U.S. patent application number 15/801854 was filed with the patent office on 2018-07-05 for biological assay of peptidoglycans.
The applicant listed for this patent is Roquette Freres. Invention is credited to Fabrice Allain, Roselyne Bernard, Marc Biguet, Mathieu Carpentier, Agnes Denys, Hela Hacine-Gherbi, Pierre Lanos.
Application Number | 20180188233 15/801854 |
Document ID | / |
Family ID | 50397130 |
Filed Date | 2018-07-05 |
United States Patent
Application |
20180188233 |
Kind Code |
A1 |
Lanos; Pierre ; et
al. |
July 5, 2018 |
BIOLOGICAL ASSAY OF PEPTIDOGLYCANS
Abstract
The present invention relates to a biological method for
assaying peptidoglycans (PGN) in a sample, particularly a sample of
glucose polymers. The PGN assay includes: a) treating the glucose
polymer sample by sonication, heating, and/or alkalizing; b)
placing the treated sample or a dilution thereof in contact with a
recombinant cell expressing an exogenous TLR2 (toll-like receptor
2) and a reporter gene directly dependent on the signaling pathway
associated with the TLR2. The reporter gene codes for a colored or
fluorescent protein or for a protein the activity of which is
measurable with or without a substrate; c) measuring the reporter
gene signal; and d) determining the amount of PUN in the sample
using a standard curve of the correlation between the amount of PGN
and the strength of the reporter gene signal.
Inventors: |
Lanos; Pierre; (La Bassee,
FR) ; Biguet; Marc; (Neuve Chapelle, FR) ;
Bernard; Roselyne; (Lestrem, FR) ; Allain;
Fabrice; (Lille, FR) ; Carpentier; Mathieu;
(Saint Andre Lez Lille, FR) ; Denys; Agnes;
(Lillie, FR) ; Hacine-Gherbi; Hela;
(Villeneuve-d'Ascq, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Roquette Freres |
Lestrem |
|
FR |
|
|
Family ID: |
50397130 |
Appl. No.: |
15/801854 |
Filed: |
November 2, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14779353 |
Sep 23, 2015 |
9857355 |
|
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PCT/EP2014/055888 |
Mar 25, 2014 |
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15801854 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2333/4722 20130101;
G01N 33/5005 20130101; G01N 2333/70596 20130101; G01N 2400/40
20130101; G01N 2400/38 20130101 |
International
Class: |
G01N 33/50 20060101
G01N033/50 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 26, 2013 |
FR |
1352732 |
Jul 22, 2013 |
FR |
1357197 |
Claims
1-14. (canceled)
15. A kit for assaying peptidoglycans (PGNs) in a sample of glucose
polymers comprising: a recombinant cell expressing an exogenous
TLR2 receptor (Toll-like Receptor 2) and a reporter gene under the
direct dependence of the signaling pathway associated with the TLR2
receptor, said reporter gene coding for a colored or fluorescent
protein or for a protein whose activity can be measured with or
without a substrate; either a calibration curve of the
correspondence between the amount of PGN and the intensity of the
reporter gene signal, or a PGN standard; and optionally,
instructions for use, and a solution for pretreating the
sample.
16. The kit of claim 15 wherein the PGN standard is derived from a
bacterium selected from Staphylococcus aureus, Micrococcus luteus,
Bacillus subtilis and Alicyclobacillus acidocaldarius.
17. The kit of claim 15, further comprising an internal standard
that is an agonist of TLR2.
17. The kit of claim 16, wherein the agonist of TLR2 is a
lipopeptide.
18. The kit of claim 17, wherein said lipopetide is
PAM3Cys-Ser-(Lys)4 trihydrochloride.
19. The kit of claim 15, wherein the recombinant cell is a stably
transformed HEK-293 cell line.
20. The kit of claim 15 further comprising a negative control.
21. The kit of claim 20, wherein the negative control comprises a
cell not expressing an innate immunity receptor.
22. The kit of claim 21, wherein the cell not expressing an innate
immunity receptor is a HEK-null cell line.
23. The kit of claim 15, wherein the PGN standard is a purified and
partially digested PGN.
24. The kit of claim 15 further comprising reagents to be used for
measuring the response of the reporter gene.
25. The kit of claim 15, wherein the reporter gene codes for an
enzyme.
26. The kit of claim 25, wherein the enzyme is luciferase or
alkaline phosphatase.
27. A method of using the kit of claim 15, and determining the
presence of PGNs.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to assay of peptidoglycans in
a sample, in particular a sample of glucose polymers.
CONTEXT OF THE INVENTION
[0002] Aseptic inflammatory episodes are major complications
observed during treatments using manufactured products for
therapeutic purposes (for example: peritoneal dialysis, parenteral
nutrition, injection by the venous route). Although some of these
inflammatory episodes are connected with a problem of a chemical
nature (accidental presence of chemical contaminants or incorrect
dosages of certain compounds), most cases result from the presence
of contaminants of microbial origin released during the
manufacturing processes. It is now clearly established that
lipopolysaccharides (LPSs) and peptidoglycans (PGNs) are the main
contaminants presenting a high risk of triggering such inflammatory
episodes when they are present at trace levels in manufactured
products.
[0003] The LAL (Limulus amebocyte lysate) assay is used routinely
by many quality control laboratories for detecting and assaying
contamination with LPS. This assay is based on recognition of the
endotoxins by a sensing complex extracted from Limulus (horseshoe
crab) hemolymph.
[0004] Other assays also based on the reactivity of extracts of
invertebrate hemolymph are currently proposed for detecting PGNs in
products for therapeutic use (SLP-Wako, Immunetics). However, these
assays have the disadvantage that they are not very specific, since
they also react with other molecules of microbial origin, such as
.beta.-glucans. Moreover, these methods require purchasing special
equipment for this use, which greatly increases the costs and
therefore limits access to these assay techniques.
[0005] Moreover, LPSs and PGNs have variable structures depending
on their bacterial origin, which is responsible for large
differences in inflammatory reactivity. That is why it is in
addition necessary to express the results of the assays in
equivalent units of standard molecules (for example, LPS of E. coli
in the LAL assay).
[0006] Moreover, these molecules are most often present in the form
of macromolecular complexes, which affects their solubility and
their inflammatory potential. For example, the PGNs are very
variable in size and are often aggregated with other molecules of
the bacterial wall, such as lipoteichoic acids and
lipopeptides.
[0007] Thus, "biological" methods have been developed solely to
take account of the inflammatory load associated with these
molecules. The effector cells of the inflammatory response possess
special sensors for recognition of molecular structures
specifically produced by infectious agents. These molecules, called
PAMPs for pathogen-associated molecular pattern molecules, are
essentially recognized by TLRs (Toll-like receptors) and NLRs
(Nod-like receptors), whose specificity is related to the molecular
structure of the different classes of inflammatory molecules. In
contrast to LPS (which is a ligand recognized by type TLR4
receptors), PGN is a ligand recognized by type TLR2 receptors.
[0008] In recent years, cellular assays in vitro have been
developed to replace the animal models of inflammatory response.
Most of these assays are based on the incubation of monocyte cells
in the presence of the contaminated products and on back-titration
of the production of inflammatory cytokines (TNF-.alpha.,
IL-1.beta., IL-6, IL-8, RANTES). However, assays using primary
cells isolated from blood are subject to considerable
inter-individual variability of the donors, which may be
responsible for experimental bias.
[0009] In contrast, the monocyte cell lines give constant
responses, which explains why they are generally preferred to
primary cells. However, these lines are not completely satisfactory
either. For example, the choice of cytokines is often criticized,
as most are expressed transiently and their concentration in the
culture medium does not always reflect the real load of
inflammatory molecules. Since all the monocyte cells express the
majority of the TLRs/NLRs, assays based on their use are not
selective for one type of contaminant, but will give an overall
inflammatory response.
[0010] Moreover, the main problem arises from the differences in
sensitivity of the cells with respect to the different inflammatory
molecules. Thus, the PGNs, TLR2 ligands, are far less reactive than
the LPSs, which makes them difficult to detect by these approaches.
In fact, the LPSs induce a significant response for concentrations
of the order of ng/mL, whereas 100 times higher concentrations of
PGN are necessary to obtain a similar response (w/w ratio).
[0011] For some years, transfected cell lines have been proposed
for replacing the above models in the biological assays for
detecting and quantifying the reactivity of inflammatory compounds.
These noninflammatory lines (for example: HEK-293) are stably
transfected by a gene coding for a specific receptor of a class of
inflammatory agonists. They also contain an expression vector for a
reporter gene coding for an enzyme (for example, luciferase or
alkaline phosphatase), whose synthesis is dependent on activation
of the inflammatory receptor. Thus, recognition of a contaminant by
the cells expressing the appropriate receptor will trigger the
synthesis of the enzyme, production of which will be followed by
transformation of its substrate into a colored or luminescent
product. As this product is easily quantifiable, this method allows
rapid assay of the inflammatory response associated with a type of
contaminant.
[0012] These cellular models have many advantages: replacement of
ELISA assays of cytokines with an enzyme assay, great
reproducibility of the assays on account of the stable character of
the lines, targeting of certain classes of inflammatory molecules
as a function of the receptor expressed, detection of contaminants
at very low thresholds.
[0013] These cellular models may therefore replace the assays of
cytokine response in vitro, as they make it possible to target
specifically the inflammatory factors that are agonists of a given
TLR or NLR, and quantify the inflammatory response associated with
this agonist. For example, cells specifically expressing TLR2 and
TLR4 have already been used for detecting contaminants in food
products (works of Clett Erridge of the Department of
Cardiovascular Sciences of Leicester--UK in British Journal of
Nutrition, Vol. 105/issue 01/January 2011, pp 15-23).
[0014] Moreover, companies such as InvivoGen now market a wide
range of cells of the HEK-293 line (HEK-Blue.TM.) transfected with
the various TLR or NRL receptors. These cells contain, as reporter,
a gene coding for a secreted form of alkaline phosphatase (SEAP:
secreted embryonic alkaline phosphatase), which allows quick and
easy colorimetric assay of the response to the inflammatory
agonists.
[0015] These HEK-Blue.TM. cells have already been used successfully
for detecting the presence of contaminants in concentrated
solutions of glucose polymer and their synergistic effect
(WO2012/143647). As the aim stated in this application is solely to
detect the contaminants that are in a form displaying inflammatory
activity, the methods described in this patent application are not
suitable for measuring the total amount of PGN contained in the
sample. In fact, the soluble PGNs (MM.apprxeq.120 kDa) are those
that induce an inflammatory response via the TLR2 receptor. Thus,
the PGNs not having a suitable size to be inflammatory or
aggregated with other molecules are not detected by the method
described in this application.
[0016] Thus, there is a constant need to develop alternative
methods of assaying total PGN in a sample, in particular a sample
of glucose polymers.
SUMMARY OF THE INVENTION
[0017] The present invention therefore relates to a biological
method for assaying the peptidoglycans in a sample, in particular a
sample of glucose polymers.
[0018] In particular, the present invention relates to a method of
assaying peptidoglycans (PGNs) in a sample of glucose polymer,
comprising: [0019] a) treating the sample of glucose polymer by
sonication, heating, and/or alkalization; [0020] b) bringing the
treated sample or a dilution thereof into contact with a
recombinant cell expressing an exogenous TLR2 receptor (Toll-like
Receptor 2) and a reporter gene under the direct dependence of the
signaling pathway associated with the TLR2 receptor, said reporter
gene coding for a colored or fluorescent protein or for a protein
whose activity can be measured with or without substrate; [0021] c)
measuring the reporter gene signal; and [0022] d) determining the
amount of PGN in the sample using a calibration curve of the
correspondence between the amount of PGN and the intensity of the
reporter gene signal.
[0023] Preferably, treating the sample by sonication, heating,
and/or alkalization makes it possible to fragment and disintegrate
the PGNs contained in the sample, in particular so as to make them
capable of activating the TLR2 receptor. In particular, the
treatment of the sample makes it possible to generate PGNs
predominantly with a size of about 120 kDa,
[0024] Preferably, the reporter gene is a secreted alkaline
phosphatase. In a preferred embodiment, the cell is a cell of the
HEK-Blue.TM. hTLR2 line.
[0025] Preferably, the calibration curve of the correspondence
between the amount of PGN and the intensity of the reporter gene
signal was prepared with PGNs derived from a bacterium selected
from Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis
and Alicyclobacillus acidocaldarius, preferably from Staphylococcus
aureus, Micrococcus luteus, and Alicyclobacillus acidocaldarius. In
particular, the method may comprise a preliminary step of
preparation of the calibration curve using PGNs derived from a
bacterium selected from Staphylococcus aureus, Micrococcus luteus,
Bacillus subtilis and Alicyclobacillus acidocaldarius, preferably
from Staphylococcus aureus, Micrococcus luteus, and
Alicyclobacillus acidocaldarius.
[0026] Preferably, the sample is diluted, if necessary, so as to
generate a signal of the reporter gene corresponding to the linear
portion of the calibration curve.
[0027] Preferably, the sample is a sample of a solution of
icodextrin.
[0028] Preferably, the calibration curve of the correspondence
between the amount of PGN and the intensity of the reporter gene
signal is standardized or calibrated with an internal standard that
is an agonist of TLR2, preferably a lipopeptide, in particular
PAM.sub.3Cys-Ser-(Lys)4 trihydrochloride.
[0029] In an alternative embodiment, the calibration curve of the
correspondence between the amount of PGN and the intensity of the
reporter gene signal was prepared with an internal standard that is
an agonist of TLR2, preferably a lipopeptide, in particular
PAM.sub.3Cys-Ser-(Lys)4 trihydrochloride. In particular, the method
may comprise a preliminary step of preparation of the calibration
curve using an internal standard that is an agonist of TLR2,
preferably a lipopeptide, in particular PAM.sub.3Cys-Ser-(Lys)4
trihydrochloride.
[0030] The invention further relates to a kit for assaying
peptidoglycans (PGNs) in a sample of glucose polymers, comprising:
[0031] a recombinant cell expressing an exogenous TLR2 receptor
(Toll-like Receptor 2) and a reporter gene under the direct
dependence of the signaling pathway associated with the TLR2
receptor, said reporter gene coding for a colored or fluorescent
protein or for a protein whose activity can be measured with or
without substrate; and [0032] either a calibration curve of the
correspondence between the amount of PGN and the intensity of the
reporter gene signal, or a PGN standard, preferably derived from a
bacterium selected from Staphylococcus aureus, Micrococcus luteus,
Bacillus subtilis and Alicyclobacillus acidocaldarius, preferably
from Staphylococcus aureus, Micrococcus luteus, and
Alicyclobacillus acidocaldarius, or an internal standard that is an
agonist of TLR2, preferably a lipopeptide, in particular
PAM.sub.3Cys-Ser-(Lys)4 trihydrochloride; [0033] optionally
instructions for use and/or a solution for pretreating the
sample.
[0034] Preferably, the kit further comprises an internal standard
that is an agonist of TLR2, preferably a lipopeptide, in particular
PAM.sub.3Cys-Ser-(Lys)4 trihydrochloride.
DETAILED DESCRIPTION OF THE INVENTION
[0035] The present invention therefore relates to a biological
method for assaying the peptidoglycans in a sample, in particular a
sample of glucose polymers.
[0036] In particular, the present invention relates to a method of
assaying peptidoglycans (PGNs) in a sample of glucose polymer,
comprising: [0037] a) treating the sample of glucose polymer by
sonication, heating, and/or alkalization; [0038] b) bringing the
treated sample or a dilution thereof into contact with a
recombinant cell expressing an exogenous TLR2 receptor (Toll-like
Receptor 2) and a reporter gene under the direct dependence of the
signaling pathway associated with the TLR2 receptor, said reporter
gene coding for a colored or fluorescent protein or for a protein
whose activity can be measured with or without substrate; [0039] c)
measuring the reporter gene signal; and [0040] d) determining the
amount of PGN in the sample using a calibration curve of the
correspondence between the amount of PGN and the intensity of the
reporter gene signal.
[0041] Preferably, the glucose polymers are intended for peritoneal
dialysis, enteral and parenteral nutrition and feeding of neonates.
In a preferred embodiment, the glucose polymers that will be tested
are icodextrin or maltodextrins. In particular, they may be
intended for preparation for peritoneal dialysis. They can be
tested at one or more stages of their preparation, and notably at
the level of the raw material, in any step in their preparation
process, and/or at the level of the end product of the process.
They may also be tested as a sample of a solution for peritoneal
dialysis.
[0042] In a first step of the method, the sample of glucose polymer
is treated by sonication, heating, and/or alkalization. The aim of
this treatment is to fragment the PGNs and/or disintegrate the PGNs
contained or trapped in aggregates, the aim being to generate PGNs
capable of interacting with the TLR2 receptors and activating them.
As stated above, this treatment should make it possible to
disintegrate the PGNs contained or trapped in aggregates and to
fragment the PGNs that are too large, notably to generate soluble
PGNs with sizes between 30 and 5000 kDa, notably of about 120 kDa.
However, the treatment must not affect the capacity of the PGNs for
interacting with the TLR2 receptors. It is preferably optimized for
maximum release of PGNs capable of interacting with TLR2 and of
activating the receptor and for storing a maximum of PGNs already
active on TLR2.
[0043] In a first embodiment, the treatment of the sample comprises
at least one sonication step. Optionally, sonication may take from
30 seconds to 5 minutes, use a power of 20 to 40 kHz and/or
comprise one or more sonication cycles, for example from 1 to 5
cycles. In a preferred embodiment, the sample will be treated by
sonication for 1 minute at 35 kHz in a single cycle. Optionally,
the treatment by sonication may be combined with treatment by
heating and/or by alkalization.
[0044] In a second embodiment, the treatment of the sample
comprises at least one alkalization step. Preferably, the
alkalizing agent is NaOH, notably at a concentration between 0.1
and 1 M. Optionally, the duration of the alkalization step may be
from 5 minutes to 60 minutes. Optionally, the alkalization step may
be carried out at a high temperature, notably a temperature between
20.degree. C. and 80.degree. C., for example at a temperature of
20, 40, 60 or 80.degree. C. Optionally, the treatment by
alkalization may be combined with treatment by sonication.
[0045] In a second embodiment, the treatment of the sample
comprises at least one heating step. Optionally, the duration of
the heating step may be from 5 minutes to 60 minutes. Optionally,
the heating step may be carried out at a high temperature, notably
a temperature between 20.degree. C. and 80.degree. C., for example
at a temperature of 20, 40, 60 or 80.degree. C. Optionally, the
heating treatment may be combined with treatment by sonication
and/or by alkalization.
[0046] The methods of sample treatment do not comprise steps of
enzymatic treatment, notably by a mutanolysin.
[0047] In a subsequent step, the sample and/or dilutions thereof
is/are brought into contact with recombinant cells expressing the
TLR2 receptor. The cells are qualified as recombinant as they are
cells that have been modified by the introduction of a nucleic acid
coding for the TLR2 receptor, preferably the human TLR2 receptor,
the initial cell not expressing TLR2.
[0048] The activity of the TLR2 receptor is detected using a
reporter gene that is under the direct dependence of the signaling
pathway associated with said receptor. Preferably, this reporter
gene codes for a colored or fluorescent protein, or for a protein
whose activity can be measured with or without substrate. In
particular, the reporter gene codes for an alkaline phosphatase.
Notably, the reporter gene may produce a secreted form of alkaline
phosphatase (SEAP: secreted embryonic alkaline phosphatase), whose
synthesis is under the direct dependence of the signaling pathway
associated with TLR2.
[0049] In a preferred embodiment, the cell line used is a HEK-10
Blue.TM. line (marketed by the company InvivoGen), modified by
stable transfection with vectors coding for human TLR2: the
HEK-Blue.TM. hTLR2 line. However, it should be noted that a person
skilled in the art may also use other lines commercially (Imgenex)
or he can prepare them.
[0050] When the cell is HEK-Blue.TM. hTLR2, the cell is preferably
used at a density of about 50 000 cells/well for a 96-well
plate.
[0051] In a particular embodiment, the sample of glucose polymers
or a dilution thereof has a concentration of glucose polymers from
5 to 50 mg/mL, preferably between 5 and 10, 20, 30 or 40 mg/mL. In
the preferred embodiment, the sample of glucose polymers or a
dilution thereof has a concentration of glucose polymers of about
37.5 mg/mL.
[0052] In another particular embodiment, the sample of glucose
polymers or a dilution thereof has a maximum concentration of
glucose polymer of 3.75% (weight/volume), preferably 3%.
[0053] For example, the samples are prepared so as to have a
concentration of glucose polymer of 3.75% (weight/volume),
preferably 3%, and the samples are submitted to the method of assay
according to the present invention, assaying the sample as well as
1/10, 1/100 and 1/1000 dilutions.
[0054] Preferably, bringing the sample of glucose polymers or a
dilution thereof into contact with the cells takes about 5 to 48 h,
preferably from 10 to 36 h, more preferably from 16 to 24 h.
[0055] Next, the method comprises measurement of the reporter gene
signal.
[0056] In a preferred embodiment using the HEK-Blue.TM. hTLR2 line,
the signal is a measure of the activity of alkaline phosphatase.
Preferably, the enzymatic reaction is carried out using a 1:3 ratio
of medium to be assayed to SEAP reagent (for example 50 .mu.L of
medium and 150 .mu.L of SEAP reagent). Moreover, a reaction time of
at least 60 minutes will be preferred.
[0057] Finally, the amount of PGN in the sample is determined using
a calibration curve of the correspondence between the amount of PGN
and the intensity of the reporter gene signal.
[0058] This curve is preferably obtained with the same cells, in
the same conditions, with increasing doses of PGNs, in particular
PGN standards.
[0059] The PGN standard may be any PGN of bacterial origin. For
example, the PGNs may be derived from the following microorganisms:
Staphylococcus aureus, Micrococcus luteus, Escherichia coli,
Bacillus subtilis and Alicyclobacillus acidocaldarius. In
particular, the standards used are purified and partially digested
PGNs. Such standards are available commercially (Invitrogen,
Catalog # tlrl-pgnec or tlrl-pgnek from E coli; Catalog #
tlrl-pgnb2 from B subtilis; Catalog # tlrl-pgnsa from S aureus)
(Wako Pure Chemical, Catalog #162-18101 from M luteus).
[0060] The PGN standard is preferably calibrated using an internal
standard that is an agonist of TLR2, so as to express the results
in equivalent units of active PGN. The internal standard may be a
lipopeptide, preferably synthetic, in particular
PAM.sub.3Cys-Ser-(Lys)4 trihydrochloride (Pam3(cys), PAM or Pam
signifying palmitic acid) (see FIG. 5). Thus, the calibration curve
of the correspondence between the amount of PGN and the intensity
of the reporter gene signal is preferably standardized or
calibrated with an internal standard that is an agonist of TLR2,
preferably a lipopeptide, in particular PAM.sub.3Cys-Ser-(Lys)4
trihydrochloride. This internal standard is preferably synthetic or
with a well-defined structure/composition. The calibration or
standardization is carried out by comparing the slopes of the
linear portions of each dose-response curve and calculating a
correction factor allowing the curve obtained with the calibration
standard and that of the PGN standard to be superimposed.
[0061] For example, the calibration curve may be obtained using
concentrations of PGNs from 0.001 to 1000 ng/mL, notably from 0.01
to 100 ng/mL.
[0062] This calibration curve may be obtained either with PGNs
only, or with a solution of glucose polymer to which defined
quantities of PGNs have been added. Notably, the solution of
glucose polymer used may comprise 3.75% (weight/volume) of glucose
polymer, preferably 3%.
[0063] This curve of the correspondence between the amount of PGN
and the intensity of the reporter gene signal can also be obtained
with an internal standard that is an agonist of TLR2, preferably a
lipopeptide, in particular PAM.sub.3Cys-Ser-(Lys)4
trihydrochloride, notably with the same cells, in the same
conditions, with increasing doses of TLR2 agonist internal
standard. This internal standard is preferably synthetic or with a
well-defined structure/composition. Just as for PGN, it can be
obtained in the absence of or, preferably, in the presence of
glucose polymer.
[0064] Typically, the calibration curve is a classical curve of
cellular response of the sigmoid type (FIG. 1). [0065] part (A)
corresponds to the responses obtained with low concentrations of
PGN, below those giving effective activation of TLR2. This
nonlinear zone therefore corresponds to the limit of detection of
the method. So as to include the variability of the method, this
detection threshold is estimated at three times the value of the
background noise (response obtained in the absence of a stimulus);
[0066] part (B) is the most interesting as a linear response is
observed. This zone with effective response makes it possible to
determine a direct relation between the cellular response and the
PGN level. This is therefore the assay zone; [0067] part (C)
corresponds to saturation of the cellular response in the presence
of excessive concentrations of PGN. There is in fact saturation of
the TLR2 receptors.
[0068] The linear part of the calibration curve is considered; this
part corresponds to a zone (part B) in which the amount of PGN is
directly proportional to the reporter gene signal.
[0069] In the case of samples likely to be heavily contaminated
with PGN, it will be necessary to perform several series dilutions
so as to still be located in the zone of linearity. Otherwise, low
concentrations of PGN require a step of concentration of the sample
if we wish to increase the sensitivity of the assay.
[0070] Optionally, the method further comprises an assay with a
control cell that does not express TLR2, more generally that does
not express an innate immunity receptor. For example, the
HEK-Blue.TM. Null2 line may be used. This is a control line, use of
which is useful for verifying that the sample of glucose polymers
does not induce production of the enzyme by an intrinsic
mechanism.
[0071] The present invention also relates to a kit for assaying
peptidoglycans (PGNs) in a sample of glucose polymers, said kit
comprising: [0072] a recombinant cell expressing an exogenous TLR2
receptor (Toll-like Receptor 2) and a reporter gene under the
direct dependence of the signaling pathway associated with the TLR2
receptor, said reporter gene coding for a colored or fluorescent
protein or for a protein whose activity can be measured with or
without substrate. Notably, the cell is preferably the HEK-Blue.TM.
hTLR2 line. As negative control, the kit may also comprise a cell
not expressing an innate immunity receptor, for example the
HEK-Blue.TM. Null2 line. [0073] either a calibration curve of the
correspondence between the amount of PGN and the intensity of the
reporter gene signal, or a calibrated PGN standard, preferably
derived from a bacterium selected from Staphylococcus aureus,
Micrococcus luteus, Escherichia coli, and Alicyclobacillus
acidocaldarius, preferably Staphylococcus aureus, or an internal
standard that is an agonist of TLR2, preferably a lipopeptide, in
particular PAM.sub.3Cys-Ser-(Lys)4 trihydrochloride. Optionally,
the kit may comprise both, i.e. a calibration curve as well as a
calibrated PGN standard derived from the same microorganism as that
used for preparing this calibration curve. [0074] optionally
instructions for use, a solution for pretreating the sample, the
reagents to be used for measuring the response of the reporter
gene, microplates, etc.
[0075] Preferably, the kit further comprises an internal standard
that is an agonist of TLR2, preferably a lipopeptide, in particular
PAM.sub.3Cys-Ser-(Lys)4 trihydrochloride.
DESCRIPTION OF THE FIGURES
[0076] FIG. 1: Theoretical curve of the cellular response as a
function of increasing concentrations of PGN.
[0077] FIG. 2: Calibration curve of the cellular response as a
function of the PGN level of S. aureus obtained with the
HEK-Blue.TM.-hTLR2 cells.
[0078] FIG. 3: Response of the HEK-Blue.TM.-hTLR2 cells as a
function of increasing concentrations of PGN from different
bacterial species.
[0079] FIG. 4: Response of the HEK-Blue.TM.-hTLR2 cells as a
function of increasing concentrations of PGN of S. aureus obtained
from different batches.
[0080] FIG. 5: Structure of PAM.sub.3Cys-Ser-(Lys)4
trihydrochloride (PAM3(cys)).
[0081] FIG. 6: Comparison of the responses induced by the PGNs of
S. aureus and PAM3(cys) in the HEK-Blue.TM.-hTLR2 cells.
[0082] FIG. 7: Response of the HEK-Blue.TM.-hTLR2 cells as a
function of the corrected PGN concentrations.
[0083] FIG. 8: Calibration curve of the response of the
HEK-Blue.TM.-hTLR2 cells as a function of the corrected active PGN
concentrations.
EXAMPLES
[0084] The assay is based on the specific recognition of PGNs by a
line expressing the TLR2 receptor and on the production of an
enzyme activity measurable via activation of the signaling pathway
associated with TLR2.
[0085] Cellular Material
[0086] For the experiments relating to this assay, two lines are
used: [0087] HEK-Blue.TM. hTLR2 line (HEK-TLR2): specific response
for the TLR2 ligands, with strong reactivity for the soluble PGNs.
[0088] HEK-Blue.TM. Null2 line (HEK-Null): nonspecific response
connected with a cytotoxic effect of the sample.
[0089] The cells are cultured according to the supplier's
recommendations (InvivoGen). At 75% confluence, the cells are
resuspended at a density of 0.28.times.10.sup.6 cells/mL. Before
stimulation, 180 .mu.L of the cellular suspension is distributed in
the culture wells (96-well plate), or 50 000 cells/well. The cells
are then stimulated for 24 h by adding 20 .mu.L of the samples of
glucose polymer at 37.5% (weight/volume) (i.e. a final dilution of
the samples at 3.75%). After 24 h of stimulation, the cellular
response is measured by quantification of the enzyme activity
produced.
[0090] 1--Establishment of the Dose-Response Curve
[0091] A dose-response curve was constructed by diluting different
amounts of PGN standard of S. aureus (FIG. 2) in a solution of
uncontaminated icodextrin prepared at 37.5% (weight/volume) (FIG.
2).
[0092] The result is a classical curve of cellular response of the
sigmoid type. [0093] part (A) corresponds to the responses obtained
with low concentrations of PGN, below those giving effective
activation of TLR2. This nonlinear zone therefore corresponds to
the limit of detection of the method. [0094] part (B) is the most
interesting as a linear response is observed. This zone of
effective response makes it possible to determine a direct relation
between the cellular response and the PGN level. This is therefore
the assay zone. [0095] part (C) corresponds to saturation of the
cellular response in the presence of excessive concentrations of
PGN. There is in fact saturation of the TLR2 receptors.
[0096] The standard curve of response of the HEK-TLR2 cells to the
PGN of S. aureus has a zone of linearity for concentrations between
0.07 and 10 ng/mL (i.e. between 2 and 267 ng/g of icodextrin).
[0097] 2--Establishment of the Calibration Curve for Biological
Assay of PGNs with an Internal Standard
[0098] The dose-response curves were constructed by diluting the
PGNs of different bacterial species in a solution of uncontaminated
maltodextrin (referenced P-11.11) prepared at 37.5%
(weight/volume). The PGNs assayed are extracted from Staphylococcus
aureus (Sigma, Cat No 77140), Micrococcus luteus (Sigma, Cat No
53243), Bacillus subtilis (InvivoGen, # tlrl-pgnb2), and
Alicyclobacillus acidocaldarius (personal preparation).
[0099] The curves obtained are classical curves of the responses
observed in the assays performed with a cellular material
(bioassay) (FIG. 3). The absorbance values below 0.2 are evidence
of PGN concentrations that are too low to induce a cellular
response, whereas values above 2 show a plateau effect connected
with saturation of the TLR2 receptors. Consequently, only the zone
between these two limit values of absorbance allows correlation of
the production of SEAP with the amount of PGN present in the
samples.
[0100] The responses observed show a large variability in the
cellular reactivity associated with each type of PGN. In fact, the
concentrations giving a response equal to 50% of the maximum
response (EC50) are .about.20 ng/mL for the PGNs of S. aureus and
B. subtilis, 1500 ng/mL for M. luteus, and more than 2000 ng/mL for
those extracted from A. acidocaldarius and E. coli K12.
[0101] However, these differences were expected, since the PGNs
have different structures depending on their bacterial origin,
which is responsible for large variations in inflammatory
reactivity. These observations emphasize the importance of defining
an internal standard so as to be able to express the results in
equivalent units of PGN.
[0102] Another factor likely to alter the response of the HEK-TLR2
cells is the size of the PGNs, which will influence their
solubility and reactivity with respect to TLR2. Thus, the procedure
for purification of these macromolecules may have a considerable
influence on the response of the cells, since the conditions of
extraction could alter the size of the PGNs, or even cause partial
degradation. To test this hypothesis, the assays were reproduced
with 3 separate batches of PGNs extracted from S. aureus: 2 Sigma
batches (Cat No 77140: batch 1, 0001442777; batch 2, BCBH7886V) and
1 InvivoGen batch (# tlrl-pgnsa).
[0103] The results show variability of reactivity between the three
batches (FIG. 4). In fact, the EC50 values are 4, 20 and 400 ng/mL
respectively for the three batches. These data indicate that there
is a risk that PGNs extracted from the same bacterial species might
show differences in reactivity, even if the batches were obtained
from the same supplier and were extracted beforehand by the same
procedure. It therefore seems necessary to introduce an internal
standard for the calibration curve, so as to avoid errors relating
to the variability of the PGNs and to express the results as amount
of "active" PGN.
[0104] PAM.sub.3Cys-Ser-(Lys)4 trihydrochloride (PAM3(cys); FIG. 5)
is a triacylated synthetic lipopeptide that mimics the structure of
the bacterial lipopeptides and acts as a strong agonist of TLR2.
Being of homogeneous structure, it is often used as positive
control for calibrating the responses of the cells expressing the
TLR2 receptor.
[0105] The experiments were therefore reproduced replacing PGN with
PAM3(cys) in our tests. As expected, the HEK-TLR2 cells show strong
reactivity with respect to this compound. Moreover, the shape of
the dose-response curve is similar to those obtained in the
presence of PGN, with EC50 estimated at 10 ng/mL (FIG. 6). These
results indicate that PAM3(cys) induces responses equivalent to
those of the most reactive PGNs, but in contrast to the latter, it
does not display structural variability. Consequently, this
synthetic lipopeptide can be used for calibrating the batches of
PGN and for establishing a standardized calibration curve, which
will allow the results to be formulated in amounts of "active" PGN,
i.e. in amounts of PGN giving TLR2 responses identical to those
obtained with the same amounts of PAM3 (cys).
[0106] Each batch of PGN is calibrated relative to PAM3(cys) by
comparing the slopes of the linear portions of each dose-response
curve, and calculating a correction factor for superimposing the
curves of the PGNs on that of PAM3(cys). In the example presented
in FIG. 6, the correction factors were estimated at 0.4, 2 and 40
for batches 1, 2 and 3, respectively. This means that 2.5 times
less PGN from batch 1 is required for obtaining responses identical
to those induced by PAM3(cys), but 2 times more PGN from batch 2
and 40 times more PGN from batch 3. After correcting the raw
quantities of PGN, it can be seen that all the points are aligned
on one and the same curve, which can be superimposed on that
obtained with PAM3(cys) (FIG. 7). Consequently, using the internal
standard makes it possible to obtain corrected concentrations for
all the batches of PGN and establish a dose-response curve
calibrated for active PGN.
[0107] By applying this method, the standard curve of response of
the HEK-TLR2 cells has a zone of linearity for concentrations of
active PGN between 0.5 and 200 ng/mL (FIG. 8), or between 13 and
5400 ng/g of glucose polymers.
* * * * *