Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
METHOD AND COMPOSITION FOR ANALYZING
A CARBOHYDRATE POLYMER
Field of the Invention
The invention relates generally to a method for analyzing molecules containing
polysaccharides and more particularly to a method for analyzing
polysaccharides based using
saccharide-binding agents such as lectins.
Background of the Invention
Polysaccharides are polymers that include monosaccharide (sugar) units
connected to
each other via glycosidic bonds. These polymers have a structure that can be
described in
terms of the linear sequence of the monosaccharide subunits, which is known as
the two-
dimensional structure of the polysaccharide. Polysaccharides can also be
described in terms
of the structures formed in space by their component monosaccharide subunits.
A chain of monosaccharides that form a polysaccharide has two dissimilar ends.
One
end contains an aldehyde group and is known as the reducing end. The other end
is known as
the non-reducing end. A polysaccharide chain may also be connected to any of
the C1, C2,
C3, C4, or C6 atom if the sugar unit it is connected to is a hexose. In
addition, a given
monosaccharide may be linked to more than two different monosaccharides.
Moreover, the
connection to the C 1 atom may be in either the a or (3 configuration. Thus,
both the two-
dimensional and three-dimensional structure of the carbohydrate polymer can be
highly
complex.
The structural determination of polysaccharides is of fundamental importance
for the
development of glycobiology. Research in glycobiology relates to subjects as
diverse as the
identification and characterization of antibiotic agents that affect bacterial
cell wall synthesis,
blood glycans, growth factor and cell surface receptor structures involved in
viral disease,
and autoimmune diseases such as insulin dependent diabetes, rheumatoid
arthritis, and
abnormal cell growth, such as that which occurs in cancer.
Polysaccharides have also been used in the development of biomaterials for
contact
lenses, artificial skin, and prosthetic devices. Furthermore, polysaccharides
are used in a
number of non-medical fields, such as the paper industry. Additionally, of
course, the food
and drug industry uses large amounts of various polysaccharides and
oligosaccharides.
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
In all of the above fields, there is a need for improved saccharide analysis
technologies. Saccharide analysis information is useful in, e.g., for quality
control, structure
determination in research, and for conducting structure-function analyses.
The structural complexity of polysaccharides has hindered their analysis. For
example, saccharides are believed to be synthesized in a template-independent
mechanism. In
the absence of structural information, the researcher must therefore assume
that the building
units are selected from any of the saccharide units known today. In addition,
these units may
have been modified, during synthesis, e.g., by the addition of sulfate groups.
Second, saccharide can be connected at any of the carbon moieties, e.g., a the
C 1, C2,
C3, C4, or C6 atom if the sugar unit it is connected to is a hexose. Moreover,
the connection
to the C 1 atom may be in either a or (3 configuration.
Third, saccharides may be branched, which further complicates their structure
and the
number of possible structures that have an identical number and kind of sugar
units.
A fourth difficulty is presented by the fact that the difference in structure
between
many sugars is minute, as a sugar unit may differ from another merely by the
position of the
hydroxyl groups (epimers).
The use of a plurality of such saccharide-binding agents, whether fixed to the
substrate and/or employed as the second (soluble) saccharide-binding agent,
characterizes the
carbohydrate polymer of interest by providing a "fingerprint" of the
saccharide. Such a
fingerprint can then be analyzed in order to obtain more information about the
carbohydrate
polymer. Unfortunately, the process of characterization and interpretation of
the data for
carbohydrate polymer fingerprints is far more complex than for other
biological polymers,
such as DNA for example. Unlike binding DNA probes to a sample of DNA for the
purpose
of characterization, the carbohydrate polymer fingerprint is not necessarily a
direct indication
of the components of the carbohydrate polymer itself. DNA probe binding
provides
relatively direct information about the sequence of the DNA sample itself,
since under the
proper conditions, recognition and binding of a probe to DNA is a fairly
straightforward
process. Thus, a DNA "fingerprint" which is obtained from probe binding can
yield direct
information about the actual sequence of DNA in the sample.
By contrast, binding of agents to carbohydrate polymers is not nearly so
straightforward. As previously described, even the two-dimensional structure
(sequence) of
carbohydrate polymers is more complex than that of DNA, since carbohydrate
polymers can
2
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
be branched. These branches clearly affect the three-dimensional structure of
the polymer,
and hence the structure of the recognition site for the binding agent. In
addition, recognition
of binding epitopes on carbohydrate polymers by the binding agents may be
affected by the
"neighborhood" of the portion of the molecule which is surrounding the
epitope. Thus, the
analysis of such "fingerprint" data for the binding of agents to the
carbohydrate polymer of
interest is clearly more difficult than for DNA probe binding, for example.
A useful solution to this problem would enable the fingerprint data to be
analyzed in
order to characterize the carbohydrate polymer. Such an analysis would need to
transform
the raw data, obtained from the previously described process of incubating
saccharide-
binding agents with the carbohydrate polymer, into a fingerprint which would
itself contain
information. The fingerprint would also need to be standardized for comparison
across
different sets of experimental conditions and for different types of
saccharide-binding agents.
Unfortunately, such a solution is not currently available.
In spite of these difficulties, a number of methods for the structural
analysis of
saccharides have been developed. For example, PCT Application No. WO 93/24503
discloses a method wherein monosaccharide units are sequentially removed from
the
reducing end of an oligosaccharide by converting the monosaccharide at the
reducing end to
its keto- or aldehyde form, and then cleaving the glycosidic bond between the
monosaccharide and the next monosaccharide in the oligosaccharide chain by
using
hydrazine. The free monosaccharides are separated from the oligosaccharide
chain and
identified by chromatographic methods. The process is then repeated until all
monosaccharides have been cleaved.
PCT Application No. WO 93/22678 discloses a method of sequencing an unknown
oligosaccharide by making assumptions upon the basic structure thereof, and
then choosing
from a number of sequencing tools (such as glycosidases) one which is
predicted to give the
highest amount of structural information. This method requires some basic
information as to
the oligosaccharide structure (usually the monosaccharide composition). The
method also
illustrates the fact that reactions with sequencing reagents are expensive and
time-consuming,
and therefore there is a need for a method that reduces these expenses.
PCT Application No. WO 93/22678 discloses a method for detecting molecules by
probing a monolithic array of probes, such as oligodeoxynucleotides,
immobilized on a VLSI
chip. This publication teaches that a large number of probes can be bound to
an immobilized
3
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
surface, and the reaction thereof with an analyte detected by a variety of
methods, using logic
circuitry on the VLSI chip.
European Patent Application No. EP 421,972 discloses a method for sequencing
oligosaccharides by labeling one end thereof, dividing the labeled
oligosaccharide into
aliquots, and treating each aliquot with a different reagent mix (e.g. of
glycosidases), pooling
the different reaction mixes, and then analyzing the reaction products, using
chromatographic
methods. This method is useful for N-linked glycans only, as they have a
common structure
at the point where the saccharide chain is linked to the protein. O-linked
glycans are more
varied, and the method has as yet not been adapted for such oligosaccharides
with greater
variability in their basic structure.
There is therefore a need for a system and method for characterizing
polysaccharides
using an accurate, high throughput method for identifying agents that bind to
the
polysaccharide.
Summary of the Invention
The invention is based in part on the discovery of a method for quickly and
accurately
identifying agents that bind a given carbohydrate polymer. Also provided by
the invention is
a method for generating a fingerprint of a carbohydrate polymer that is based
on its pattern of
binding to saccharide-binding agents.
In one aspect, the invention features a method for characterizing a
carbohydrate
polymer. The carbohydrate polymer is contacted with a surface that includes at
least one first
saccharide-binding agent attached to a predetermined location on the surface
under
conditions allowing for the formation of a first complex between the first
saccharide-binding
agent and the carbohydrate polymer. The surface is then contacted with at
least one second
saccharide-binding agent under conditions allowing for formation of a second
complex
between the first complex and the second saccharide-binding agent. The first
saccharide-
binding agent and second saccharide-binding agent are then identified, thereby
characterizing
the carbohydrate polymer.
Also provided by the invention is a method of generating a fingerprint of a
carbohydrate polymer by contacting a carbohydrate polymer with a first
saccharide-binding
agent, determining whether the carbohydrate polymer binds to the saccharide-
binding
reagent, contacting the carbohydrate polymer with a second saccharide-binding
agent, and
4
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
determining whether the carbohydrate polymer binds to the second saccharide-
binding
reagent. Identification of the first and second saccharide-binding agent is
used to generate a
fingerprint of the carbohydrate polymer.
Unless otherwise defined, all technical and scientific terms used herein have
the same
meaning as commonly understood by one of ordinary skill in the art to which
this invention
belongs. Although methods and materials similar or equivalent to those
described herein can
be used in the practice or testing of the present invention, suitable methods
and materials are
described below. All publications, patent applications, patents, and other
references
mentioned herein are incorporated by reference in their entirety. In case of
conflict, the
present specification, including definitions, will control. In addition, the
materials, methods,
and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the
following
detailed description and claims.
Brief Description of the Drawings
Fig. 1 is an illustration of the Glycomolecule identity (GMID) cards obtained
for
pasteurized goat's milk (A and B), non-pasteurized goat's milk (C and D) and
bovine milk
(E).
Fig. 2 is a reproduction of the GMID cards obtained for various
lipopolysaccharide
samples. Cards A to E correspond to LPS# 1, 7, 10, 15 and 16 respectively.
Fig. 3 is a high-level logic flowchart that illustrates an algorithm for
choosing a set of
colored lectins.
Fig. 4 is a flowchart of an exemplary method according to the present
invention for
performing a fingerprint assay with a GMID card.
Detailed Description of the Invention
Provided by the invention is a method for characterizing a carbohydrate
polymer by
systemically assembling a representation of information that describes the
binding status of
the carbohydrate polymer with respect to saccharide-binding agents.
5
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
To assess binding status, the carbohydrate polymer is added to a surface that
includes
at least one saccharide-binding agent attached to a predetermined location on
the surface.
The carbohydrate polymer is incubated with the surface under conditions
allowing for the
formation of a complex between the first saccharide-binding agent and the
carbohydrate
polymer. The surface can then be washed if desired to remove unbound
carbohydrate
polymer. The surface is then contacted with a second saccharide-binding agent
under
conditions allowing for formation of a second complex between the first
complex and the
second saccharide-binding agent. The second agent preferably carries a
detectable label to
allow for detection of the second complex. Detection of the second complex at
a location on
the substrate corresponding to the location of a predetermined binding-agent
allows for the
identification of the first and second binding agents as agents that bind to
the carbohydrate
polymer. Detecting the first and second-binding agents provides structural
information about
the carbohydrate polymer.
While the method has been described by first contacting the carbohydrate
polymer
with the surface and then adding a detectable label, it is understood that
this order is not
obligatory. Thus, in some embodiments, the second agent is mixed with the
carbohydrate
polymer, and this complex is added to the surface.
In some embodiments, a plurality of saccharide-binding agents are attached to
the
surface. Similarly, a plurality of second detectable saccharide-binding agents
may be used.
In preferred embodiments, a plurality of both first and second saccharide-
binding agents are
used.
Thus, in various embodiments, at least, 5, 10, 15, 25, 30, or 50 or more first
saccharide-binding agents are attached to the surface. Preferably, each the
first saccharide-
binding agents are attached at spatially distinct regions of the substrate.
In other embodiments, at least 5, 10, 15, 25, 30, or 50 of more second-
saccharide
binding agents are used. Preferably, each of the second-saccharide have
attached thereto
distinguishable labels, i.e., labels that distinguish one-second saccharide-
binding agent from
another second saccharide-binding agent.
As used herein, a "carbohydrate polymer" includes any molecule with a
polysaccharide component. Examples include polysaccharide, a glycoprotein, and
glycolipid.
While a carbohydrate polymer includes any saccharide molecule containing two
or more
linked monosaccharide residues, it is understood that in most embodiments, the
carbohydrate
6
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
polymer will include 10, 25, 50, 1000, or 10,000 or more monosaccharide units.
If desired,
the carbohydrate polymer can be added to the surface after digestion with a
saccharide-
cleaving agent. Alternatively, the carbohydrate polymer can be added to the
surface, allowed
to bind to a first saccharide-binding agent attached to the surface, and then
digested with a
saccharide-cleaving agent.
In general, any agent that binds to a polysaccharide can be used as the first
or second
saccharide-binding agent. As is known in the art, a number of agents that bind
to saccharides
have been described. One class of agents is the lectins. Many of these
proteins bind
specifically to a certain short oligosaccharide sequence. A second class of
agents is an
antibody that specifically recognize saccharide structures. A third class of
saccharide-
binding agents is proteins that bind to carbohydrate residues. For example,
glycosidases are
enzymes that cleave glycosidic bonds within the saccharide chain. Some
glycosidases may
recognize certain oligosaccharide sequences specifically. Another class of
enzymes is
glycosyltransferases, which cleave the saccharide chain, but further transfer
a sugar unit to
one of the newly created ends.
For the purpose of this application, the term "lectin" also encompasses
saccharide-
binding proteins from animal species (e.g. "mammalian lectins"). Thus,
carbohydrate
polymers, like DNA or proteins, clearly have an important biological function
which should
be studied in greater detail.
A saccharide-binding agent is preferably an essentially sequence-specific
agent. As
used herein, "essentially sequence-specific agent" means an agent capable of
binding to a
saccharide. The binding is usually sequence-specific, i. e., the agent will
bind a certain
sequence of monosaccharide units only. However, this sequence specificity may
not be
absolute, as the agent may bind other related sequences (such as
monosaccharide sequences
wherein one or more of the saccharides have been deleted, changed or
inserted). The agent
may also bind, in addition to a given sequence of monosaccharides, one or more
unrelated
sequences, or monosaccharides.
The essentially sequence-specific agent is usually a protein, such as a
lectin, a
saccharide-specific antibody or a glycosidase or glycosyltransferase.
Examples of saccharide-binding agents lectins include lectins isolated from
the
following plants: Conavalia ensiformis, Anguilla anguilla, Triticum vulgaris,
Datura
stramoniuim, Galanthus nivalis, Maackia amurensis, Arachis hypogaea, Sambucus
nigra,
7
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
Erythrina cristagalli, Lens culinaris, Glycine max, Phaseolus vulgaris,
Allomyrina
dichotoma, Dolichos biflorus, Lotus tetragonolobus, Ulex europaeus, and
Ricinus communis.
Other biologically active carbohydrate-binding compounds include cytokines,
chemokines and growth factors. These compounds are also considered to be
lectins for this
patent application.
Examples of glycosidases include a-Galactosidase, (3-Galactosidase, N-
acetylhexosaminidase, a-Mannosidase, ~i-Mannosidase, a-Fucosidase, and the
like. Some of
these enzymes may, depending upon the source of isolation thereof, have a
different
specificity. The above enzymes are commercially available, e.g., from Oxford
Glycosystems
Ltd., Abingdon, OX14 1RG, UK, Sigma Chemical Co., St. Lois, Mo., USA, or
Pierce, POB.
117, Rockford, 61105 USA.
The saccharide-binding agent can also be a cleaving agent. A "cleaving agent"
is an
essentially sequence-specific agent that cleaves the saccharide chain at its
recognition
sequence. Typical cleaving agents are glycosidases, including exo- and
endoglycosidases,
and glycosyltransferases. However, chemical reagents capable of cleaving a
glycosidic bond
may also serve as cleaving agents, as long as they are essentially sequence-
specific. The term
"cleaving agent" or "cleavage agent" is within the context of this
specification synonymous
with the term "essentially sequence-specific agent capable of cleaving".
The cleaving agent may act at a recognition sequence. A "recognition sequence"
as
used herein is the sequence of monosaccharides recognized by an essentially
sequence-
specific agent. Recognition sequences usually comprise 2-4 monosaccharide
units. An
example of a recognition sequence is Ga1~31-3 GaINAc, which is recognized by a
lectin
purified from Arachis hypogaea. Single monosaccharides, when specifically
recognized by
an essentially sequence-specific agent, may, for the purpose of this
disclosure, be defined as
recognition sequences.
The reaction conditions for the various essentially sequence-specific agents
are known in the art. Alternatively, the skilled person may easily perform a
series of tests with each essentially sequence-specific agent, measuring the
binding activity thereof, under various reaction conditions. Advantageously,
knowledge of reaction conditions under which a certain essentially sequence-
specific agent will react, and of conditions under which it remain inactive,
may
be used to control reactions in which several essentially sequence-specific
8
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
reagents are present. For example, the second and third sequence-specific
reagents may be added to the reaction simultaneously, but via a change in
reaction conditions, only the second essentially sequence-specific agent may
be
allowed to be active. A further change in reaction conditions may then be
selected in order to inactivate the second essentially sequence-specific agent
and
activate the third essentially sequence-specific agent. Some illustratix~e
examples of reaction conditions are listed in the Table 1 below. In addition
to the
pH and temperature data listed in Table 1, other factor, e.g. the presence of
metals such as Zn, or salts of cations such as Mn, Ca, Na, such as sodium
chloride salt, may be investigated to find optimum reaction conditions or
conditions under which certain essentially sequence-specific agent will be
acti.~e
while others are inactive.
9
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
Table 1:
Reaction conditions for some essentially sequence-specific agents
codes for Condition pH Temp Enzymes)
condition sets serial (C)
number
1 3.5 30 Jackbean (3-
galactosidase
2 ' 5.0 37 Endo a-N
Acetylgalactosidase
a 1,2 Fucosidase
(31,2 galactosidase
3 5.0 25 Bovine kidney a
Fucosidase
4 7.2 25 Coffee bean a
galactosidase
5 5.8 55 B. Fragilis
Endo (3-galactosidase
6 6.2 25 Chicken egg lysozyme
7 4.3 37 Bovine testes /3 1-3,4,6,
Galactosidase
from 2- 50 Gly 001-02
Biodiversa 9.5
from 3.0- 50 Gly 001-04
Biodiversa 8.0
from 2-11 50 Gly 001-06
Biodiversa
Symbols represent enzyme groups which are separable by external conditions.
Diversa Corp. produces Thermophilic Endo/Exo glycosidases with a wide
variety of activity in various pH and Temperatures
CA 02407272 2002-11-O1
WO 01/84147 PCTNSOOI30402
The first saccharide-binding agent may be immobilized using any art-recognized
method. For example, immobilization may utilize functional groups of the
protein, such as
amino, carboxy, hydroxyl, or thiol groups. For instance, a glass support may
be
functionalized with an epode group by reaction with epoxy silane, as described
in the above
PCT publication. The epode group reacts with amino groups such as the free s-
amino groups
of lysine residues. Another mechanism consists in covering a surface with
electrometer
materials such as gold, as also described in the PCT publication. As such
materials form
stable conjugates with thiol groups, a protein may be linked to such materials
directly by free
thiol groups of cysteine residues. Alternatively, thiol groups may be
introduced .into the
protein by conventional chemistry, or by reaction with a molecule that
contains;one or more
thiol groups and a group reacting with free amino groups, such as the N-
hydroxyl
succinimidyl ester of cysteine. Also thiol-cleavable cross-linkers, such as
dithiobis(succinimidyl propionate) may be reacted with amino groups of a
protein: ~A
reduction with sulfhydryl agent will then expose free thiol groups of the
cross-linker.
The label attached to the second detectable label can be any label that is
detected, or is
capable of being detected. Examples of suitable labels include, e.g.,
chromogenic label, a
radiolabel, a fluorescent label, and a biotinylated label. Thus, the label can
be, e.g., colored
lectins, fluorescent lectins, biotin-labeled lectins, fluorescent labels,
fluorescent antibodies,
biotin-labeled antibodies, and enzyme-labeled antibodies. In preferred
embodiments, the
label is a chromogenic label. The term "chromogenic binding agent" as used
herein includes
all agents that bind to saccharides and which have a distinct color or
otherwise detectable
marker, such that following binding to a saccharide, the saccharide acquires
the color or other
marker. In addition to chemical structures having intrinsic, readily-
observable colors in the
visible range, other markers used include fluorescent groups, biotin tags,
enzymes (that may
be used in a reaction that results in the formation of a colored product),
magnetic and isotopic
markers, and so on. The foregoing list of detectable markers is for
illustrative purposes only,
and is in no way intended to be limiting or exhaustive. In a similar vein, the
term "color" as
used herein (e.g. in the context of step (e) of the above described method)
also includes any
detectable marker.
The label may be attached to the second saccharide-binding agent using methods
known in the art. Labels include any detectable group attached to the
saccharide or
11
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
essentially sequence-specific agent that does not interfere with its function.
Labels may be
enzymes, such as peroxidase and phosphatase. In principle, also enzymes such
as glucose
oxidase and (3-galactosidase could be used. It must then be taken into account
that the
saccharide may be modified if it contains the monosaccharide units that react
with such
S enzymes. Further labels that may be used include fluorescent labels, such as
Fluorescein,
Texas Red, Lucifer Yellow, Rhodamine, Nile-red, tetramethyl-rhodamine-5-
isothiocyanate,
1,6-diphenyl-1,3,5-hexatriene, cis-Parinaric acid, Phycoerythrin,
Allophycocyanin, 4',6-
diamidino-2-phenylindole (DAPI), Hoechst 33258, 2-aminobenzamide, and the
like. Further
labels include electron dense metals, such as gold, ligands, haptens, such as
biotin,
radioactive labels.
The second saccharide-binding agent can be detected using enzymatic labels.
The
detection of enzymatic labels is well known in the art of ELISA and other
techniques where
e~ymatic detection is routinely used. The enzymes are available commercially,
e.g., from
companies such as Pierce.
In some embodiments, the label is detected using fluorescent labels.
Fluorescent
labels require an excitation at a certain wavelength and detection at a
different wavelength.
The methods for fluorescent detection are well known in the art and have been
published in
many articles and textbooks. A selection of publications on this topic can be
found at p. O-
124 to O-126 in the 1994 catalog of Pierce. Fluorescent labels are
commercially available
from Companies such as SIGMA, or the above-noted Pierce catalog.
The second saccharide-binding agent may itself contain a carbohydrate moiety
and/or
protein. Coupling labels to proteins and sugars are techniques well known in
the art. For
instance, commercial kits for labeling saccharides with fluorescent or
radioactive labels are
available from Oxford Glycosystems, Abingdon, UK. Reagents and instructions
for their use
for labeling proteins are available from the above-noted Pierce catalog.
Coupling is usually carried out by using functional groups, such as hydroxyl,
aldehyde, keto, amino, sulfliydryl, carboxylic acid, or the like groups. A
number of labels,
such as fluorescent labels, are commercially available that react with these
groups. In
addition, bifunctional cross-linkers that react with the label on one side and
with the protein
or saccharide on the other may be employed. The use of cross-linkers may be
advantageous
in order to avoid loss of function of the protein or saccharide.
12
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
The label can be detected using methods known in the art. Some detection
methods
are described in the above-noted WO 93/22678, the disclosure of which is
incorporated
herein in its entirety. Particularly suitable for the method of the present
invention is the CCD
detector method, described in the publication. This method may be used in
combination with
labels that absorb light at certain frequencies, and so block the path of a
test light source to
the VLSI surface, so that the CCD sensors detect a diminished light quantity
in the area
where the labeled agent has bound. The method may also be used with
fluorescent labels,
making use of the fact that such labels absorb light at the excitation
frequency. Alternatively,
the CCD sensors may be used to detect the emission of the fluorescent label,
after excitation.
Separation of the emission signal from the excitation light may be achieved
either by using
sensors with different sensitivities for the different wavelengths, or by
temporal resolution, or
a combination of both.
In some embodiments, the method further includes acquiring one or more images
of
the first saccharide-binding agent and the saccharide-binding agent. The
information can be
is stored, e.g., as a photograph or digitized image. Alternatively, the
information provided by
the first and second binding image can be stored in a database.
The invention also includes a substrate that includes a plurality of
complexes. Each
complex includes a first saccharide-binding agent bound to a predetermined
location on the
substrate. The substrate can also optionally include a saccharide bound to the
first
saccharide-binding agent and/or a detectable second saccharide-binding agent.
In some
embodiments, the substrate is provided in the form of a solid support that
includes in a pre-
defined order a plurality of visual or otherwise detectable markers
representative of a
saccharide or saccharide sequence or fragment.
If desired, a substrate containing a plurality of first saccharide-binding
agents can be
provided in the form of a kit. Diagnostic procedures using the methods of this
invention may
be performed by diagnostic laboratories, experimental laboratories,
practitioners, or private
individuals. This invention provides diagnostic kits which can be used in
these settings. The
presence or absence of a particular carbohydrate polymer, as revealed by its
pattern of
reacting with saccharide binding agent, may be manifest in a provide sample.
The sample
can be, e.g., clinical sample obtained from that an individual or other
sample.
Each kit necessarily comprises saccharide-binding agent or agents which
renders the
procedure specific. The reagent is preferably supplied in a solid form or
liquid buffer that is
13
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
suitable for inventory storage, and later for exchange or addition into the
reaction medium
when the test is performed. Suitable packaging is provided. The kit may
optionally provide
additional components that are useful in the procedure. These optional
components include
buffers, capture reagents, developing reagents, labels, reacting surfaces,
means for detection,
control samples, instructions, and interpretive information.
The kit may optionally include a detectable second saccharide-binding agent
and, if
desired, reagents of detecting the second binding agent. The plurality of
first saccharide-
binding agents are preferably attached at predetermined location on the
substrate and a
detectable second saccharide-binding agent. In other embodiments, the kit is
provided with a
substrate and first saccharide-binding agents that can be attached to the
substrate, as well as
second saccharide-binding agents.
Generating fin,~erprints of carbohydrate polymers
The method and reagents described above can be used to generate a fingerprint
of a
carbohydrate polymer. As used herein, a fingerprint of a carbohydrate polymer
is a
compilation of information about the binding status of the carbohydrate
polymer and a
plurality of scattered-binding agents. In some embodiments, the fingerprint is
a numeric
representation of the detection of the presence of binding by the saccharide-
binding agents to
the carbohydrate polymer.
The fingerprint of the carbohydrate polymer can be generated by contacting the
carbohydrate polymer with a first saccharide-binding agent and determining
whether the
carbohydrate polymer binds to the saccharide-binding reagent. The carbohydrate
polymer is
also contacted with a second saccharide-binding agent, and a determination is
made as to
whether the second binding-agent binds to the carbohydrate polymer.
The carbohydrate polymer is preferably contacted with at least five saccharide-
binding agents, and a determination is made as to whether the carbohydrate
polymer binds to
each of the at least five saccharide-binding reagents. In preferred
embodiments, the binding
of the carbohydrate polymer to at least 10, 15, 20, or 25 or more agents is
determined.
In preferred embodiments, binding of the first and second saccharide-agent is
determined by providing a surface comprising at least one first saccharide-
binding agent
attached to a predetermined location on the surface and contacting the surface
with a
carbohydrate polymer under conditions allowing for the formation of a first
complex between
14
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
the first saccharide-binding agent and the carbohydrate polymer. Unbound
polymer is
removed if desired and the surface is contacted with at least one second
saccharide-binding
agent under conditions allowing for formation of a second complex between the
first complex
and the second saccharide-binding agent. The first and second saccharide-
binding agent are
S then identified, and the information generated provides a fingerprint for
the carbohydrate
polymer. By including a plurality of first and/or second saccharide-binding
agents, it is
possible to generate a detailed fingerprint of the carbohydrate polymer. Of
course, it will be
apparent to one of ordinary skill in the art that the absence of binding of a
first or second
saccharide-agent to a carbohydrate polymer will also contribute to the
fingerprint generated
for the polysaccharide.
The second saccharide agent preferably contains a detectable label. When the
second
saccharide-binding agent is labeled, the identity of the second label
determines the identity of
the second saccharide-binding agent. The position of the second label on the
substrate in turn
reveals the identity of the first saccharide-binding agent.
The invention will be further illustrated in the following examples, which do
not limit
the scope of the appended claims.
Example 1
G(ycomolecule analysis using antibodies as first and second sequence-specific
agents
This example further illustrates the technique of analyzing glycomolecules
according
to the invention. As a first and second sequence-specific agent. antibodies
are used. The
following tables lists the results of reactions with two different saccharides
denoted for
purposes of illustration, HS and NS.
The structure of the sugars is as follows:
MFLNH-II (HS):
X Fuca(1-3)
Le I Gal(3(1-4) lcNAc(3 (1- ~, Gal(3(1-4) Glc ~---
T
SUBSTITUTE SHEET (RULE 26)
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
NS:
Lex FUCa(1-3) ~ICNAC(3 (1-
Gal 1-4
Gal~3(1-4) Gic ~~~
Leb Fuca(1-2)Gal~(1- ~ICNAc(i (1-
Table 2 lists the results of the reaction between the saccharide and the first
and second
essentially sequence-specific agents, which are antibodies against T-antigen,
Lewis" (Le"), or
Lewisb antigen (Leb). The first essentially sequence-specific' agent is
immobilized on a
matrix, preferably a solid phase microparticle. The second essentially
sequence-specific agent
is labeled with a fluorescent agent, i.e., nile-red or green color. In
addition, the reducing end
of the saccharide is labeled, using a label clearly distinguishable from the
nile-red or green
color label which act as markers for the second essentially sequence-specific
agents. Table 2
lists the reactions for the saccharide HS, while Table 3 lists the reactions
for the saccharide
NS.
Table 2
On the matrix anti T-antigen Anti - Le anti - Le
Saccharide boundHS HS
Second mAb nile-red anti
- Le"
Signal rile-red, reducingReducing end none
end
Table 3
16
SUBSTITUTE SHEET (RULE 26)
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
On the matrix anti T-antigen Anti - Le anti - Le
Saccharide bound NS NS
Second mAb Green anti-Le nile-red anti
- Le"
Signal Green, reducingnile-red, reducing
end end
In summary, the following signals are now detectable in the reactions of the
saccharide HS or NS (rows) when using the indicated antibodies as first
essentially sequence-
specific agent (columns):
Table 4
On the matrix anti T-antigen Anti - Le anti - Le
HS nile-red, reducingReducing end
end
NS Green, reducingnile-red, reducing
end end
NS Green, reducingnile red, reducing
end end
After the label has been detected and the result recorded for each reaction, a
third
essentially sequence-specific agent is added. In this example, two independent
reactions with
a third essentially sequence-specific agent are used. The solid phase carrying
the sugar
molecule may now be advantageously divided into aliquots, for reaction with
either al-2
Fucosidase or Exo (3 galactosidase (third essentially sequence-specific
agents). Alternatively,
three sets of reactions with a first and second essentially sequence-specific
agent may be
carried out.
Table 5:
reactions after applying al-3,4 Fucosidase:
On the matrix anti T-antigen Anti - Le anti - Len
17
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
HS reducing end
NS
Table 6:
reaction after applying Exo (3 galactosidase from D. pneumoniae (EC 3.2.1.23
catalog
number 1088718 from Boehringer Mannheim, 68298 Mannheim, Germany)
On the matrix anti T-antigen Anti - Le anti - Le
HS nile-red
NS Green nile-red
Table 7:
reactions after applying al-2 Fucosidase:
On the matrix anti T-antigen Anti - Le anti - Le
HS nile-red, reducingReducing end
end
NS Reducing end
From the data gathered as explained above, a glycomolecule identity (GMID)
card
can now be created. An example for such information is listed in Table 8 for
saccharide HS
and in Table 9 for saccharide NS.
18
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
Table 8
On the matrix anti T-antigen Anti - Le" anti - Le
0 nile-red, reducingReducing end
end
1 reducing end - -
2 nile-red
3 nile-red, reducingReducing end
end
Table 9
On the matrix anti T-antigen Anti - Le~' anti - Le
0 Green, reducingnile red, reducing
end end
1 - - -
2 Green nile red
3 Reducing end
The identity of the second and third essentially sequence-specific agents need
not be
disclosed in such a data list. For the purpose of comparison, it is sufficient
that a certain code
number (1, 2 or 3 in the above tables) always identifies a certain combination
of reagents.
Example 2
A scheme for the sequential labeling of reducing ends
As has been indicated in the description and example above, the method of the
invention advantageously uses labeling of the saccharide to be investigated at
its reducing
end. However, this labeling technique may be extended to sites within the
saccharide, and
19
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
thus contribute to the method of the invention, by providing more information.
As it is
possible to label the saccharide within the chain, by cleavage using an
endoglycosidase
followed by labeling of the reducing end, it is therefore possible to obtain a
labeled reducing
end within the saccharide chain. As that reducing end is necessarily closer to
the binding sites
for the first, second and third essentially sequence-specific agents, compared
to the original
reducing end, the use of an internally created labeled reducing end provides
additional
information. Moreover, it is possible, by sequentially labeling of reducing
ends according to
the method described further below, to identify the sites for distinct
glycosidases in sequential
order on the chain of the saccharide to be investigated.
The method of sequential labeling of reducing ends is now described in more
detail in
the following steps:
1. Blocking:
A polysaccharide having a reducing end is incubated in a solution containing
NaBH4 /
NaOH at pH 11.5.
This treatment blocks the reducing end, so that the polysaccharide is now
devoid of a
reducing end (RE).
2. Exposing
The polysaccharide of step 1 is treated with an endoglycosidase. If the
recognition site
for that endoglycosidase is present within the polysaccharide, a new reducing
end will be
created by cleavage of the polysaccharide. The solution now contains two
saccharides: the
fragment with the newly exposed RE in the endoglycosidase site, and the second
fragment
whose RE is blocked.
3: Labeling of the reducing, end
This reaction may be carried out using e.g., 2-aminobenzamide (commercially
available in kit form for labeling saccharides by Oxford Glycosystems Inc.,
1994 catalog, p.
62). After the reaction under conditions of high concentrations of hydrogen
and in high
temperature (H+/T), followed by reduction, has been completed, the mixture
contains two
fragments, one of which is labeled at its reducing end, while the other
remains unlabeled due
to the fact that its reducing end is blocked.
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
Another way to label reducing ends is by reductive amination. Fluorescent
compounds containing arylamine groups are reacted with the aldehyde
functionality of the
reducing end. The resulting CH=N double bond is then reduced to a CH2-N single
bond, e.g.,
using sodium borohydride. This technology is part of the FACE (Fluorophore
assisted
Carbohydrate Electrophoresis) kit available from Glyko Inc., Novato, CA, USA,
as detailed
e.g., in the Glyko, Inc. catalog, p. 8-13, which is incorporated herein by
reference.
4. Reaction with a second endog_lycosidase
A second endoglycosidase may now be reacted with the saccharide mixture. The
new
reaction mixture has now three fragments, one with an intact reducing end, a
second with a
reducing end labeled by 2-aminobenzimide, and a third with a blocked reducing
end.
Example 3
Derivation of structural information from a series of reactions with
essentially
sequence-specific agents
This example further illustrates the method of the invention, i. e., the
generation of
data related to the structure of the saccharide by using a set of reactions as
described further
above. The example further demonstrates that sequence information can be
deduced from
said set of reactions.
In some cases, the reagents used may not react exactly as predicted from
published
data, e.g. taken from catalogs. For instance, the lectin Datura stramonium
agglutinin as
described further below is listed in the Sigma catalog as binding GlcNac.
However, in the
reactions detailed further below, DSA is shown to bind to Coumarin 120-
derivatized Glc
(Glc-AMC). It appears that Glc-AMC acts like GlcNac for all purposes, because
of the
structural similarity between these compounds. Further, as apparent from the
results below,
the endogalactosidase used cleaves not only at galactose residues, but also
the bond
connecting the Glc-AMC group to the rest of the saccharide.
It is apparent that the essentially sequence-specific agents used in the
practice of the
invention may in some cases have fine specificities that vary from the
specificity of these
agents given in published material, e.g., catalogs. Such reactions can quickly
be identified by
21
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
using the method of the invention with saccharides of known structure. The
results found
may then be compared with expected results. and the differences will allow the
identification
of variant specificities of the essentially sequence-specific agents used.
Such variation from
published data in fine specificities of essentially sequence-specific agents
may then be stored
for future analysis of unknown saccharides structures using these agents.
In the following, the method of the invention is illustrated using an end-
labeled
pentasaccharide and various lectins and glycosidases. The pentasaccharide has
the structure
Gal-[3(1,4)[Fuc-a(1,3)]-GIcNAc-[i(1,3)-Gal[i(1,4)-Glc. The pentasaccharide is
branched at
The GIcNAc position having fucose and galactose bound to it in positions 3 and
4
respectively. The pentasaccharide is labeled at its reducing end (Glc) with
Coumarin-120 (7-
amino-4-methyl coumarin, available, e.g., from Sigma, catalog No. A 9891). The
coupling
reaction may be carried out as described above for the labeling of reducing
ends by using
arylamine functionalities. Coumarin-120, when excited at 312 nm emits blue
fluorescence.
As first and second essentially sequence-specific agents, Endo-(3-
Galactosidase (EG,
Boehringer Mannheim) and Exo-1,3-Fucosidase (FD, New England Biolabs) are
used. The
reaction conditions for both reagents are as described in the NEB catalogue
for Exo-1,3-
Fucosidase.
Three reactions were carried out. The first included Fucosidase (FD) and Endo-
Galactosidase (EG), the second, FD only, and the third, EG only. A fourth
reaction devoid of
enzyme served as control.
In order to ascertain that the enzymes had digested the saccharide, the
various
reactions are size-separated using thin-layer chromatography (TLC).
After separation, the saccharides on the TLC plate may detected by exposing
the plate
to ultraviolet light. The results are shown in the following illustration.
v',..
~ ''~'
2 3 4
22
SUBSTITUTE SHEET (RULE 26)
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
In reaction 4, no glycosidase was added, so the saccharide is intact and moves
only a
small distance on the plate. The fragment of reaction 2 is second in molecular
weight, while
the fragments of reactions l and 3 appear to be equal. From these data, it can
be concluded
that the sequence of the glycosidase sites on the saccharide is FD--EG--
reducing end
(coumarin-label).
The above pentasaccharide is now tested by a set of reactions as described
further
above. As first and second essentially sequence-specific agents, lectins were
used. The lectins
(Anguilla Anguilla agglutinin (AAA), catalog No. L4141, Arachis Hypogaea
agglutinin
(PNA), catalog No. L0881, Ricinus communis agglutinin (RCA I) catalog No.
L9138, Lens
Culinaris agglutinin (LCA) catalog No. L9267, Arabs Precatorius agglutinin,
(APA). catalog
No. L9758) are available from Sigma. Lectins are also available from other
companies. For
instance, RCA I may be obtained from Pierce, catalog No. 39913. Lectins are
immobilized by
blotting onto nitrocellulose filters.
The reaction buffer is phosphate-buffered saline (PBS) with 1mM CaCI and 1mM
MgCI. After binding of the lectins, the filter was blocked with 1% BSA in
reaction buffer. As
controls, reactions without lectin and with 10 ~.g BSA as immobilized protein
were used.
The results of the reactions are indicated in Table 10. A plus indicates the
presence of
312 nm fluorescence, which indicates the presence of the coumarin-labeled
reducing end. The
numerals 1-4 in the table indicate reactions as defined above.
Table 10
AAA PNA LCA DSA RCAI
1 ++
2 ++ ++ ++
3 ++
4 ++ ++ ++ ++
From the results as listed in Table 10 (reaction 4-control) it is evident that
lectins
AAA, PNA, DSA and RCA-I bind the saccharide. Therefore, Fucose, Gal(1-
3)GIcNAc,
23
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
GIcNAc, and Galactose/GaINAc must be present in the saccharide, as these are
the respective
saccharide structures that are recognized by AAA, PNA, DSA and RCA-I. It is
further
evident that the above described glycosidases Fucosidase and Endo-(3-
Galactosidase
recognize cleavage sequences in the saccharide. These sequences are Fuc (1-3/1-
4) GIcNAc
and GIcNAc(3(1-3)Gal(3(1-3/4)Glc/GIcNAc, respectively.
It can further be deduced that both glycosidase sites are located between the
fucose
sugar and the reducing end, as said end is cleaved by either glycosidase when
AAA (which
binds to fucose) is used as immobilized lectin. The reaction with DSA, on the
other hand,
allows the deduction that either the GIcNAc monosaccharide is located between
the
glycosidase sites and the reducing end, or that Glc is directly bound to the
coumarin, as
neither glycosidase cleaves off the reducing end when DSA is used as
immobilized agent.
Moreover, the reaction with PNA as immobilized agent shows that the reducing
end is
cleaved only if Endo-~iGalactosidase is used (reactions 1 and 3). This
indicates that the Endo-
(3Galactosidase site is located between the site for PNA and the reducing end.
On the other
hand, the Fucosidase site must be located between the PNA site and the other
end of the
saccharide.
When taking into account the above data, it is now possible to propose a
sequence of
the saccharide as follows:
Fuca(1-3,1-4)GIcNAc(1-3)Gal(1-4)Glc/GIcNAc-~~~~~reducing end
The above experiment clearly demonstrates that the method of the invention can
yield
a variety of data, including sequence information, based upon relatively few
reactions. Some
details in the sequence information may not be complete, such as the (1-3) or
(1-4)
connection between Fucose and GIcNAc in the above saccharide. Had the
monosaccharide
composition of the pentasaccharide been known, then the above analysis would
have yielded
all of the details of said pentasaccharide. Nevertheless, the information
gained even in the
absence of the monosaccharide composition data is very precise compared to
prior art
methods.
24
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
Example 4
Derivation of partial or complete sequence information
The method of the invention is suitable for automation. Thus, the steps
described
above, for example, in examples 1 to 3, may be carried out using an automated
system for
mixing, aliquoting, reacting, and detection. The data obtained by such an
automated process
may then be further processed in order to "collapse" the mapping information
to partial or
complete sequence information. The method for such data processing is
described in further
detail below.
After all data have been collected, a comparison is made between detection
signals
obtained from reactions prior to the addition of glycosidase, to signals
obtained after the
addition (and reaction with) of glycosidase: Those signals that disappear
after reaction with
glycosidase are marked. This may advantageously be done by preparing a list of
those
signals, referred to hereinafter as a first list. The identity of two sites on
the polysaccharide
may now be established for each such data entry. The position in the
(optionally virtual) array
indicates the first essentially sequence-specific agent. If a signal has been
detected before
reaction with the glycosidase, the recognition site for that agent must exist
in the
polysaccharide. The disappearance of a signal, for instance, of the signal
associated with the
second essentially sequence-specific agent, now indicates that the glycosidase
cleaves
between the recognition sites of the first and second essentially sequence-
specific agents. The
sequence of recognition sites is therefore (first essentially sequence-
specific agent)-
(glycosidase)-(second essentially sequence-specific agent). If the signal for
the reducing end
is still present after digestion with the glycosidase, then the relative order
of the recognition
sequences with respect to the reducing end can be established; otherwise, both
possibilities
(a-b-c and c-b-a) must be taken into account. For the purpose of illustration,
the term
"recognition site of the first essentially sequence-specific agent" shall be
denoted in the
following "first recognition site", the term "recognition site for the second
essentially
sequence-specific agent" shall be denoted "second recognition site", and the
term
"recognition site for glycosidase" shall be denoted "glycosidase".
It is now possible to create a second list of triplets of recognition sites of
the above
type (type 1 triplets):
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
(first recognition site)-(glycosidase)-(second recognition site).
Similarly, a third list can now be created relating to (optionally virtual)
array
locations where all signals remain after addition of glycosidase (type 2
triplets):
(glycosidase)-(first recognition site)-(second recognition site)
Obviously, a sufficient number of triplets defines a molecule in terms of its
sequence,
i.e., there can only be one sequence of saccharides that will contain all of
the triplets found. A
lower number of triplets may be required when information on the length of the
molecule is
available. The number of required triplets may be even lower if the total
sugar content of the
molecule is known. Both saccharide molecular weight and total monosaccharide
content may
be derived from prior art methods well known to the skilled person.
The process of obtaining sequence information, i.e., of collapsing the
triplets into a
map of recognition sites, is described below.
The second and third lists of triplet recognition sites are evaluated for
identity (three
out of three recognition sites identical), high similarity (two out of three
recognition sites
identical), and low similarity (one out of three recognition sites identical).
For the purposes of
illustration, it is now assumed that the polysaccharide is a linear
polysaccharide, such as, for
example, the saccharide portion of the glycan heparin.
The above second and third lists are then used to prepare therefrom a set of
lists of
triplets wherein each list in said set of lists contains triplets that share
the same glycosidase
recognition sequence. By comparing all triplets containing a certain
glycosidase recognition
sequence with all triplets containing a second glycosidase recognition
sequence, it is now
possible to divide the polysaccharide sequence into four areas, ranging from
the first end of
the molecule to glycosidase 1 (fragment a), from glycosidase 1 to glycosidase
2 (fragment b),
and from glycosidase 2 to the second end of the molecule (fragment c):
<first end> <glycosidase 1 > <glycosidase2> <second end>
Identical recognition sites within triplets of type 2 with different
glycosidase sites,
wherein said recognition sites are located in the same direction in relation
to the respective
26
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
glycosidase site, are candidates for the location within either the area a or
c, depending on
said location. Identical recognition sites within triplets of type 2 with
different glycosidase
sites, wherein said recognition sites are located in different directions
(e.g., one in the
direction of the reducing end, in the other triplet, in the direction of the
non-reducing end),
are candidates for the location within the area b, i.e., between the two
glycosidase sites.
Identical recognition sites within triplets of type 1 with different
glycosidase sites are
candidates for the location of one of the first or second recognition sites in
area a (or c), and
the other of said first or second recognition sites being located in the area
c (or a). That is, if
one of the first or second recognition sites is located in area a, then the
other of said first or
second recognition sites must be located in area b, and vice versa. None of
the said first or
second recognition sites may be located in area b.
Identical recognition sites within triplets of type 1 with different
glycosidase sites,
wherein a given recognition site is located in one of the triplets, in the
direction of the
reducing end and in the other triplet, in the direction of the non-reducing,
are candidates for
the location of said recognition site within area b.
Having established the above positional relationships for a number of
recognition
sites within the triplets, the total of the recognition sequences can now be
arranged in a
certain order using logical reasoning. This stage is referred to as a sequence
map. If a
sufficient number of recognition sequences are arranged, the full sequence of
the saccharide
may be derived therefrom. As the method does not determine the molecular
weight of the
saccharide, the chain length is unknown. Therefore, if the degree of overlap
between the
various recognition sites is insufficient, there may be regions in the
sequence where
additional saccharide units may be present. Such saccharide units may be
undetected if they
do not fall within a recognitiomsite of any of the essentially sequence-
specific agents used.
However, the entire sequence information may also be obtained in this case, by
first obtaining
the molecular weight of the saccharide, which indicates its chain length, and
secondly its total
monosaccharide content.
Another possibility of closing gaps in the sequence map is the method of
example 2,
wherein sequential degradation by glycosidase is employed to derive sequence
information.
The existence of branching points in the saccharide may complicate the method
as
outline above. One remedy to that is to use glycosidases to prepare fractions
of the molecule,
and analyze these partial structures. The extent of branching in such partial
structures is
27
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
obviously lower than in the entire molecule. In addition, reagents may be
employed that
specifically recognize branching points. Examples for such reagents are e.g.,
the antibodies
employed in example 1 above. Each of these antibodies binds a saccharide
sequence that
contains at least one branching point. Moreover, certain enzymes and lectins
are available
that recognize branched saccharide structures. For instance, the enzyme
pullanase (EC
3.2.1.41 ) recognizes a branched structure. In addition, antibodies may be
generated by using
branched saccharide structures as antigens. Moreover, it is possible to
generate peptides that
bind certain saccharide structures, including branched structures (see e.g.,
Deng SJ,
MacKenzie CR, Sadowska J, Michniewicz J, Young NM, Bundle DR, Narang;
Selection of
antibody single-chain variable fragments with improved carbohydrate binding by
phage
display. J. Biol. Chem. 269, 9533-38, 1994) .
In addition; knowledge of the structure of existing carbohydrates will in many
cases
predict accurately the existence of branching points. For instance, N-linked
glycans possess a
limited number of structures, as listed at p. 6 of the oxford Glycosystems
catalog. These
structures range from monoantennary to pentaantennary. The more complicated
structures
resemble simpler structures with additional saccharide residues added.
Therefore, if
monoantennary structure is identified, it is possible to predict all of the
branching points in a
more complicated structure, simply by identifying the additional residues and
comparing
these data with a library of N-linked glycan structures.
Moreover, it will often be possible by analyzing data gathered according to
the
method of the invention, to deduce the existence and location of branching
points logically.
For instance, if two recognition sites, denoted a and b, are located on
different branches, then
digesting with a glycosidase whose site is located between the reducing end
and the
branching point will result in loss of the reducing end marker. The markers
for both
recognition sites a and b, however, will remain. If a glycosidase located
between the
branching point and recognition site a is used, then the marker for
recognition site b and the
reducing end marker will be cleaved off. Not taking into account the
possibility of branching
points, this would indicate that the recognition site b is located between the
recognition site a
and the reducing end. However, if a glycosidase located between the
recognition site b and
the branching point is used, the reducing end marker and recognition site a
will be cleaved
off. Again, not taking into account the possibility of branching, this would
indicate that
recognition site a is located between the reducing end and recognition site b.
These
28
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
deductions are obviously incompatible with one another, and can only be
resolved if one
assumes that recognition sites a and b are located on two different branches.
The branching
point is located between the recognition sites a and b and the first of the
above glycosidases.
The other above glycosidases used are located on a branch each, between the
branching point
S and the respective recognition site (a or b).
Therefore, when using agents that recognize branched structures in the method
of the
invention, as essentially sequence-specific agents, it is possible to derive
information on the
existence and location of branching points in the saccharide molecule. This
information can
then be used to construct sequence maps of each branch of the structure,
yielding a sequence
map of the entire branched structure. The gaps in such a structure may then be
closed as in
the case of unbranched saccharides, according to the invention, i.e., by using
additional
reactions, by digestion with glycosidases, whereby the regions of the molecule
where gaps
exist are specifically isolated for further analysis according to the method
of the invention;
and by sequential glycosidase digestion as described further above.
In summary, a method for determining the sequence of a saccharide and/or for
mapping the structure of said saccharide according to the invention comprises
the steps of:
1. collecting triplets of type l and type 2
2. sorting said triplets according to similarity
3 . comparing triplets with different glycosidase recognition sites
4. arranging the triplets in the order of occurrence on the saccharide
5 . arranging the glycosidase recognition sites
6. checking the compatibility to the triplets
7. arranging recognition sequences of glycosidases and of first and second
essentially
sequence-specific agents in a single file order
29
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
8. translating the recognition sequences (sites) into polysaccharide sequence
9. correcting "overlap" problems
10. outputting a sequence
11. checking against all available data
After the above step 5 has been carried out, a preliminary order of
glycosidase sites
has been established. In step 6, it is now checked for each triplet whether
predictions based
thereon are in agreement with that order. Then, based on contradiction in the
data, a new
model is generated that fits the data of the triplet. This model is then
tested against the data of
all triplets. Furthermore, additional reactions may be carried out, in order
to extract additional
vectorial information regarding the recognition sites that involve said
triplet.
After the above step 8, wherein the sequentially arranged recognition sites
are
translated into a sequence of actual monosaccharide units, a model of the
saccharide sequence
can be suggested. In order to test said model, a number of questions needs to
be answered.
The first of these is, what is the minimum sequence that would still have the
same sequence
map? At this stage, information on molecular weight and monosaccharide
composition, if
available, is not taken into account. This approach merely serves the creation
of a sequence
which incorporates all of the available data with as few as possible
contradictions. In that
respect, the second question to be answered is, does the minimum sequence
still agree with
all of the data available at that point (excluding optional molecular weight
and
monosaccharide composition data)? The third question to be answered is, do
other sequences
exist that would fit the sequence map as established? In the affirmative, the
additional
sequences may then be tested using the question: How does each sequence model
agree with
the triplet information, and with additional optional data, such as
information on the
molecular weight, monosaccharide composition, and model saccharide structures
known
from biology.
Finally, the sequence model that has been found to be best according to the
steps 1-10
described above, will then be tested against all triplets, monosaccharide
composition, prior
knowledge on the molecular weight and structural composition of the
saccharide, and
predictions from biologically existent similar structures. By such repeated
testing, the
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
contradictions between the available data and the sequence model are
identified, and if
possible, the sequence model is adapted to better represent the data.
Example 5
Glycomolecule identity (GMID) analysis of milk samples
The aim of this example is to demonstrate the application of the GMID
technique to
the analysis and comparison of milk samples.
A. Membranes and 1 S' layer lectins:
The supporting surface used in the experiments described hereinbelow is a
nitrocellulose membrane. The membranes were prepared as follows:
1. Nitrocellulose membranes were cut out and their top surface marked out into
an array of
9x6 squares (3mm2 each square). The membranes were then placed on absorbent
paper and
the top left square of each one marked with a pen.
2. Lyophilized lectins were resuspended in water to a final concentration of 1
mg/ml. The
resuspended lectins (and a control solution: 5% bovine serum albumin) were
vortex mixed
and 1 p,1 of each solution is added to one of the 28 squares on the blot,
indicated by shading in
the following illustrative representation of a typical blot:
The lectins used in this experiment are listed in Table 11.
31
SUBSTITUTE SHEET (RULE 26)
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
Table 11
Lectin Manufacturer Cat. No.
WGA Vector MK2000
SBA Vector MK2000
PNA Vector MK2000
DBA Vector MK2000
UEA I Vector MK2000
CON A Vector MK2000
RCA I Vector MK2000
BSL I Vector MK3000
SJA Vector MK3000
LCA Vector MK3000
Swga Vector MK3000
PHA-L Vector MK3000
PSA Vector MK3000
AAA - -
PHA-E Vector MK3000
PNA Leuven LE-408
LCA Sigma L9267
DSA Sigma L2766
APA -
WGA Leuven LE-429
Jacalin Leuven LE-435
5% BSA Savyon M121-033
3. The prepared blots were placed in 90 mm petri dishes.
4. The blots were blocked by adding to each petri dish 10 ml of any suitable
blocking
solution well known to the skilled artisan (e.g. 5% bovine serine albumin).
5. The dishes containing the blots in the blocking solution were agitated
gently by
rotation on a rotating table (50 rpm) for 2 hours at room temperature (or
overnight at 4° C,
without rotation).
6. The blots were then washed by addition of 10 ml washing solution to each
petri dish.
32
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
Any commonly available buffered solution (e.g. phosphate buffered saline) may
be used for
performing the washing steps. The dishes were washed by rotating gently (50
rpm) for 5
minutes. The procedure was performed a total of three times, discarding the
old washing
solution and replacing with fresh solution each time.
B: Addition of milk samples:
The milk samples used were as follows:
1. Bovine UHT long-life milk (3% fat) obtained from Ramat haGolan dairies,
Israel (lot
522104);
2. Pasteurized goat's milk, obtained from Mechek dairies, Israel (lots 1 and
2);
3. Non-pasteurized goat's milked obtained as in 2. (lots 3 and 4).
The milk samples were diluted to 10 % v/v and approximately Sml of each sample
applied to separate blots.
Duplicate blots were prepared for each of the aforementioned milk samples. In
addition a further pair of blots were prepared without the addition of
saccharides (negative
control).
The blots were then incubated at room temperature with agitation for one hour.
C. Colored lectins:
From prior knowledge of the monosaccharide composition of the milks tested,
and by
application of a computer program based on the algorithm described hereinbelow
in Example
7, the following colored lectins were chosen: Con A, VVA.
A mixture of these two lectins was prepared in washing solution, such that the
concentration of each colored lectin was 2 mg/ml.
500 p1 of each lectin mix was incubated on the blots prepared as described
above.
Each blot was read both by measuring the fluorescence of fluorescein at 520
nm, and, in the
case of the biotinylated lectin, measuring the signal of the TMB blue color
produced
following reaction of biotin with an HRP-streptavidin solution
The results obtained for the FITC-labeled and biotin-labeled lectins are given
in
Tables 12 and 13, respectively. The results presented in these tables are
measured on a 0 to 3
scale, wherein 0 represents a signal that is below the noise level, and
wherein results of 1-3
represent positive signals (above noise) following subtraction of the results
obtained in the
no-saccharide control.
33
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
Glycomolecule identity (GMID) cards obtained from these results for
pasteurized
goat's milk (lots 1 and 2), non-pasteurized goat's milk (lots 3 and 4) and
bovine milk are
shown in Fig. 1 (A to E, respectively). The positions of lectins 1 to 24 are
shown in one row
from left to right at the top of each card 1.
D. Interpretation of results:
The bovine milk sample yielded a GMID indicating that the polysaccharide in
the
sample contains saccharides that yield positive results for lectins specific
for:
a. glucose/mannose (ConA, PSA and LCA);
b. GlcNac (WGA and DSA).
The pasteurized goat milk samples yielded positive results for:
a. glucose/mannose (conA, PSA and LCA);
b. GlcNac (DSA).
No difference in lectin reactivity between the lots tested was observed.
The non-pasteurized goat milk sample gave a positive reaction for:
a. glucose/mannose (ConA, PSA and LCA);
b. GlcNac (DSA).
In summary, the bovine milk differed from the goat's milk in that only the
former
reacted with WGA. There was essentially no difference between the pasteurized
and non-
pasteurized goat's milk samples, with the exception that the signal intensity
was significantly
lower in the pasteurized samples.
Example 6
Glycomolecule identity (GMID) analysis of lipopolysaccharides
A GMID analysis was performed on five different bacterial lipopolysaccharides
obtained from Sigma Chemical Co. (St. Louis, Missouri, USA)(LPS#1, 7, 10, 15
and 16),
essentially using the method as described in Example 5, above. The colored
lectins used
were ECL, WGA, VVA and SBA.
The GMID cards obtained for samples LPS# 1, 7, 10, 15 and 16 are shown in Fig.
2
(A to E, respectively). It may be seen from this figure that the GMID cards
provide unique
"fingerprints" for each of the different lipopolysaccharides, and may be used
for identifying
the presence of these compounds in samples containing bacteria or mixtures of
their products.
34
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
Example 7
Method for selecting colored lectins
S A number of factors must be taken into consideration when selecting colored
lectins
for use in the method of polysaccharide analysis illustrated in Examples 5 and
6. Among
these considerations are the need for each of the chosen lectins to have a
distinguishable color
or other detectable marker, and for the need to reduce interactions between
lectins. A flow
chart illustrating an algorithm for use in colored marker selection is shown
in Fig. 3. The
algorithm shown in Fig. 3 begins with the selection of n colored lectins (or
other detectable
markers) 101, said initial selection being made in accordance with information
obtained
about the partial or full monosaccharide composition of the saccharide to be
analyzed.
In the next step 102, the colors of the selected lectins are examined in order
to check
for identity/non-identity of the colors selected. If there are identical
colors in the selected
group, then the process proceeds to step 103, otherwise the flow proceeds with
step 104. In
step 103, one of the lectins that has been found to have a non-unique color is
replaced by
another lectin that belongs to the same binding category (that is, one that
has the same
monosaccharide binding specificity); the flow proceeds to step 102.
In step 104, the n selected lectins are tested in order to detect any cross-
reactivity with
each other, and with the non-colored lectins used in the first stage of the
method described
hereinabove in Example 5. If cross-reactivity is found, then the process
continues to step
105, otherwise the flow proceeds to step 106, where the algorithm ends.
In step 1 O5, one of the lectins determined to cross-react with another lectin
is replaced
by a lectin which does not cross-react; the flow then proceeds to 102. The
algorithm ends
with step 106.
It is to be emphasized that while for values of n which are small, and for
saccharides
with a simple monosaccharide composition, the above-described algorithm may be
applied by
the operator himself/herself manually working through each step of the
selection procedure.
Alternatively (and especially for cases where n is a larger number or the
monosaccharide
composition is more complex), the algorithmic processes described hereinabove
may be
performed by a computer program designed to execute said processes.
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
The above examples have demonstrated the usefulness of the method described
herein. However, they have been added for the purpose of illustration only. It
is clear to the
skilled person that many variations in the essentially sequence-specific
agents used, in the
reaction conditions therefor, in the technique of immobilization, and in the
sequence of
labeling, reaction and detection steps may be effected, all without exceeding
the scope of the
invention.
Other Embodiments
The above Examples describe particular types of fingerprint assays and methods
according to the present invention. These assays may optionally be performed
with a variety
of different configurations for "wet" or experimental assay devices, hardware
and software
programs for gathering and analyzing the data. Figure 4 is a schematic block
diagram of an
exemplary method according to the present invention for performing a
fingerprint assay with
the GMID card, which illustrates one type of systemic configuration and
operation according
to the present invention for performing the fingerprint assay. It should be
noted that this
description is intended as an example only and is not meant to be limiting in
any way.
As shown, in step 1, optionally and preferably, the saccharide-binding agents
are
examined for efficacy before they are used in the assay with the GMID card. In
this example,
the saccharide-binding agents are described as lectins, although of course
other such agents
could optionally be used within the scope of the present invention. More
preferably, each
such lectin is examined for positive activity, most preferably through
reactivity with a
standard glycomolecule. Such reactivity shows that the lectin is capable of
binding to such a
standard glycomolecule in a reproducible manner. Additionally and also
preferably, the
lectin should be tested for its ability to operate as either saccharide-
binding agent in the
preferred embodiment of the assay, whether attached to the surface of the
solid support, or
alternatively present in a solubilized form.
In step 2, the lectins are optionally and preferably examined for their
ability to bind to
the solid support for the GMID card for the immobilized saccharide-binding
agent. In
addition, optionally and preferably, the solubilized form of the saccharide-
binding agents is
examined in order to determine if there is any non-specific binding to the
solid support,
which may increase levels of background lectin binding, thereby degrading the
signal of the
specifically bound lectins.
36
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
In addition, more preferably the solid support for the GMID card is itself
examined
for various types of behaviors, such as generation of background signals in
the absence of
specific lectin binding, and/or quenching of such signals. A particularly
preferred solid
support for the GMID card of the present invention is a porous or semi-porous
membrane,
such as nitrocellulose for example. Alternatively, the solid support could be
a nitrocellulose
coated solid surface such as a glass slide, for example, or any other suitable
solid surface
which has been coated with a porous or semi-porous material.
In step 3, once the set of lectins has been selected for immobilization on the
solid
support for the GMID card, and the support itself has also been selected, then
the GMID card
is prepared with the immobilized lectins. Optionally, the GMID card may be
prepared with
"arrayer" or "spotting" devices, which are able to place relatively small,
precise amounts of
lectins in a specific array on the solid support, to form an array of a
plurality of "spots".
These devices are also known as "microdispensing systems", as they deposit
volumes of
material which are typically measured in nanoliters, for example with an array
of pins for
depositing such small volumes of material. Examples of suitable devices which
are operative
with the present invention include, but are not limited to, HydraTM (Robbins
Inc., USA),
MicroGrid II/TAS/ProTM (BioRobotics Ltd., United Kingdom) and GMS417TM
(Genetic
Microsystems Affymetrix Inc., USA).
Optionally and preferably, the lectins are pretreated before being immobilized
to the
solid surface or incubated with the GMID card in the solubilized form. For
example, such
pretreatment could optionally include periodation of the lectins in order to
improve the signal
to noise ratio.
In step 4, optionally and more preferably, before being incubated with the
GMID
card, the glycomolecules are treated to maximize the efficiency of specific
binding to the
immobilized lectins on the support, and also to decrease non-specific binding
to the
immobilized lectins, the support and the solubilized lectins. In addition,
preferably the
glycomolecules are mixed with an appropriate buffer in order to form the
sample solution.
In step 5, the sample solution is contacted with the solid support containing
the
immobilized lectins. Optionally, before the sample solution is contacted with
the solid
support, the solid support is washed with the sample buffer alone. The sample
solution with
the glycomolecules is then incubated with the solid support for an appropriate
period of time.
37
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
Optionally, a control solution is also incubated with at least a portion of
the solid support, as
a measurement for non-specific binding.
In step 6, the solid support with the complexed glycomolecules is then
preferably
washed at least once with an appropriate washing buffer, as well as with an
appropriate
blocking buffer. In step 7, the solid support is then incubated with the
solubilized, labeled
lectins as the second saccharide-binding agent. In step 8, again an
appropriate washing
procedure is preferably performed.
In step 9, the signal from the labeled lectins is detected with an appropriate
detection
device. For example, if the label is chromogenic, then the detection device
could be a CCD
(charge-coupled device) camera. Clearly, one of ordinary skill in the art
could select the
appropriate detection device according to the type of label on the lectin.
According to preferred embodiments of the present invention, the label is a
fluorescent dye, as previously described. For such a preferred embodiment, the
detection
device would also preferably include a light source of an appropriate wave
length, for
exciting the fluorescent dye label, and also an appropriate filter set for
optionally filtering the
light from the light source and for filtering the resultant signal. It should
be noted that such
filters are not required for monochromatic light sources, such as lasers for
example. The
possibility of photobleaching and the efficiency cofactor of each dye or
fluorochrome is
preferably considered in the analysis phase, as described in greater detail
below.
The image of the entirety or at least a significant majority of the GMID card
could
optionally be obtained (as opposed to the detection of a plurality of single
signals, for
example). Examples of suitable detection devices include "scanners" for
obtaining at least a
portion of the image of the GMID card, with multiple signals from a plurality
of "spots".
Such devices may optionally be single band (light of a single wavelength is
detected); double
band (light of two separate wavelengths is detected); or spectrum devices
(light is detected of
at least two, but preferably a large number of, wavelengths).
In step 10, most preferably, the various signals from one or more control
"samples"
are analyzed in order to determine the appropriate threshold for the signal
for the specifically
bound lectins, as well as for determining signal to noise ratios, and so
forth. In addition,
these various signals can optionally be compared to the results from previous
assays, in order
to verify the quality of the assay for example.
38
CA 02407272 2002-11-O1
WO 01/84147 PCT/US00/30402
In step 11, optionally and preferably the signals are examined in order to
determine
the level of specific binding, if any, for example by subtraction of
background noise and by
comparison to the threshold for specifically bound lectins. The background
noise is
preferably determined as a function of the average noise, ~ the standard
deviation.
Steps 9-11 are optionally and preferably performed with a software program for
controlling the process of capturing the signal, for example in the form of
image data;
analyzing the control signals; and then analyzing the sample signals in order
to obtain the
actual assay data. Examples of suitable software programs include, but are not
limited to,
GeneToolsTM (BioRobotics Ltd., United Kingdom); GenePix Pro 3.OTM (Axon
Instruments
Inc.) and QuantArrayTM (GSI Lumonics Inc.). Alternatively, these steps could
be optionally
performed with firmware and/or hardware, or some combination thereof.
According to preferred embodiments of the present invention, these steps
preferably
include the step of first defining the array for the "spots". Such an array is
optionally and
more preferably defined automatically, and includes the definition of a grid
for determining
the expected location of any specific signal from the "spots". Next, the
initial location of the
spots is preferably determined in relation to the grid. Each individual spot
is then centered,
after which edge detection is preferably performed to locate the boundary of
each spot. Edge
detection is optionally performed according to a free form determination of
the size and shape
of the spots; a fixed form determination for the size and shape; or
alternatively a fixed size
but free shape determination process. Any of these steps may be performed
automatically or
alternatively may be performed manually.
Next, the intensity of the signal for each spot is determined. Such an
intensity is
preferably determined relative to the background signal and to the signal to
noise ratio, for
example by subtracting the background signal from the raw signal data which is
detected by
the detection device.
It is to be understood that while the invention has been described in
conjunction with
the detailed description thereof, the foregoing description is intended to
illustrate and not
limit the scope of the invention, which is defined by the scope of the
appended claims. Other
aspects, advantages, and modifications are within the scope of the following
claims.
39
