Note: Descriptions are shown in the official language in which they were submitted.
.; l~Z931~
RZ II
The present invention relates to an in vitro testing
method for the diagnosis of malignant tumours, prefe-
rably human tumour~ comprising determining the mo~ility
varlation of cellular blood components contacted with
a migra~ion inhibition factor (MIF), wherein the MIF
is secreted from lymphocytes being sensitized against
tumour tissue. Such MIF is not secreted when the
lymphocytes come from patients who do not suffer from
malignant tumo~,,r(malignoma) since these lymphocytes
are not sensitized and, therefore, fail to release
MIF when contacted with tumourantigen.
It is kno~m from literature (~ouwes, F. R. et al.;
Verh. Deutsche Ges. inn. Med., Vol. 82, J. F. Bergmann-
Verlag, Munich) that the early diagnosis of malignant
diseases can be made by means of the so-called electro-
phoresis mobility test (EMT). The principle of the
EMT is based upon the fact that the velocity of migration
(mobility) of ~pecific bodies li. e. macrophagesJ
granulozytes and other blood corpuscles) visible in
the electric ield of a special microscope (zytophero-
meter) is only varied when these bodies come lnto
., .. , . . .......... ~ . , .
'
'~
1:1~3 ~9 `
- 2 -
contact with lymphokines of sensitized lymphocytes.
It is hereby assumed that lymphocytes which are sen-
sitized against a specific antigen (so-called T-
lymphocytes) release a series of soluble factors, i.
e. the so-called lymphokines. An 1mportantone among
these factors is the migration inhibition factor (MIF3.
This factor affects the capability of migration of
blood corpuscles, especially of macrophages and
granulocytes, and provides for their arresting and
accumulation at the place where the antigen inducing the
sensibilization is present. In vivo, this represents
a possibility to destroy the tumour.
Lymphocytes of patients suffering from malignoma
react in specific manner by releasing the so-called
soluble factors, when contacted with a basic protein
obtained from the brain substance on the one hand,
or from carcinoma tissue on the other hand. The
basic proteins causing the specific reactions of the
lymphocytes, are termed encephalitogenous factor (EF)
and basic protein from carcinoma tissue (CaBP). They
are produced in accordance with known per se methods
(Caspary, E. A. et al., Brit. Med. 1971, pages 61~
- 617, and other publications; compare Douwes F. R.,
loc. ~it.).
.
. :;
3L1293~,~
-- 3 --
The results of blood test with the EMT as described
in literature reveal that the test allows an almost
100 % differentiation of the samples to be made so
as to determine whether the samples have been taken
from an organism affected by malignant (tumour) diseases
or from a healthy organism or an organism affected
by non-malignant diseases, respectively. All of the
patients tested and suffering from (malignant) tumour
provided a positive lymphocyte response, i. e. the
lymphocytes yielded a factor resulting in a measurable
and characteristic variation of the mobility of indicator
cells (compare Douwes, F. R., loc. cit.). Control
diagnoses with patients affected by non-malignant
diseases did not provide such lymphocyte response.
Incorrect results could be traced back to specific
causes. Accordingly, the EMT can be considered to be
the presently most sensitive test for cancer.
It is considered to be disadvantageous in the perform-
ance of the test that qualified control of an instrument
being relatively complicated to operate, i. e. the
zytopherometer, is required. The personnel okserving
and measuring the speed of migration must be highly
trained and supervised very carefully. Owing to di-
minishing personnel attention, the operators can be
31~29;~i9
engaged with the work for a limited period of time only.
Accordingly, the use of the EMT is restricted to highly
specialized institutes. The analysis of the test is made
while the blood corpuscles are in motion, i.e. dynamically.
"Stopping" of the experiment is not possible; therefore,
repeated measurements cannot be taken, either.
It is the object of the prescnt invention to provide a
testing method for the diagnosis of malignomae, which method
is as general and unspecific as possible a manner with respect
to the type of the malignomae and with a high degree of
accuracy or reliability signals the presence of malignomae,
but owing to its priciple may be used or performed in any
medical laboratory, while lending itself particularly for use
in mass examination for preca~tional diagnosis of cancer. The
result of the test should be readily verifiable. The test
should be adapted to the analyzed statistically and recorded
without any intermediate protocolling.
Thesc objects are solved in a testing method as defined
above by subjecting as said cellular blood components the
lymphocytes as a part of a leucocyte suspension to be examined
prior to a known per se migration inhibition test wherein the
, . ~
. :
1129319
-- 5 --
lymphocytes are contacted with an antigen capable
of stimulating them to produce MIF, and inhibiting and
fixing the leul~ocyte migration after a predetermined
period of time.
Various migration inhibition tests are known which
are principally suitable for the examination in
accordance with the basic object. Among other methods,
the so-called capillary technique by S0BORG and
BENDI~EN is known. For the realization of the inventive
idea, it is necessary to detect the mobility of the
indicator cells by "freezing" a state of migration
reached a~ter a given period of time.
According to the invention, preferably the propagation
or variation of mobility of the lQ~ocytes within
an extremely fine "open" capillary layer of about 1O
microns ~m) thickness is measured. A test system has
been found to be particularly suitable, which comprises
a dual-layer element, namely a carrier or substrate
made of an inert medtum such as e.g. glass, and an
agar-agar being saturated with nutrients for ~lood
cells. A particularly suitable agar is one sold under
the tradename Agarose as produced by the firm Behring-
Werke of Marburg (Germany).
'
llZ93~9
-- 6
Preferably, the intermediate layer between the agar
and the carrier layer is partially filled with liquid.
This layer has a thickness of about 1 to 20 microns.
Advantageously, an incubation period in the MIT of
between 10 and 30 hours is chosen.
The test is performed with a sample of leucocyte-rich
supernatant phase ("buffycoat") obtained from human
blood and containing a fixed number of leucocytes of
between 104 and 107 per sample. These standard values
are approved and tried values which do not require
an excessive quantity of blood to be taken from the
patients.
The buffycoat sample is mixed with an antigen solution
in a ratio of from 1 : 50 to 1 : 10. To this end,
antigen solutions may be used which have been obtained
by a method disclosed by CASPARY and FIELD or by means
of a 3 M KCl extraction according to MELTZER. As the
starting material, human malignoma tissue from surgical
operations or human brain substance are employed.
However, it is also feasible to yield antigen solution
from tumour cells grown in vitro.
The testing method according to the invention i6 not
only applicable to an unspecific malignoma examination,
llZ~3~
but may be extended also to an organo-specific
examination. If antigens are employed which have
been recovered from carcinomous tissue of specific
~rgans, lymphocytes of patients affected by the
same organic carcinomae react in the indicated manner.
It has been disclosed in literature that antigen
solution obtained, for instance, from mammary
carcinoma according to CASPARY and FIELD or MELTZER,
result in a specific reaction exclusively ~ith
respect to lymphocytes coming from persons affected
by mamary carcinoma.
In the case that a positive immune response in a
general test is present, i.e. in the case of suspected
carcinoma, it is also possible to perform a series of
tests by us1ng organo specific antigens, and thereby
to determine within a relatively short per~od of
time, what organs might be affected by carcinoma.
It is of great importance that the so-called immune
response, i.e. the production of the slowing effect
in the mo~ement of the indicator cells in the course
of existing tests, can be suprisingly enhanced by
adding to the sample solution a ~olution containing
the so-called transfer factor. Such factor is known
.
~129319
from immunology (compare e.g.B.M. Rosenthal; "Der
Transferfaktor und seine therapeutische Anwendung";
Schweiz. med. Wschr., Vol. 104, 1974; pages 1501 -
1506). The transfer factor may be produced from
normal leucocytes of the blood. This factor represents
a subcellular, dialyzable, non-immunogeneous leuco-
cyte factor which is responsible for the T-lymphocyte
responsive reaction. Transfer factor preparations are
commercially available (SCHURA, Krefeld/Germany)
which have been prepared by fractionation in liquid
oxygen and subsequent gradual ultrafractionation.
One commercially available unit contains transfer
factor of about 5 x 101 leucocytes. The preparation
is stabilized by means of 1 % solution of human albumin.
It is found that the addition of transfer factor may
yield an increase in the immune response. Thus, the
discrlmination of the various sample tests is
substantially improved by the add~tion of the transfer
factor.
Further, the present invention relates to a test dish
or plate for carrying out the method, which plate
comprises a planar carrier plate of an inert materlal,
e.g. glass, havlng coated thereon a solidifled agar
,
;-
112931~:1
- g
layer of from 2 to 5 mm thickness and having at
least two substantially circular punched holes of
a diameter of the order of from o,5 to 5 mm
~preferably from 1,1 to 3,0 mm). Each punched hole
has a capacity of from 5 to 20 and preferably 8
microliters of test substance.
It is essential that the punched holes are cut with
such a configuration that uniformity of the capillary
migration layer is provided even in the transition
area between the punched hole and the layer.
Below, the invention is explained in Examples, and
furthermore, the following Figures are added:
igure 1 shows in plan view a test plate according
to the invention; and
igure 2 shows a test plate in cross-sectional
view.
Exam~le 1
A) Prepaxation of the le~cocyte suspension
30 ml of human blood are heparinized with 300 USP
units of Na heparinate. The heparinized human blood
~Z~3 ~
-- 10 --
(from a patient) is substrated within a plastic ferrule
with 8 ml of 6 percent by weight dextran solution in
physiological normal salt solution (molecular weight
of the dextran: 75,000). Thereupon, the erythrocytes
are allowed to sediment (settle) in a heating cabinet
for about 45 minutes at 37 C. The leucocyte enriched
supernatant phase (buffyco2t) is pipetted off and
mexed with an equal volume of 0,9 percent by weight
NaCl solution and then centrifuged at a centrifugal
force of 750 G's. The pellet deposited in the centrif-
uge is again washed for two tLmes in 10 ml of Hank's
solution (pH 7.2) each and suspended in a Tc 199 medium
(producer: Serva, Heidelberg). During washiny and
suspending, the cells are aqitated by means of a
pipette until lumps of cells are no longer visible
to the naked eye. Upon dyeing with Turk's solution,
the leucocytes arecounted in the Neubauer compartment
and asjusted to 2 x 105 cells/microliter in Tc 199
medium. In the average, the sample contains 25 % of
mononuclear cells (lymphocytes and monocytes) as well
as 75 % of granulocytes. The erythrocyte contamination
i~ le s than 1 to 1.
B) Preparation of the anti~en solution
For the preparation of the antigen solution, the
llZ93~9
- 11 -
encephalitogenous factor (EF) or the basic protein
of carcinoma tissue (CaBP) has to be isolated.
The tumour or the brain, respectively, are processed
as rapidey as possible under sterile conditions
after the surgical removal. Connective tissue and
healthy tissue are removed from the tumour. There-
after, the tumour or brain tissue it cut into small
pieces with sterile scissors (scalpel) and further
disintegrated in a bath of ice in a homogenizer. The
bumour tissue must not be heated during such processing.
The homogenate is suspended in four times the volume
of distilled water and then centrifuged at a force
of 23.000 G's. The thus produces pellet is again
suspende~ in a fourfold volume of 0.9 ~ NaCl, homogenized
and centrifuged at 23.000 G's for 30 minutes. The pellet
again suspended in four times the ~olume of dis~illed
water is thereafter freeze-dried. The freeze-dried
powder is blended with a mixture (2 : 1) of chloro-
form and methanol in a ratio of 1 : 10 and agitated
for 30 minutes. The mixture is centrifuged for 10
minutes at 600 G's. The supernatant phase is discar-
ded.
This process ~tep i8 repeated twlce. The pellet is
~ ~2~31~
- 12 -
then air dried. The dried pellet is resuspended in
the fivefold volume of a 5 % NaCl solution in water,
and centrifuged at 23,000 G's for 30 min. By adding
N/100 NaCl, the pellet again resuspended in a five-
fold volume of distilled water is adjusted to a pH
of 3.5. The suspension is slightly shaked and there- ;
after again centrifuged at 23,000 G's for 30 minutes.
The supernant phase is discarded. Again, the pellet
is resuspended in a fivefold volume of distilled
water which is adjusted to a pH of 2.6 with N/100
~Cl, and allowed to stand for from 3 to 18 hours.
The pH value is not allowed to vary during this period.
Then, the suspension is centrifuged at 23,000 G's
for 30 minutes. The supernant phase is stored, and
the pellet is again washed in N/100 NaCl at a pH of
2.6 and centrifuged. The supernatant phase resulting
fr~m this step is stored. The two lastmentioned super-
natant phaSeS are combined and then dialyzed~distilled
water. Hereupon, the product ls subjected to freeze-
drying.
An antigen test solution is obtained by dissolving
0.~ mg/ml of the freeze-drled substance in dlstilled
water. Principally the same process of preparation is
employed for obtaining a solution of the baslc protein
11~2931~
- 13 -
from tumour tissue (CaBP).
C) Preparation of the test plate
(compare also Figures 1 and 2)
0.5 g of a specific culture agar (agarose; producer:
Behring-Werke of Marburg) are weighted into a 50 ml
Erlenmeyer bulb or flAsk. 22.5 ml of sterile aqua
bidestillata are added to the mass.
22.5 ml of the double concentrated Tc 199 medium and
5 ml of plasma obtained from human blood are charged
into a second Erlenmeyer bulb. The agarose is dissol-
ved in a boiling water bath by being supported by a
bracket, under slight circulatory agitation. Hereby,
the Erlenmeyer bulb is sealed with a film or stopper
to prevent a part of water from evaporating.
When the agarose is completely dissolved, the substance
is cooled to 47 C in a water bath. In the s~me bath,
the medium-plasma mixture is preheated and added to
the agarose under shaking, such that there are now
pre ent 50 ml of 1 % agarose ln plain Tc 193 medium
containing 10 ~ of plasma.
~ r ~ ~
llZ93~
- 14 -
With the ald of a preheated discharge pipette, 5 ml
each of the agar are filled into circular tissue
culture dishes (Petri dishes) 10 having a diameter
of 50 mm, which dishes are shown in plan view in
Figure 1. Upon solidification of the agar, holes 11
of 2,5 mm diameter are punched out from the agar
by means of a telescoping punching canule ~ehring-
Werke, Marburg) with the aid of a template. In the
punching work, care has to be taken that the agar
layer is not released from the glass layer in the
punching area, in order that the capillary layer is
not increased such that, migration of the leucocytes
would no longer be possible.
The pattern of the holes 11 appears from Figure 1.
Of course other configurations are imaginable.
Figure 2 illustrates an enlarged sectional view of
the lower part of dish 10. ~s indicated, the agar
layer 1 is separated from the glass layer of the
dish 10 by a thin capillary layer 3. The various
columns of holes are provided e. g. for the following
purposes: I
~A) Control (standard)
(B) Test ~uspension contain$ng general antigen ;.
(C) Test suspension conatining organo-specific antigen
112931~
- 15 ~
(D) Test suspension containing organo-specific anti-
gen and transfer factor.
D) Reaction, incubation and fixation
,
The following preparations are provided for a test:
1) A preparation containing each 100 microliters of
leucocyte suspension according to (A) and 8 micro-
liters of antigen suspension according to (B);
2) a preparation containing each 100 microliters of
leucocyte suspension and 8 microliters of N/100
NaCl solution (for reference value).
The mixed preparations 1 and 2 are subjected to incu-
bation for 60 minutes in a heating cabinet at 37 C
and at 100 ~ humidity. Then, 8 microliters of leuco- :
cyte- and antigen-containiny substance or standard
substance, respectively, of the incubated solution
are filled into each punched hole. Depending on the
configuration of the test plate (number of punched
holes~, from 2 to 5 measurement results and from
2 to 5 reference values are available for the analysis
of the leucocyte migration and the effect of the factors
produced by the lymphocytes and delaying the migration.
~he cell~ contained in the suspension and introduced
112931~
, ~
lnto the punched holes at the start of the test, migrate
into the intermediate layer 3 between the agar and
the survace of the dish. By incubating the dishes at
a temperature of 37 C in the heating cabinet for a
period of 18 houxs, such migration is maintained
across a definite distance. The leucocytes do not
migrate into the agar, but rather take their nutrients
therefrom. Incubation is effected in a humidified
cabinet at a relative humidity of 100 %.
Following the incubation, the plates are coated with
methanol for 15 minutes, and subsequently with for-
malin solution (37 percent by weight of methanal) for
10 minutes. Thereafter, the agar is carefully stripped
from the bottom of the dish as a membrane. The cells
migrated in the intermediate layer are now fixed to
the surface of the dish. Upon drying, the cells are
dyed ~n accordance with the PappenheLm method, where-
by dark-violet halos 4 (m~gratlon halos) are formed on
the plate surface (compare Figure 1).
E~ Measuring and determinin~ the results
~slng a graduated mag~ifying glass (Bausch & Lomb),
the dlameter of the migration halo is measured. In
llZ93~9
- 17 -
such measurement, the polymorpho-nuclear cells discre-
tely positioned in the periphery should be detected,
too. The arithmetic average of a plurality of mea-
suring steps each is formed.
The quotient of the migration area F1 with antigen and
of the migration area F2 without antigen yields a
"migration index" MI:
MI F1/F2.
A migration index smaller than 0.85 means inhibition
of the leucocytes to significant degree and, thus, a
positive cell response. The following Table illustra-
tes one example of measurement:
Column 0 (mm) F (mm2~ MI
__ _ . _
B 6,75 F1 = 35,8 0,80
A 7,55 F2 = 44~75 (1)
1~93~
- 18 -
Initial experiments with a total of 97 female patienb
were conducted to demonstrate the effectiveness of
the migration inhibition test in the case of mammary
carcionmae. 27 among 46 female patients tested and
affected by mammary carcinoma showed an inhibition
higher than 15 ~ (= 58.7 %), i. e. the migration
index MI is less than 0.85. Among 23 female patients
tested and suffering from mastopathia, only four patients
showed such inhibition ~= 17 %), whereas in the case
of female patients affected by fibroadenoma (fibroma),
one female patient out of five (=20 %) showed an inhi-
bition. The following Table provides a general summary:
Diagnosis Number of patients Migration index
(n)(inhibited) (normal)
.
Controls 20 0 (O %) 20 (100 %)
Mastopathia cystica 23 4 (17 %) 19 (83 %)
Fibroadenoma 5 1 (20 ~) 4 (80 %)
mammary cysts 4 0 ( 0% ) 4 (100 ~)
Other malignant
diseases 9 3 (33 %) 6 ~67 %~
Mammary carcinoma 46 27 (58.7%) 19 (41.3 %)
Example 2
Employment of the transfer actor for increasing the
lmmune respon~e.
1~29319
1 9
It has already been noted that an increase of the im-
mune response may be cbtained by the addition of a solu-
tion containing transfer factor (producer: SCHURA,
Xrefeld3. It is found that with such addition the
factor MI is more accentuated as compared to Test -
Example 1.
The following test compositions were prepared:
1) 100 microliters of leucocyte suspension + 8 micro-
liters of antigen solution
2) 100 microliters of leucocyte suspension + 8 micro-
liters of antigen solution + 20 microliters of
transfer factor solution (prepared by SCHURA;
1 microliter corresponding approximately to the
transfer factor of 5 x 10 leucocytes)
33 100 microliters of leucocyte suspension
4) 100 microliters of leucocyte suspension + 20 micro-
liters of transfer factor solution as in composition
2).
Incubation and preparation of the dish are made in
the same manner as in Example 1. A dish having four
t$mes f$ve punched holes (diameter 2.5 mm) is pre-
pared. One column of 5 holes each is charged with
10 microllters o~ test compositlons 1 to 4.
~l~g3~9
- 20 -
Results (Average of five values each):
Composition Column Diameter (mm) F (mm2) MI
_ _ __ _ _
1 B 6,90 37,4 0,79
2 C 6,05 28,7 0,61 (incl.
transfer-
factor)
3 A 7,75 47,2 1,0
standard!
4 D 7,70 46,6 0,99
The low MI of columns of B/C indicates a malignoma case.
Results of the migration inhibition test by using the transfer
factor in the determination of gastrointestinal tumors:
Measurements were made in accordance with the m~asuring
instruction according to Example 2. No appreciable inhibition
was found in a (health) group of patients; the migration index
was of the order of 1.02 * O.l. The trans~er fractor did not
affect these results. In the group of patients by colonic
carcinomae, the migration index amounted to 0.65 ~ 0.2.
` ~ .
l~Z9319
Actually, 11 out of 15 cases tested showed a migration index
smaller than 0.85. Upon addition of the transfer factor, the
migration index was of the order of 0,61 ~ 0.09. Furthermore,
14 out of 15 patients exhibited an MI of less than 0.85 after
the addition of the transfer factor, such that a significant
improvement of the test result was obtained in this instance,
too. No substantial inhibition could be observed in other
groups tested.
Example 3
An antigen solution may be extracted also from tumour cells
which have been grown in v1tro in a fermentation culture, with
the aid of a buffer and with repeated disintegration of the
tissues.
Cells of cerebral tumour are grown _ vitro as follows:
Special care has to be exercised to malntain sterile
operating conditions. Preferably, the cells are washed
for about 30 seconds in a Han~'s balancing solution.
Agitation should be avoided. All walls and surfaces
of the bulb, preferabley a 250 ml bulb of glass, are
washed carefully. The solution is made free of the cells
';~
`d
~Z93~
- 22 -
by centrifuging under cold conditions for a period of
10 minutes. The medium is poured into a beaker glass.
A small quantity of buffered proteinaise encyme solution
is added and purged rapidly in order to avoided decom-
position of the cells. In the preferred method, up to
2 ml of trypsin solution (EDTA) are added in a bulb.
The trypsin solution is poured away after a purging
period of 10 seconds.
Subsequently, the same volume of fresh typsin solution
is added. Incu~ation is conducted until it can be seen
by observation under the microscope that the cells
.
separate from the walls of the chamber. This takes nor-
mally from 5 to 10 minutes. A suitable growing medium
is added, e. g. 50 ml of a solution of 7 to 10 %
solution of the serum of calf foetuses in 100 ml of
F-10 nutrient.
25 ml of the fresh medium including the cells are
transferred into a new growing chamber (bulb) for re-
productio~. Both chambers are placed into an incubator
at 35 C for about seven days. In accordance with the
process of this Example, a culture of the artificial
tumor celLs ~s divided up to this point into a pair
of fresh culture~ approximately every seven days.
1~293~
- 23 -
The mode of process may be repeated in intervals of
seven days. Accordingly, the number of cells grown in
vitro doubles every seven days.
The medium containing the cells is transferred into
a centrifuge tube and centrifuged at 3,000 rpm (~about
2200 G's) in the cold for 10 minutes. The medium is
discarded. The cells remaining in the growing chamber
are scraped from the chamber walls and washed or purged
into the centrifuge tubes with a neutral buffer so-
lution. The cells are washed twice with a neutral
buffer solution and then again centrifuged under cold
conditions at 3,000 rpm (- about 2200 G's). The medium
is disposed of. The washed cells are suspended in 10
ml of neutral phosphate buffer until they are prepared
for extraction. Then, the washed cells are treated under
cold conditions with a sonic dtvice for 20 seconds in
order to b~ak up the cells. However, denaturation of
the proteins must be avoided as far as posslble. Follo-
wing the acoustic irraditaion, the remains of the cells
are centrifuged at 30,000 rpm (- about 220 000 G's) for
30 minutes. The supernatant product is decanted. 10 ml
of buffer solution are employed for washing the remaining
cell residues. Another acoustic irradiation and centrl-
fugation are performed~ and the supernatant products
~29319
- 24 -
axe combined. This method is repeated once. The combined
supernatant product is prevaporized in order to reduce
the volume from about 30 ml to about 6 to 7 ml. A
corresponding portion is removed for the total protein
analysis.
By means of a liquid column chromatograph, and with
filtering through Millipore Filters, the preparation is
than made free of contaminants. Details of the respec-
tive process may be gathered from Example 2 of the laid-
open German patent application 2,606,257. A polypeptide
having a molecular weight of about 10.000 may be ob-
tained. An antigen solution is prepared having a con-
centration of about 100 mg (milligrams) of the antigen
per liter.
The experiments according to Examples 1 and 2 are carried
out with the artificially produced antigen. It is found
that the artificially produced antigen provides the same
effect as the one obtained from body tissue.
le_4
Use of an organo-specific antigen.
.. ,- ~ . .
11'~93~5~
- 25 -
A) The production of the leucocyte suspension is effec-
ted in the same manner as in Example 1 - ~.
B) Preparation of an organo-specific antigen
solution.
.
Preferably fresh tumour tissue of a mammary carcinoma
is cleaned of blood, fatty tissue and binding tissue,
and cot into small cubical pieces or strips. All of
the subse~uent method steps are performed at a tem-
perature of 4 C. The ~issue pieces are disintegrated
to pieces of about the size of peas in a threefold
volume of a 3 molar potassium chloride solution. The p~
of this coarse suspension is adjusted to 7.4.
Thereafter, the tissue is disintegrated in an Ultra-
turrax (Trademark) homogenizer (20,000 rpm). The homo-
genisate is allowed to stand for 24 hours. Then, the
homogenisate is centrifuged, The supernatant phase ob-
tained after 60 minute~ of centrifugation at 4000 G's
is dialyzed for 2 hours against a tenfold volume of
aqua destillata, and thereafter dialyzed for further
24 hours against a S0-fold volume of 0.9 NaCl solution.
The proteins are precipitated from the dialysate by
adding 2 molar ammonium sulfate during a period of
1 hour~, and then separated by centrifugation for
20 minutes at 4,000 G's. Upon suspending the prote~ns
llZ93~9
:`
- 26 -
in a 0.9 ~ N~aC1 ~olution, a dialysis against the five-
fold volume of aqua destillata for one hour, and a
further dialysis against the 50-fold volume of 0.9 %
NaCl are carried out. Following a sterile filtering
step through a 0.45 Millipore Micropore filter, the
antigen extract is filled into small ~mpules and
stored at -20 C. Prior to use, the protein content
of every charge is determined by the Biuret method
after a preceding TCA precipitation.
C) The same test Plate as in Example 1 is e~ployed.
Three compositions are prepared for the t-st, as
follows:
1) Each containing 100 microliters of leucocyte sus-
pension from patient blood according to Example
1 - A, + 8 microliters of N/100 NaCl solution
. (for column A, standard value).
2) Each containing 100 microllters of leucocyte
suspension + 8 microliters of antigen suspension
- according to Example 4 - B. I<
3) Each containing 100 microliters of leucocyte
su~pension + 8 micxoliters of antigen suspension
11~9319
- 27 -
according to Example 4 - B + 15 microliters of
transfer factor solutlon.
The test plate having a hole diamter of 2,5 mm is
loaded with 8 mlcxoliters each of the test substance
1 to 3 incubated in accordance with Example 1. The
results, each representing the average of five
measurements, are as follows:
Composition Column Diameter F (mm2) MI
1 A 7,70 46,5 1,0
2 C 7,00 38,1 0,82
3 D 6,10 29,3 0,63
The result indicates a mammary carcinoma since the
MI is clearly below 0.85 and the result becomes still
more prominent when the transfer factor is employed.
~,~
l~Z9319
- 28 -
Further examinations show that the results in the
case of female patients, as explained in Example 1 t
vary still more significantly. Fema~ patien s affected
by mammary carcinoma, when the method is applied with
the aid of the transfer factor, for more than 9~ %
show a migration index smaller than 0.80. It is only
in 10 %of the patients affected by mammary carcinoma
that the migration index is above 0.85.
Example 5
The mobility change of lymphocytes, foreign macro-
phages and other indicator cells is determined in a
capillary test according to S0BORG and BENDIXEN. To
this end, an opPn capillary of 10 microns thickness
is used. The results correspond to those according to
Examples 1 and 2, wherein principally the same prepa-
rations or compositions are used.
Concluding remarks
The testlng method cannot be applled to cert~in
aspects of case. Incorrect by negative results were
obtained ln each case fo ~o-called leucoses. At present,
93i9
- 29 -
this phenomenon cannot be explained because it is
just leucemic cells that contain plenty of basic
proteins. Also, the cellular immunity of such patients
is not affected so remarkably that this re~ult could
be explained with an affection of the immune system.
Thus, it is also striking that the chronic lymphatic
leucemia, same as the acute lymphatic leucemia, fail
to yield a reaction whereas lymphosarcomae and lympho-
granulomatoses show the same reaction as other mali-
gnomae. Prior to the chemotherapy, however, the
patients showed a distinct inhibiti~n. This inhibition
is eliminated, however, already after the first chemo-
therapy cycle, and converted into a incorrectly po-
sitive value in the second cycle. Further, wrong
results are obtained from patients in tumour cachexy.
Patientæ with rapidly progrediently growing tumours,
or patients in the tumour cachexy exhibit wrong re-
sults in partO as the cellular immunity is affected
tn such instances. Experiments to clear up this
question are still being carried out.
Incorrectly positive results were obtained ~n first
line in the case of inflammatory and degenerative
neurologic disea~es, for instance multiple sclero31s.
The reason was seen by Caspary and Field in a cross
~ J ~;~ i""~
1129319
- 30 -
reaction between the EF and the sensibilization of
the lymphocates by brain tissue.
Bowever, wrong results may be easily eliminated by
pre-examination such that the abovementioned dise-
ases do not represent any impediment to using the
test.
~P
.
