concen-
tration with amounts determined to be indicative of cancer.
TAG products are also useful for diagnosing the presence of
tumor cells in a histology section, which comprises applyiny
TAG conjugated with a labeling substance such as dyes and
radioactive substances, to said section, whereby staining or - ~
radioactive labeling occurs only with tumor cells. TAG `
pro~ucts additionally have been found to be cytotoxic to tumor
cells. TAG products are also useful for directing the delivery `
of diagnostic, nutritional and therapeutic agents to specific
cells or sites by introducing said agents in complexed form with
. . .
the TAG product.
Normal cell division in plants or animals is
restricted or inhibited when the cells come to occupy fully a
particular space. The mechanisms (a) by which normal cells
"recognize" that they have filled the space available to them,
and (b) by which the operation of this recognition mechanism
in turn inhibits cell division, have both been unknown. The
inventor has produced a group of compounds whose precursors
are increased in concentration when normal recognition and
learning occur, and which relate to recognition and learning in
particles and cells, and with the connection of cells to each
other. These compounds are termed RECOGNINS by the inventor.
1 .^ 30 By attempting to produce these compounds from normal cancer
cells, the inventor has discovered that they are absent as such,
and tha~ changes in their molecular structure have occurred at
, ~ :
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the same time that the cancer cells have lost their ability ~a)
to recognize that they have filled their normal volume, and/or
(b) to stop dividing when they have filled their normal volume.
The inventor has discovered novel compounds and
methods for producing such compounds. These new compounds are ~
termed RECOGNINS by the inventor. RECOGNINS are novel compounds ~ -
; which have physicochemical characteristics which mimic those
configurations characteristic of cancel cells in terms of their
failure to recognize and stop cell division. The use of
RECOGNINS goes beyond insight into the cancer mechanism, for
immediate products and method are thereby provided which are
useful in the diagnosis and treatment of cancer, and for its
prevention.
I have discovered methods by which artificially
cultured cells can be used to produce MALIGNI~s for the first
time. One advantage of the methods disclosed herein is that ;
MA~IGNINS and new products from them can now be manufactured ;~
efficiently in virtually limitless quantities.
` This invention transcends the field of cancer
research and is immediately applicable to any and all biological
systems in which it is desired to influence all growth and
metabolism. Thus by the manufacture of the particular compound
or compounds of appropriate cell type in artificial culture~
and the further manufacture of products from these substances
specific influence may for the first time be brought ~o bear
on any tissue, cell, cell organelle, sub-organelle molecule or
molecular aggregate in any living system. Thus specific
nutritional influences at critical times in development,
specific diagnostic, preventative and treatment methods, and
. .
the construction of artificial bioelectrical systems (as in
tissue or organ transplants) can all be affected for the first
time. These artificial bioelectrical systems can now be made
to bear the characteristics of the specific RECOGNIN, MALIGNIN
.. ~ .
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or their CHEMORECIPROCALS of the normal tissue or component
which they will neighbor and -thus avoid being "recognized" as
"foreign" and thus avoid the reactions to alien substances,
including rejection.
Another aspect of this invention is the production
of a valuable specific antibody-like product (Anti-Astrocytin)
; to a specific brain product ~Astrocytin), permitting the use of ~ ~
this antibody-like product to specifically complex with and, as ~ -
a specific delivery vehicle to, specific points in the nervous
system of all species. MALIGNINS and ASTROCYTIN are RECOGNINS.
Still another aspect of this invention is the
production from biological fluids of two new products, TARGET- ~ .
ATTACHING-GLOBULINS (TAG), which are so named because they are
produced by two reactions, the first reacting biological fluids
with a synthetic complex containing physicochemical configurations .
which mimic those of the MALIGNINS and called TARGET, the second,
cleaving the specific TAG from the complex, and by the measure of
the TAG so produced obtaining a ~uantitative indication from the
biological fluids of living organisms whether there is present ; :
a tumor in that organismi hence a diagnostic test for tumors.
Because TAG products and ANTI-MALIGNIN are physicochemically
complimentary to MALIGNINS, they are termed CHEMORECIPROCALS.
I have further discovered that two ~uantitatively
and ~ualitatively distinct TAG products can be produced de- ;
pending upon the time permitted for the reaction of serum with
the specific TARGET reagent used, and depending upon the time `~
permitted for the cleavage of the product which has been
complexed.
.:i . .
After examining the amounts of these products
30 which could be produced from a number of different individuals .
with brain tumors and various other medical disorders, as well
as in those with no apparent disease process, it became apparent
. . ',
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that the amounts of these two new produc-ts which could be
produced in a given individual was indicative of whether that
individual had a brain tumor, hence a serum diagnostic test
for brain tumors, the first to my knowledge.
The utility of these new products, in addition to
their use to diagnose from serum and other biological fluids
the presence of brain and other tumors, is illustrated by the
demonstration that TAG and anti-RECO~NIN compounds attach to
glial tumor cells preferentially in histological sections of
brain tumor and surrounding tissue removed at surgery of the
brain tumor. This preferential labelling by TAG and Anti-
RECOGNINS of tumor cells is demonstrated through standard
immunofluorescent techniques. Thus a new method is also available
for determining through histological examination with a new
degree of certainty whether tumor cells are present in the
tissue removed, and whether these tumor cells have penetrated ;
to thevery edges of the tissue removed indicating the likeli-
hood that tumor still remains in the brain or other organ, or
that tumor cells are absent from the periphery of the tissue
removed, indicating the possibility that all of the tumor has
been removed from the brain or other organ. In addition,
TAG and Anti-RECOGNINS produced as described have been found
to be cytotoxic for glioma brain tumor cells grown in tissue
culture in vitro. This high affinity for tumor cells in another
medium, here grown in tissue culture, is further evidénce of
the specific-coupling potential of the new product TAG, and
explains the adoption of the name TARGET-ATTACHING-GLOBULINS
(TAG) as do TAG's properties in regard to the synthetic product ~
,: :
TARGET, and to tumor cells in histolo~ical section. Further, ~
the cytotoxicity of TAG and anti-RECOGNINS for tumor cells ;
provides an additional new diagnostic test for seru~ of patients
who are suspected of suffering from a tumor. Thus, for example,
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the serum or other body fluid of these patients is reacted with
TARGET -to produce l'AG and the product TAG is tested in tissue
culture growths of tumor cells for cytotoxicity. Both the
concentration of TAG and the degree of cytotoxicity manifested
by the TAG which can be produced from a yiven individual ' 5 serum
may be not only diagnostic but also of value in tracing the
course of the disorder preoperatively and postoperatively in
a given patient. Coupling of radioactive and dye tracers to
TAG provides new TAG products which are useful in viv~ in the
diagnosis of tumors and in their exact localization. Thus the
injection of suitably labelled TAG elther intraarterially or
intravenously, into the cerebrospinal ~luid, or directly into
brain tissue or its cavities, permits the demonstration by
radioactive means, or by visualization of the coupled dye, of
` the presence of a brain tumor, for it is only to the tumor cells
that the TAG specifically attaches. Further, this method permits
the precise visualization of the location of the brain tumor.
This can be seen to be an improvement of this in vivo diagnostic
method using anti-ASTROCYTIN produced in rabbit blood to label
the brain tunor, because the use of TAG produced from human
serum avoids the possibility of foreign protein reactions. Since
TAG and anti-RECOGNINS have the chemical specificity which per-
mits preferential attachment to ASTROCYTIN precursor containing
tumor cells both in vi~ro and in vivo, these products may also
be used therapeutically, as well as diagnostically, when coupled, ~-
e.gO, with radioactive proton capture agents, or other toxic
physical or chemical agent.s, so that these toxic substances may ~ '
be localized preferentially through these compounds' specificity
of attachment in the tumor cells as compared to their neigh-
; 30 boring normal cells. This selectivity is universally recognized
as the crucial, or at least one crucial factor for achieving
effective chemical or physical therapy of tumors, and a factor
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which has hit}lerto not been achieved~ Thus T~G has demonstrated
efficacy in attaching preferentially to the tumor cells, and
should have promise as a new therapeutic product for these
reasons.
In the serum of patients with malignant tumors, as
will be seen in the examples below, one type of TAG, SLOW-TAG
(S-TAG) as distinguished from FAST-TAG (F-TAG), can be produced
in relatively greater amounts from a given volume of serum than
in patients without such tumors. This suggests that either one
of TAG's naturally occurring precursors (P-TAG) is increased in
concentration or tha-t other factors exist which favor the
relative in vitro production of S-TAG over F-TAG.
The possible relationship of the function of the
actual synthetic products TARGET and TAG to their precursors,
and in turn to functions of postulated but not demonstrated
cell "antigens" and circulating "antibodies" to them which may
exist in vivo has yet to be elucidated. Thus for example, in
antibody-like fashion, F-TAG and S-TAG produce single discrete
lines of reaction with ASTROCYTIN in Ouchterlony gel diffusion,
and the injection of TARGET in rabbits induces an increase in
the yield of TAG products from rabbit serum after reacting with
TARGET. The finding that there may be a normal level of a
precursor resembling circulating antibody to a cell antigen
which is hidden in the non-dividing cell raises a question as
to the possible function of the pair. It is here proposed that
TAG precursor (P-TAG) and TARGET-like substances exist in vivo
which function in the control of cell proliferation and cell
death~ Thus, for example, the exposure of a cell constituent
which normally is not directly exposed to serum proteins may
occur during cell division. The exposure of this cell ;~
constituent could result in that constituent becoming converted
-to a TARGET-like substance to which the attachment of a P-TAG- ~ ;
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~35i716~
like molecules from serum may then occur, which would stimulate
cell division or inhibit it. Alternatively, a non-dividing
cell which is injured or malfunctioning may expose a TARGET-
like substance to which the attachment of P-TAG-like molecules
may be reparative. However, under certain cell conditions
the attachment of P-TAG-like molecules may induce the
destruction of the cell (e.g. ANTI-GLIOMA-TAG synthetically
produced as here described is markedly cytotoxic to glioma
tumor cells growing in tissue culture). This could thus represent
a mirror of a normal mechanism for the control of cell division,
and for either the repair or the removal of individual cells in
the body throughout the life of the organism. If the exposure
of cell constituents is abnormally increased so that abnormally
large amounts of cell TARGET-like substances are ormed, as
may occur in rapidly dividing cancer cells such as in brain
gliomas, an increase in the concentration of one type of serum
P-TAG relative to another may be induced.
Whatever the actual function of the precursors,
the increase in the relative amount of predominately one type of
TAG, SLOW-TAG (S-TAG) which can be produced in ~itro by the
methods here described from the serum of patients with malignant i~;
tumors is the basis of the serum diagnostic test described in
the examples which follow. -
The following examples illustrate the invention.
EXAMPLE I
~, ;-:
Production of Crude ASTROCYTIN~Precursor-Contain-
ing Fraction.
Human brain glioma tumor tissue/ removed at
surgery, is dissect~d free as possible of surface blood vessels
and normal brain tissue. For a typical amount of dissected
tumor tissue of 11 grams, the tissue lS weighed into six 1.5 g.
and two 1.0 g. aliquots. Each aliquot is then treated as follows: .~
- .''' ' ' .
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Each aliquot is homogenized in neutral buffer
solution by sonification or other mechanical means. For
example, each aliquot is homogenized in 100 cc per g. of tissue
of 0.005 M phosphate buf~er solution, pH 7, in a Waring* blender.
Homogenization should be done in the cold to prevent denaturation
of proteins. For example, the blender should be precooled in a
cold room at 0-5C and operated for about only three minutes.
The homogenate is then centrifuged for clarifi-
cation, for example at 80,000 times ~ravity for 30 minutes in a
refrigerated ultracentrifuge. The soluble supernatant is
decanted and kept in the cold. The insoluble residue is re-
homogenized with a further 100 cc of neutral buffer and centri-
fuged as before, and the second soluble extract combined with
the first. Best yields are obtained when this procedure of
homogenization and centrifugation is repeated until less than 50
micrograms of protein per ml. of solution are obtained in the
supernate. With most tissues this is accomplished by the fifth
extraction.
The solutions thus obtained are combined and
concentrated by perevaporation with subsequent dialysis, as by
dialysis against 0.005 M phosphate buffer in the cold to
produce a volume of 15 ml. The volume of this solution is
noted, an aliquot is taken for total protein analysis, and
the remainder is fractionated to obtain the protein fraction
having a pK range between 1 and 4. The preferred method of
fractionation is chromatography as follows.
The solution is fractionated in the cold room
(4C) on a ~EAE cellulose (Cellex*-D) column 2.5 x 11.0 cm.,
which has been equilibrated with 0.005 M sodium phosphate
buffer. Stepwise eluting solvent changes are made with the
following solvents (solutions): Solution (1) 4.04 g. NaH2PO
; and 6.50 g. Na2HPO4 are dissolved in 15 litres of distilled
H2O (0.005 molar, pH 7); Solution (2) 8.57 g. NaH2PO4 is ~;
*Trade}nark -16-
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dissolved in 2480 ml. of distilled El2O; Solution (3) 17.1 g.
of NaH2PO4 is dissolved in 2480 ml. of distilled H2O, (0.05
molar, pH 4.7); Solution (4) 59.65 g. of NaH2PO4 is dissolved in
2470 ml. distilled H2O (0.175 molar); Solution (5) 101.6 g of
NaH2PO4 is dissolved in 2455 ml. distilled H2O (0.3 molar, pH 4.3);
Solution (6) 340.2 g. of NaH2PO4 is dissolved in 2465 ml. of
distilled H2O (1.0 molar, pH 4.1); Solution (7) 283.64 g. of 80
phosphoric acid (H3PO4) is made up in 2460 ml. of distilled H2O
(l.0 molar, pH l.0).
Add nervous tissue extract, 6 to lO ml. volume.
Let it pass into column. Then overlay with Solution (l) and
attach a reservoir of 300 ml. of Solution (1) to drip by gravity
onto the column. Three ml. aliquots of effluent are collected
by means of an automatic fraction collector. The subsequent ~ `
eluting solutions are exchanged stepwise at the followiny
elution tube numbers. Solution (2): at tube 88, bring solution `~
on column to top of resin, then overlay and attach reservoir f s
50 ml. of Solution ~2); Solution (2): at tube 98, bring solution
on column to top of resin, then overlay and attach reservoir
of 75 ml. of Solution (3); Solution (4): at tube 114, bring
solution on column to top of resin, then overlay and attach
reservoir of 150 ml. of Solu~ion (4); Solution (5): at tube 155,
bring solution on column to top of resin, then overlay and -
attach reservoir of 125 ml. of Solution (5); Solution(6): at
tube 187, bring solution on column to top of resin, then overlay
and attach reservoir of 175 ml. of Solution (7); continue
eluting until at tube 260, elution is complete. Use freshly
prepared resin for every new volume of tissue extract. Each
effluent tube is quantitatively analyzed for protein. The ~ ~
elutes ln the tube numbers 212 to 230 are combined, and contain ~ ~;
the crude products from which ASTROCYTIN will be produced.
While data has been published on this crude
., ", `~ .
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l~S'~
material, called ~raction 10B ln the past, [Protein ~etabolism
of the Nervous System, pp. 555-69 (Pleum Press, 1970); Journal
of Neurosurgery, Vol. 33, pp. 281-286 (September, 1970)~ the
cleavage from fraction 10B of the specific product herein called
ASTROCYTIN has now been accomplished. Crude fraction 10B can
be prepared as a product in amounts between 0.1 and 10 mg. per
gm. of original fresh nervous system tissue from which it was
obtained. In addition to an ASTROCYTIN-precursor it contains
varying amounts of covalently bound carboh~drate residues
including a number of hexoses, namely glucose, galatose, mannose;
hexosamines, including ~lucosami~ne, galatosamine and mannosamine;
and occasionally other sugars, such as fucose, ribose and perhaps
rhamno~e. It also contains large molecular weight protein pro-
ducts, several lipids and nucleic acids.
EXAMPLE 2
Production of Purified ASTROCYTIN from Crude
ASTROCYTIN-Precursor-Containing Fraction.
The ASTROCYTIN-Precursor-Containing fraction is
further isolated rom contaminants. In the preferred embodiments,
the material from Example 1 is chromatographed on Sephadex* G-50
resin with a typical column of 40 cm. long, 2.5 cm. diameter,
and 196 ml. volume. The pressure used is 40 mm. Hg.; the flow
rate is 35 ml. per hour, and the buffer is 0.05 molar phosphate
buffer solution, pH 7.2. The first (flow-through) peak contains
ASTROCYTIN-Precursor together with impurities, whereas subse-
quent peaks contain only impurities.
In the preferred embodiment, the products in the
above first flow-through peak are then concentrated on Sephadex*
G-15, then passed onto a column of Cellex-D with the same
solutions, tl) through (7~ as Example l, and the same elution
steps as per~ormed in Example 1. The product ASTROCYTI~ is
present as a sharp peak in the same tubes (numbers 212-230) as
' ~
*Trademark
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before, thus maintaining its behaviour on Cellex-D chroma-
tography without the presence of a large number of contaminants.
Low molecular weight contaminants may then be
removed by techniques known to the art, such as millipore disc
filtration. In the preferred method, the product ASTROCYTIN is
freed of salt and other small molecular weight contaminants by
filtration through Millipore Pellicon Disc* No. 1000, 13 mm.,
which retains substances of molecular weight greater than 1000
and permits to pass through those of molecular weight less than
1000. The product ASTROCYTIN remains on the Pellicon Disc, and
is recovered from it by washing with 5Olution (l) of Example l.
ASTROCYTIN is then obtained by isolating the
compound havinga molecular weight of about 8000 from the above
solution. A preferred method uses thin layer gel (TLG)
chromatograph as follows:
The apparatus used is the commercially available
one designed by Bochringer Mannheim GmbH; Pharmacia Fine Chemicals
and CAMAG (Switzerland). The resin 2.5 g of Sephadex G-200 ~;
superfine is prepared in 85 ml. of 0.5~ NaC1 in 0.02 M. `
Na2HPO4KH2PO4 Phosphate Buffer pH 6.8 (6.6-7.0). Allow to swell
two or three days at room temperature with occasional gentle
mixing. (Magnetic and other stirrers should not be used). The ;
swollen gel is stabilized for three weeks at refrigerator
temperature; however, bacterial and fungal growth may interfere
with the swollen gel. If the gel is to be kept for longer
periods of time, a small amount of a bacteriostatic agent should
be added (sodium Azide 0.02%) 2.5 g. of dry gel are used to make
two 20 x 20 cm. glass plates of 0.5 mm thick. The plates are
either allowed to dry at room temperature for 10 minutes and
transferred to a moist chamber where they can be stored for
about two weeks, or they are used immediately after appropriate
pre-equilibration. (Usually during the night for a minimum of
', .: ' '
*Trademark
:~ -19-
~35'~6B~
12 hours). The main Eunction o equilibration is to normalize
the ratio between the stationary and mobile phase volumes. With
the pre-equilibrated plates in a horizontal position, substances
to be determined are applied with micropipettes as spots or as a
streak at the start line. 10 ml. to 20 ml. of 0.2-2~ protein
solution is placed on the edge of a microscopic cover slide
(18 x 18 mm.) and held against the gel surface. In a few seconds
the solution will soak into the gel. All samples are first
prepared on the cover slides and then quickly applied. If not
enough material is used, it is difficult to locate individual
spots after separation. If too much material is applied no
defined separation occurs. The samples are diluted with buffer ~ ;
for easier handling and the separation of samples is carried in
a descending technique ~ith the plate at a~ angle of 22. The
flow rate of about 1-2 cm/hour is most suitable. Marker sub-
stances (such as cytochrome C, haemoglobin, myoglobin or bromo-
phenol Blue labeled albumin) are applied at different positions
across the plate to give a check on possible variation of flow
across the plate and also to serve as reference proteins for
calculation of relative distance (mobility) of unknowns. After
application of samples, the plates are replaced in the apparatus
and the paper wick pushed slightly downwards to ensure good con-
tact with the gel layer. The paper wick must not drip. Excess
moisture is wiped of. The liquid solvent in the reservoir is
kept constant at 1 cm. from the upper end of the vessel. The
runs are usually completed in 4 to 7 hours depending on the pro-
~ress o separation. With colored substances separation follows
directly. The separated spots of protein are easily made visible
by transferring them to a paper sheet replica of TLG plate after
the chromatographic separation has been completed, and by stain-
ing th~m on the prewashed methanol + H2O + acetic acid -
90 5:5, for 48 hours. The paper sheet is 3 mm. filter paper. A
-20
~S7ti~
sheet of paper 20 x 18 cm. is placed over the gel layer and
pressed trolled) just enough to ensure contac-t with the gel.
Care is taken not to trap air under the paper (replica) and not
to disturb the gel layer. The liquid phase is soaked off from
the gel layer by the paper and removed ater about one minute,
immediately dried in an oven at a 60 temperature for 15 minutes
and stained in the normal way with any o~' the routine stalning
procedures. Staining is performed by spraying the replica-paper
with 0.03% diazotized sulfanilic acid in 10% Sodium Carbonate
(Pauleyls Reagentj. Staining can also be accomplished with a ;
saturated solution of Amido Black in Methanol-Acetic Acid ;
(90:10,v/v is used); the staining time is 5-10 minutes~ For ;
destaining, rinse with two ~olumes of the 90:10 methanol and
acetic acid solution mixed with one volume of H2O. It is
difficult to obtain low background staining without very
., .
extensive washing. The plates themselves may also be dried at
about 60C (in an oven with air circulation) but only if the
ASTROCYTIN is to be stained. For isolation purposes, the plate
should only be air dried at room temperature. Over-heating `~
can lead to cracXing, but this can usually be avoided with at
j
50-60~C temperature which dries a Sephadex G-200 plate in `
15-30 minutes. The dry plates are allowed to swell for 10
minutes in a mixture of methanol ~H2O ~ acetic acid (75,20:5)
and stained in a saturated Amido Black in the same solvent
system for five hours and subsequently washed by bathing for
two hours in the same solvent before they are dried. For
molecular weight determinations the distance from the starting
line to the middle of each zone is measured with an accuracy
;l '
;, of 0.05 mm. either directly on the print (replica) or on the
- 30 densitogram. The result is e~pressed by the Rm value defined as
~ ,
the ratio of the migration distance of the tested protein (dp) ;~
to that of cytochrome C or myoglob,in (dm) which is used as the
reference protein: ~elating migration distance of tested
.': ,-
-21-
... ~ .
;~05'7~
substance to standard is the formula ( Rm~ dp~. A straight
d
m
calibration line is obtained by plotting the lo~ithm of the
molecular weight of the standards used against the Rm. From
this line the molecular weight of the unknown protein can be
obtained. For most exact results six equal parts of the protein
sample solution with standard, in this case, Cytochrome C, before ;~
applying to the plate. By the above TLG procedure the product
ASTROCYTIN is observed as a discrete spot at a distance of
approximately 0.83 - 0.02 with reference to the standard
Cytochrome C, yielding an approximate molecular weight of
8000 for ASTROCYTIN. Several discrete products are separated
in this procedure from ASTROCYTIN on the basis of slight
differences in chemical structure and large differences in
molecular weight. Thus, three products carried as contaminants
to this point with molecular weight of approximately 64,000,
148,000 and 230,000, and one occasionally of molecular weight
32,000, have been detected and removed by the TLG methods
described above. The product ASTROCYTI~ is aspirated with the
gel in which it is contained, in dry form, dissolved in Solution
(l) and freed of resin by centrifugation or other similar means.
The product ASTROCYTIN which has been produced
at this stage is soluble in distilled water, soluble at neutral
and acid pH, and insoluble at alkaline pH and has a spectro-
photometric absorption peak wavelength of 280 m ~. It is a
polypeptide with molecular weight, as stated above, of
approximately 8000. Its covalently linked amino acids are shown
by hydrolysis with 6N HCl then quantitative automatic determin
ation to have the following average composition of amino acids:
Approximate Number
o~ residues
Aspartic acid 9
Threonine 5
., ;
-22-
~s~
Approximate Number
of residues
Serine 6
Glutamic acid 13
Proline 4
Clycine 6
Alanine 9
Valine 4
1/2 Cystine 2
Methionine 1
Isoleucine 2 `
Leucine 8 ~;
Tyrosine 2 ~ `~
Phenylalanine 3 ~ ~ `
Lysine 8
Histidine 2
Arginine 4
Approximate ;~
Total 88
Cysteic acid, hydroxyproline, norleucine, ammonia, isodesmosine,
`
desmosine, hydr~xylsine, lysinonorleucine and gamma-aminobutyric ~ -
acid are all absent in detectable amounts, but a trace of gluco~
samine may be present.
From lI grams of the starting brain tumor tissue
in EXAMPhE 1, approximately 3 mg. o puriied ASTROCYrrIN is
produced by the above methods.
E~AMPLE 3 ;~
Production Oe MALIGNIN-Precursor in Artificial
Cancer Cell Culture.
30Generally, sterile technique is scrupulously
maintained.
All solutions [e.g. Hank's Balanced Salt ~BSS),
F-10 Nutrient medium, fetal calf serum, trypsin solution] are
-23-
., ~
~5 7~8~
incubated at about 35C in a water bath for approximately 20
minutes or more before use.
Cells are removed rom tumor tissue and grown
in vitro for many generations using a suitable medium, such as
described below. Pre-rinse ~eakers to be used with a sterilizing
solution, for example 12-proponal plus Amphyl or creolin solution
In the preferred embodiment, the arti~icial cancer
cells (i.e., cells grown in vitro for many generations) are grown
in 250 ml. flasks. The liquid medium in which the cells are
growing is discharged into the pre-rinsed beakers. The cells
are then washed gently with 5-10 ml. of Hank's BSS or other
similar solution for about 30 seconds. Avoid agitation. All
walls and surfaces are washed. The solution is clarified of
cells by centrifugation in the cold from 10 minutes at 3,000
rpm. The medium is poured into a beaker as above. Add a small
amount of buffered proteinase enzyme solution and rinse quickly
to avoid digestion of the cells. In the preferred method, 1-2 ml.
of trypsin solution (EDTA) is added and rinsed for only 10 seconds. ` ;
Pour off the trypsin solution.
Add a similar volume o fresh trypsin solution
and incubate until the cells are seen to be separated from the
walls of the chamber through microscopic observation. This
usually requires 5-10 minutes. Add a suitable growth medium,
such as 50 ml. of a solution of 7-10 percent solution of fetal
calf serum in 100 ml. of F-10 Nutrient medium.
T~enty-five ml. of the fresh medium with cells is
transferred to a new growth chamber for propagation and the
remaining 25 ml. is kept in the first chamber for propagation.
Both chambers are placed in an incubator at 35C for approx~
imately seven days. By the procedure of this Example to this
point, an artificial cancer cell culture is divided into two
fresh cultures approximately every seven days. This entire
'-
~ -24
.
~ 5i7~
procedure may be repeated as often as desired, at approximately
seven-day intervals, for each growth chamber. Thus, the
number of cells growing in vitro may be doubled approximately
every seven days.
The cells may be extracted for the production of ~;
MALIGNIN after approximately seven days of growth. For example,
cells growing in each 250 ml. growth chamber as described above, -
may be recovered as follows:
The medium is transferred to a centrifuge tube and
centrifuged at 3,000 rpm in the cold for 10 minutes. The medium
is discarded. The cells remaining in the growth chamber are ;
scraped from the chamber walls and washed into the centrifuge
tubes with neutral buffer solution. The cells are washed twice
with neutral buffer solu-tion, centriuged again at 3,000 rpm
in the cold, and the medium is discarded. The washed cells are
suspended in 10 ml. of neutral phosphate buffer until ready for
extraction of crude MALIGNIN-Precursor-Containing fraction.
EXAMPLE 4
Production of Crude MALIGNON-Precursor-Containing
Fraction.
Washed cells suspended in neutral bufer from
EXAMPLE 3 are mechanically disrupted under conditions which
avoid denaturation of most proteins. In the preferred method,
the washed cells are treated in the cold with a sonifier for
20 seconds.
After sonification the cell residues are
centrifuged at 30,000 rpm for 30 minutes and the supernatant
decanted. Ten ml. aliquots of buffer solution are used to
wash remaining cells from the chamber and these are added to
the remaining cell residues. Sonify and centrifuge as above and
combine the supernatants. Repea~ the process once more.
The combined supernatant is perevaporated to
~,
, .
-25-
:, , ,, , ., , . .. ,., ., . . , . . . .: .
~135'7~
reduce the approximate 30 ml. volume to about 6-7 ml. An
aliquot is taken for total protein analysis and the remainder `~
is fractionated according to the methods of EXAMPLE 1 for
ASTROCYTIN Precursor.
EXAMPLE 5 ~ -
Production of Purified MALIGNIN Product from
Crude MALIGNIN-containing Fraction.
The product MALIGNIN is further isolated from `~
contaminants by the methods of EXAMPLE 2 for ASTROC~TIN.
In the TLG step of the preferred embodiment, -~
the product MALIGNIN is obse~ved as a discrete spot at a distance
of approximately 0.91 - 0.02 with reference to ~he standard
cytochxome C, yielding an approximate molecular weight of
10,000 for MALIGNIN.
The pxoduct MALIGNIN which has been produced at ~`;
this stage is soluble in distilled water, soluble at neutral
or acid pH, and insoluble at alkaline pH and having a spectro- `~
photometric absorption peak of 280 m ~. It is a polypeptide
with molecular weight of approximately 10,000. Its covalently
20 linked amino acids are shown by hydrolysis with 6N HCl then ,
quantitative determination to have the following average
, composition of amino acids:
I Approximate Number
I of residues
Aspartic Acid 9
Threonine 5
Serine 5
Glutamic Acid 13
Proline 4
Glycine 6 `
Alanine 7
Valine 6
~ ,, .
' ~; 1/2 Cystine 1 ~ ~ ~
, ; :,
-26- ~ ~
.. . , ,. .. .. , , . ~ .... ~ . . . .. .
6~ -
Approximate Number
of residues
Methionine 2
Isoleucine 4
Leucine 8 ~;;
. . ~ .
Tyro sine 3
Phenylalanine 3
Lysine 6
Histidine 2 ~ ~
Arginine 5 ~;
Approximate
Total 89
the amino acids cysteic acid, hydroxyproline, norleucine,
i ammonia, isodesmosine, desmosine, hydroxylysine, lysinonor-
J leucine and gamma-aminobutyric acid being absent in detectable
? amounts.
t: A typical yield of pure MALIGNIN from twelve
250 ml. reaction chambers of EXAMPLE 3 together is approximately
~ 1 mg. of MALIGNIN.
i I EXAMPL~ 6
2~0 ~ ~ Production of TARGET Reagents from RECOGNINS.
. i ~
ASTROCYTIN,~ prepared as in EXAMPLE 2 above, or
MALIGNIN, prepared as in EXAMPLE 5 above, is complexed with a
carrier to produce TARGET reagent.
;,:
In the preferred embodiment, ASTROCYTIN or ~
MALIGWIN is dissolved in 0.15 M NaH2PO4 - citrate buffer, pH 4Ø ;;
A bromoacetyl-resin, for example bromoacety1cellulose (BAC)
having 1.0 to 1.5 miIliequivalents Br per gram of cellulose,
stored in the cold, is prepared in 0.15 M Na~I2PO4 buffer, pH 7.2.
Convert the buffer to pH4~by pouring off the p~ 7.2 buffer
~1 30 solution and adding 0.15~ M Na~2PO4 - citrate buffer, p~l 4Ø ;~
The ASTROCYTIN or MALIGNIN solution and the BAC solution are
stirred together (10:1 BAC~to RECOGNIN ratio) for 30 hours at
:,
i room temperature, then centrifuged.
,, ! ; ~,
~, -27-
.~:~ .:;
: 1 ;
7~
It is preferred that all sites on the BAC which are
available to bind to RECOGNIN be bound. This may be accomplished
as follows. The supernatant from the immediately preceding step
is lyophilized and the protein content determined to indicate
the amount of ASTROCYTIN or MALIGNIN not yet complexed to BAC~
The complexed BAC-ASTROCYTIN (or BAC-MALIGNIN) is resuspended
in 0.1 M bicarbonate buffer pH 8.9, stirred 24 hours at 4 to
permit the formation of chemical bonds between the BAC and the
ASTROCYTIN or MALIGNIN. After the 24 hours, the suspension
is centrifuged and supernatant analyzed for protein. The
complexed BAC-ASTROCYTIN or BAC-MALIGNIN is now resuspended in
0.05 M aminoethanol - 0.1 M bicarbonate buffer pH 8.9 in order
to block any unreacted bromine. The suspension is centrifuged,
and the supernatant is kept but not analysed because of the
presence of aminoethanol. Removal of all unbound ASTROCYTIN or
MALIGNIN is then accomplished by csntrifugation and resuspension
for three washings in 0.15 M NaCl until no absorbance is measured
on the spectrophotometer at 266 m ~ . The BAC-ASTROCYTIN or
BAC-MALIGNIN complex is now stirred in 8 M urea for 2 hours
at 38C, centrifuged, then washed (three times usually suffices)
with 8 M urea until no absorbance is shown in the washings at ~
266 m~. The complex is then washed with 0.15 M NaCl two times "~;
to rid of urea. The complex is then stirred at 37C in 0.25 M
acetic acid for 2 hours to demonstrate its stability~ Centrifuge
and read supernatant at 266 m ~ no absorbance should be
present. This chemically complexed BAC-ASTROCYTIN or BAC- -
MALIGNIN is therefore stable and can now be used as a reagent
in the methods described below; in this stable reagent form it ~ `
is referred to as TARGET (TOPOGRA~HIC-ANTIGEN-LIKE-REAGENT-
TEMPLATE) because it is a synthetically produced complex whose
physical and chemical properties mimic the stable cell-bound
precursor of ASTROCYTIN or MALIGNIN when it is in a potential ` ;~
-28-
~ .
3 ~5~7~
reactive state with serum components. For storing, TARGET
reagent is centrifuged and washed until neutralised with 0.15 M
NaH2PO~ buffer pH 7.2.
TARGET reagents may be prepared from bromoacetyl
liganded carriers other than cellulose, such as bromoacetylated
resins or even filter paper.
: .
` EXAMPLE 7
:
Production of antisera to Astrocytin, Malignin -
and TARGET.
Antisera to Astrocytin, Malignin or TARGET reagents
may be produced by inducing an antibody response in a mammel to
them. The following procedure has been found to be satisfactory. ~;
One mg. of RECOGNIN (Astrocytin or Malignin) is ~-
injected into the toe pads of white male rabbits with standard
Fruend's adjuvant, and then the same injection is made intra-
3.
.~ .
peritoneally one week later, again intraperitoneally ten days ~ -
a~nd, if necessary, *hree weeks later. Specific antibodies may
be detected in the blood serum of these rabbits as early as
one week to ten days after the first injection. The same
; ~ 20 procedure is followed or TARGET antigen by injecting that
:j :
amount of TARGET which contains 1 mg. o Astrocytin or Malignin
i as determined by Folin-~owry determination of protein.
The speci~ic antibody to Astrocytin is named
Anti-Astrocytin. The specific antibody to Malignin is named
,
Anti-Malignin. Similarly, the specific antibody to TARGET re-
1 agent is named Anti-Target.
} These antibodies show clearly on standard
Ouchterlony gel diffusion tests for antigen-antibody reactions
with specific single sharp reaction ~lines produced with their
specific antigen.
The presence of specific antibodies in serum
can also be tested by the standard quantitative precipitin test ;~
;j :
-29-
', ~
~S761~ :
for antigen-antibody reactions. Good quantitative precipitin
curves are obtained and the micrograms of specific antibody
can be calculated therefrom.
Further evidence of the presence of speciic
antibodies in serum can be obtained by absorption of the specific
antibody Anti-Astrocytin onto Bromoacetyl-cellulose-Astrocytin
(BAC-Astrocytin) prepared above. The antiserum containing
specific Anti-Astrocytin can be reacted with BAC-Astrocytin.
When the serum is passed over BAC-Astrocytin only the specific
antibodies to Astrocytin bind to their speciic antigen
Astrocytin. Since Astrocytin is covalently bound to Bromo-
acetyl-cellulose the specific antibody, Anti-Astrocytin, is now
bound to BAC-Astrocytin to produce BAC-Astrocytin-Anti-Astro-
cytin (BAC~Anti-Astrocytin~. This is proved by testing the
remainder of the serum which is washed free from BAC-Astrocytin.
On standard Ouchterlony diffusion no antibodies now remain in
the serum which will react with Astrocytin. It is therefore
concluded that all specific antibodies (Anti-Astrocytin)
previously shown to be present in the serum, have been absorbed
to BAC-Astrocytin. Furthermore, when Anti-Astrocytin is
released from its binding to BAC-Astrocytin it is thereby
r isolated free of all contaminating antibodies. This release
of Anti-Astrocytin may be accomplished by washing the BACA-Anti-
Astrocytin complexed with 0.25 M acetic acid (4C., 2 hrs)
1 which has been shown above not to break the BAC-Astrocytin bond. -
'~ Still further evidence of the presence of specific -
i antibodies in serum can be obtained by adsorption of the specific
antibody Anti-Malignin onto Bromoacetyl-cellulose-Malignin
~, (BAC-Malignin) prepared above. The antiserum containing specific
Anti-Malignin can be reacted with BAC-Malignin. When the serum
;J is passed over ~AC-Malignin only the specific antibodies to
; Malignin bind to their specific antigen Malignin. Since Malignin
:'
,
:` :
1~5~7~
is covalently bound to Bromoace-tyl~cellulose the specific
anti-body, Anti-Malignin, is now bound to BAC-Malignin to
produce BAC-Maliynin-Anti-Malignin (sAcM-Anti-Malignln). This
is proved by testing the remainder of the serum which is
washed free from BAC-Malignin. On standard Ouchterlony
diffusion no antibodies now remain in the serum which will
react with Malignin. It is therefore concluded that all specific
antibodies (Anti-Malignin) previously shown to be present in ; ;
the serum, have been absorbed to BAC-Malignin. Furthermore,
when Anti-Malignin is released from its binding to BAC-Malignin ~
it is thereby isolated free of all contaminating antibodies. ~ ;
This release of Anti-Malignin may be accomplished by washing
the BACM-Anti-Malignin complex with 0.25 M acetic acid (4C.,
2 hrs) which has been shown above not to break the BAC-
Malignin bond.
The antibodies to TARGET show clearly on standard
Ouchterlony gel diffusion tests for antigen-antibody reactions
with specific single reaction lines produced with TARGET which
show a line of identity with the line of reaction to ANTI~
ASTROCYTIN or ANTI-MALIGNIN antisera (i.e. that produced to the
injection of ASTROCYTIN or MALIGNIN them~elves). Some rabbits,
it has been noted, have levels o ANTI~TARGET in their blood
prior to being injected with TARGET. These ANTI-TARGET sub-
stances, when reacted speciically with TARGET reagent as to be
described in tests of humansera, lead to the production of
approximately equivalent amounts of the two types of TAG, S-TAG
and E-TAG (see later EXAMPLES).
EXAMPLE 8
Detection of Malignant Tumors by Quantitative
Production in vitro of TARGET ATTACHING-GLOBULINS (TAG) from
Blological Fluids.
TARGET reagent prepared in accordance with
-31-
.-
8~ ~
EXAMPTE 6 is washed to remove any unbound RECOGNIN which may ~ ;
be present due to deterioration. The following procedure is
satisfactory. TARGET reagent is stirred for two hours at 37C.
with acetic acid, centrifuged, the supernatan-t decanted, and the
optical density of the supernatant read at 266 m ~. If there is
any absorbance, this wash is repeated until no further material
is solubilized. The TARGET is then resuspended in phosphate
buffered saline, pH 7.2. (Standard S-TAG and F-TAG purified
from previous reactions of human serum by the procedure
described below can be used if available, as reference standards
to test the TARGET reagent, as can whole rabbit serum which has
been determined to contain S-TAG and F-TAG by other T.ARGET . -~
preparations~
The Slow-Binding ( S-TAG) determination is per-
formed as follows: Frozen serum stored more than a few days
should not be used. Serum is carefully prepared from freshly
obtained whole blood or other body fluid by standard procedures
in the art. The following procedure has been found to be
satisfactory. Blood is allowed to clot by standing for 2 hours
at room temperature in a slass test tube. The clots are
separated from the walls with a glass stirring rod, and the
blood allowed to stand at 4C for a minimum of 2 hours (or
overnight). The clots are separated from the serum by
centrifuging at 20,000 rpm at 4C for 45 minutes. The serum
, is decanted into a centrifuge tube and centrifuged again at
2000 rpm at 4C for 45 minutes. The serum is decanted and a
1% Solution of Methiolate (1 g. in 95 ml. water and 5 ml
! 0.2 M bicarbonate buffer pH 10) is added to the extent of 1% -
. of the volume of serum.
Serum samples, prepared by the above or other
procedures, of 0.2 ml. each are added to each of 0.25 ml.
aliquots of TARGET suspension reagent containing 100-200
` ~ -32-
;, ,
,.,. , ~
~57~
micrograms oE RECOGNIN per 0.25 ml. TARGET reagent, in duplicate
determination. The suspension is mixed at 4C in a manner to
avoid pellet formation. For example, a small rubber cap rapid
shaken may be used for 1 2 seconds and then, with the tubes
slightly slanted, they may be shaken in a Thomas shaker for
about 2 hours or more. The TARGET reagent and protein bound to
it are separated from the serum. One of the procedures which has
been found to be satisfactory is the following. The tubes are
then centrifuged at 2000 rpm for 20 minutes at 4C, the super-
natant decanted, the pellet which is formed by centrifugation ; ;
washed 3 times by remixing and shaking at room temperature with
0.2-0.3 ml. of 0.15 M Saline, centrifuged and the supernatants
discarded.
The protein which remains attached to the TARGET
is cleaved therefrom and quantitatively determined. For example,
0.2 ml. of 0.25 M acetic acid is added, the suspension shaken
for 1 to 2 seconds with a rubber cup shaker, then in a Thomas
shaker for about 2 hours in a 37C incubator. The tubes are
centrifuged at 2000 rpm at 4C for 30 minutes. The super-
natant is carefully decanted to avoid transferring particles and
the optical density of the supernatant is read at 280 m ~. The
value of the optical density is divided by a factor of 1.46
j for results in micrograms per ml. serum protein (S-TAG).
. . ,
Duplicate determinations should not vary more than 5%. Any
other procedure effective for determining protein content may
~ be used, such as Folin-Lowry determination, but standards must
i be specified to determine the range of control and tumor values
of S-TAG minus F-TAG concentration.
The Fast-Binding (F-TAG) determination is per-
formed as follows: Frozen serum stored more than a few days `
should not be used. Serum is carefully prepared from freshly
obtained whole blood or other body fluid by standard procedures
-33-
-,: ; :
~I)S"~8~
in the art. Th~ procedure given above in this EXAMPLE for
serum preparation is satisfactory.
Serum samples, prepared by the above or other
procedures are allowed to stand at 4C for 10 minutes less than
the total time the S-TAG serum determinations were allowed to
be in contact with TARGET reagent above [e.g. 1 hour 50 minutes
if a "two hour" S-TAG determination was made]. This procedure
equilibrates the temperature histories of S-TAG and F-l~AG
determinations.
Add 0.~ ml. samples of the temperature equilibrated
serum to each of 0.25 ml. aliquots of TARC7ET suspension reagent ~-~
containing 100-200 micrograms of RECOGNIN per 0.25 ml. TARGET
reagent, in duplicate determination The suspensiQn is then
. .
mixed at 4C for approximately 10 minutes in a manner to avoid
pellet formation. For example, a small rubber cap rapid shaken `
may be used for 1-2 seconds and then, with the tubes slightly
slanted, they may be shaken in a Thomas shaker for approximately
10 minutes. The TARGET reagent and protein bound to it are
separated from the serum. One of the procedures which has been
:~ ;
found to be satisfactory is the following. The tubes are then
centrifuged at 2000 rpm for 20 minutes at 4C, the supernatant
decanted, the pellet which is formed by centri~ugation washed
3 times by remixing and shaking at room temperature with 0.2-
0.3 ml. of 0.15 M Saline, centrifuged and the supernatants ~ ~
discarded. ~ -
J The protein which remains attached to the TARGET
is cleaved therefrom and quantitatively determined. The procedure
described above in this EXAMPLE for determining S-TAG concen- ~?~
tration is satisfactory. Any other procedure effective for
determining protein content may be used, such as Folin-Lowry
determination, but standards must be specified to determine
the range of control and tumor values of S-TAG minus F-TAG`~
'i concentrations.
~ -34-
~5~
The final results are expressed as TAG micrograms
per ml. of Serum, and equal S-TAG minus F-TAG. TAG values in
non-brain-tumor patients and other controls currently range
from zero (or a negative number~ to 140 micrograms per ml. of
serum. TAG values in brain tumor patients currently range from ;
141 to 500 micrograms per ml. of serum. In the first "blind"
study of 50 blood samples conducted according to the procedures
of this EXAMPLE utilizing TARGET reagent prepared from
Astrocytin and bromoacetyl-cellulose, 11 of 11 brain tumors and
28 of 32 normals were correctly identified. One of the 4
supposed normals (l.e. non-brain tumor controls) turned out to
have a cancel of the tyroid gland which had apparently been -
successfully treated some years before. The -three remaining
normals were individuals aged 60-70 who were in poor health,
- possibly having nondiagnosed cancer. Of the remaining 7 samples,
three out of three cases of Hodgkin's Disease were correctly
identified; one sample in the tumor range (141-500 ug. TAG/ml.)
corresponded to a patient having a severe gliosis, and thre0
j samples in the non-tumor range (0-140 ug. TAG/ml.) corresponded
;~ 20 to patients having respectively, an intracranial mass diagnosis
uncertain but non-tumor, and oateosarcoma (non-brain tumor) and
a melanotic sarcoma ~non-brain tumor).
A subsequent study conducted according to the
procedures of this example utilizing TARGET reagent prepared
3 from MALIGNIN and bromoacetylcellulose correctly identified
three out of three malignant brain tumors and all normals.
EXAMPLE 9
Diagnosis of Tumor Cells by Immunafluorescence
' The compounds Anti-Astrocytin, Anti-Malignin, and
S-TAG have been shown to attach preferentially to tumor cells. ;
This specificity permits use of these compounds to diagnose
tumor cells in histology sections by conjugating dyes or radio-
., :
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7~;84
active substances to An-ti-Astrocytin r Anti-Malignirl~ or S-TAG.
Standard labeling techniques may then be used. A procedure
using S-TAG is as follows~
One procedure which has been found satisfac~ory
is amodified St. Marie procedure. Human brain tumor specimens
are frozen and 5 micron thick sections cut. These are stored in
a moist container at minus 70C for 4 to 8 weeks before staining.
The conjugate may be a standard antiserum such as goat anti-
rabbit conjugate. The conjugate is labeled by techniques known
in the art with fluoresein or other labeling substance. Fluores~
cein labeled goat anti-rabbit conjugate as commercially
available may be used. The fluorescent technique used was a
standard one in which a 1:2V0 to 1:400 solution of TAG is
incubated for about 30 minutes or more on the tumor section,
followed by washes to remove unattached TAG. Three washes with
phosphate buffered saline has been found satisfactory. Conjugate
incubation with 1uorescein-labeled conjugate followed by
washes is then perormed, followed by microscopic inspection. -
! Normal cells and their processes fail to stain both in tumor
sections and in control sections of normal non-tumor brain.
Fluorescence is brightly present in tumor glial cells and their
processes.
EXAMP~E 10
Demonstration that Anti Astrocytin, Anti-Malignin
and S-TAG are Cytotoxic to Tumor Cells Growing in Tissue Culture.
Standard tests for determining cytotoxicity may
be used. Generally, the number of cells in a fixed aounting
chamber, usually arranged to contain about 100 live cells, is
counted. These cells are then treated with the agent being
tested and the number of cells which are still alive is counted.
In a standard test of cytotoxicity of S TAG
Solution obtained in accordance with the methods of EXAMPLE 8
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., .
against cells in tissue culture derived from a patient with
a yliblastoma Grade III-IV, we]l characterized as ofglial origin,
S-TAG produced death of all cells in the counting chamber even
when in high dilution of 1:100 and 1:1000, representing as
little as 0.2 and 0.02 ug. of S-TAG per ml. of solution. Similar
results are obtained with high dilutions of Anti-Astrocytin
and Anti-Malignin.
Both the specificity exhibited in EXAMPLE 9 and
the cytotoxicity demonstrated in EXAMPLE 10 are highly relevant
to the therapeutic possibilities of Anti-Astrocytin,r Anti-
Malignin and S-TAG for brain tumors in man. While these
therapeutic uses are in the future, the practical diagnostic
potential of both of these phenomena for tumor tissue removed
at operation but requiring diagnosis by histology is already
demonstrated herein.
EXAMPLE 11
Hydrolytic Cleavage of RECOGNINS
A solution of RECOGNIN, in this case either
~,
Astrocytin or Malignin at pH between 1 and 2 is allowed to stand
~ 20 in the cold. After 7 to 14 daysl TLG chromatography shows the
product to have been reduced in molecular weight by approximately ~;
200. When the solution is allowed to stand longer, further
units of approximately 200 molecular weight are cleaved every
7 to 10 days~ Thus with Astrocytin the molecular weight is
reduced from 8,000l and with MALIGNIN the molecular weight is
, reduced from 10,000, in each case by units of approximately
,~ 200 sequentially. `
The physicochemical specificities of Astrocytin
, are retained by each product down to approximately 4,000
molecular weight. The physicochemical specificities of
Ma~ignin are retained by each product down to approximately
5,000 molecular weight. This is shown by Ouchterlony gel
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diffusion tests against An-ti-Astrocytin and Anti-Malignin,
respectively.
This cleavage can also be accomplished
enzymatically, as with trypsin and other proteinases, with
similar results.
The molecular weights of these compounds
prepared by hydrolytic cleavage o RECOGNINS may be approximately
defined by the following formulae~
For products having the physicochemical
specificities of Astro~ytin; 4000 + 200 x = Y.
,
For products having the physicochemical ;
specificities of Malignin; 5Q00 + 200 x = Y
wherein Y is the molecular weight of the product and X is an
integer from 0 to ]9.
EXAMPLE 12
Production of Artificial Tissue or
Organ with RECOGNINS
A rigid walled tube of plastic, metal, or other
suitable rigid material is dipped in or impregnated with a
i 20 highly concentrated, [i.e., 10 mg./ml.l viscous solution of
; RECOGNIN, in this case either Astrocytin or Malignin, until
all surfaces are fully coated with the RECOGNIN. Alternately, ~
., RECOGNIN solution is passed through and around the tube under `
pressure until all surfaces are fully coated. The tube is
~ then dried in air or in vacuo, or lyophilized. The process
i, of coating is repeated several times in order to build up `
multiple molecular layers of RECOGNIN coating.
The tube is now ready to be placed in a cavity
or in a tissue which contains Astrocytin or Malignin-like
. 30 precursors in the neighboring tissue or fluid of a living
;~ mammal. This artificial tissue or organ may be used to
minimize or eliminate reaction which foreign substances without ;
, '.'
-38- ~
'. ~
1~57&j89~
RECOGNIN coating would incite.
Artificial tissues or organs of other geometries
may similarly be produced.
1 0
. 3g_
:' ~
., .,, .. . .. , . . .... . . . ~.... . ~ . ~ , . . .
