Note: Descriptions are shown in the official language in which they were submitted.
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5013381,J.48
P~tonoclonal antibodies against the human 'I"NF-binding
protein I (TNF-BP 2)
The present inventi,an relates to monoclonal antibodies
against the TNF-binding protein I (TNF-BP I) and to
hybridoma cell lines which secrete them<
The two structurally related cytokines tumour necrosis
factor (TNF-a) and lymphotoxin (TNF-~) were originally
discovered as a result of their cytotoxic 'air vitro
activity against tumour cells and their ability to
induce haemorrhagic necroses of tumours in a mouse
model. The cloning of their eDNAs and their expression
in E.coli have made these proteins available in
virtually unlimited quantities and have made it possible
to develop highly specific antibodies and sensitive
immunoassays. There is a wealth of information o1 the
biological activities of these proteins, their
physiological roles as pleotropic mediators of
inflammatory processes and their participation in
pathological conditions. In particular, an increased
production of TNF-a has been linked with the
pathogenesis of, for example, septic shock, tissue
damage in the "Graft-versus-host" disease and cerebral
malaria and cachexia (Beutler, 1988; Paul and Ruddle,
1988; Beutler and Cerami, 1989).
The possible undesirable effects of TNF-a have led to a
search for natural inhibitors of this cytokine. A
protein which binds to TNF-a and thereby inhibits the
activity thereat was originally identified in the urine
of patients with kidney failure (Peetre et al., 198'8)
and fever patients (Seckinger et al., 1988). This
protein with an apparent molecular weight of about
30 kDa was purified in order to homogenise it, partially
i~~
- 2 -
sequenced (EP-A2 308 378) and the cDNA was cloned
(Olsson et al., 1989. Engelmann et al., 1989; E~Iimmler et
al., 1990; Schall et al., 1990). The structure of the
cDN~ showed that this protein, designated TNF-BP, is the
extracellular fragment of a TNF receptor (TNF-R). (Tt
was assumed that this fragment is released by
proteolytic cleaving.) These results were confirmed by
isolation of the intact membrane receptor for TNF-a,
which was carried out independently by other working
groups (hoetscher et al., 1990). The entire receptor
protein consists of 455 amino acids (55 - 60 kDa);
TNF-BP makes up the majority of the extracellular domain
of the receptor and contains all three N-glycasylation
sites. It was found that TNF-BP isolated from urine is
heterogeneous at the N-terminus as a result of
proteolytic cleaving and is therefore a mixture of two
molecular farms consisting of 161 amino acids (main
fraction) or 172 amino acids (Himmler et al., 1990).
Recently, the existence of a second TNF-binding protein
was demonstrated, which is a fragment of a second TNF
receptor type having a higher molecular weight
(75 - 80 kDa: Engelmann et al., 1990a: Smith et al.,
1990 Kohno et al., 1990). The two receptors and
binding proteins were then designated TNF-R I/TNF-BP I
and TNF-R II/TNF-BP TI (fox the 60 kDa and 80 kDa
receptors, respectively). Sequence comparison showed
that the two proteins are structurally related; in
particular, the number and distribution of the cysteine
groups is very similar. However, the two receptors
differ from each other immunologically (Engelmann et
al., 1990x; Brockhaus et al., 1990).
The human 50 kDa TNF-receptor plays an essential part in
TNF-a-signal transmission. The activity~of the receptor
is subjected to several regulatory effects on a protein
basis: the treatment of cells with phorbol esters or
other activators of protein kinase C results in a rapid
_~ ~ r1 e~ ~ ~~
3 _
decrease in the number of cellular binding sites for
TNF-a. This is linked with the release of the
extracellular part of the receptor, corresponding to
TNF-BP I, by proteolytic cleaving. similar effects,
albeit with different kinetics, are caused by various
other substances, particularly by the physiological
ligands TNF-a and TNF°~. The cleavage sites for the
protease on human and rat TNF-R I are conserved: they
differ in structure from the specificity of all known
professes. It is therefore probable that a highly
specific proteolytic enzyme is part of a regulating
circuit which controls the sensitivity of cells to TNFs;
the exact mechanism of these events i: not yet known.
Recently this phenomenon has also been observed in vivoe
it was found that the administration of TNF°u to cancer
patients resulted in a significant increase of TNF-BP I
in the serum (Taantz et al., 1990a).
The concentration of TNF-BP T in culture residues or
body fluids is therefore an indicator of the activation
of the TNF receptor system in vitro or in vivo as a
result of interactions with the ligands or
transmodulation by other mediators; the TNF-BP I
concentration in body fluids can thus be regarded as a
useful marker for various diseases.
There was therefore a need for efficient and sensitive
methods of detecting TNF-BP I and for kits which can be
used for such detection methods.
Monoclonal antibodies against TNF-R T have been -
descr3.bed which were prepared by immunising with the
solubilised receptor (Brockhaus et al., 1990;
EP A2 334 1f>5; Thoma et al., 1990) or with purified
TNF-BP.I (Engelmann et al., 1990b); however, it was not
shown that the antibodies are suitable for use in
immunoassays for TNF-BP I.
1~.~"~3~t)
_~_
Lantz et al. (1990x) have developed a competitive ELTSA
in which, in a three-step test method, test plates
coated with TNF-BP I, polyclonal rabbit antibodies
against TNF-BP I, biotin-labelled goat antibodies
against rabbit immunoglobulin and avidin-coupled
alkaline phosphatase were used. By means of this assay,
the presence of TNF-BP I in serum from normal donors was
detected and increased concentrations of TNF-BP I were
detected in sera from patients suffering from kidney
failure or cancer patients who had been treated with
TNF-a.
EP A1 412 486 describes monoclonal antibodies against
TNF-BP I. One of these antibodies was used in a
sandwich ELISA as a coating antibody: polyclonal rabbit
anti-TNF-BP I antibody was used as the second antibody
and polyclonal goat anti-rabbit antibody was used as the
third, enzyme-coupled antibody.
The known assays are complicated in their structure and
procedure required and moreover the use of polyclonal
antibodies involves the use of animals, which is
something which is increasingly to be avoided.
The aim of the present invention was.to prepare
monoclonal antibodies with specificity for human
TNF-BP I, which are suitable for use in a simple and
highly sensitive immunoassay for detecting TNF-BP I.
The present invention relates to monaclonal antibodies
against human TNF-BP I entitled tbp-l; tbp-2 and tbp-6;
active fragments thereof and the hybridoma cell lines
TBP-1, TBP-2 and TBP-6 which produce these antibodies.
The hybridoma cell lines designated TFiP-1 and TBP-2 were
deposited on 5th June 1991, and the cell line designated
TBP-6 was deposited on 28th November 1991 at the
CA 02107340 2002-04-22
27855-48
_5_
European Collection of Animal Cell Cultures (ECACC;
Salisbury, United Kingdom) in accordance with the Budapest
Agreement on the depositing of microorganisms for patent
purposes (TBP-1: deposit number 91060555, TBP-2: deposit
number 91060556, TBP-6: deposit number 91112811).
In one aspect, the present invention provides a
monoclonal antibody against human TNF-BP I, selected from
tbp-1 and tbp-6, which are secreted by the hybridoma cell
lines TBP-1 (ECACC 91060555) and TBP-6 (ECACC 91112811),
respectively, and active fragments thereof.
In another aspect, the present invention provides
a hybridoma cell line selected from TBP-1, TBP-2 and TBP-6
deposited at ECACC under deposit numbers 91060555 (TBP-1),
91060556 (TBP-2) and 91112811 (TBP-6), respectively.
In a further aspect, the present invention
provides a process for determining TNF-BP I in a sample,
wherein the sample is brought into contact with the antibody
tbp-1 as described herein and the formation of a binary or
ternary complex between the TNF-BP I contained in the sample
and the tbp-1 is determined.
In a further aspect, the present invention
provides use of the process described herein for diagnosing
pathological conditions of the human body which involve
activation of the TNF-receptor system, particularly
conditions in which the TNF-a concentration falls more
rapidly than the TNF-BP I concentration.
In a further aspect, the present invention
provides a sandwich immunoassay kit for carrying out the
process described herein, wherein it contains tbp-1 as
CA 02107340 2002-04-22
27855-48
-5a-
described herein in one container and the antibody tbp-2 as
described herein in another container, one of the two
antibodies optionally being bound to a solid carrier, whilst
the other antibody has a measurable label and, optionally,
tbp-2 is replaced by another antibody which has the ability
to form an antibody/antigen/antibody complex with tbp-1.
In a further aspect, the present invention
provides a process for determining TNF-BP I in a sample,
wherein the sample is brought into contact with the
antibodies tbp-1 and tbp-6 as described herein and the
formation of a ternary complex between the TNF-BP I
contained in the sample and the antibodies is determined.
In a further aspect, the present invention
provides a sandwich immunoassay kit for carrying out the
process described herein, wherein it contains the antibody
tbp-1 as described herein in one container and the antibody
tbp-6 as described herein in another container, whilst one
of the two antibodies is optionally bound to a solid carrier
and the other antibody has a measurable label.
In a further aspect, the present invention
provides a use of one or both of the monoclonal antibodies
tbp-1 and tbp-6 as described herein, for intensifying the
protective effect of endogenous or exogenous TNF-BP I
against TNF-a or TNF-~ or both.
CA 02107340 2002-04-22
27855-48
- 5b -
The hybridoma cell lines according to the invention were
obtained by immunising mice with TNF-BP I highly
purified from human urine (Olsson et al., 1989) using
methods known per se and spleen cells from mice with a
positive antibody reaction were used for fusing with
myeloma cells in order to obtain hybridoma cells which
secrete monoclonal antibodies against TNF-BP I. The
first cell fusion yielded two culturas which produce
monoclonal antibc~tlies against TNF-BP I: a second cell
fusion yielded a third antibody-producing culture. The
antibodies obtained were designated tbp-1, tbp-2 and
tbp-6. The antibodies were purified and characterised:
tbp-1 and tbp-6 are IgGI-antibodies whilst tbp-2 is an
IgG2b-antibody. All three antibodies were capable of
recognising TNF-BP I in Western blots, tbp-1 showing the
strongest reactivity. tbp-2 reacted more weakly whilst
tbp-6 yielded the faintest colouring. In order to ~-w "
characterise the epitopes which are recognised by the
antibodies, the three antibodies and a fourth antibody
designated H398, obtained by immunisation with
solubilised TNF-receptor (Thama et al., 1990) were
investigated. The antibodies investigated recognise
three different epitopes on the TNF-tiP I molecule, tbp-2
and H398 recognising the same or overlapping epitopes.
The sandwich ELISAs carried out in the presence of TNF-a
in various arrangements with the antibodies lead one to
conclude that the epitopes recognised by~H398 and tbp-2
are involved in the binding of TNF-a, whilst those
recognised by tbp-1 and tbp-6 are not connected to the
ligand binding site. It was found, surprisingly, that
1 0 rf cJ lk D
some monoclonal antibodies against TNF-BP I are not only
capable of binding TNF-BP I in the presence of ~xcess
TNF-a, but moreover significantly increase the
protective effect of TNF-SP I against the cytotoxic
activity of TNF-a and TNF-~. This effect was observed
with the two antibodies tbp-1 and tbp-6. (These two
antibodies belong to the group of antibodies against
TNF-BP I, which do not compete with TNF-a and TNF-~ for
the binding of TNF-BP I. By contrast, the antibodies
tbp-2 and H398, which compete with TNF-a for binding to
TNF-BP I, bloc3e the activity of TNF-BP I).
according to another aspect, the present invention
relates to the use of tbp-1 and/or tbp-2 for detecting
TNF-BP I in body fluids or cell culture residues by
immunoassay.
(The terms tbp-1 and tbp-2, in connection with the use
of these antibodies, also includes hereinafter the
active fragments thereof which bind to TNF-HP I.
Experts in the relevant field will be familiar with
methods of preparing active antibody fragments (Fab-
fragments), e.g. by means of enzyme ;iigestion.)
The present invention also relates to the use of tbp-1
and tbp-6 for detecting TNF-BP I in body fluids or cell
culture residues by means of immunoassay. The antibody
combination tbp-1/tbp-fi is particularly suitable for use
in samples in which there are very high concentrations
of TNF-a and/or TNF-Q, which disrupt the measurement of
TNF-BP I in other test systems.
The immunoassays which can be used within the scope of
the present invention are based on standard methods'with
which experts in the field will be familiar and of which
there are a large number available. These methods are
based on the formation of a complex between the
antigenic substance to be measured and one or more
antibodies. One or more of the complex partners is
labelled so that the antigen can be detected and/or
quantitatively measured. Labelling may take the form,
for example, of a coupled enzyme, radioisotope, metal
chelate or a fluorescent, chemiluminescent or
bioluminescent substance.
In the case of a competitive immunoassay, the antigen in
the sample to be analysed competes with a known quantity
of labelled antigen for binding to the antibody binding
sites. The quantity of labelled antigen bound to the
antibody is therefore inversely proportional 'to the
quantity of antigen in the sample.
In tests using labelled antibodies, the quantity of
labelled bound antibody is directly proportional to the
quantity of antigen.
Assays based on the formation of an
antibody/antigen/antibody complex, using two antibodies
which do not prevent each other from binding to the
antigen, are known as °'sandwich" immunoassays.
Since monoclonal antibodies are available in unlimited
amounts with a constant quality, immunoassays using
monoclonal antibodies have crucial advantages, thanks to
their constant quality and reproducibility, over assays.
which use polyclonal antibodies. Morever, they avoid
the disadvantage connected with polyclonal antibodies,
namely that animals have to be used constantly in order
to produce them.
Preferably, the monoclonal antibodies according to-~the
invention are used in a sandwich immunoassay,
particularly a sandwich ELISA.
g
the monoclonal antibodies according to the invention can
be used in sandwich immunoassays as coating- and
7.abelling-coupled antibodies and thus make it
unnecessary to use polyclonal antibodies.
According to another aspect the present invention
relates to immunoassay kits, particularly sandwicta
immunoassay kits containing tbp-1 and/or tbp-2.
A preferred embodiment of the present invention is a
sandwich immunoassay kit, preferably a sandwich ELISA
kit, in which tbp-1 is the coating antibody and tbp-2 is
the labelled, preferably enzyme-coupled antibody.
2t is possible within the scope of the present
invention, in a sandwich immunoassay, to replace tbp-1
or tbp-2 with another antibody which is capable of
forming an antibody/antigen/antibody complex with tbp-2
or tbp-1.
2f tbp-1 or tbp-2 is replaced by another antibody, it is
preferable to use an antibody which recognises the same
epitope of TNF-BP I or part thereof as the tbp-1 or
tbp-2 which is to be replaced.
Sandwich immunoassays can be used to determine which
antibodies are suitable as a replacement for tbp-Z or
tbp-2 in the immunoassay on the basis of their ability
to form an antibody/antigen/antibody complex with tbp-2
or tbp-1. Immunoassay plates are coaeted with tbp-1 or
tbp-2, the antigen added and the labelled antibodies to
be investigated applied. Antibodies which are capable
of forming an antibody/antigen/antibody complex with
tbp-1 or tbp-2,'which can be determined by measuring the
labelling of the test antibody, recognise a different
epitope on the antigen from tbp-1 or tbp-2. Antibodies
which are not capable of forming a sandwich with tbp-1
r
-
or tbp-2 show the same or an overlapping epitope
recognition as these antibodies.
If one of the antibodies tbp-1 or tbp~2 is replaced in
the sandwich immunoassay, preferably t;he labelling-
coupled antibody tbp-2 is replaced by an antibody which
has the same or overlapping epitope specificity as
tbp-2m ~n example of a suitable antibody is the
monoclonal antibody H398 described by Thama et al.
(1990), which has been shown, within the scope of the
present invention, to bind to an identical or
overlapping epitope.
With the help of a sandwich ELTSA according to the
invention based on tbp-1/tbp-2, it has been possible to
detect TNF-BP I in human serum, plasma, urine and cell
culture residues with a sensitivity of about 200 ng/1
and an accuracy of more than 10%.
It has been found, surprisingly, that the natural
ligands for the TNF-receptor, TNF-a and TNF-,B, do not
affect the predictive qualities of the immunoassay
according to the invention in which tbp-1 and tbp-2 are
used: TNF°~B has, no measurable influence on the assay
whilst TNF-a only causes a measurable change ~to the
signal at concentrations of >-10 ~eg/1. Since the TNF-~
concentrations in healthy people are normally <_20 ng/1
and, even in serious pathological conditions, the TNF-o:
concentrations only rarely exceed 1 ~Cg/1 (e. g.
Lahdevirta et al., 1988; Offner et a.., 1990), '
falsification of the immunoassay according to the
invention by endogenous TNF-a secreted under natural
conditions can be ruled out.
In view of its sensitivity, an immunoassay based an the
monoclonal antibodies tbp-1/tbp-2 is also capable of
detecting concentrations of TNF-BP I which deviate
- 10 -
downwards from the normal levels. Thus, functional
disorders of the body which are accompanied by a reduced
production of TNF--DP I can be deteotec~ diagnostically.
In addition to detecting TNF-BF I in serum, plasma and
urine and in cell culture residues, the monoclonal
antibodies tbp-1 and tbp-2 can also be used for
detecting TNF-BHP I in other body fluids, e.g. in
cerebrospinal fluid or in bronchoalveolar secretions.
In the experiments carried out within the scope of the
present invention it was found, surprisingly, that the
signal obtained in a sandwich ELISA with a combination
of tbp-1 and tbp-5 was not even .affected by extremely
high TNF-a or TNF-~i concentrations in the region of
mg/l.
The antibody combination tbp-1/tbp-6 is preferably used
in a sandwich immunoassay, especially in a sandwich
ELISA, whilst tbp-~. and tbp-6 may both be used as a
coating antibody and as a labelled antibody.
Examples of the use of the pair of antibodies
tbp-1/tbp-6 for. detecting TNF-BP I include samples from
in vitro experiments in which cells such as leukocytes
are stimulated with lipopolysaccharides, or serum
samples from experimental animals treated with large
quantities of lipopolysaccharides or bacteria. Under
these conditions, TNF-a is secreted in extremely large
amounts.
Thus, with the aid of the present invention, a highly
sensitive method of detecting TNF-BF I is provided, by
means of which it is possible to determine the -
activation of the TaF receptor by its physiological
ligands TNF-a and TNF-,0 and its transmodulation by other
mediators in various pathological conditions or in i,r~
~~~~~J~~
vitro models. The detection of '.~ldF-Bp I in body fluids
is thus particularly useful for diagnosing pathological
conditions which are accompanied by an increased TNF-a
production, or for confirming such diagnoses.
One advantage of TNF-BP Z determination, as compared
with TNF-a determination, is the fact that normal levels
of TNF-BP I are found in the serum, thus making it
possible to detect even slightly raised levels and
thereby reach a diagnosis.
Tn spite of the clear correlation between the induction
of TNF-a and the occurrence of septicaemic and
endotoxaemic reactions in animals, the measurement of
TNF-a in patients suffering from severe gram-negative
infections has produced conflicting results. This would
appear to be connected with the mechanisms which control
the synthesis and secretion of TNF-a. When activated by
a suitable stimulus, TNF-a is rapidly secreted by
macrophages, after which the~macrophages should be
resistant to further stimulation. Moreover, the half-
life in the plasma is short, being only 15 to 17 minutes
in humans. These phenomena would appear to be the
reason why the appearance of TNF-a in the blood system
is rapid and short--lived and therefore difficult to
detect (Michie et al., 1988). Therefore, if blood is
not taken from the patient at the right time, it is
possible that the increase in the TNF-a concentration
will not be detectable at the moment of analysis. Lantz
et al. (1990x) have established that, after infusion
with TNF-a, the TNF-HP I level in the serum drops much
more slowly than the serum TNF level. This finding
indicates that the measurement of TNF-BP I
concentrations as a basis for diagnosis is particularly
advantageous if one is setting out to diagnose
pathological conditions connected to activation of the
TNF-receptor system, particularly by TNF-a, and wherein
~. 0 '~ 3 ~~ ()
- 12 -
the TNF-a level falls more rapidly by comparison with
the TNF-BP I level.
Examples of diseases which can be diagnosed with the aid
of the measurement of TNF-BP z include gram-negative or
general bacterial infections, septic shock, tissue
damage in "Graft-versus-host°° disease and cerebral
malaria as well as cachexia.
The immunoassays according to the invention are
particularly beneficial in diagnosing septic shock,
which is a life threatening disease if treatment is not
carried out promptly.
With the aid of an immunoassay according to the
invention based on the monoclonal antibodies tbp-Z and
tbp-2, TNF-BP I could be detected in the serum of normal
donors at an average concentration of about 2 ug/1.
Raised levels were detected in the serum of patients
suffering severe burns; very~high levels were detected
in dialysis patients, whereas the sera of patients with
chronic polyarthritis showed no increased TNF-BP I
concentrations.
The immunoassays according to the invention also
provide, for the first time, a simple immunological
method of detecting TNF-BP I in the urine. in urine
samples from normal donors, an average TNF-BP I
concentration of about 2 ~Cg/1 was detected.
The immunoassays according to the invention can thus be
used to diagnose pathological conditions in which there
is a correlation between raised TNF-BP I concentrations
in the urine and in the serum, as was found in cases of
kidney failure, by determining the concentration of
TNF-BP I in the urine. This method has the advantage of
requiring no blood sample and of being able to provide a
~1~'~~~~0
- 1 a
diagnosis on the basis of urine analysis, which is
considerably more pleasant for the patient.
The term "diagnosis", for which the immunoassays
according to the invention can be used, also covers the
monitoring of the course of a disease which is connected
with activation of the 'fNF-receptor system, or the
course of therapy for treating such a disease, e.g.
treatment with antibodies against TNF-a. The use of the
immunoassay according to the invention is also
convenient for monitoring the progress of therapies in
which TNF-a or TNF-/~ are administered. In the course of
these diagnostic applications, a sample of body fluid is
generally taken from the patient at regular intervals
and examined for its content of TNF-BP I.
Monoclonal antibodies against TNF-BP I, which do not
compete with TNF-a and/or TNF-(3 for binding to TNF-BP I
and which increase the protective effect of TNF-BP I,
may be used according to the~invention to reinforce the
effect of TNF-BP I within the scope of the treatment of
diseases in which TNF-a and/or TNF-R has a damaging
effect. (It has been found that the protective effect
of TNF-BP I against TNF-a is relatively weak (Lantz et
al., 1990b, Loetscher et al., 1991)).
In order to make use of the reinforcement of the
protective effect of these antibodies both for
endogenous TNF-BP I and also for exogenous TNF-BP I,
administered therapeutically, the antibodies may be used
per se or combined with TNF-BP I in suitable
preparations as a therapeutic agent.
Examples of antibodies of this kind which enhance the
protective effect of TNF-BP I against TNF-a and TNF-/3
include tbp-1 and tbp-6. The suitability of other
antibodies against TNF-BP I far this purpose can be
~:I~'~3~~0
_1~_
established by testing the antibodies far their effect
on TNF°BP I in bioassays. The dosage of monoclonal
antibodies is adjusted according to the dose of TNF-BP I
and is appropriately in the range from ec~uimolar to an
approximately lU0--times molar excess, based on the
amount of TNF-BP I.
Another fiald of application of the present invention is
the use of the monoclonal antibodies tbp-1, tbp-2 and
tbp-6 in immobilised form for purifying TNF-BP I by
affinity chromatography.
In addition, the monoclonal antibodies according to the
invention, especially tbp-l, can be used for detecting
TNF-BP I in immunoblots.
Summary of Figures
Fig. 1: ELTSAs for TNF-BP I' using monoclonal
antibodies
Fig. 2: Representative calibration curves for TNF-BP I
ELISAs of serum and urine samples
Fig. 3: Effect of TNF-cx on the immune reactivity of
TNF-BP I
Fig. 4: TNF-BP T concentrations in human serum and
urine
Fig. 5: Effect of TNF-a on detecting TNF-BP I in
sandwich ELISAs
Fig. 6: Effect of monoclonal antibodies on the
protective effect of TNF-BP I in cytotoxic
bioassay
~aaP~~~~a
The invention is further .i.llustrated by the following
Examples:
Example 3.
Preparation of monoclonal antibodies with specificity
for human TNF-BP I
a) Immunisation
3 female BABL/c mice about 6 weeks old were immunised
with TNF-BP I, purified to homogeneity, according to the
method described in EP A2 393 X38, in accordance with
the following plan:
1st immunisation: 9 fag of TNF-BP I per mouse in
complete Freund's adjuvant by
intraperitoneal route
2nd immunisation: 9 ~Cg of TNF-BP I per mouse in
incomplete Freund's adjuvant, by
intraperitoneal route, 3 weeks after
the 1st immunisation
3rd immunisation: 9 ~Cg of TNF-BP I ;aer mouse in
incomplete Freund's adjuvant, by
intraperitoneal route, ~ weeks after
the 2nd immunisation.
8 days later, serum samples were takers from the mice and
investigated by sandwich ELTSA for the formation of
antibodies against TNF-BP I. In order to do this,
TNF-BP T was bound to test plates coated with rabbit -
antibodies against TNF-BP I and .specific antibodies were
detected with peroxidase-ooupled rabbit antibodies
against mouse immunoglobulin. The test sera were '
applied in dilutions of 1:102, 1.:103, 1:104 and 1:105.
All three mice exhibited positive reactions (absorption
more than twice the background) at dia_utions up to 105.
z~op~~~o
- 16 -
The mouse with the highest ta.tre was given a booster,
about 8 weeks after the 3rd immunisation, on three
successive days, with 4 ~,g of TNF-BP T in PBS; the
spleen cells of this mouse were used the next day for
fusion with hybridoma cells. A second immunisation was
carried out in a similar manner, except that the mouse
used was additionally given a fourth dose of TNF-BP I
(15 ~Cg) 8 months after the 3rd immunisation and was
given a booster 7 weeks later.
b) Fusion:
The spleen of the mause was removed under sterile
conditions one day after the last/ injection,
mechanically chopped up and washed with serum-free
culture medium (RPMI 1640). About 10e spleen cells were
fused using the method of Kohler and Milstein (1975) in
the presence of PE3 4000 (Merck, 40% in serum free
culture medium] with about 5x107 P3?S63Ag8.653 BALB~c-
myeloma cells (Kear:rey et al., 1979). The cells were
then suspended in HAT-selection medium (RPMI 1640 -
coinpleted with 100 U/ml of sodium penicillin G, 50 U/ml
of streptomycin and 20% FCS - with 10'4 M hypoxanthine,
4x10'7 M aminopterin and 1.6x10°5 M thymidine) and
distributed into 16 96-well microtitre plates containing
peritoneal mouse cells as the "Feeder Layer". After 11
days, supernatants were removed and tested for antibody
production. Of the 1500 cultures which were grown in
selective medium, more than 90% showed growth of
hybridomas.
c) Screening of hybridoma culture residues
Unless otherwise specified, the following buffers°caere
used for all ELISA axperiments:
Coating buffer: 0.05 M sodium carbonate pH 9.6
CA 02107340 2002-04-22
27855-48
.. - 17 -
Washing medium: phosphate-buffered saline solution pH
7.4 (PBS) containing Tween 20 in an amount of 0.5 g/1
Test buffer: PBS with bovine serum albumin (5 g/1) and
Tween 20 (0.5 g/1)
Substrate solution: tetramethylbenzidine dihydrochloride
(0.1 g/1) and sodium perborate (0.05 g/1) in 0.05 M
potassium citrate pH 5.0
Stopping solution: 2 M sulphuric acid
96-well immunoassay plates were coated with rabbit
antibodies against TNF-BP I, partially purified by
ammonium sulphate precipitation (50% saturation), in
coating buffer at a concentration corresponding to a
1:3000 serum dilution (50 ~1/well, incubation overnight
at 4'C or for one hour at 37'C). The plates were washed
once and blocked with test buffer at ambient temperature
for one hour. Then hybridoma residues (50 ~C1) were
added together with the antigen (50 ~C1, 10 ~cg TNF-BP I/1
in test buffer) and the~dishes were incubated for 2
hours at ambient tea~perature.~ They were washed once, a
solution of peroxidase-coupled rabbit antibodies against
mouse immunoglobulins (DAKO, Denmark, 1:5000 dilution in
test buffer, 50 ~cl/well) was added and the plates were
incubated for 2 hours at ambient temperature. They were
then washed 3 times and 200 ~1 of substrate solution
were added to each well. After 20 to 40 minutes the
reaction was stopped by the addition of 50 ~cl of
stopping solution. The absorption of the solution was
measured in an ELISA reader at a wavelength of 450 nm
(reference: 690 nm), using culture medium as the
negative control and dilute mouse immune serum as
positive control. Only two of the cultures investigated
from the first immunisation showed any antibody
production (TBP-1 and TBP-2); the second'fusion yielded
a third antibody-positive cell line (TBP-6). The
positive cultures were transferred after about 25 days
from HAT-medium into HT-medium and after a further 10
*Trade-mark
27855-48
CA 02107340 2002-04-22
- 18 -
days they were transferred to normal culture medium
(RPMI 1640 complete, 1% antibiotics, 10% L-glutamine,
10% FCS ) .
d) Cloning of the-hybridomas: '
The positive cultures were cloned by the limiting
dilution method. Dilution was carried out so that
100 ~1 of culture medium contained 1 cell, after which
the wells in a 96-well dish were filled with this volume
(a total of 3 dishes were prepared, in which each well
was charged with 100 ~,l of mouse peritoneal macrophage
suspension on the previous day). The culture residues
were tested by ELISA as described above: positive clones
from each culture were pooled, expanded and frozen.
e) Production of monoclonal antibodies
In order to produce antibodies ',fin vivo, about 10T cells
were taken from each hybridonia culture and injected
intraperitoneally into BALB/c mice which had been.
treated 2 or 3 days previously with 0.5 ml of incomplete
Freund's adjuvant. After about to to 14 days the
Ascites fluid was removed. The monoclonal antibodies
formed were purified by ammonium sulphate precipitation
followed by affinity chromatography over carrier-bound
protein-G.
For antibody production in cell culture, foetal calves'
serum (FCS) was purified by chromatography on protein-G-
sepharose in order to eliminate bovine IgG: this serum
preparation was used in a concentration of 5% for the
hybridoma cultures. The antibodies were again isolated
from the culture residues using protein-G-sepharose.
Alternatively, the cells were grown in serum-free medium
(serum-free and protein-free hybridoma medium, Messrs.
SIGMA, Catalogue No. 5-2772: USA) and the antibodies
*Trade-mark
z~~~3~~~
- 19 -
were likewise isolated by chromatography on protein-C-
sepharose.
Example ?.
Characterisation of the monoclonal antibodies
The antibody subisotypes were determined using
peroxidase-coupled rabbit antibodies (Serotec, ~xford,
GB): tbp-1 and tbp-6 are IgG1-antibodies whilst tbp-2 is
an IgG2b-antibody.
In the Western blot, all three antibodies recognised
TNF-BP I: tbp-~. showed a strong reactivity, tbp-~
reacted less strongly whilst tbp-f yielded only a very
slight coloration.
In order to determine the epitopes which were recognised
by the antibodies, 'the three antibodies tbp-1, tbp-2 and
tbp-6 and the antibody H~98 developed by Thoma et al.
(1990) by immunising with solubilised TNF-receptor were
coupled to horseradish peroxidase and characterised by
ELxSA: test plates were each coated with one of the
unlabelled antibodies (10 mg/1), after which a series of
dilutions of the antigen ware added and then one of the
enzyme coupled antibodies was applied. This experiment
was carried out in every possible arrangement [~'ig. 1:
A: tbp-1; B: tbp-2, C: tbp-6, D: H398. The symbols
indicate the peroxidase conjugates: tbp-1 (solid
circles), tbp-2 (solid squares), tbp-6 (solid
triangles), H398 (open circles)]. Tt was found that
none of the individual antibody species was capable of
forming a '°sandwich'°, which indicates that the antigen
is present in the form of a monomer. Combinations of
the antibodies tbp-1 with tbp-z, tbp-6 or H398 and
combinations of tbp-2 with tbp-6 or tbp-6 with H398 were
- 2U -
capable of producing dosage-dependent signals, whereas
tbp-2 in conjunction with ti398 did not react. From this
it was concluded that the antibodies recognise three
different epitopes on the TNF-BP I molecule; tbp-~ and
H398 bind to identical or overlapping epitopes. For
further develapment a test arrangement was chosen in
which tbp-1 constitutes the coating antibody and tbp-2
the peroxidase-coupled antibody.
lExample 3
Development of an en2yme linked immunosorbent assay
(Sandwich--ELISA)
In the light of the use of ELISAs for detecting TNF-BP I
in human serum, first of all various media were
investigated for their suitability as dilution media for
samples and a standard. Whereas both FCS and also
calves' serum yielded useable dosage-activity curves,
pooled normal human serum showed a very high blank
value. In order to check whether this phenomenon can be
put down to non-specific reactivity or to the presence
of antigen, human serum was passed over an affinity
column containing immobilised TNF-a. As the throughflow
of the chromatography column was used as a diluting
medium, the blank value was approximately equal to that
obtained with calves' serum. This indicated that normal
human serum contains immune-reactive and biologically
active TNF-BP I. The concentration of TNF-BP I in the
serum pool used was estimated at about 1 ~g/1. Standard
curves drawn up with 50A calves' serum could not be
distinguished from those with 50o human serum from which
TNF-BP I had been removed. Therefore, the former-medium
was used for all subsequent tests (of all the supplies
of calves' serum used obtained from various companies,
only two proved suitable; all the others exhibited poor
- 21 -
reproducibility.
The tests were set up and carried out according to
standard methods.
First of all, in preliminary tests for developing an
assay for measuring TNF-E3P T in serum, it was found that
a so-called twa-step assay, i.e. a method in which
incubation of the sample is followed by a washing step
and the incubation with the antibody-enzyme conjugate is
carried out separately, has no advantage over the faster
and more convenient ane step method, Furthermore,
preliminary tests were carried out in order to vary the
concentration of the coating antibody and to vary the
incubation time for the immune reaction.
The optimised assay was carried out in order to
determine TNF-BP I in serum as follows:
96 well immunoassay plates were coated with the
monoclonal antibody tbp-1 at~a concentration of 3 mg/1
in coating buffer (overnight at 4°C or for 1 hour at
ambient temperature; 100 ~c1/well). The wells were
washed once and the binding sites remaining were blocked
with 200 ~,1 of test buffer at ambient temperature for 1
hour. After another washing cycle the wells in rows 2
and 11 were filled with 100 ~,1 of 50% calves' serum/50%
PBS. A standard solution of TNF-BP I (see Example la,
20 ~Cg/1 in 50% calves' serum/50% PBS, 100 ~C1) was
pipetted into wells A2 and All; serial dilutions in the
ratio 1:2 were made directly in the wells in rows 2 and
11. All other wells were given 50 u1 of PHS and the
serum samples were each pipetted out twice (50 ~1/well).
50 ~S1 of a solution of peroxidase-coupled antibody tbp-2
in test buffer were placed in all the wells after-a
suitable dilution had been established in preliminary
trials. (The coupling of the antibodies to horseradish
peroxidase (Boehringer Mannheim) was carried out in
f
- 22 -
accordance with the method described by Wilson and
Nakane (1978)). The plates ware incubated for 3 hours
at ambient temperature on a plate vibrating apparatus.
Z'he plates were then washed three times, substrate
solution was added (200 u1), the reaction was stopped by
the addition of 50 dal of stopping solution and the
absorption values were determined as described above.
The concentrations of TNF-BP Z in the samples were
calculated using the Titercalc program (Hewlett
Packard).
zn a similar way an assay was set up for analysing cell
culture residues, except that, when plotting the
calibration curve, cell culture medium containing 10%
FCS was used and the samples were used undiluted.
A modified (two step) method was developed for measuring
TNF°BP T in the urine. The need for this arose because
the low pH value of some samples and other unexplained
factors interfered with the test. The effect caused by
the pH value could not be compensated by means of the
standard test buffer owing to the insufficient buffer
capacity. A strong phosphate buffer (0.5 M) was
necessary in order to ensure that even the most acidic
samples (pH 5) could be brought up to a neutral pH.
This measure was still not sufficient as some samples
still showed poor reproducibility. This pxoblem was
solved by using a two-step test method (successive
incubation of samples and conjugate): the plates were
coated, blocked and washed as described for the serum
assay. Then a solution consisting of bovine serum
albumin (5 g/1) and Tween 20 (0.5 g/1) in 0.5 molar
sodium phosphate buffer pH 7..4 was pipetted into the
wells in the plate (50 ~,1/well). The calibration-curve
was drawn up with the same solution. Then urine samples
(50 ~1/well; measurements carried out twice) were added
and the plates were incubated for 2 hours on a vibration
~l~lrl~~~D
23 _
apparatus. The plates were then washed and 100 ~W of a
solution of enzyme conjugate in test buffer was added,
after which incubation was carried otat for a further 2
hours. The final treatment of the plates and the
quantitative measurement of TNF-OP I were carried out as
described above for the serum test.
A typical calibration curve is shown in Fig. 2 (solid
circles: serum samples; open circles: urine samples); it
permits quantitative measurement of TNF-BP x in a
concentration range of between 0.3 and 10 ~Cg/1. '.the
detectable minimum quantity in the serum, defined as the
concentration which produces a signal corresponding to
the blind value signal plus three standard deviations,
was determined at 0.2 psg/1 (average of 4 independent
assays). At concentrations of up to 1 mg/1 (loo-fold
excess compared with the .normal test range) no "high-
dose hoo7c°' effect (excess antigen) could be detected.
The sensitivity of the test for urine samples was
comparable. The sensitivity~for cell culture samples is
around 0.1 ~ag/l, since these samples are used undiluted.
The accuracy of the serum assay was tested by analysing
serum samples containing TNF-BP Z at concentrations of 2
and ZO ~Sg/1 in six different tests. The intra-assay
variation coefficients were 4.7% and 5.9%, respectively;
the inter-assay variation coefficients were ~.6% and
7.5%, respectively. The linearity was determined by
investigating a series of external two-fold dilutions of
TNF-BP I in the serum (concentrations between 0.3 and
~tg/1) and the linear regression of the values
determined experimentally was calculated by comparison
with the expected values. In three independent tests,
correlation coefficients of between 0.99 and 1 we're
obtained.
The detection of TNF-BP I by serum assay was
- 24 -
investigated by adding exogenous TNF-BP I to the serum
of 7 normal donors in two different concentrations (1
and 5 ,ug/1). This experiment was made more difficult by
the fact that all the samples contained endogenous
"fNF-BP I in various concentrations: these values
therefore had to be subtracted before the detectian rate
could be calculated. The average detection rate was
found to be 93 ~ 15% at 1 ~Sg/1 and 85 ~ 13% at 5 ~g/1
(range: 62 - 101% and 65 - 105%, respectively).
The detection of TNF-BP I by means of the modified urine
assay in 14 urine samples to which exogenous TNF-BP I
was added was investigated in the same way as for the
serum samplesa the average detection rate for a nominal
concentration of 5 ~Cg/1 was 83 ~ 15% (range: 52 ° 204%).
In order to determine whether the physiological ligands
of the TNF-receptor influence the interaction of
TNF-BP I with the antibodies, the ELISA was carried out
at a constant concentration of TNF-BP I (5 ~ag/1) in the
presence of increasing concentrations of recombinant
TNF-a (Gray et al., 1984) and recombinant TNF-,~ (Pennica
et al., 1984), in each case from E.E. coli and with a
purity of >99%. As can be seen from Fig. 3 (G shows the
assay background in the absence of TNF-BP I), TNF-a
inhibits the reactivity of TNF-BP I with the antibodies
at concentrations >_ 10 ~Sg/11 by contrast, TNF-/3
exhibited no activity even at very high concentrations
(up to loo mg/lj.
25 -
Example 4
Stability of TNF-BP I
a) Stability in the serum
Filter-sterilised samples of pooled normal serum were
stored for 24 hours at various temperatures: the samples
were also subjected to several freezing/thawing cycles,
As can be seen from Table l, neither the incubation at
temperatures of 37°C nor the repeated freezing and
thawing affected the immune reactivity of TNF-BP I. The
samples consisted of pooled human serum containing
endogenous TNF-BP I (sample 1: 1.2 ~Cg/1) and the same
serum sample with the addition of exogenous TNF-BP I
(sample 2: final concentration 5 ~eg/1).
b) Stability in the urine
The tests were carried out as specified under a): three
samples were investigated which represented a broad
spectrum of pH values. As can be seen fram Table l,
TNF-BP I was stable in all the samples after freezing
and thawing. All the samples could be stored for 24
hours.at temperatures up to 37°C without losing any
activity. The urine samples originated from three
different donors (sample 1: pH 5.1, 1.9 ~cg/1: sample 2:
pH 5.9, 4.0 ~Cg/1; sample 3: pH 7.2, 3.7 ~tg/1) . "nd"
indicates ''not determined".
example 5
Detection of TNF-BP I in human serum ~ '
Sera from 42 normal donors (blood donors and laboratory
staff) were investigated using the serum sandwich ELISAs
- 26 -
described in Example 3. TNF-BP T concentrations varied
between 0,5 and 5.4 ~g,/1, with an average of 2.1 ~Sg/1
(standard deviation 1.0 ~ag/1; Fig. 4). Serum, EDrfA-
plasma, citrate plasma and heparin plasma obtained at
the same time, were available from two people; the
TNF-BP I concentrations did not differ significantly
(donor A: 1.7, 2.0, 1.6 and 1.9 ~ag/1; donor B: 1.8, 1.8,
3.8 and 1.9 ~g/1). The TNF-BP I concentrations in sera
from 15 patients with chronic polyarthritis slid not
differ significantly from those of healthy people (2.3 ~
0.79 ~g/l; range: 1.2 ° 3.9 ~ag/1). Significantly raised
levels were found in sera from patients with severe
burns (6.5 r 1.7 ~g/1; ranges 3.1 - 9.1 ~Cg/l; n = 10).
Patients with kidney failure (n = 6) showed remarkably
high concentrations in a range from 20 - 69 ug/1
(average ~ standard deviation; 49 ~ 17 ~ag/1).
Example 6
Using the sandwich ELISA developed in Example 3
specifically for urine samples, the TNF-BP I
concentrations of urine samples from 16 normal donors
were determined. They varied between 0.78 and 4.3 ug/1
(average ~ standard deviation: 2.2 ~ 1.2 ~,g/1). There "
was no significant difference between female (2.1 ~
1.4 ~cg/l; n = 9) and male (2.2 t 1. o ~cg/1; n = 7)
donors.
Example 7
Determination of TNF-BP I in culture residues from human
cell lines -
In order to determine whether the developed E1LISA is
suitable for detecting TNF-BP I produced by human cell
- 27
cultures, a series of cell lines were investigated.
Culture media (containing 10$ FCS) were harvested from
dense cultures, generally several days after dilution
with fresh medium or after the passage of adhering
cells. Culture medium containing 10% FCS was used as
the standard diluent; the assays were carried out by the
one-step method. TNF-BP T was detectable in residues of
the cell lines A549 (lung cancer; 1.1 ~g/'1), HeLa
(cervical cancer; 0.38 ~Sg/1) and Namalwa (Burkitt's
lymphoma; 0.25 ~ag/1), but was below the detection limit
(10D ng/1) in the cell lines U937 (histiocyte lymphoma),
EoL-3 (eosinophilic leukaemia), Raji (Burkitt's
lymphoma), HL-60 (myeloid leukaemia), U256 (myeloma) and
LuKII (B-l.ymphoblastoid cell line, immortalised by
Epstein-Barr virus). In accordance with earlier results
(Lantz et al., 1990b), it was observed that HL-60 cells
cultivated in the presence of TNF-~ (10 ,~g/1) released
increased quantities of TNF-BP I; after 4 days of this
treatment a concentration of 0.45 ~g/1 was achieved.
Example 8
a) Determining the influence of TNF-cx on the binding of
the antibodies to TNF-BP I
In order to determine whether TNF-a affects the binding
of the antibodies to TNF-BP I, a one-step sandwich. ELISA
was carried out in a number of different arrangements.
One of the monoclonal antibodies was used as a coating
antibody for mopping up the antigen, a constant quantity
of TNF-BP T was used in the presence of various
concentrations of TNF-cx,.and a second monoclonal
antibody labelled with horseradish peroxidase was used.
The ELTSAs were carried out substantially as described
in Example 3: the plates were coated with 10 ~Cg/ml of
antibody in coating buffer, washed and blocked with test
~~o r~~o
_ 28
buffer. The one-step incubation with TNF-BP I in a
final concentration of 5 ng/ml (in the presence of
varying concentrations of TNF-a) and with pero~tidase-
coupled antibody was carried out in test buffer at
ambient temperature for 3 hours. The plates were then
washed, developed with substrate solution, then the
reaction was stopped and absorption was measured at
45o nm in a plate reader, subtracting the absorption at
69p nm. The results of these experiments are given in
Fig. 5: Tables A to D show the results of plates which
were coated with the antibodies tbp-1, tbp-2, tbp-6 and
FI398; the symbols represent the peroxidase conjugates of
the antibodies tbp-1 (solid circles), tbp-2 (open
circles), tbp-6 (solid rectangles) and H398 (open
rectangles); the background signal (absence of TNF-BP I)
is shown at C. It was found that the signal obtained
with combinations of the antibodies tbp-1 and tbp-6 was
not even affected by the highest TNF-a concentrations
tested (l0 ~g/ml). By contrast, all the combinations
containing tbp-2 ar H398 were sensitive to TNF-a,
although with different dosage-activity ratios. (These
results indicate that H398 and tbp-2 recognise epitopes
which are connected with the TNF-a binding site, whereas
tbp-1 and tbp-6~define epitopes which are independent
thereof.)
b) Determining the influence of TNF-~ an the binding of
the antibodies to TNF-BP I
The assay was carried out with the monoclonal antibodies
tbp-1 and tbp-6 for TNF-/~, as described in a). Tn the
case of TNF-~, also, it was found that concentrations of
l0 pag/ml do not interfere with the assay.
9
- 29 -
Example 9
Cytotoxic bioassay for determining the biological
activity of the monoclonal antibodies
a) Tnfluence of the antibodies on the protective effect
of TNF-BP x against TNF-a
The cytotoxic activity of TNF-cx was determined
essentially using the methad described by ICramer and
Carver, 198b, For this purpose, mouse L-M cells (ATCC
CC1.. 1.2) were cultivated overnight in microtitre plates,
TNF-a preparatians were added in serial two-fold
dilutions and actinomycin D was added to give a final
concentration of 1 ~g/ml. The plates were incubated at
33°C for 18 to 20 hours, the cells were stained with
0.5~ crystal violet and absorption was determined at
540 nm. (The cell controls and blank values were
provided 4-fold on each plate). By definition, a
solution which kills off 50% of the cells contains 1
unit/ml. Under the assay conditions, the specific
activity of the recombinant human TNF-a used was 5 x 107
units/mg of protein; the reference preparation for
TNF-a, 86/659 (NIBSC, England) with a fixed activity of
4 x 104 E/ml, showed an activity of 5 x 104 E/ml. The
neutralisation assays were carried out by pre-incubating
serial dilutions of TD1F-BP I in culture medium with a
constant quantity of TNF-a (final concentration 20 E/ml)
and/or monoclonal antibodies for 1 hour at 37°C. Then
the culture liquid was removed from the plate and the
pre-inculcated TNF-a/TNF BP I/antibody solution was added
to the cells in a quantity of 100 ~Cl. The plates were
incubated and stained as described above. TNF-BP I
concentrations exhibiting a.50~ protective effect on the
cells (ED50) were determined graphically. The results
of the bioassay carried out are given in Fig. 6; the
symbols indicate the following results: TNF-BP Z on its
r1 t~
- 30 -
own (solid circles; broken line), TNF-BP I combined with
the antxbadzes at 0.01 ~g/ml (open triangle), 0.1 ~Sg/ml
(solid triangles) , 1 ~sr~/ml (apen rectangles) , 10 ,~g/ml.
(solid rectangles) or 100 ~g/ml (open circles).
It was faund that, in the presence of a constant
quantity of TNF-a (400 pg/ml corresponding to 20
cytotoxic units/ml) half-maximum protection is obtained
at TNF-BP T concentrations of 270 ~ 44 ng/ml (average of
6 independent tests). When the monoclonal antibody
tbp-2 was added together with TNF-BP I, the protective
effect of TNF-BP I was reduced: at antibody
concentrations of 1, 10 or 100 ~eg/ml, the concentrations
of TNF-BP I required for half-maximum protection
increased to 380, 1,000 and 12,000 ng/m1, respectively.
H398 reduced the protective effect of TNF-BP Z to a
similar degree (final values of 840, 3,800 and
> 20,000 ng/ml). (The treatment of the cells with these
antibodies in amounts of up to 100 ~cg/ml in the absence
of TNF did not produce any cytotoxic activities of any
kind; it was also found that the antibodies do not
protect the cells against the toxicity of TNF in the
absence of TNF-BP T.)
Surprisingly, however, the antibody tbp-1 did increase
the protective effect of TNF-BP I. At antibody
concentrations of up to 100 ng/ml, the cells were
completely protected by TNF-BP I at doses as low as
40 ng/ml. tbp-6 showed a similar but quantitatively
smaller activity (this antibady also showed a lower
activity in the EbISAs and presumably has a lower
affinity), zn the presence of a large excess of
antibody (10 ~Cg/ml) half-maximum protection could be
found at a TNF-BP I concentratian of 7.3-1 0.5 ng/"ml
(tbp-1) or 53 1 13 ng/ml (tbp-6), corresponded to an
approximately 40-fold or 5-fold increase in the
protective effect (which was established by titrations
~:~~'~3~J
- 31 -
carried out in 4 experiments). In control tests which
were carried out at the same TNF-a concentration but in
the absence of TNF-HF X, the antibodies showed no
activity.
b) Influence of the antibodies on the protective effect
of TNF-~P I against TNF-~
1Cn the bioassay with L-M cells carried out as described
in a), the cytotoxic activity of TNF-p is greater than
that of TNF-a (300 E/ng as against 50 E/ng). TNF-~P I
also exhibited an inhibition of the cytotoxic effect
against the same cytotoxic dose of TNF-~ (20 E/ml,
corresponding to 66 pg/ml), but the dosages required
were at least ten times as great (E~50 = 3,00 ng/ml,
compared with 270 ng/ml for TNF-a). The addition of the
antibodies tbp-1 and tbp-6 (10 ~,g/ml) intensified the
protective effect to a similar extent as for TNF-a; a
half-maximum protective effect was achieved at 53 ng/m1
and 500 ng/ml, respectively.
~~0"13~~
- 32 -
TABLE 1
Stability o~ TNF-f3P I in serum and urine
Treatment % Detection
Serum Utrine
Sample l~Sample 2 Sample 1 Sample 2 Sample 3
Storage
at
-2~C loo 100 100 100 100
~- 4 98 100 97 105 105
C
+2oc zoo 98 100 ~ 112 l09
+37C 98 91 110 107 89
Freezing/
thawing
cycles
0 100 100 100 100 100
1 101 105 99 92 101
3 95 103 107 98 g9
92 104 nd 94 99
~~.~'~~~i)
_ 33
f3ib1 iography:
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