Title: Immunoadsorber for use in sepsis therapy
Abstract: The invention relates to immunoadsorbers for use in sepsis therapy, in particular for removal of complement factors and lipopolysaccharides (LPS) and, if need be, further sepsis mediators such as TNF and interleukins from body fluids, methods for their production and their use.
Patent Number: 6,881,408 Issued on 04/19/2005 to Heinrich, et al.
| Inventors:
|
Heinrich; Hans-Werner (Riemserort, DE);
Hahn; Hans-Jurgen (Karlsburg, DE);
Meyer; Udo (Hastorf, DE);
Kruschke; Peter (Greifswald, DE);
Wagner; Heinz-Jurgen (Berlin, DE)
|
| Assignee:
|
Bioserv AG (Rostock, DE)
|
| Appl. No.:
|
937126 |
| Filed:
|
December 19, 2001 |
| PCT Filed:
|
March 23, 2000
|
| PCT NO:
|
PCT/DE00/00927
|
| 371 Date:
|
December 19, 2001
|
| 102(e) Date:
|
December 19, 2001
|
| PCT PUB.NO.:
|
WO00/58005 |
| PCT PUB. Date:
|
October 5, 2000 |
Foreign Application Priority Data
| Mar 26, 1999[DE] | 199 13 707 |
| Current U.S. Class: |
424/140.1; 424/139.1; 604/5.01; 604/5.02; 604/5.04 |
| Intern'l Class: |
A61K 039//39.5 |
| Field of Search: |
424/140.1,139.1
604/5.01,5.02,5.04
|
References Cited [Referenced By]
U.S. Patent Documents
| 5626843 | May., 1997 | Surkovich et al. | 424/140.
|
| 5853722 | Dec., 1998 | Rollins et al. | 424/145.
|
| 6074642 | Jun., 2000 | Wang et al. | 424/145.
|
| 6193681 | Feb., 2001 | Davidner et al. | 604/6.
|
| 6287516 | Sep., 2001 | Matson et al. | 422/44.
|
| Foreign Patent Documents |
| WO 98/34959 | Aug., 1998 | WO.
| |
Primary Examiner: Saunders; David
Attorney, Agent or Firm: Norris McLaughlin & Marcus PA
Claims
What is claimed is:
1. An immunoadsorber for blood treatment use in sepsis therapy, the
immunoadsorber comprising a carrier of organic or synthetic polymers to
which are immobilized antibodies that are specific to C3a and/or C5a and
to lipopolysaccharides (LPS) and wherein,
a) the antibodies to C3a are specific for at least one peptide selected
from the group consisting of SEQ ID NO: 1, 2, and 3; and
b) the antibodies to C5a are specific for at least one peptide selected
from the group consisting of SEQ ID NO: 4, and 5.
2. The immunoadsorber according to claim 1, wherein the antibodies are
polyclonal antibodies.
3. The immunoadsorber according to claim 2, wherein the antibodies are
avian antibodies of type IgY.
4. The immunoadsorber according to claim 1, further comprising at least one
immobilized antibody specific for at least one sepsis mediator selected
from the group consisting of TNF, 1L1, 1L6 , IL8 and/or IL10.
5. The immunoadsorber of claim 4, wherein the immobilized antibodies
comprising the immunoadsorber are varied as a function of the actual
content of sepsis mediators in the blood.
6. The immunoadsorber according to claim 4, wherein the immobilized
antibodies are specific for at least one of the following peptide
sequences of interleukins 1.alpha. and 1.beta.
IL1.alpha.: NH2-NCYSENEEDSSSID-COOH SEQ ID NO. 6
NH2-GAYKSSKDDAKIT-COOH SEQ ID NO. 7
NH2-WETHGTKNYFTS-COOH SEQ ID NO. 8
IL.beta.: NH2-RISDHHYSKGFRQA-COOH SEQ ID NO. 9
NH2-VQGEESNDKIPVA-COOH SEQ ID NO. 10
NH2-ESVDPKNYPKKKMEKRF-COOH SEQ ID NO. 11.
7. The immunoadsorber according to claim 4, wherein the immobilized
antibodies are specific for at least one of the following peptide
sequences of interleukin 6:
IL6: NH2-APHRQPLTSSERIDKQI-COOH SEQ ID NO. 12
NH.sub.2 -QNRFESSEEQARA-COOH SEQ ID NO. 13
NH2-AITTPDPTTNAS-COOH SEQ ID NO. 14.
8. The immunoadsorber according to claim 4, wherein the immobilized
antibodies are specific for at least one of the following peptide
sequences of interleukin 10
IL10: NH2-SPGQGTQSENSCT-COOH SEQ ID NO. 15
NH2-QMKDQLDNLLLKES-COOH SEQ ID NO. 16
NH2-MPQAENQDPDIKA-COOH SEQ ID NO. 17
NH2-LPCENKSKAVEQ-COOH SEQ ID NO. 18.
9. The immunoadsorber according to claim 4, wherein the immobilized
antibodies are specific for at least one of the following peptide
sequences of TNF.alpha.
TNF.alpha.: NH2-VRSSSRTPSDKPVA-COOH SEQ ID NO. 19
NH2-KSPCQRETPEGAEAKPW-COOH SEQ ID NO. 20.
10. The immunoadsorber according to claim 1, wherein the organic or
synthetic polymers further comprise membranes or particles of one or more
of the group consisting of polystyrenes, carbohydrates, cellulose, agarose
derivatives, and acrylates.
11. The immunoadsorber according to claim 1, wherein the immobilized
antibodies are covalently bound to the carrier.
12. The immunoadsorber according to claim 1, wherein the immobilized
antibodies are attached to the carrier via spacers or linkers.
13. A method for the production of immunoadsorber according to claim 1,
wherein antibodies specific for C3a and/or C5a and LPS and, optionally,
against further sepsis mediators are covalently or adsorptively coupled to
the carrier.
14. A method according to claim 13, wherein the antibodies are produced by
immunisation of mammals or birds with the corresponding antigens.
15. The method of claim 14 wherein the antibodies are raised by immunizing
one or more animals selected from the group consisting of mice, rats,
rabbits or chickens.
16. A method of treating blood plasma or serum using the immunoadsorber of
claim 1, the method comprising the steps of,
providing an amount of blood plasma or serum in need of sepsis therapy; and
contacting the blood with the immunoadsorber of claim 1;
and recovering the contacted blood plasma or serum from the immunoadsorber.
17. The method of claim 16, wherein the blood plasma or serum has not been
subjected to hemofiltration prior to contacting the immunoadsorber.
Description
The invention in question relates to an immunoadsorber for use in sepsis
therapy, in particular for removing complement factors and
lipopolysaccharides (LPS) as well as, if need be, TNF and interleukins
from body fluids and methods for their production and their use.
Every year, about 3.5 million patients suffer from sepsis in the USA, Japan
and the EU. With a total number of inhabitants of 785 million, the
incidence for these countries is less than 0.5%. But when hospitalised
patients are examined with regard to the frequency of suffering,
2.0.+-.0.16 cases of sepsis are found per 100 hospital admissions. The
enormous health political and individual importance can also be seen from
the observation that about 25% of these patients also suffer the syndrome
of a septic shock, characterised by the lethality rate of >45%, even
with most intensive medicinal care by highly qualified specialists in
institutions with modern equipment (intensive care units).
The risk of suffering a septic shock is very high especially with
poly-traumatised patients (traffic accidents, burns, serious operations).
Alongside infection from the outside, breaking through the intestinal
barrier for gram-negative bacteria normally occurring in the intestines as
a result of a partial loss of function of the immune system of these
patients and thus an infection "from the inside" can be detected.
In more than 50% of the cases, gram-negative bacterial or their cell-wall
components, endotoxins (lipopolysaccharides, LPS), cause the septic shock.
The LPS released by bacterial binds to a serum protein (LBP) and is then
absorbed by the LPS receptors of the monocytes/macrophages (CD14). The
CD14+ cells activated in this way produce cytokines (TNF.alpha.,
Interleukin-1' (IL-1), IL-6, IL-8), which have their effect via cytokine
receptors of the target cells.
Parallel to the stimulation of the monocytes and macrophages, the
complement system is activated. It is an integrated part of the
immunological defence of mammals for direct and unspecific combating of
bacterial micro-organisms and foreign particles. Of the complement
proteins occurring in the blood serum, primarily proenzymes activated by
proteolytic fission, the C3 protein with a serum concentration of about 1
g/l plays a central role. After contact of the micro-organisms with the
C3, the complement protein C3a is split off and, on the one hand, the
formation of C5 convertase is initiated by the resultant C3b (alternative
way of complement activation) and, on the other hand, the reaction is
amplified by the C3B converting to C3 convertase due to depositing of
serum factors. The complement protein C5 also occurring in serum is now
proteolytically fissured by the C5 convertase, which is provided in larger
amounts, also forming C5a. Further complement proteins (C6-C9) deposit on
the resulting C5b until finally the polymeric hydrophobic membrane attack
complex (MAC) is formed, settling in the bacteria membrane
(opsonidisation) and forming pores, which lead to phagocytosis and thus to
the elimination of the micro-organisms (and the bound MAC). The complement
factors C3a and C5A (anaphylatoxins) released in the process of the
complement activation result in stimulation of the phagocytising cells to
the location of the bacterial attack by increasing the vascular
permeability and the release of chemotoxins induced thereby. The reduction
of the number of bacteria results in a reduction of the activation of the
complement system. This direct and unspecific reaction is closely
connected with the other immunological defence systems insofar as the
synthesis and release of the cytokines essential for cellular defence is
regulated, for example by complement factors. In order to bring about the
inflammatory effect, C3a and C5a are bound to specific cell-based
receptors, which for their part are expressed in different strengths as a
function of the immune reactivity. In order to keep the immune defence
permanently ready for activity, activated complement factors are
detectable not only after an attack with micro-organisms, but also an
integrated part of the serum of standard persons with a concentration of
1-10 ng/ml.
The plasma levels of the anaphylatoxins can be increased by a factor of
more than one thousand, particularly in a developed sepsis, acute
pulmonary failure and in moribund patients.
Almost exclusively on the basis of in vitro examinations, there exist
various, mainly unspecifically effective variations of solutions in order
to eliminate the effects of various complement factors, which however can
hardly be tested under in vivo conditions on account of the side effects
to be expected (e.g. WO-A-98/34959).
In ex vivo methods for the prevention of complement activation by
artificial, extracorporal surfaces (e.g. surface coatings), an unspecific
complement activation was successfully carried out. Further, selective
removal of activated complement factors making use of specific C5
antibodies is known from U.S. Pat. No. 5.853,722 and certainly also to be
preferred, especially as highly affined antibodies have been generated in
the meantime against all the components of the complement system.
The functional cascade manifested is primarily used to eliminate the
bacteria penetrating into the organism. But as soon as a discrepancy
occurs between the number and/or virulence of the penetrating bacteria and
the elimination capacity of the immune system (e.g. in post-traumatic
immune deficiency), an excessive activation is observed, subsequently
accompanied by a massive release of "shock mediators" (interleukins,
thrombocyte activation factor (PAF), but also oxygen radicals,
prostagiandins and their metabolic products), thus further limiting the
elimination capacity for LPS. In addition, CD14-negative cells (e.g.
endothellae) are also activated by the LPS, as soluble CD14 (sCD14) exists
in the blood plasma as an LPS trapper, facilitating binding to these cells
and inducing the formation and release of further shock mediators, thus
reinforcing the circulus vitiosus. As the shock mediators act selectively,
but not specifically, function restrictions in various cells and organs
are observed (blood coagulation system, circulation, complement system),
with the result that the inflammation reactions attacking the entire
organisms initiate shock genesis, leading to irreversible organ damage, to
circulation collapse and death.
In order to break through this chain of functions, various therapy
strategies have been studies.
Interruption of the cascade with antibodies interrupting the LPS binding to
proteins (LBP, sCD14), to the receptor (CD14), to released cytokines or to
cytokine receptors or with antagonists blocking the functional areas of
the receptors did achieve impressive success in various sepsis models in
animal experiments, but there are still no clinically tested, successful
prevention and/or therapy studies.
It was not possible to fulfil the high expectations, as it was increasingly
seen that LPS also influences and changes the functional condition of
cells and tissue which are not impaired by these therapeutic approaches.
In addition, it must be taken into account that an LPS (immune complex)
inactivated by an antibody/antagonist must be eliminated in order to
exclude a biological reactivity on a permanent basis. But the elimination
is also a function of the immune system, which, as it is greatly weakened,
can hardly or only very imcompletely fulfil this task.
The development of the septic shock is a very dynamic occurrence of
primarily varying genesis, in which various mediators cause highly
differing reactions within a short period of time, these quickly leading
to the expression of the septic shock by dysregulation after an initial
life-maintaining function.
Therefore, the invention was based on the task of developing an
immunoadsorption system of modular construction, in particular for
extra-corporal detoxification, enabling a reduction of the plasma and
tissue levels specific to the patient.
Inter alia, the invention is based on the knowledge that TNF.alpha. has a
key role to play in this regulation system. It is released inter alia by
macrophages as a result of various "external" influences such as injuries,
inflammations, infections, septicaemia and induces a local and systemic
activation of the unspecific and specific defence system via a cytokine
cascade (IL-1, IL-6). Clinically, a massive TNF.alpha. release is
expressed by increased body temperature, lack of appetite and all the
subsequent symptoms of a catabolic metabolism situation. In pathogenesis
of the sepsis, activation of the macrophages and thus the release of
TNF.alpha.- appears to be of essential importance for a survival of the
patient in the early phase of this disease, whereas the continued state of
activation results in the de-compensation of all defence reactions in the
further course.
The task of the invention was solved by an immunoadsorber for use in sepsis
therapy. The immunoadsorber according to the invention is particularly
used for the removal of complement factors and lipopolysaccharides (LPS)
as well as the removal of further sepsis mediators, and also TNF and
interleukins from body fluids, if need be. It is characterised by carrier
materials of organic or synthetic polymers, to which both poly or
monoclonal antibodies aimed against the complement factors C3a and/or C5a,
and also antibodies aimed against lipopolysaccharides (LPS) are bound. In
a preferred embodiment, antibodies aimed against further sepsis mediators
are also bound to the carrier.
Preferably, these are polyclonal antibodies, particularly preferably avian
antibodies of type IgY. The antibodies against sepsis mediators are
contained according to the state of the dysregulation.
According to this invention, these are antibodies aimed against TNF, IL1,
IL6, IL8 and/or IL 10.
Preferred antibodies against the complement factor C3a manifest specific
activity against at least one of the following peptide sequences:
NH.sub.2 -KCCEDGMRQNPMR-COOH (SEQ ID NO: 1)
NH.sub.2 -RFSCQRRTRFISL-COOH (SEQ ID NO: 2)
NH.sub.2 -ITELRRQHARAS-COOH (SEQ ID NO: 3)
Preferred antibodies against the complement factor C5a possess specific
activity against at least one of the following peptide sequences:
NH.sub.2 -QADYKDDDDKLPAE-COOH (SEQ ID NO: 4)
NH.sub.2 -DDKLPAEGLDIENS-COOH (SEQ ID NO: 5)
Preferred antibodies against IL1 .alpha./.beta. possess specific activity
against at least one of the following peptide sequences:
NH.sub.2 -NCYSENEEDSSSID-COOH (SEQ ID NO: 6)
NH2 GAYKSSKDDAKIT-COOH (SEQ ID NO: 7)
NH.sub.2 -WETHGTKNYFTS-COOH (SEQ ID NO: 8)
NH.sub.2 -RISDHHYSKGFRQA-COOH (SEQ ID NO: 9)
NH.sub.2 -VQGEESNDKIPVA-COOH (SEQ ID NO: 10)
NH.sub.2 -ESVDPKNYPKKKMEKRF-COOH (SEQ ID NO: 11)
Preferred antibodies against IL6 possess specific activity against at least
one of the following peptide sequences:
NH.sub.2 -APHRQPLTSSERIDKQI-COOH (SEQ ID NO: 12)
NH.sub.2 -QNRFESSEEQARA-COOH (SEQ ID NO: 13)
NH.sub.2 -AITTPDPTTNAS-COOH (SEQ ID NO: 14)
Preferred antibodies against IL10 possess specific activity against at
least one of the following peptide sequences:
NH.sub.2 -SPGQGTQSENSCT-COOH (SEQ ID NO: 15)
NH.sub.2 -QMKDQLDNLLLKES-COOH (SEQ ID NO: 16)
NH.sub.2 -MPQAENQDPDIKA-COOH (SEQ ID NO: 17)
NH.sub.2 -LPCENKSKAVEQ-COOH (SEQ ID NO: 18)
Preferred antibodies against TNF.alpha. possess specific activity against
at least one of the following peptide sequences:
NH.sub.2 -VRSSSRTPSDKPVA-COOH (SEQ ID NO: 19)
NH.sub.2 -KSPCQRETPEGAEAKPW-COOH (SEQ ID NO: 20)
The immunoadsorber according to the invention manifests membranes or
particles customary per se of organic or synthetic polymers as carrier
materials, e.g. of polystyrenes, carbohydrates such as cellulose or
agarose derivatives, or of acrylates, with the specific antibodies being
covalently linked to them or fixed to them via spacers or linkers.
The production of the immunoadsorbers according to the invention is done by
methods known per se in that the antibodies aimed against C3a and/or C5a
and LPS and, if need be, against further sepsis mediators are coupled
covalently or adsorptively to the carrier materials or organic or
synthetic polymers.
The specific antibodies are produced by immunisation known per se,
preferably of small mammals such as mice, rats or rabbits, or birds, such
as chickens, with the corresponding antigens.
The object of the invention is also the use of the immunoadsorbers in
appliances for the removal of complement factors, LPS and, if need be,
further mediators from body fluids such as blood plasma as a function of
the patient-specific situation.
Preferably, the immunoadsorbers are used in sepsis therapy for
piasmapherese in patients with sepsis or septic shock.
Although antibodies are available for most substances and are coupled to
the various carriers by known methods, avian antibodies are preferably
used, as they do not activate the complement system, unlike mammal
antibodies. As the activating properties are bound to the F.sub.c part of
the mammal antibodies, the F.sub.ab fragment fissured with papain can
principally also be used.
According to the current state of knowledge, immobilised avian antibodies
have no kind of unspecific effects on the human defence system. Birds,
preferably chickens, are immunised with customary methods with or without
the use of adjuvants. The specific immunoglobulins are excreted in the egg
yolk and can be isolated from it with customary methods. They are
covalently bound to micro-particles or membranes via the Fc part with
known methods.
With the immunoadsorption system for extra-corporal detoxification
according to the invention, there exists for the first time a selective
system which can be used patient-specifically and by which dysregulations
of the immune system can be rectified.
The invention is explained in more detail by the following examples:
EXAMPLE 1
Production of polyclonal antibodies by means of immunogenic peptides:
TABLE I
The peptides listed in Table I are produced by means of
a solid phase synthesis
Peptide sequence Antigen
KCCEDGMRQNPMR (SEQ ID NO: 1) C3a
RFSCQRRTRFISL (SEQ ID NO: 2)
ITELRRQHARAS (SEQ ID NO: 3)
QADYKDDDDKLPAE (SEQ ID NO: 4) C5a
DDKLPAEGLDIENS (SEQ ID NO: 5)
SPGQGTQSENSCT (SEQ ID NO: 15) IL10
QMKDQLDNLLLKES (SEQ ID NO: 16)
MPQAENQDPDIKA (SEQ ID NO: 17)
LPCENKSKAVEQ (SEQ ID NO: 18)
NCYSENEEDSSSID (SEQ ID NO: 6) IL1 .alpha.
GAYKSSKDDAKIT (SEQ ID NO: 7)
WETHGTKNYFTS (SEQ ID NO: 8)
RISDHHYSKGFRQA (SEQ ID NO: 9) IL1 .beta.
VQGEESNDKIPVA (SEQ ID NO: 10)
ESVDPKNYPKKKMEKRF (SEQ ID NO: 11)
APHRQPLTSSERIDKQI (SEQ ID NO: 12) IL6
QNRFESSEEQARA (SEQ ID NO: 13)
AITTPDPTTNAS (SEQ ID NO: 14)
VRSSSRTPSDKPVA (SEQ ID NO: 19) TNF.alpha.
KSPCQRETPEGAEAKPW (SEQ ID NO: 20)
These peptides are covalently bound to a carrier (KLH) according to a
standard recipe. The conjugate dissolved in PBS is mixed in equal shares
with Freund's adjuvant. The individual inoculation dose is set in such a
way that it contains 200 pg of the peptide belonging to the antigen in
question. 15-week-old young hens are im-
EXAMPLE 2
Production of Polyclonal Antibodies by Means of Lipopolysaccharides (LPS)
Cleaned LPS (SIGMA) of E. coli J5 are dissolved in PBS and mixed in equal
shares with Fueund's adjuvant. 15-week-old young hens are immunised with
this mixture. The LPS dose amounts to 1 mg of LPS per immunisation.
Boostering is done 4 times at intervals of 4 weeks.
EXAMPLE 3
Obtaining the Antibodies (IgY) from Egg-Yolk:
The eggs from the clutches of the immunised hens are collected. After
separation of the egg-yolk containing antibodies, there is storage at
-20.degree. C. According to requirements, the yolks are thawed and treated
according to the following plan (C. SCHWARZKOPF, B. THIELE (1996) ALTEX 13
Suppl. 16, 35-3):
A TBS: 20 mM Tris/HCl, pH 7.5, 0.5 M NaCl
B 10% (w/v) dextra sulphate in A Solutions
C 1 M CaCl.sub.2
D 0.5 M EDTA, pH 7.5
E saturated ammonium sulphate solution
The egg yolk (corresponds to a volume of 10-20 ml/egg-yolk) is suspended in
100 ml TBS per egg-yolk. Lipids and lipoproteins are precipitated with
dextran sulphate (6 ml B per 100 ml TBS/egg yolk suspension) and Ca.sup.++
(15 ml C per 100 ml TBS-egg yolk suspension), stirred for 30 to 60 min. at
room temperature and centrifuged off at 5,000 g. The pellet is washed with
a small volume of TBS (approx. 20 mlg/egg yolk) and centrifuged again.
The combined supernatants are filtered through a paper filter, then 0.5 M
EDTA is added to the filtrate up to a final concentration of approx. 30 mM
EDTA (6 ml per 100 ml), in order to bind remaining Ca.sup.++ ions. After
this, the supernatant is mixed with 24.3 g of ammonium sulphate per 100 ml
(corresponds to 40% saturation) and incubated at +4.degree. C. for 30
min.. The resultant precipitation (IgY) is firstly washed with 30%
(NH.sub.4).sub.2 SO.sub.4 (30 ml E+70 ml dist. water), centrifuged, then
dissolved in the smallest possible volume of TBS (approx. 10 ml/egg-yolk
used) and dialysed against TBS.
The content of IgY is determined photometrically at 275 nm.
EXAMPLE 4
a) Activation of a Carrier:
The IgY cleaned according to Example 3 are covalently bound to a suitable
carrier. For example, sepharose can be activated as described below for
this purpose (H.- F. Boeden, W. Buttner, C. Rupprich, B. Buttner, S.
Heinrich, M. Becker, M. Holtzhauer (1992) Makromol. Chem. 193, 865-887):
The agarose carrier is gradually transformed, i.e. with an amount of
acetone increasing in steps of 20%. Finally, the carrier is left to stand
in an enclosed container in a quintuple bed volume with water-free acetone
overnight, again washed with 5 to 10 Vol. water-free acetone and briefly
sucked off on a G2 slice. 400 mg
N-(Chlorcarbonyloxy)-5-norbornen-2,3-dicarboximid (CICOONB) in 10 ml
water-free acetone p.a. are added to 10 ml sedimented carrier. Within 15
minutes, a solution of 280 .mu.l triethylamine and 20 mg
4-dimethylamino-pyridine (DMAP) in 5 ml dry acetone is added drop by drop
(mol ratio CICOONB:triethylamine:DMAP 1:1.2:0.1) with shaking. After this,
there is further shaking for 15 minutes, after which the carrier is washed
with about 200 ml water-free acetone.
b) Coupling of the IgY to a Solid Carrier:
The polysaccharide matrix (gel) activated according to Example 4a) is
gradually transformed into a watery medium and then immediately stirred
into the coupling solution containing the ligand. Citrate buffer pH 4.2 is
used as a coupling buffer. The coupling is done with gentle shaking for 2
h at room temperature. Free bindings are subsequently blocked by addition
of ethanolamine. Table 2 shows the concrete conditions for the individual
antibodies.
TABLE 2
Ab ml coupling Ethanol-
solu- buffer amine moist
Gel Chicken- mg/ tion (Citrate, 0.1 1 M gel
No. Ab (lgY) mg ml (ml) M, pH 4.2) (ml) (g)
1 ChaIL1 9.5 13.5 0.7 4.3 0.5 5.55
2 ChaIL6 9.8 9.8 1.0 4.0 0.5 5.58
3 ChaIL10 9.2 7.4 1.2 3.8 0.5 5.55
4 ChaTNF 11.0 11.6 1.0 4.1 0.5 5.56
5 ChaLPS 11.6 13.7 0.9 4.2 0.5 5.60
6 ChaC3a 6.9 10.7 0.6 4.4 0.5 5.57
7 ChaC5a 11.3 11.1 1.0 4.0 0.5 5.55
8 Control 0.0 0.0 0.0 5.0 0.5 5.61
EXAMPLE 5
The antibodies immobilised according to Example 4 are used in order to
remove lipopolysaccharides, interleukins, TNF or complement factors from
liquid media such as buffer solutions, serum or blood plasma.
For this, the carriers are washed, transformed into a physiological buffer
(PBS) and packed in plastic or glass pillars free of air bubbles. The
arrangement is completed by connection to a chromatography appliance. The
sample material to be adsorbed (buffer doted with the antigens, serum or
blood plasma samples, doted or with natural antigen content) can now be
guided by gravity or with a suitable pump via the immobilised antibodies
specific for the antigens stated. The existing antigens are recognised,
firmly bound and thus removed from the medium flowing through the column
by the IgY. The detection of the effectivity is done by analysis (ELISA)
of the column throughflow, the antigen content of which is reduced. After
washing of the column with a physiological buffer, there is desorption of
the bound antigen with suitable elution agents (0.1 M citrate buffer pH
3), fractioning and analysis of the eluate. Quantitative detection of the
antigens is used to determine the capacity of the immunosorbent.
SEQUENCE LISTING
<100> GENERAL INFORMATION:
<160> NUMBER OF SEQ ID NOS: 20
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 1
<211> LENGTH: 13
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence designed
synthetical peptide of the complement factor C3a
<400> SEQUENCE: 1
Lys Cys Cys Glu Asp Gly Met Arg Gln Asn Pro Met Arg
1 5 10
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 2
<211> LENGTH: 13
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence designed
synthetical peptide of the complement factor C3a
<400> SEQUENCE: 2
Arg Phe Ser Cys Gln Arg Arg Thr Arg Phe Ile Ser Leu
1 5 10
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 3
<211> LENGTH: 12
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence designed
synthetical peptide of the complement factor C3a
<400> SEQUENCE: 3
Ile Thr Glu Leu Arg Arg Gln His Ala Arg Ala Ser
1 5 10
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 4
<211> LENGTH: 14
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence designed
synthetical peptide of the complement factor C5a
<400> SEQUENCE: 4
Gln Ala Asp Tyr Lys Asp Asp Asp Asp Lys Leu Pro Ala Glu
1 5 10
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 5
<211> LENGTH: 14
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence designed
synthetical peptide of complement factor C5a
<400> SEQUENCE: 5
Asp Asp Lys Leu Pro Ala Glu Gly Leu Asp Ile Glu Asn Ser
1 5 10
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 6
<211> LENGTH: 14
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence designed
synthetical peptide of the interleukin 1 alpha
<400> SEQUENCE: 6
Asn Cys Tyr Ser Glu Asn Glu Glu Asp Ser Ser Ser Ile Asp
1 5 10
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 7
<211> LENGTH: 13
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence designed
synthetical peptide of the interleukin 1alpha
<400> SEQUENCE: 7
Gly Ala Tyr Lys Ser Ser Lys Asp Asp Ala Lys Ile Thr
1 5 10
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 8
<211> LENGTH: 12
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence designed
synthetical peptide of the inerleukin 1 alpha
<400> SEQUENCE: 8
Trp Glu Thr His Gly Thr Lys Asn Tyr Phe Thr Ser
1 5 10
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 9
<211> LENGTH: 14
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence designed
synthetical peptide of the interleukin 1beta
<400> SEQUENCE: 9
Arg Ile Ser Asp His His Tyr Ser Lys Gly Phe Arg Gln Ala
1 5 10
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 10
<211> LENGTH: 13
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence designed
synthetical peptide of interleukin 1beta
<400> SEQUENCE: 10
Val Gln Gly Glu Glu Ser Asn Asp Lys Ile Pro Val Ala
1 5 10
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 11
<211> LENGTH: 17
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence designed
synthetical peptide of interleukin 1beta
<400> SEQUENCE: 11
Glu Ser Val Asp Pro Lys Asn Tyr Pro Lys Lys Lys Met Glu Lys Arg
1 5 10 15
Phe
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 12
<211> LENGTH: 17
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence designed
synthetical peptide of interleukin 6
<400> SEQUENCE: 12
Ala Pro His Arg Gln Pro Leu Thr Ser Ser Glu Arg Ile Asp Lys Gln
1 5 10 15
Ile
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 13
<211> LENGTH: 13
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence designed
synthetical peptide of interleukin 6
<400> SEQUENCE: 13
Gln Asn Arg Phe Glu Ser Ser Glu Glu Gln Ala Arg Ala
1 5 10
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 14
<211> LENGTH: 12
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence designed
synthetical peptide of interleukin 6
<400> SEQUENCE: 14
Ala Ile Thr Thr Pro Asp Pro Thr Thr Asn Ala Ser
1 5 10
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 15
<211> LENGTH: 13
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence designed
synthetical peptide of interleukin 10
<400> SEQUENCE: 15
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr
1 5 10
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 16
<211> LENGTH: 14
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence designed
synthetical peptide of interleukin 10
<400> SEQUENCE: 16
Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser
1 5 10
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 17
<211> LENGTH: 13
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence designed
synthetical peptide of interleukin 10
<400> SEQUENCE: 17
Met Pro Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala
1 5 10
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 18
<211> LENGTH: 12
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence designed
synthetical peptide of interleukin 10
<400> SEQUENCE: 18
Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln
1 5 10
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 19
<211> LENGTH: 14
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence designed
synthetical peptide of TNF alpha
<400> SEQUENCE: 19
Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala
1 5 10
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 20
<211> LENGTH: 17
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence designed
synthetical peptide of TNF alpha
<400> SEQUENCE: 20
Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro
1 5 10 15
Trp
*
