Note: Descriptions are shown in the official language in which they were submitted.
~16~5~3
The invention relates to a method of producing a blood-
coagulation-promoting preparation comprising human proteins,
including coagulation factors II, VII, IX and X and factor-VIII-
inhibitor-bypassing-activity, and a blood-coagulation-promoting
preparation produced by the method.
Blood-coagulation-promoting preparations exhibiting
factor-VIII-inhibitor-bypassing-activity, in short "FEIBA"
(Factor-Eight-Inhibitor-Bypassing-Activity), are known. In
United States patent No. 4,160,025 the production of such a pre-
paration is described. It is successfully applied for the treat-
ment of patients suffering from haemophilia A and whose blood
contains an inhibitor directed against factor VIII. The chemical
structure of the FEIBA factor so far has been unknown. It is
only known that a protein having a molecular weight of approx-
imately lO0,000 ~s involved. The production of the preparation
according to the above-mentioned patent specification was carried
out by generation from human plasma containing citrate lons in
the absence of free calcium ions by treatment with water-insoluble
inorganic coagulation-phys~ologically-surface-active substances,
such as silicagel or kaolin, and subsequent adsorption and
elution, wherein a mixture of factors II, VII, IX and X, of
factor FEIBA and of other proteins IS obtained, whose composition
has not been described so far.
Although as ment~oned above, the preparation according to
United States patent No. 4,160,025 has proved valuable for the
treatment of factor V~II inhibitor patients, there is the task
of enlarging the sphere of application and further improving the
safety of FEIBA preparations, in particular of reducing to a
minimum undesired side react~ons, such as thrombogenic and
vasoactive effects.
~ccording to the inventIon, there is provided a method of
~ 168583
producing a blood-coagulation-promoting preparation comprising
human proteins, including coagulation factors II, VII, IX and X
and having factor-VIII-inhibitor-bypassing-activity, which
preparation having the following characteristics:
(a) the preparation is free of thrombogenic activity in doses of
up to at least 2 units of FEIBA per ky rabbit in the thrombosis
inducing activity test according to Wessler,
(b) the preparation is free of kallikrein activity and free of
prekallikrein activator-activity, measured in an aqueous solution
of said preparation with a FEIBA concentration of up to at least
10 units per ml,
(c) the preparation is affinity-chromatographically separable on
dextran sulphate agarose by means of an NaCl gradient so as to
obtain an eluate containing a protein with factor IX activity and
an eluate contain~ng a protein with FEIB-~activity, said protein
with factor IX activity eluting at a lower NaCl concentration
than said protein with FEIB-activity, which method comprises
treating human plasma with sulphated high-polymer carbohydrates
and/or with basic ion exchangers so as to obta~n a protein mixture
with generated FEIB-activity, adsorbing said protein mixture,
and recovering said preparation by elution and concentration.
The present invention also provides a blood-coagulation-
promoting pxeparation comprising human proteins including
coagulat~on factors II, VII, IX and X and factor-VIII-inhibitor-
bypassing-activity (FEIBA), when prepared by the above method.
The above-defined character~stic features of the prepar-
ation, i.e. the absence of thrombogenic activity and of
kallikrein activity or prekallikrein activator activity - the
latter being responsible for vasoactive effects -, suggest that
the preparation has an excellent safety. The thrombosis
inducing activity test and the test on kallikrein activity and
1 168~83
prekallikrein activator activity are known to one skilled in the
art. They will be described in more detail after the Examples.
The third characteristIc feature of the preparation
according to the invention, i.e. the separability by way of
affinity-chromatography on dextran sulphate agarose by means of
an NaCl gradient is illustrated in Figure 1 of the drawings by
way of an example. The method of affinity-chromatography on
dextran sulphate agarose ~s known to one skilled in the art (D.S.
Pepper and C. Prowse, Thrombosis Research 11 (1977), 687-692).
Dextran sulphate (,molecular weight 500,000) ~s coupled to CNBr-
activated sepharose* 4 B (Pharmacia F~ne Chemicals ~B~ Uppsala !
Sweden). The dextran sulphate sepharose thus o~tained is equi-
librated in a 0.4~ w/v tr~sodiumcitrate.2H20 solut~on (pH = 7,4)
and filled into a column. The sample to be exami,ned is applied
onto the column and then fract~onally eluted with an NaCl
solution in 0.4% w~v trisod~umcitrate.2H20 (pH = 7,4) with an
increasing NaCl concentration.
I~n F~gure l the fraction numbers of the eluates are
plotted on the abscissa. On the left ord~nate the activities
of the coagulation factors are plotted in units per ml and on
the right ordinate the concentrat~on of the sodium chloride
gradient 's plotted in mol~l. The l~near course of the gradient
is entered as line G. As can ~e seen from Figure l, the
protein w~th factor IX act~v~ty elutes in the regi,Gn of
0.1 to 0.5 molar NaCl, w~th a maximum at 0.3 molar, the protein
with FEIB-activity elutes in the reg~on of 0~3 to 0.5 molar
NaCl, with a maximum of 0.4 molar. The increas~ng NaCl concen-
tration is revealed by the NaCl gradient G reaching from O molar
to 0,65 molar NaCl.
Ftnally, as the fourth characterist~c feature of the
* Trade Mark
preparation according to the invention also the content of
globulins when electrophoretically separated is typical, which
is explained by way of Figure 2 of the drawings. The upper
section of Figure 2 illustrates, by way of an example, the
electrophore-tic separation trace of the eluates of the chroma- -
tographic separation on dextran sulphate sepharose according
to the invention containing proteins with factor IX activity
and with FEIB-activity, whereas the lower section of Figure 2
reflects the electrophoretic separation trace of a native human
plasma. As will be seen from the traces in F-~gure 2, in this
example, for a preparation in accordance with the invention,
the main peak in the ~-globulin region amounts to 70% of the
total protein. Downstream of the main peak there follows a
shoulder ~n the a-globulin reg~on, amounting to 14% of the total
protein content, and thereafter one observes a small peak in
the ~-globul~n region, which amounts to 16% of the total
protein,
Advantageously a further characteristic feature of the
preparat~on according to the invention resides in the fact that
the FEIB-acti~lty, after a one~h~ur incubation ;n factor
VIII inhibitor plas~a, IS` preserved by at least 50%. This
property ind~cates a longer lasting efficacy when applied to
factor YIII inh~bitor pat~ent$.
Advantageously a further characteristic of the preparation
according to the invention resides in the fact that the factor
IX activity, after a one-hour incubation in factor IX deficient
plasma, is preserved by at least 50%. This means that the
preparation contains little or only a very slight port~on of
activated factor IX. It is known that activated factor IX is
inactivated in human plasma. ~ctivated factor IX would cause
-- 4 --
8 3
detrimental thrombogenic effects. It is known from the
literature (Proc. Natl. Acad. Sci. USA, Vol. 74, No. 7,
3028-3032, July 1977, "In vitro and in vivo correlation of
clotting protease activity: Effect of heparin" by S.N. Gitel,
R C. Stephenson and S. Wessler) that it is exactly factor IXa
that possesses the highest thrombogenic efficacy as opposed to
other activated coagulation factors, such as Xa and IIA
(thrombin)~
In a preferred embodiment of the preparation according
to the invention, the preparation further comprises inter-alpha-
trypsin-~nhibitor (ITI) in an amount of 0.05 to 5 mg per
FEIBA un;t.
The content of IT~ causes the thrombogenicity of the
preparation of the invention to be low in the ~essler test.
The method of producing the new blood-coagulation-
promoting preparat~on compr~sing human proteins, including
c~agulation factors II, VII~, IX and X and having a factor-
VIII-inhibitor=bypassing-actlvity, is characterized in that
human plasma is treated with sulphated , high molecular weight
polymer carbohydrates and/or w~th basic ion exchangers, and
the resultant protein mixture having generated FEIB-activity
is adsorbed, and the preparation is recovered by elution and
concentration.
When carrying out the method it is to be taken care
that the plasma and the reactants be kept free from substances
that are capable of increas~ng the antithrombin III act~vity,
such as heparin or heparinoids.
According to one embodi~ent of the method of the
present invention, the plasma at first is briefly treated w~th
sulphated, h~gh ~olecular weight polymer carbohydrates, the
~; ;
~ "~
~8~83
protein mixture having generated FEIB-activity is then adsorbed
on an ion exchanger having a dextran structure and eluted and
concentrated immediately thereafter.
According to another embodiment of the method of the
present invention, the plasma is treated with an ion exchanger
having a dextran structure; after at least two hours of exposure
the protein mixture with generated FEIB-activity, adsorbed on
the ;on exchanger, is eluted and then concentrated. With this
embodiment, the generation of the FEIB-activity depends on
the period of exposure. This may last up to 48 hours,
The ind~vidual s-teps of the production process are not
critIcal and may vary within a large range; thus the pH may be
from 6 to 9, the temperature may range from 0 to 40C, the
amounts of dextran sulphate used may be from 0.1 to 500 mg~l
plasma, and DEAE-Sephadex* may be used ~n an amount of from
0.01 to 10 g/l plasma. As a starting material for the method
of the invent~on, not only nati~e human plasma, but also plasma
fractions, e.g., cryosupernatant and Cohn-I-supernatant
(8% v/v alcohol) may be used.
The preparation according to the ~nvention and -the
method for its production wIll be explained in more detail by
the follow-ing examples, the methods of determ~nation applied
~eing explained and the res~ults ~e~ng shown in the
* Trade Mark
~16~583
Tables following the Examples.
Example 1:
1,000 l of fresh frozen human citrated plasma are
thawed at 0 to +4C and the resultiny cryoprecipitate
is separated by centrifugation at +2C. To the resulting
"cryosupernatant" 10 g of dextran sulphate (molecular
weight 500,000) are added at a native pH of 7.7 and
stirred for 15 minutes at + 4 C, with the substance
FEIBA being generated.
Thereafter 500 g of the anion exchanger DEAE-Sephadex
A-50 (Pharmacia Fine Chemicals AB, Uppsala, Sweden) are
added and stirred at +4 C for half an hour, the generated
FEI~A-substance together with the factors of the prothrom-
bin complex (II, VII, IX, X) and inert proteins being ad-
sorbed on the insoluble DEAE-Sephadex.
The DEAE-Sephadex is separated immediately after the
adsorption procedure by centrifugation or filtration; the
supernatant plasma may be used for recovering gamma-glo-
bulin and albumin.
The DEAE-Sephadex is subjected to a double washing
process; at first the DEAE-Sephadex is stirred with 50 l
of a solution consisting of 4 g/l trisodiumcitrate.2H20,
7 g/l sodium chloride and 18 g/l disodium hydrogen phos-
phate.12H20 in distilled water, pH 7.5,for 15 minutes at
+4 C. After separation by filtration the DEAE-Sephadex is
stirred with 50 l of a solution consisting of 4 g/l tri-
sodiumcitrate.2H20 and 7 g/l sodium chloride in distilled
water, pH 7.5, for 15 minutes at + 4 C and then again is
separated by filtration.
For elu-tion the DEAE-Sephadex is stirred with 25 l of
-- 7 --
1 ~8583
a solution consisting of 30 g/l sodium chloride and 1 g/l
trisodiumcitrate.2H20 in distilled water, pH 7.0, for
20 minutes at +4 C. The eluate con-taining the generated
FEIBA substance, the factors of the prothrombin complex
(II, VII, IX, X) as well as inert protein, is gained by
filtration, the DEAE-Sephadex is discarded. The eluate
is dialyzed over night against 1,000 l of distilled water
at +4 C, then frozen and subjected to a first lyophili-
zation process. In the resulting bulk-material the FEIB-
activity is determined according to the method describedin U.S. patent No. 4,160,025.
.For the production of the pharmaceutically applicable
preparation with FEIB-activit~ the bulk-material is dis-
solved in so much distilled pyrogen-free water that the
FEIB-activity amounts to between 10 and 50 FEIBA units
per ml (in the present case 25 FEIBA units per ml). After
the addition of the salts required for establishing isoton-
icity and adjusting the pH to between 7.0 and 7.5 the so-
lution is cleared through membrane filters and at last is
sterile-filtered through a 0.2 ~um membrane filter.The so-
lution is filled into the final containers under sterile
conditions in 20 ml portions, deepfrozen and lyophilized.
Example 2:
1,000 l fresh frozen human citrated plasma are thawed
at 0 to +4 C and the resulting cryoprecipitate is sepa-
rated by centrifugation at +2 C. To the resultin~ "cryo-
supernatant" 500 g of the anion exchanger DEAE-Sephadex
A-50 (Pharmacia Fine Chemicals AB, Uppsala, Sweden) are
added at a native pH of 7.7 and stirred for half an hour
at +4 C, the factors of the prothrombin complex (II, VII,
-- 8 --
~1~85~3
IX, X) and inert proteins being adsorbed on the DEAE-
Sephadex.
Thereafter the mixture is allowed to stand for 12
hours at +4 C; during this "contact time" the FEIBA sub-
stance is generated.
The DEAE-Sephadex, after a 12-hour "contact time", is
separated by centrifugation or filtration; the supernatant
plasma may be used for recovering gamma-globulin and al-
bumin.
The further processing of the DEAE-Sephadex (double
washing, elution etc.) is effected in the same manner as
described in E~ample 1.
The thrombosis induci:ngactivity test according to
Wessler, which is described in the literature, i.e. in
J. Appl. Physiol 14 (1959), 943-946, "Biologic Assay of a
Thrombosis-Inducing Activity in Human Serum" by Stanford
Wessler, Stanley M. Reimer and Mindel C. Sheps, is per-
formed in the following manner:
3 rabbits are used per test. The animals are narcotized
with Nembutal; after an additional local anesthesia the
heart-side vena jugularis is laid open, two ligatures
being prepared at a distance of 1 to 2 cm.
The preparation to be tested is now injected within 15
seconds in the desired dosis into the ear vein opposite
the vena jugularis laid open. Within 10 to 25 seconds after
the injection of the preparation the prepared ligatures
are contracted. The isolated vein segment now remains in
situ in the rabbit for 10 minutes. Then the vein section is
removed from the animal and dissected in a Petri dish in
vfv~
~,~ 30 a 5 ~ sodium citrate solution, the contents being evaluated
_ g _
1 168583
according to the following scheme.
0 = no clot
1 = few macroscopically visible fibrin particles
2 = some small thrombi
3 = two or more large thrombi
4 = one single thrombus filling up the entire isolated
vein segment.
The test is valued positive in the case of a ~-re-
action~ It is essential for the preparation according to
the invention that upon injection of the preparation which
contains at least 2 units of FEIBA per kg of experimental
animal, no 4-reaction takes place.
The determination of the kallikrein activity and the
prekallikrein activator activity is carried out in the
following way.
KALLIKREIN:
1. Method:
Kallikrein amidolytically splits para-nitroanilin (pNA)
from a specific chromogenic substrate. The concentration
of pNA is photometrically measured at a wave length of
405 nm.
2. Reagents:
Buffer:
Solution A: 3.03 g "TRIS" and 1.7 g imidazole are dis-
solved in 500 ml 0.1n hydrochloric acid and
water is added up to 1,000 ml.
Solution B: 4.04 g "TRIS" and 2.27 g imidazole are dis-
solved in 500 ml 0.1n hydrochloric acid and
water is added up to 1,000 ml.
Solution C: 11.69 g sodium chloride are dissolved with
- 10 -
~1~85~3
water to 1,000 ml.
Solutions A and B are mixed until a pH of 7.9 is reached.
To this mixture the same volume of solution C is added.
Chromogenic substrate S-2302 (Kabi/ Stockholm): H-D-
d~
prolyl-L-phenylalanyl-L-arginine-p-~aR~d-dihydro-
chloride
1 millimolar solution of S-2302: 25 mg in 41 ml of water.
Sample:
The sample is dissolved in the original volume and used
in the test undiluted.
3. Test:
In a water bath at a temperature of 37 C
1.0 ml buffer preheated to 37 C
0.1 ml sample
0.2 ml chromogenic substrate S-2302
are pipetted into a plastic tube. This mixture is charged
into a photometer heated to 37 C and the increase of the
optical density per minute (~OD/min) at a wave length of
405 nm with a path length of 10 mm is measured. The ac-
0 tivity of a sample is expressed in ~OD/min.PREKALLIKREIN ACTIVATOR:
1. Method:
From a purified prekallikrein preparation (PKK) kallikrein
(KK) is generated by means of a prekallikrein activator
(PKKA). The kallikrein amidolytically splits para-nitro-
aniline (pNA) from a specific chromogenic substrate. The
concentration of pNA is photometrically measured at a
wave length of 405 nm.
2. Reagents:
Buffer and chromogenic substrate correspond to the re-
- 11 -
~168583
agents described in connection with the kallikrein deter-
mination.
Prekallikrein preparation:
The production of the preparation is effected according
to a prescription of Harpel, modified by M. S. Horowitz
(New York Blood Center). Therein human citrated plasma
is treated with a DEAE-cellulose. The fraction that
is not bound to the DEAE-cellulose contains the pre-
kallikrein.
Positive control (standard):
As standard (= reference value) an albumin preparation of
the Burau of Biologics (soB) of the Food and Drug Ad-
ministration, Bethesda, Maryland 20205, U.S., is used. This
preparation contains a prekallikrein activator. The kalli-
krein generation with this BoB-standard represents the re-
ference value 1 and is equated with 100 %.
Sample:
The sample is dissolved in the original volume and used in
the test undiluted.
3. Test:
In a water bath at a temperature of 37 C
0.05 ml prekallikrein preparation
0.05 ml sample a) BoB-standard for the reference value
b) test sample (in a second test mixture)
are pipetted into a plastic tube. After an incubation of 10
minutes at 37 C
0.7 ml buffer solution
0.1 ml chromogenic substrate S-2302
are pipetted. This mixture is charged into a photometer
heated to 37C and the increase in the optical density
- 12 -
5 ~ 3
per minute (aOD/min) is measured at a wave length of 405 nm
with a path length of 10 mm. The activity of a sample
OD/min) is expressed in % of the BoB-standard.
The characterization of the preparation according
to the invention by an affinity-chromatographic separation
of thepreparation on dextran sulphate sepharose has
already been described in connection with Fig. 1.
The electrophoretic separation of the proteins with
factor IX activity and with FEIB-activity, which is re-
ferred to in connection with Fig. 2, can be carried outin the following manner.
As the carrier for the electrophoretic separation
of the various proteins a cellulose-acetate membrane
serves, which is wetted with the electrophoresis buffer
(p~ 8.6, ionic strength 0.075). On this membrane the
samples to be analyzed - together with a normal human
plasma as standard - are applied and then separated in
the electric field; the separation is effected in that
differently charged proteins migrate at different speeds
in the electric field. To this end the membrane provided
with the samples is treated in a special cell filled with
the electropharesis buffer for 16 to 18 minutes at a
voltage of 250 volts and an initial current intensity
of 4 to 6 milliamperes.
After termination of the separation procedure the
various proteins are rendered visible by putting the mem-
brane into a fixing and dyeing solution. After some rinsing
baths the membrane is made transparent in a iurther bath,
applied onto a glass plate and dried in a drying chamber
at 100 C.
- 13 -
~16~583
The dried membrane is then analyzed in an automati-
cally registering and integrating densitometer, which
produces separation curves as illustrated in Fig. 2.
The various proteins appear as different peaks whose
areas are proportional to the relative percentage
values of the corresponding proteins, these areas being
determined by the automatic integration of the densito-
meter.
The allocation of the various peaks or proteins
of the test sample to defined proteins or protein
groups is effected by comparing them with the peaks
of the normal human plasma simultaneously analy~ed as
standard. During the electrophoretic analysis the
latter is separated ir,to5protein groups which by de-
finition are designated as follows - in the order of
decreasing mobility in the electric field: albumin,
~-globulins, ~-globulin, fibrinogen, and ~-globulin.
The definition of the FEIBA unit as well as
its determination (potency test) is described in
U.S. patent No. 4,160,025. The determination of the
activity of the coagulation factors II, VII, IX
and X is also described in the above mentioned
U.S. patent No. 4,160,025.
The determination of the residual activity of
FEIBA after incubation in factor VIII inhibitor plasma
is carried out in den following manner.
1. Reagents:
The reagents to be used are described in U.S. patent
No. 4,160,025 in connection with the determination
- 14
llfi8583
of the FEIBA units (potency test).
2. Test:
From a preparation adjusted to 50 FEIBA units/ml t'ne
following dilutions are prepared with a solution of 7 g/l
sodium chloride and 7 g/l sodium citrate.2H20 as diluent:
1 : 2, 1 : 4, 1 : 8, 1 : 16, 1 : 32 and 1 : 64. From these
6 dilutions a 1 : 10 dilution in factor VIII inhibitor
plasma (0.05 ml pre-diluted sample+-0.45 ml factor-VIII-
inhibitor plasma) is each prepared.
These 1 : 10 dilutions in factor VIII inhibitor plasma
("incubation mixtures") are then analyzed immediately and
after one hour of incubation at 37 C according to the
following test scheme:
0.1 ml "incubation mixture"
0.1 ml phospholipid-kaolin suspension
incubation at 37 C for 1 minute
0.1 ml m/40 calcium chloride.
The time from the addition of calcium chloride to the clot
formation is taken with a timer like in the potency test.
3. Calculation of the residual activity:
Analogously to the description in the potency test, a cali-
bration curve is established with the coagulation times of
the immediately determined dilutions (undiluted s~nple =
50 FEIBA units/ml). The activities (FEIBA units/ml) of the
various dilutions incubated for one hour are then calculated
by using the calibration curve and are expressed in percent
of the individual activities of the non-incubated dilutions.
The mean values of the activities thus calculated produce the
average residual activity of the sample after an hour of
incubation, expressed in percent of the initial activity
- 15 -
1~8583
prior to incubation.
The determination of the residual activity of factor
IX after incubation in factor IX deficient plasma is
carried out in the following manner:
1. Reagents:
Factor IX deficient plasma: citrated plasma of a patient
suffering from severe haemophilia B (factor IX below 1 %).
Phospholipid/kaolin suspension: PTT reagent of Immuno Dia-
gnostica Ges.m.b.H. For the test the required amount of
factor IX deficient plasma is mixed with an equal volume
of the phospholipid/kaolin suspension, incubated for 5
minutes at 37 C and then stored in an ice bath during
the test period.
Citrate/saline solution as diluent for samples: 7 g/l
trisodium citrate.2H20, 7 g/l sodium chloride.
Calcium chloride m/20 (0.05 molar): storing at 37 C during
the test period.
2. Test:
From a preparation adjusted to 50 factor IX units/ml 7
geometric dilutions (1 : 2, 1 : 4 etc. until 1 : 128) are
prepared with the citrate/saline solution. From the undi-
luted sample as well as from the 7 geometric dilutions
a 1 : 10 dilution in factor IX deficient plasma is each
prepared (0.05 ml prediluted sample + 0.45 ml factor IX
deficient plasma).
These 1 : 10 dilutions in factor IX deficient plasma
("incubation mixtures") are then analy~ed immediately and
after an hour of incubation at 37 C according to the
following test scheme, each incubation mixture being di-
luted 1 : 10 with the citrate/saline solution prior to
- 16 -
1 ~6~583
determination:
0.2 ml mixture of factor IX deficient plasma and phospho-
lipid/kaolin
0.1 ml "incubation mixture", 1 : 10 diluted with citrate/
saline solution
incubation at 37 C for 1 minute
0.1 ml m/20 calcium chloride
The time from the addition of calcium chloride to the
clot formation is taken by a timer.
3. Calculation of the residual activity:
A calibration curve is established with the coagula~ion
times of the immediately determined dilutions (undiluted
sample = 50 factor IX units/ml) by plotting the coagulation
times against the corresponding dilutions on double logarith-
mic graph paper. The activities (factor IX units/ml) of the
various dilutions incubated for one hour are then calculated
by using the calibration curve and are expressed in percent
of the respective activities of the non-incubated dilutions.
The mean values of the activities thus calculated produce
the average residual activity of the sample after one hour
of incubation, expressed in percent of the initial activity
prior to incubation.
Immunologic determination of the inter-alpha-trypsin-
inhibitor (ITI):
1. Method:
The determination is effected according to the Ouchterlony- -
technique, wherein a specific antibody diffuses in an agar
medium against an antigen-containing sample. The antigen
specifically reacts with the antibody, forming an immune
0 precipitation p-e-ak that is valued as positive reaction.
- 17 -
1~6~8~
2. Reagents:Rabbit antiserum against ITI, Behringwerke AG, Marbury/
Lahn, sRD
Agar: A solution of 1.25 g agar, 0.9 g sodium chloride
and 100 mg sodium azide in 100 ml water is briefly boiled,
and the hot homogenous solution is poured onto plates
yielding a layer of about 2 mm thickness. Into the cooled
solidified gel holes having a size of about 2 mm are
punched in two rows at a distance of 5 mm.
Standard and sample:
As calibration substance a protein standard serum of Beh-
ringwerke with a definea content of ITI serves. From this
reference serum a geometric dilution series in a physiologic
sodium chloride solution (9 g NaCl/l) is prepared. The sample
to be tested is treated like the standard.
3. Test:
Into one hole row of the agar the dilutions of the cali-
bration substance or sample to be tested are charged, in
the adjacently arranged hole row the undiluted specific
antiserum is pipette-d. The agar plate thus charged is in-
cubated at 37 C for 15 hours. Afterwards the reading of
the immune precipitations is effected.
~. Calculation of the ITI concentration:
As a measure for the ITI concentration of a sample the di-
lution step is chosen at which a precipitation is just
visible ("titer" of the sample). The ITI concentration
of the sample to be tested is calculated as follows:
titer of test sample
x ITI concentratlon of standard
titer of standard
The ITI concentration is indicated in mg ~ (mg/100 ml).
- 18 -
The preparations produced according to Examples 1 and
2 were analyzed according to the preceding assay methods;
the results are summarized in the following Table.
The illustrations in Fig. 1 (affinity-chromatographic
separation) and Fig. 2 (electrophoretic separation) of the
drawings correspond to the data of Example 2 in the follow-
ing Table.
- 19 -
_ __ _ .,
C~
Z Z Z Z
a) .~
o ~ ~ o U~ ~ ~ ~ o o o o
C~ ~ . . . . . I
~, ~ ~ ~ ~9 1 ~ ~
~d X t`l ~`1 N ~ `J H O O O O
~ 1:':1 IL~ O O
O
~ ~l
O ,Y
.~ ~ ~ ~ O C.) t_)
:z æ ~;
O G~ O ~ ~ ~
a) ~ ~D ~ ~ ~ ~ ~ ~ ~
~ X ~ ~ ~
~ ~1 H O O O O
m ~ ~r ~
~ o o o
_ . _ _
E~ . ~ O
o ~ a
o
a
~ ~ U~ ~ ~ ~
~ ~ o ~
~ ~ ~ ~ ~ ~ O O ~ ~ ~ .
v o ~ v
~1 Ul ~ ~ Q O ~
~1 ~ ~ ~) O ~ IJ O ~1 1
,~,J v o ~ s, u~ v ~ ~ m
U~ ~ ~ ~ ~ ~ ~ c) rl H
~J ~ H ~ ~ ~ O O S~
,1 H H ~C a) ~ ~ I ~ X ~1 ~
H ~ H X t~ >1 ~I H ~)
~ , O ~J (d ~ ~
S~ Q O ,1
,~C (j O ~3 o s:~ a) o
m ~ o ~ ~ ~ m
H V V V V S l O IH V X H X
~1 ~ ~ ~! ~ ~1 ~ (a td ~ Id
E~ ~ ~ ~ O ~
_ _
o
-- 20 --
1168S83
~ ~ ~ ~ ~ C~
X ~D 00 ~_ Ln ~
o
h _ __ . .. ... . r :--
r~l ~
~ )C
~ ~ o\ d~ o\ o\ o\
h ~ ~ ~ O O Ln O
H ~:1
1:4-- -- ~ O
_ _ _ __
4~ ~ ~ ~ ~ C~
o ~ m~ .~ ~
~ 1l:l ~1 H R
~ m ~ H X ~
~d H O ~1 ~ ~1 1--1 ~::
I .Y ~ O O ,~
h ~ h a~ ~1 5-1
~a~ ~ ~ ~ ~ o ~ o
a) ~ ~ ~ ~ ,, ~ ~ ~ ~ ,J
~_ U~ ~ ~ ~ }~ r~ O rl O ~a
1~ ~ O ~ 1
O 0 ~1 0 rl t~ ~1 ~H rJ 4
~S ~ ~ ~ ~ ~ ~1
):: ~ H ~ u~ ~ ~ ~:
h h` `
~ O O~ ~ ~ ~ h ~1 ~ ~ ~ ,~:
O ~: ~ rl rl ~1 r~ X 1~ 0 rd O P~
O ~ O ~ ~ ~ rl ~S rl ~ rl
O ~ h ~1 ~)
O h 4-1 Q Q Q X ~1 ~ S ,1 ~
~ ~ O O O rl (1~ u~ Q u~ Q h
Q o ~ ~I X a) ~ a)
~ a~ ~ ~ ~ ~ ~ ~ ~ o
E~ ~ ~1 1 1 I aS h ~: ~
3 ~ ~ . o\O .,1 o\O .,1 H
o
- 21 -
