Note: Descriptions are shown in the official language in which they were submitted.
WO94/10191 2 14 7 7 4 1 PCT/DK93/00344
Title: LOW ALLERGENIC PROTEINS
FIELD OF THE I-NV ~:N~1~1ON
This invention relates to low allergenic proteins and methods
for changing proteins, especially industrially produced
proteins including enzymes, physically or chemically to become
less allergenic than their precursors. Furthermore the inven-
lO tion relates to compositions containing such proteins, and theuse of the low allergenic proteins or the compositions in
various fields including household, industry food/feed and
medicine.
BACKGROUND OF THE lN V ~:N-'l'lON
An increasing number of proteins are being produced indus-
trially, for use in industry, household, food/feed or medicine.
20 Being proteins they are capable of stimulating the immune
systems of animals including humans.
Depending on the way of presentation proteins can elicit the
production of different kinds of antibodies and/or a cellular
25 response. At least one of these routes can give adverse effects
in animals including humans.
The production of IgE (in humans, and molecules with comparable
effects in animals) can lead to an allergic state, giving
30 symptoms like rhinitis, conjunctivitis or other.
Generally, most animals including humans are not exposed to
these proteins to a degree that will generate adverse effects,
but certain risk groups exist for which these phenomena are of
35 significant importance.
The risk groups can be personnel in industrial production or
WO 94/10191 21 4 7 7 4 ~ PCT/DK93/00~
research departments etc.
The increased use of such protein products could, however, lead
to persons using the proteins as consumers or otherwise being
5 affected. Furthermore, the effect of these proteins after their
use and disposal on the environment and the animals including
humans living there is expected to increase.
With industrial proteins present methods to avoid problems in
10 connection with allergic reactions, generally consist of
various ways of immobilizing, granulating, coating or dissolv-
ing the proteins, to avoid especially protein in dust form from
stimulating the immune system.
15 There will anyhow still be a risk of having protein dust or
dissolved protein in aerosol form. Therefore some release of
protein can occur leading to a possible allergic reaction in
those sensitive to such exposure.
20 Another way of diminishing the problem has been to select
proteins of human origin for production, f.ex. in bacteria.
This may alleviate some problems for humans, but not for
animals. Furthermore, it will in many cases not be possible to
find proteins with the desired properties of human origin,
25 wherefore other origin has to be considered. This can be either
human proteins that are altered in one or more positions in the
molecule, giving performance that is desired. Or it might be
molecules from other species, including bacteria, mold etc. All
the latter groups of products will have potency for immune
30 stimulation.
A further proposition for decreasing allergenicity has been to
reduce the size of the protein molecules (see e.g. JP Patent
Publication No. 4112753, or Research Disclosure No. 335102).
35 This is, however, a solution that is only available when the
activity of the protein is without importance, or in such rare
cases, where the activity of the protein in question is
WO94/10l91 2 I 4 7 7 4 I PCT/DK93/00344
retained in spite of a breakdown of the protein.
Recently the use of protein engineering has been suggested to
reduce the allergenicity of proteins through epitope mapping
5 and subsequent change of the allergenic epitopes (see Interna-
tional Patent Publication NO. WO 92/10755). This procedure,
however, usually requires a large investment in work and
development.
10 In the medicinal field suggestions have been made of diminish-
ing the antigenicity or allergenicity of proteins through the
attachment of one or more polymer molecules to the protein.
This usually has the effect of interfering with the interac-
tions of the protein with other macromolecular structures.-
Such a conjugate may also exhibit novel properties: e.g. EPPatent No. 38 154 discloses conjugates of allergens with
polysarcosine which have immunosuppressive properties.
20 It has been found that the attachment of proteins to polymers
in general has the effect of lowering the activity of the
protein or interfering with the interaction between the protein
and its substrate.
25 EP Patent No. 183 503 discloses a development of the above
concept by providing conjugates comprising pharmaceutically
useful proteins linked to at least one water-soluble polymer by
means of a reversible linking group.
SUMMARY OF THE I-NV~N'1'10N
The primary object of the invention is to provide novel low
allergenic proteins, especially low allergenic proteins
35 comprising an oligomeric form of the parent monomeric protein
which oligomer has substantially retained its activity.
W094/10191 214 7 7 41 PCT/DK93/Oa
The invention furthermore relates to methods for changing
proteins, especially industrially produced proteins including
enzymes, physically or chemically to become less allergenic
than their precursors. The change is brought about through a
5 change of the effective size of the protein to a size above the
possible size for penetration of the membrane barrier of the
wet epithelias, while substantially retaining the activity of
the protein for its desired use.
lO According to the invention the change in size is effected
through various methods that are more or less known per se in
order to obtain oligomeric forms of the protein in question,
whereafter the oligomeric proteins are tested for their desired
activity. If this is satisfactory the proteins are subsequently
15 tested for their allergenicity.
The invention in this aspect also relates to the selection of
methods for increasing the size of proteins, without disabling
- the later use of these proteins. This is achieved either by
20 leaving the activity/functionality intact during the binding/-
linking of the protein(s), or through the protein in question
resuming the original activity/functionality after a reversion
to its monomeric form, e.g. by splitting off the binding or
linking molecule(s).
According to one special aspect of the invention the increase
in size is obtained through the insertion of a DNA sequence
coding for a protein comprising a multiplicity of the "orig-
inal" protein, e.g. a di-, tri-, or tetramer of said protein
30 into a suitable host, and subsequent expression of this low
allergenic polymeric form of the protein. Alternatively a DNA
sequence can comprise a multitude of codings for different
enzymes, arranged in one combined sequence giving one combined
protein. This DNA sequence is inserted into a suitable host,
35 with subsequent expression of this low allergenic polymeric
form of the combined protein.
WO94/10191 21 4 7 7 41 PCT/DK93/00344
The invention furthermore relates to the production and
commercialization of such modified proteins. Of special
interest is here industrial enzymes, food/feed proteins,
proteins for use in householding and medicine.
A third aspect of the invention relates to compositions
comprising the low allergenic proteins of the invention. Such
as food/feed compositions, detergent compositions, or composi-
tions used in therapy and/or diagnosis in the human or animal
10 body.
A fourth aspect of the invention relates to the use of above
mentioned compositions.
BRIEF DESCRIPTION OF THE DRAWING
The invention is described in more detail in the following
parts of the specification with reference to the Examples and
20 the Figures, where
Figures 1 to 5, show proteins according to the invention where
the size of protein is increased through the methods of the
invention.
In Fig.1. two identical proteins are being reversibly bound
through a "linker" protein or peptide, Whereby the total size
of the combined molecule eyce~c the limit for penetration of
the membrane.
Fig.2. shows the reversible linkage of three identical pro-
teins.
Fig.3. shows the irreversible linkage of three identical
35 proteins.
Fig.4. shows a number of monomers, identical to the original
WO 94/10191 2 ~ 4 ~ ~ 4 1 PCT/DK93/00
molecule, linked together via a spacer consisting of a number
of amino acids.
The linking amino acids will be breakable or degradable, f.ex.
using enzymatic digestion specifically reacting with this
5 sequence of amino acids.
Fig.5. shows a structure where the protein and the linking
molecule are co-produced and react with each other to form
larger molecular units.
DET~TnT~n DESCRIPTION OF THE I~v~NllON
The invention relates generally to the allergic potential of
15 proteins.
Proteins introduced to the wet epithelia will if they penetrate
the membrane barrier pose the risk of stimulating the immune
system. The stimulation of immunocompetent cells in these
20 regions will often lead to production of IgE in humans, and
antibodies with comparable action in animals. If IgE is being
produced there is a risk of developing symptoms of allergy. On
the other hand, the same molecules introduced subcutaneously or
intraperitoneal will normally stimulate production of IgM and
25 IgG. Therefore the way of presentation of the protein is of
importance for the risk of developing symptoms of allergy.
Essential criteria for a protein stimulating the immune system
are:
A) it is presented to the immunocompetent cells in the body,
B) it is foreign to the body,
C) it is of a size that allows cell-cell cooperation in the
immune system leading to a reaction.
All three criteria will in many cases be fulfilled with
proteins being produced by conventional present day production
WO94/10191 21 ~ 7 7 ~ 1 PCT/DK93/00344
methods.
For many industrially produced proteins the criteria (A) and
(B) cannot be eliminated and the present invention therefore
5 focus on the size of the molecule. The presentation of f.ex.
dust form protein will be through the wet epithelia of espe-
cially the nose, but also pharynx, larynx, lungs and the
gastro-intestinal system.
10 According to the invention protein molecules are increased in
size so as to disable the penetration of the membrane barrier
of the wet epithelia.
The size increase can basicly be obtained in five different
15 ways:
1) By combining the protein with a reversibly binding protein
or peptide, e.g. an enzyme inhibitor or an antibody or
parts hereof, that together with the protein has a size too
large to penetrate the membrane barrier. This can be
achieved either by simple combination of one protein
molecule with a protein/peptide binding molecule, or using
protein/peptide binding molecules with 2 or more binding
sites for protein molecules. In either way the total size
will have to exceed the size for penetration in order to
qualify for being a low allergenic protein of the inven-
tion. In later use of the protein the protein/peptide
binding molecule is split off. The object protein persists
or resumes its original activity/functionality. See Fig.
1 where two identical object proteins are being reversibly
bound by another "binding" protein or peptide. Hereby the
total size of the combined molecule is exceeding the limit
for penetration of the membrane. Any other combination of
object protein and binding protein/peptide can be used. The
important parameter, besides the size, is the reversibility
of the binding and the persistent or resumed activity/-
functionality of the object protein.
WO94/10191 214 7 7 41 PCT/DK93/On
2) By binding a reversible di- oligo- or multimerization
linker molecule to the protein molecules. The proteins will
therefore still be functioning as proteins with or without
the linker split off, but their size will exceed the
penetration possible size. See Fig. 2 where a reversible
linkage of three identical object pr-~oteins has been made.
The size of the linker can or can-not be of importance.
There can be any number from dl- to multimerization of
proteins to the linker. The important parameter again,
besides the size, is the reversibility of the linking and
the persistent or resumed activity/functionality of the
object protein.
3) By binding a small molecule/ligand to the protein, this mo-
lecule/ligand being recognizable by a di- oligo or multi-
merization linker molecule. The linking molecule will bind
such molecule/ligand and hereby link two or more molecules
together reversible or irreversible.
20 4) By binding a non-reversible di- oligo- or multimerization
linker molecule to the protein molecules. The proteins will
still be functioning in the linked conformation, but will
be unable to penetrate the wet epitheliae. See Fig. 3,
where an irreversible linkage of three identical object
proteins has been made. The size of the linker can or
cannot be of importance. There can again be any number of
proteins on the linker from di- to multimers. The important
parameter, besides the size, is the irreversible linking
and the persistent activity/functionality of the linked
object proteins. The oligomerization can be by chemical,
biochemical or enzymatic means.
5) By changing the genomic material to include more than one
coding DNA sequence for the protein/peptide. This will
preferably be within the one and same initiation and
termination regulatory signals in the replicable entity.
In between the single coding regions for the molecule can
WO94/10191 214 7 7 4 1 PCT/DK93/003~
be non-informative basepairs, basepairs coding for extra
amino acids, or the coding regions can be consecutive.
The optional extra amino acids may act as a linker or
spacer between two copies of the original molecule. The
extra amino acids will preferably not interfere with the
3-dimensional structure or the activity of the original
molecule incorporated. Flexible, uncharged amino acids like
Glycine, Alanine or Serine are preferred. The multiple
1o copies are therefore at the mRNA or at the protein level
joined to make one molecule with increased size.
The larger molecule can or cannot be breakable into smaller
subunits, corresponding to the molecule of origin. This
lS will enable the same or nearly same activity as the
molecule of origin. In Fig. 4 the eventual molecule
corresponds to a number of monomers, identical to the
original molecule, linked together via a spacer consisting
- of a number of amino acids. These new linking amino acids
are preferably small uncharged amino acids, and will give
small or no change the activity and the 3 dimensional
structure of the monomers. The change is in the genome, and
hereby the number of monomers in the new molecule is
determined strictly to the desired number.
The linking amino acids will be breakable or degradable,
f.ex. using enzymatic digestion specifically reacting with
this seguence of amino acids.
The activity of the new oligo- or multimere is identical
or comparable to the original monomer. After a possible
splitting of the oligo- or multimere, to become monomer
with or without "tails" of degraded linker amino acids, the
activity is identical or comparable to the original
monomer.
6) By including one or more extra molecules in the replicable
WO94/10191 21 ~ 7 7 ~ ~ PCT/DK93/00
organs of the host organism in which the object molecule
is produced. This change of host will eventually mean
production of the object molecule together with another
molecule, that preferably will bind to the object molecule,
reversibly or irreversibly. Therefore the product both
before and after purification will-be larger than the
original molecule. In Fig. 5 the organism producing the
original molecule is changed to become producer of another
molecule too. The change therefore is in the genome of the
organism. The co-produced molecules react with each other
and form larger molecule units. Any numbers can be used in
combination.
The important parameter besides the molecular size, is the
reversibility of the bonding, and the sustained or resumed
activity of the original molecule linked or unlinked to the
added molecule.
7) The size increase can include any of the above mentioned
methods (l) to (5) alone or in combination, and can involve
more than one type of protein being bound/linked, f.ex. two
different enzyme molecules being linked to one linker.
The methods for binding proteins in (l) can for example be
25 using antibodies of parts hereof, or using biotinylation of the
protein and bridgeformation with the quadrovalent avidin or
streptavidin, or any other conventional reversible binding, or
in the case of enzymes an inhibitor to the enzyme(s).
30 The methods for linking proteins in (2) and (3) can in example
be using methods like the ones described in :"Ultrogel,
Magnogel and Trisacryl. Practical guide for use in affinity
chromatography and related techniques. (1983) LKB+IBF. Reactifs
IBF- Societé Chimique Pointet-Girard. 35, avenue Jean-Jaures,
35 92390 VILLENE W E-LA-GARENNE, FRANCE.
The reversible bonds and links will be breakable through one of
WO94/10191 21 4 7 7 41 PCT/DK93/00344
11
the following procedures:
a) change of pH,
b) change of temperature,
c) change of ionic strength,
5 d) change of molarity, e/di-lution,
f) addition of competitor for binder/linker,
g) degradation of binder/linker molecule, or
h) any combination of these.
10 Test of sustained activitY of proteins or pePtides
The molecules from any of the procedures (1) through (6) above
is tested for activity.
In the procedures giving irreversible binding of the original
15 molecule, or where multiple copies of the molecule is repre-
sented in the genome giving molecule oligo- or multimeres, the
activity must be sustained directly as such in the larger
molecule.
20 In the procedures giving reversible or breakable binding or
linking of original molecules, the activity of the molecules
must be a/sustained in the larger molecule, or, b/resumed after
splitting the larger molecule into molecules identical or
comparable to the molecule of origin.
In the above mentioned pr~cedures (3), (4) and (6) the activity
is measured with the larger molecule, whereas molec~les from
procedures (1), (2), (5) and (6) the activity is measured with
both the larger molecules, and with the monomers after revers-
30 ing or breaking the bonds/links.
Test of reduced immunologic/allergenic potential
The molecules are after change tested for their immunologic/allergenic potential, and selected according to reduction
35 hereof. The new molecule(s), together with molecule of origin
and control material is presented to test animals through one
or more of the following ways : a/intra dermal b/sub cutaneous,
W094/lolgl 21 4 7 7 41 PCT/DK93/00-
12
c/intra venous, d/intra nasal(inhalation), e/per oral, f/intra
tracheal or g/intra peritoneal.
The allergenic potential will be measurable with preferably d/
5 and f/ or partwise e/, whereas the immunologic potential will
be measurable in all techniques.
For means of dosing the test material, please refer to standard
protocols and for f/ to examples later in this text.
10 The response of the animals is measured, and the responses
compared to evaluate the effect of change of molecule size. The
responses can be measured as : differential count of blood/-
lymphoid cells, lymphocyte stimulatory index, specific anti-
bodies in animal sera (the quality and quantity), or any other
15 means of measuring the immunologic status of the stimulated
animal.
The molecules are bound either to another protein or peptide or
to each other through a linker. After this bonding or linking
20 the new molecule will have a size, that impedes or stops any
penetration over the membrane barrier in the wet epithelia.
The molecules are monitored for immunologic stimulatory
potentials with and without the change on the wet epithelia and
25 as control by injection. The optimal change(s) is selected for
use and implemented in either production or processing of the
proteins. The final product is again tested for immunologic
potential.
30 Methods for monitoring and testing the potentials are immuno-
logical and proteochemical, both in vivo and in vitro methods
can be used.
The changes selected for size increase of proteins, can be
35 implemented either in the production step of the proteins, or
in any later processing step of these proteins. It can there-
fore be implemented on the genomic level of the production
2147741
WO94/10191 PCT/DK93/00344
13
organism, included in the food/medium for growing these
organisms, or used to change proteins post-production, before
or after a possible purification of the protein(s).
EXAMPLES
EXAMPLE l
lO In this example the irreversible coupling of proteins to each
other is discussed. The coupling will preferably not change the
molecular activity.
The means of coupling is through the use of bivalent linker
15 molecules i.e. homo- or heterobifunctional reagents. For
exhaustive information on bifunctional reagents see among
others ref.s l & 2.
Any one of these coupling methods can be used. The selection of
20 which one to choose depends on the chemical composition and
stability of the molecule to be coupled.
Method
Glutaric acid is reacted with EEDQ (= N-ethoxycarbonyl-2-
25 ethoxy-1,2,dihydroquinoline) (ref l). A mixed anhydride is
formed, that in its turn will react with a primary amino group
in a peptide or protein. Through a condensation reaction the
glutaric acid is coupled to the peptide or protein.
Hereby glutaric acid acts as a bridging molecule linking two
30 f.ex. peptides.
The reactions are illustrated in the following figs, taken from
ref l (where the discussion in on protein binding g~s).
WO94/10191 21 4 7 7 ~ l PCT/DK93/003
14
-OHt ~O-C,ll, ~C-O-`lCl-
C--O--C,h.
C~OamC O
~CER GEL EED~ ~XEO Al . I 't
~c_~ -E~
I~I~D ~ r+ ~ 3 P ' -~ DF~l L~
l. In the first step glutaric acid is reacted with EEDQ in
excess in ethanol. After one hour of reaction formic or
acetic acid is added (equimolar to EEDQ) to stop the
reaction. The mixed anhydride is now formed, and further
reaction with excess EEDQ is stopped.
.
2. The peptide or protein is dissolved in a waterphase or
mixed water/ethanol solution. The peptide or protein is
added to the mixed anhydride. The peptide or protein must
be in molar excess compared to the initial glutaric acid.
The reaction is incubated at least one hour, preferably
over night with or without agitation.
In this reaction f.ex. two proteins are being linked
together via glutaric acid.
3. The product of step 2 is purified using size or other
chromatography. The correct fraction is selected according
to molecular size.
If necessary the procedure is run over with the purified
product of step 3 as the ligand, in replace of peptide or
WO94/10191 21 ~ 7 7 ~ 1 PCT/DK93/00344
protein as listed there.
In one or more runs of the procedure, the molecular weight
is increased to exceed the limit for being penetrable over
the wet epithelias.
EXAMPLE 2
lO The theory behind this is identical to example l.
Method
A dicarboxylic acid like glutaric, ketoglutaric, sebacic or
like acid is used for linking peptides or proteins. In the
15 following sebacic acid is be used.
A carbodiimide is reacted with sebacic acid, whereupon an
isourea ester is formed. Residual carbodiimide is exhausted
with a monocarboxylic acid like formic or acetic acid. The
20 peptide or protein is introduced, and through a condensation
reaction this is bound to the former sebacic acid in a chemical
bond.
Hereby sebacic acid is linking two f.ex. proteins together
25 chemically.
The reactions are illustrated in the following figs (see ref l)
~ C~ ~ R~
C;AI~X`~L~ R^S~Uf3~F~
SPAC~6~L ~n~ L ~S~UP~E~ ~ST~R
WO94/10191 214 7 7 41 PCT/DK93/00
16
2 .
~so~n~C~ E5~ N ~ C--NH - . Ll~iAND
la h ~ o LIGAN~
l. Sebacic acid is diluted in water to the desired molarity,
pH is adjusted to slightly acidic. EDCI (= l-ethyl-3-(3-
dimethylaminopropyl)-carbodiimide hydrochloride, or, CMCI
(= l-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide-metro-p-
toluene-sulphonate is added, either in excess. The isourea
ester is now formed.
Leave to react for one hour. Formic or acetic acid is added
to exhaust residual carbodiimide.
15 2. The peptide or protein to be coupled is added. This reacts
with the isourea ester through a condensation reaction
where peptide or protein is chemically bound to the former
sebacic acid. As sebacic acid is bifunctional it will link
to two peptides or proteins acting as a bridge between
these.
3. The product of step 2 is purified using size or other
chromatography. The correct fraction according to molecular
size is selected.
If necessary the procedure is run over, with the purified
product of step 3 as the ligand, as replace for peptide or
protein mentioned there.
In one or more runs of the procedure, the molecular weight
is increased to exceed the limit for being penetrable over
the wet epithelias.
W O 94/10191 :- 214 7 7 4 1 PC~r/D K93/00344
17
EXl~PLE 3
In this example the irreversible linking of another, small
molecule to be object molecule is used as a preactivation step.
5 Hereafter the preactivated molecule is linked using a di-
oligo- or multimerization linker molecule that binds reversible
or irreversible.
The activity of the object molecule is retained during the
10 manipulation or, resumed after monomerization of the object
molecule by splitting off the linker.
Method
The object molecule is being biotin labelled using Biotin N-
15 HydroxySuccinimide (BNHS) reagent. Hereby the molecule is addedthe small biotin molecule (mw 241 D) in low incorporation
quantity, preferably ratio 1 biotin to 1 object molecule.
The residual unused BNHS reagent is extinguished using an amino
20 containing molecule like Glycin, and the free biotin molecules
are removed.
Next step is addition of the linking Streptavidin or Avidin,
that will be able to bind the biotin labelled object molecule
25 in reversible bonds, although with very high binding strength.
1. The object molecule containing free amino groups is reacted
with 8NHS reagent at neutral to alcaline pH. The molecular
ratios must be more than 1:1, preferably more than 1:15
during the reaction, to give an incorporation of 1:1
eventually.
2. A solution of Glycin is added to the reaction mixture of
point 1 above. The molecular ratios BNHS to Glycin must
3 5 exceed 1:1, preferably 1:8.
3. Dialyse or gelfiler the product of point 2 above to remove
W094~101~ 214~ PCT/DK93/00
18
free uncoupled Biotin from the reaction mixture.
4. Aliqout the reaction mixture of point 3 above, add to a few
of these linking Strptavidin or Avidin giving mixing ratios
of linker to object molecule of 1:100, 1:40, etc. Find the
best ratio using sized exclusion chromatography or electro-
phoresis or other mw determining procedure. Use mixing
ratios will possibly give linked products of ratios linker
to object molecule of 1:1,-i:2, 1:3 and 1:4, wherefore a
spectrum of molecular sizes can be obtained.
5. Purify the eventual product of point 4 above by e.g. size
exclusion chromatography, verify the mw by e.g. electropho-
resls.
6. The correct size is selected according to molecular size.
Further examples, usinq chemical cou~linq
Homo- or heterobifunctional reagents is used together with
20 f.ex. dicarboxylic- or diamino containing compounds acting as
linkers.
The bifunctional reagents will be any one listed in the
references or any other.
The linkers will be any compound capable of being reacted upon
by the bifunctional reagents. Examples are putrescine, spermi-
dine or spermidine (all diamino compounds), or, glutaric,
ketoglutaric (alpha/beta) or spermine (all dicarboxylic acids),
30 or any other compound with thiol- hydroxyl- carboxyl- or amino
groups combined in one linker, or with two identical groups in
the same linker.
Exam~les usinq breakable linkages
35 Any one of the above listed methods, using a linker with
scissile bond, or, a linker that is breakable. All linkages
will preferably not change the activity of the peptide or
21~7741O94/10191 PCT/DK93/00344
19
protein.
A scissile bond is broken by changing the dilution(i.e. the
molarity), temperature, ionic strength, pH or other.
A breakable linker is a linker that itself breaks apart by
changing the temperature, ionic strength, pH or other.
The break can also be induced by enzymes capable of digesting
10 or splitting the linker, preferably without altering the
peptide or protein coupled to the linker.
Furthermore the linkage can be obtained using another peptide/-
glycopeptide or protein/glycoprotein. This molecule will be
15 active like f.ex. an antibody or an enzyme inhibitor, preferab-
ly without having any immunological potential of its own.
The bond to the active molecule can be broken by any of the
above mentioned means.
W094/10191 21 ~ PCT/DK93/00:
REFERENCES
1. Ultrogel, Magnogel and Trisacryl. Practical guide for use
in affinity chromatography and related techniques. 2nd
Edition. Réactifs IBF - Sociéte Chimique Pointet-Girard.
35, Avenue Jean-Jaurés. 92390 Villeneuve-La-Garenne. France
(book).
2. Tae,H.Ji (1983) Bifunctional Reagents. Methods in
Enzymology. Vol. 91. p.580-605 (article).
10 3. Hudson, L. and Hay,F.C. (1991). Third edition. Practical
Immunology. Blackwell Scientific Publications (book).
4. Henderson,R.F.(1988). Use of Bronchoalveolar Lavage to
Detect Lung Damage. in : Toxicology of the Lungs. Eds.
Gardner, D.E.; Crapo,J.D. and Massaro, E.J.. Raven Press
N.Y. (book).
