Note: Descriptions are shown in the official language in which they were submitted.
- 3~2~3~
10B145-378
ANTIBODIES AGAINST BACTERIAL PEPTIDOGLYCANS
This invention relates to antibodies against
bacterial peptidoglycans and to processes for their
quantitative determination.
As described in DE-A-32 31 204 ~eqUiVa1ent
to Canadian Patent Application 434968) filed Aug. 19,1983,
antibodies against the peptide sub-unit pentapeptide
L-Ala-D-Glu(L-Lys~D-Ala-D-Ala) of peptidoglycan
are indicators Eor acute or chronic bacterial infections.
It has been found that, in addition to the
specific antibodies of the immunoglobulin class
IgG which can be detected in the later stage of
an infection, specific antibodies of the immunoglobulin
classes IgAI IgM and IgE also occur during infection.
Thus, antibodies of the class Ig~ are found
in the initial stages of an infection and in some
cases even before any clinical symptoms occur.
Antibodies of class IgA can be detected in infections
which initially manifest themselves essentially
in the mucous membranes. Antibodies of class IgE
are found in diseases which involve allergic reactions.
According to one aspect, the present invention
thus provides antibodies against the peptide sub-
unit pentapeptide L-Ala-D-Glu(L-Lys-D-Ala-D-Ala)
of peptidoglycans, characterized in that said antibodies
are from the class of immmunoglobulins A, M or
E. The antibodies of the invention are preferably
isolated from other immunoglobulins and/or are
in sterile solution and are suitably those characteri2ed
by
a) special binding to pentapeptides of formula
I
R - Al - D-Ala (I)
(wherein
,
, .
3~
R represents an amino acid component with 1, 2
or 3, preferably 3, amino acid residues selected
from the group comprising Gly, L-Ala~ D-Glu
and L-Lys; and
Al represents one of the amino acid residues D-Ala,
D-Ser, D-Val or D-Leu); and/or
b) the ability to bind to bacterial cell walls
or cell wa~l material which contain pentapeptides
of formula I; and/or
c) the ability to bind to Gram-positive bacteria
which contain pentapeptides of for~ula I.
The antibodies of the invention, which can
be used as standards for or as positive controls
in the quantitative de~ermination of antibodies
of the same class, can be extracted from plasma
e.g. by affinity chromatograph~- as described hereinafter.
According to a further aspect, the invention
provides a process for the quantitative determina~ion
o~ antibodies from the class of immunoglobulins
A, M and E against the peptide sub-unit pentapeptide
L-~la-D-Glu(L-Lys-D-Ala~D-Ala) of peptidoglycans
in a biological test material which process comprises
contacting said test material with an antigen of
formula I which is optionally labelled and optionally
substrate bound. The process of the invention
is conveniently performed according to immunochemical
procedures known to the art, e.g. the double antibody
ra~ioimmunoassay, the double antibody enzyme-immunoassay,
the nephelometric method t the solid-phase sandwich
radioimmunoassay, the solid-phase enzyme immunoassay,
the solid~phase fluorescence immunoassay and the
latex agglutination test.
Thus ~or example a general ~est scheme might
involve: (a) coupling a pentapeptide of formula
I bonded to a carrier polymer (e.g. albumin) to
a solid phase (such as polystyrene~; (b~ contacting
the solid phase with the test sample (e.g~ patient's
3~
serum) or a standard; (c) washing to remove unbound
immunoglobulins; (d) contacting the solid phase
on which the immunoglobulin is bound with corres-
ponding anti-antibodies (anti-IgA, an~i-IgM or
anti-IgE) which are coupled to an indicator system;
and (e) washing again to remove unreacted anti-
antibodies. The indicator moieties can then be
measured on the solid carrierO The tests for determining
the differen~ types of immunoglobulin require different
conditions which are described in detail in the
Examples.
According to a further aspect, the invention
provides a method of extracting the antibodies
of the invention from human plasma which method
comprises: (a) coupling a pentapeptide of formula
I to a solid phase; (b) contacting said solid phase
with IgA, Ig~ or XgE containing human plasma whereby
said antibodies selectively bind to said pentapeptide
coupled to said solid phase; and (c) subsequently
selectively eluting said antibodies from said solid
phase.
Standardisation of the test
.
l. Obtainina a standard from s~ecific IaM and
IgM and IgA are isolated by conventional
methods (e.g. FPLC - ~ast Performance Liquid Cbromato-
graphy) from sera which contain antibodies with
specificity against the peptide sub-unit pentapeptide
of formula I and the specific antibody fraction
is obtained by affinity chromatography (with matrix-
bonded peptides of formula I). After the specific
antibodies have bound to the affinity matrix, any
non-bound serum protein is removed by washing with
buffer solutions. The elution of the antibodies
bound to the soIid matrix is effected by specific
desorption of the bound immunoglobulins with peptides
..~.,
of formula I. Desorption may also be effected
by methods which have been published in principle,
such as the use of low pH values (such as for example
l-molar acetic acid) or high salt concentrations
(such as for example 6-molar guanidinium chloride,
8-molar urea or 3-molar NaCl). After dialysis
with buffer solutions, the content of specific
antibodies against the peptide sub-unit pentapeptide
o~ peptidoglycan is quan~ified by means of the
immunoglobulin content of the purified fraction.
-
2. Standardlsation of the determination of specific
IgE
This is done by using a pooled serum from
test subjects with a high specific IgE and subsequently
comparing the sample measured with a standard curve
(corresponding dilution).
3. Design of test
Quantitative determination of specific antibodies
of the above immunoglobul}n classes against the
peptide sub-unit pentapeptide of peptidoglycans
can be effected by two fundamentally different
methods:
a) quantitative determination in a soluble system
tmeasurement in a homogeneous phase); and
b) quantitative determination by measurement in
a heterogeneous system (measurement in a heterogeneous
phase; solid-phase immunoassay).
3.1. Measurement in the homo~en ous phase
3.1.1. Quantitative determination of antibodies
against the peptide sub-unit pentapeptide of peptido-
glycans in a double antibody radioimmunoassay.
Peptides of formula I are radioactively labelled
and ~he test mixture is prepared according to the
following scheme:
~0
*Pept. + Ab ~ ~~ *Pept.-Ab
(wherein *Pept. represents the labelled peptide
and Ab represents an antibody thereto.)
The separation of the radioactive free antigen
from the radioactive antibody-bound antigen is
effected by the double antibody method by specific
precipitation of the immunoglobulins using a second
anti-antibody according to the following scheme:
*Pept.-Ab ~ Anti-Ab -~ *Pept.-Ab m~(Anti-Ab)m-
complexes
(wherein Anti-Ab represents the anti-antibody).
Radioactive labelling of the antigen may
be efected, for example:
a) by coupling tyrosine to the N-terminal end of
the peptide and subsequentl-y iodinating with 125I
by conventional methods
b) by iodinating with ~N-succinimidyl-3-(4-hydroxy-
5-(125I)iodophenyl)propionate
c) by tritiation of the antigen with N-succinimidyl
(2,3~ H)propionate.
Xt is advisable to purify the anti-antibodies
used by affinity chromatography on matrix-bonded
peptides ~f ~ormula I before use in order to remove
any antibodies against peptidoglycans which they
may contain, or preferably to use monoclonal anti-
antibodies.
3.1.2. Quantitative determination of antibodies
against the peptide sub-unit pentapeptide in a
double antibody enzyme-immunoassay.
The test procedure o~ ~.1.1. above is modified
, ~,
~ ~3~
by effecting the labelling of the anti-antibodies
by means of an enzyme such as alkaline phospha~ase,
peroxidase, glucose-oxidase-peroxidase, glucose-
6-phosphate~dehydrogenase, malate dehydrogenase,
etcO, rather than labelling with a radioactive
isotope.
3.1.3. Quantitative determination of antibodies
against the peptide sub-unit pentapeptide of peptidoglycans
by nephelometric methods.
Peptides of formula I are bound-, via their
N-terminal amino group, to a soluble matrix, P.g~
proteins, polyvinyl pyrrolidone, etc., and the
test is carried out according to the following
scheme:
Matrix--(Pept.)n ~ Ab ~ Matrix-(Pept.)n-Ab-complexes
turbidity
The coupling of the peptides to the carrier
material may be effected, for exampleO
1. by binding the iodoacetylated peptides to the
matrix;
2. by the carbodiimide method;
3. by means of bifunctional reagents, e.g. diisocyanates,
glutardialdehyde, etc.
3 D 2'~ Measur~ment_ in the heterogeneous phase
Peptides of formula I are bound via their
N-terminal amino group to a solîd insoluble matrix
such as paper, plastics, latex, etc. Coupling
is effected by conventional methods. The peptides
may be bound either directly to the matrix or via
so called "spacers". These "spacers" may be aliphatic
hydrocarbon chains, proteins such as albumin, RNase,
etc.
.
,.
~ 7 --
3.2.1. Quantitative determination of antibodies
against the peptide sub-unit pentapeptide of peptido-
glycans in a solid phase "sandwich" radioimmuno
assayO
3.2.2. Quantitative determination of antibodies
against the peptide sub-unit pentapeptide of peptido-
glycans in a solid phase enzyme-immunoassay.
The test procedure of 3.2.1. above is modified
by effecting the labelling of the anti-antibodies
by means of an enzyme such as those ~entioned in
3.1.2. above rather than by labelling with a radioactive
isotop~.
I
3.2.3. Quantitative determination of antibodies
against the peptide sub-unit pentapeptide of peptidoglycan
in a solid phase 1uorescence immunoassay.
The test procedure of 3.2.1 above is modified
by labelling the antibody by means of a fluorescen-t
dye (cf~ also FI~ ~ - Sti ~ system~.
3.2.4. Quantitative determination of antibodies
against the peptide sub-unit pentapeptide o~ peptidoglycan
by means of a latex agglutination test.
Peptides of formula I are coupled to latex
particles either directly or via "spacers" such
as proteins.
In the methods of quantitative determination
discussed at 3.1~ and 3.2. above the quantification
is effected by means of a calibration curve which
can be plotted with the aid of the standard solutions
of antibodies described in 1 above.
Eor the determination of specific IgE a relative
quantification over the pooled serum standard described
at 2 above i5 effected.
The follo~ing Examples are provided to illustrate
the scope of the invention without serving to restrict
the scope of protection sought therefor-
~243~
Example 1Determining specific antibodies of class
a) Pre~ration of the conju~ate (antigen)
Equimolar quanti~ies of peptides of formula
I and iodoacetylsuccinimide ester in dioxan/water
(1:2 v/v), containing 100 mmol/l of sodium hydrogen
carbonate, are incubated at 4C for 20 hours with
gentle stirring. The dioxan is then eliminated
_ vacuo and ~he reaction product formed is precipitated
from the aqueous phase by acidification with HCl.
After washing in a hydrochloric acid medium, the
product is lyophilised.
100 mg of iodoacetylated peptide and carrier
protein, e.g. 250 mg of human serum albumin, are
incubated for 72 hours at 37C in 8 molar urea,
containing 0.1 mM sodium bicarbonate. After 3
days' dialysis with distilled water (the dialysi-
~liquid beiny changed 6 times) the peptide coupled
to the albumin is lyophilisedO With the process
described, on average 10 moles of peptide per mole
of carrier protein are covalently bound.
b) Adsorption_of the conjugate on plastics surfaces,
e.~. polystyrene
In this example, polystyrene tubes were used
having a capacity of barely 2 ml, a height of 4 cm
and a diameter of 1 cm. Microtitre plates, halls,
dishes, etc. of similar plastics material are also
~ suitable for bindingO
100 ~1 of conjugate solution (corresponding
to 100 ng of conjugate in 0.1 molar bicarbonate
buffer, pH 9.6) are pipetted into the tubes and
dried at 37C for 24 ~hours. The dry tubes are
each washed once with 200 microlitres of 10 mmol/l
phosphate buffer (pH 7.2) in isotonic common salt
solution containing 0.1% Twee ~ 80 and after the
wash solution has been removed by suction filtering
the tubes are again rinsed with 200 microlitres
3~
g
of distilled water and then suction filtered again.
The tubes coated with the conjugate are stored
in a drying cupboard at 37C.
c) Test procedure
lQ0 microlitres of specific standard solution
(see the preparation of the standard) or serum
with a known content of specific IgA are pipetted
into the tubes coated as described above and incubated
at 20C for 2 hours. ~n order to stay within the
measuring range of the standard curve-, samples
of biological material, e.g. patients' sera, are
diluted in the ratio of 1:5 to 1:500, depending
on the titre, with PBS ~phosphate buffered saline)/Tween~
80 buffer and are used in the same volume. The
incubation mixture is suction filtered and then
washed twice with 200 microlitres of PBS/'I'ween~
80 buffer and then suction filtered again. 100 ~1
(anti-human IgA) - immunoglobulin (e~g. from goats)
labelled with peroxidase or alkaline phosphatase
are pipetted in~o the tubes and incubated for 1
hour at a constant temperature of between 20C
and 37C. Then the tubes are washed twice more,
each time wi~h 200 microlitres of PBS/Twee ~ buffer.
If conjugated peroxidase is used as the indicator
system, o~pheny}enediamine and H~02 are added as
su~strate in a citrate buffer (0.1 mol/l, p~ 5.0)
and the tubes are incubated for 30 minutes at constant
temperature.
To stop the reaction, 50 pl of 5N H~SO4 are
added~ The extinction at 492 nm is proportional
to the ~uantity of immunoglobulin used.
Example 2
Determinin~ specific antibodies of class I~M
a) Preparation of the coniugate tantigen)
The conjugate is prepared as described in
Example 1.
~2~3~
-- 1() --
b) Adsorption of the conjugate on plastics surfaces,
e g. polystyrene
The adsorption of the conjugate on plastics
surfaces is also carried out as described in Example
1, except that instead of lO0 ng of conjugate 50 ng
of conjugate in 0.1 molar bicarbonate buffer (p~I
9.6) are used.
c) Carrying out the test
Pre-treat~ent of the sera: in order to exclude
interference by l'rheumatic factors" t~e sera are
treated on latex-bound IgG (e.g. latex-RF reagen
before being used in the test system.
lOO~ul of specific standard solution (see
preparation of standard) or serum with a known
conten~ of specific IgM are pipetted into the tubes
which have been coated as described above and then
incubated for 2 hours at 20~C. In order to stay
within the measuring range of the standard curv~,
sample-s of biological material, e.g. patients'
serar are diluted with 0~3 mol/l of glycine-NaCl
buffer, pH 8.2, depending on the titre, and used
in the same volume. The incubation mixture is
suction filtered and then washed twice with 20~ ~l
of 3.3 mol/l glycine-NaCl buffer, pH 8.2, and then
suction filtered again. lO0/ul ~anti-human IgM)
immuno~lobulin ~e.g. from goats) labelled with
peroxidase or alkaline phosphatase are pipetted
into the tubes and incubated for l hour at a constant
temperature of between 20C and 37C. Then the
tubes are washed twice more each time with 200/ul
of 0.3 mol/l of glycine-NaCl buffer, p~ 8.2. If
conju~ated peroxidase is used as the indicator
system, o~phenylenediamine and H~O2 are added as
substrate in a citrate buffer (0~1 mol/l, pH 5.0),
and the tubes are incubated for 30 minutes at constant
temperature.
To stop the reaction, SO~ul of 5N H2SO4 are
~3~
added. The extinction at 492 nm is proportional
to the quantity of immunoglobulin used.
~ le 3
Determining specific antibodies of class I~E
a) Preparation of the conju~ate (antigenJ
The conjugate is prepared as described in
Example 1.
b~ Adsorption of the conjugate on plastics surfaces,
e.~. polystyrene
The adsorption of the conjugate on plastics
surfaces is also carried out as described in Example
1, except that instead of 100 ng of conjugate 200 ng
of conjugate in 0.1 molar bicarbonate buffer (p~
9.6) are used.
c) Carrying out the test
In order to arrive in the measuring range
of the po~led -serum standard, patients' sera are
used undiluted or in a dilution of up to 1:20.
100 ~1 of serum are pipetted with 100 ~1 of PBS/Tween~
80 buffer into the tubes coated as described above
and then incubated for 3 hours at 20C. The incubation
mixture is suction filtered and subsequently washed
twice with 200 ~1 of PBS/Twee ~ 80 buffer and suction
filtered again. 100 pl (anti-human IgE) immunoglobulin
(e .~ . f rom horses), labelled with peroxidase or
alkaline phosphatase, are pipetted into the tubes
and incubated for 2 hours at constant temperature
of between 20C and 37C. Then the tubes are washed
twice more each time with 200 ~1 of PBS/Twee ~
80 buffer. If conjugated peroxidase i5 used as
the indicator system, o-phenylenediamine and H2O2
are added as substrate in a ci~trate buffer of 0.1 mol/l,
pH 5.0, and the tubes are incubated for 30 minutes
at constant temperature.
To stop the reaction, 50 ~1 of 5W H2SO4 are
, ,
, .
~3~
added. The extinction at 492 nm is proportional
to the quantity of immunoglobulins used.
Example 4
Obtaining a specific human immuno~lobulin-A standard
_ _
~i~'' J A. ~eling the albumin-(L-Ala-D-G]
~` Ala-D-Ala)) con~ugate to Sepharose 4B
Synthesis of the conjugate is carried out~
as described for the test. 10 ml of ~ ~ es4~
gel are washed 5 times with a 4- to 5-fold excess
of distilled water and then suspended in 10 ml
of water. 430 mg of bromocyanogen, dissolved in
8 ml of water, are added thereto. The reaction
takes place at ambient temperature and the pH i5
kept constant at 11.0 by the addition o~ 2 mol~l
of sodium hydroxide solution. After 15 minutes'
reaction with gentle stirring, the gel is suction
filtered on to a BUchner funnel and washed with
60 ml of 0.1 mol/l of sodium-hydrogen carbonate
solution. The sepharose is stirred into 7O5 ml
of 0.1 mol/l of sodium hydrogen carbona~e containing
50 mg of albumin-peptide conjugate. Coupling is
effected overnight a~ 4C with gentle stirring.
After the reaction time, the substance is suction
filtered again and the coupled gel is wasbed twice
with each of the following: 30 to 40 ml of water,
then 0.1 mol/l of sodium hydrogen carbonate, 0.2 mol/l
of sodium acetate buffer, pH 4.5, 0.2 mol/l of
sodium phosphate buffer, p~ 7.2, and 0.01 mol/l
of PBS, pH 7.~. The affinity gel is taken up in
PBS buffer, pH 7.2, at 4C.
B. Procedure for affinity chromatography
The gel is placed in a suitable column and
serum containing antibodies or an isolated immuno-
globulin-A fraction is added. The charged column
is washed with PBS buffer ~without Tween) until
3g~
the extinction at 28~ nm in the eluate falls to
below 0.03 (consumption of about twice as much
buffer as serum).
~lution is carried out with acetic acid (0.1
to 1 mol/l)~ The antibody is eluted with the first
fractions vf the acetic acid, the fractions containing
antibodies are combined and neutralised, then dialysed
and concentrated.
Alternatively, standardisation may be carried
out as follows:
The concentration of specific I~A in a pooled
serum is determined by affinity chromatographyO
For this, the isolated IgA fraction from the poolecl
serum is added to the column and washed as described
above. Total elution o~ the specifically bonded
Ig~ is carried out with 6 mol/l of guanidinium
chloride. After dialysis the protein content of
the eluate is determined.
~0 Example 5
Determinin~ the content of specific IgM in a pooled
serum standard
A. Couplin~ the albumin-(L-Ala-D-Glu(L-Lys-D-
Ala-D-Ala)) conjugate to Sepharose 4 B
Synthesis is carried out as described for
the tes~. 10 ml o~ sepharose gel are wa~hed 5
times with a 4- to 5-fold excess of dis illed water
and then su~spended in 10 ml of water. 430 mg of
bromocyanogenl dissolved in 8 ml of water, are
added thereto. The reaction takes place at ambient
temperature and the pH is kept constant at 11.0
by the addition of~2 moI/l of sodium hydroxide
solution~ After 15 minutes' reaction with gentle
stirring, the gel is suction filtered on to a B~chner
funnel and washed with 60 ml o 0.1 mol/l of sodium
hydrogen carbonate solution. The sepharose is
stirred into 7.5 ml of 0.1 moljl of sodium hydrogen
,,
-~ ~43~
-- 14 --
carbonate containing 50 mg of albumin-peptide conjugate.
Coupling is effected overnlght at 4C with gentle
stirring. After the reaction time, the substance
is suction filtered again an~ the coupled gel is
washed twice with each of the following: 30 to
40 ml of water, then 0.1 mol/l of sodium hydrogen
carbonate, 0.2 mol/1 of sodium acetate buffer,
pH 4.5, 0.2 mol/l of sodium phosphate buffer, pH
7~2, and 0.01 mol/1 of PBS, pH 7.2. The affinity
gel is taken up in PBS buffer, p~l 7.2, at 4C.
B Procedure for affinity chromatography
The gel is placed in a suitable column and
isolated IgM is added. The charged column is washed
with PBS buffer (without Tween) until the extinction
at 280 nm in the eluate falls to below 0.03 (consumption
of about twice as much buffer as serum).
Total elution of the specifically bound IgM
is carried out with 6 mol/l guanidinium chloride.
After dialysis, the protein content of the eluate
is determined.
