U.S. patent application number 13/545118 was filed with the patent office on 2012-11-01 for disease diagnosis according to saccharide binding.
This patent application is currently assigned to Procognia, Ltd.. Invention is credited to Ofer Markman, Assaf (Assaph) Peretz Oron, Chana Rothmann-Schertz.
Application Number | 20120277116 13/545118 |
Document ID | / |
Family ID | 37985841 |
Filed Date | 2012-11-01 |
United States Patent
Application |
20120277116 |
Kind Code |
A1 |
Markman; Ofer ; et
al. |
November 1, 2012 |
Disease diagnosis according to saccharide binding
Abstract
Disclosed is a method for characterizing carbohydrate polymer by
identifying at least two binding agents that bind to the
carbohydrate polymer. Binding is preferably determined by
contacting the carbohydrate polymer with substrate that contains a
plurality of first saccharide-binding agents affixed at
predetermined locations on the substrate. The carbohydrate polymer
is allowed to contact the substrate under conditions that allow for
formation of a first complex between the first saccharide-binding
agent and the carbohydrate polymer. In one aspect, the method is
used in the diagnosis of a disease, including a viral disease, an
autoimmune disease, a bacterial infection, or cancer.
Inventors: |
Markman; Ofer; (Rehovot,
IL) ; Oron; Assaf (Assaph) Peretz; (Seattle, WA)
; Rothmann-Schertz; Chana; (Petach Tikva, IL) |
Assignee: |
Procognia, Ltd.
Ashdod
IL
|
Family ID: |
37985841 |
Appl. No.: |
13/545118 |
Filed: |
July 10, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12850205 |
Aug 4, 2010 |
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13545118 |
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12176959 |
Jul 21, 2008 |
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12850205 |
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11402646 |
Apr 11, 2006 |
7407773 |
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12176959 |
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10275222 |
Sep 9, 2003 |
7132251 |
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PCT/US00/30402 |
Nov 3, 2000 |
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11402646 |
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Current U.S.
Class: |
506/9 ; 435/7.1;
436/501 |
Current CPC
Class: |
G01N 2400/00 20130101;
G01N 33/5308 20130101 |
Class at
Publication: |
506/9 ; 436/501;
435/7.1 |
International
Class: |
C40B 30/04 20060101
C40B030/04; C12Q 1/70 20060101 C12Q001/70; G01N 33/574 20060101
G01N033/574; G01N 33/569 20060101 G01N033/569; G01N 33/566 20060101
G01N033/566; G01N 33/53 20060101 G01N033/53 |
Claims
1-47. (canceled)
48. A method for characterizing a carbohydrate polymer, the method
comprising adding the carbohydrate polymer to a surface that
includes at least one saccharide binding agent attached to a
predetermined location on the surface; incubating the carbohydrate
polymer with the surface under conditions allowing for the
formation of a complex between the first saccharide-binding agent
and the carbohydrate polymer, and systemically assembling a
representation of information describing the binding status of the
carbohydrate polymer with respect to the plurality to determine a
fingerprint, thereby characterizing the carbohydrate polymer.
49. The method of claim 48, further comprising washing the surface
to remove unbound carbohydrate polymer.
50. The method of claim 48, further comprising correlating said
representation t to a disease in a subject from whom the
carbohydrate polymer is obtained, wherein the disease is selected
from the group consisting of a vial disease, an autoimmune disease,
a bacterial infection, and cancer.
51. The method of claim 48, further comprising contacting the
surface 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.
52. The method of claim 50, wherein said disease is a viral
disease.
53. The method of claim 50, wherein said disease is an autoimmune
disease.
54. The method of claim 50, wherein said disease is a bacterial
infection.
55. The method of claim 50, wherein said disease is cancer.
56. The method of claim 50, wherein the fingerprint is at least
partially collapsed to form at least partial sequence information
about the carbohydrate polymer sequence.
Description
[0001] This application is a continuation of U.S. Ser. No.
12/850,205, filed on Aug. 4, 2010, which is a continuation of U.S.
Ser. No. 12/176,959, filed on Jul. 21, 2008, which is a
continuation of U.S. Ser. No. 11/402,646, filed on Apr. 11, 2006,
which is a continuation of U.S. Ser. No. 10/275,222, filed on Sep.
9, 2003, which is a national state application filed under 35 USC
.sctn.371 of PCT/US00/30402, filed on Nov. 3, 2000. The contents of
these applications are each incorporated herein by reference in
their entireties.
FIELD OF THE INVENTION
[0002] 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
[0003] 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.
[0004] 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 C1 atom may be in either the .alpha. or .beta.
configuration. Thus, both the two-dimensional and three-dimensional
structure of the carbohydrate polymer can be highly complex.
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 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.
[0009] Second, saccharide can be connected at any of the carbon
moieties, e.g., a the C1, C2, C3, C4, or C6 atom if the sugar unit
it is connected to is a hexose. Moreover, the connection to the C1
atom may be in either .alpha. or .beta. configuration.
[0010] 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.
[0011] 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).
[0012] 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.
[0013] 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 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.
[0014] 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.
[0015] 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.
[0016] 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.
[0017] 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 surface, and the reaction thereof with an analyte
detected by a variety of methods, using logic circuitry on the VLSI
chip.
[0018] 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.
[0019] 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:
[0020] 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.
[0021] 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.
[0022] 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 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.
[0023] 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.
[0024] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] 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).
[0026] 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.
[0027] FIG. 3 is a high-level logic flowchart that illustrates an
algorithm for choosing a set of colored lectins.
[0028] 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
[0029] 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.
[0030] 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.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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.
[0035] 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 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.
[0036] 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.
[0037] 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.
[0038] 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.
[0039] The essentially sequence-specific agent is usually a
protein, such as a lectin, a saccharide-specific antibody or a
glycosidase or glycosyltransferase.
[0040] 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,
Erythrina cristagalli, Lens culinaris, Glycine max, Phaseolus
vulgaris, Allomyrina dichotoma, Dolichos biflorus, Lotus
tetragonolobus, Ulex europaeus, and Ricinus communis.
[0041] Other biologically active carbohydrate-binding compounds
include cytokines, chemokines and growth factors. These compounds
are also considered to be lectins for this patent application.
[0042] Examples of glycosidases include .alpha.-Galactosidase,
.beta.-Galactosidase, N-acetylhexosaminidase, .alpha.-Mannosidase,
.beta.-Mannosidase, .alpha.-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.
[0043] 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".
[0044] 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 Gal.beta.1-3 GalNAc,
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.
[0045] 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 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 illustrative 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 active while others are
inactive.
TABLE-US-00001 TABLE 1 Reaction conditions for some essentially
sequence-specific agents Condition codes for serial Temp condition
sets number pH (C.) Enzyme(s) 1 3.5 30 Jackbean .beta.-
galactosidase 2 5.0 37 Endo a-N Acetylgalactosidase .alpha.1,2
Fucosidase .beta.1,2 galactosidase 3 5.0 25 Bovine kidney .alpha.
Fucosidase 4 7.2 25 Coffee bean .alpha. galactosidase 5 5.8 55 B.
Fragilis Endo .beta.-galactosidase 6 6.2 25 Chicken egg lysozyme 7
4.3 37 Bovine testes .beta.1-3,4,6, Galactosidase from 2-9.5 50 Gly
001-02 Biodiversa from 3.0-8.0 50 Gly 001-04 Biodiversa 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
[0046] 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 .epsilon.-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.
[0047] 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.
[0048] 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 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 .beta.-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 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.
[0049] 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 enzymatic detection
is routinely used. The enzymes are available commercially, e.g.,
from companies such as Pierce.
[0050] 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.
[0051] 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.
[0052] Coupling is usually carried out by using functional groups,
such as hydroxyl, aldehyde, keto, amino, sulfhydryl, 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.
[0053] 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.
[0054] 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.
[0055] 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.
[0056] 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.
[0057] 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
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.
[0058] 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 Fingerprints of Carbohydrate Polymers
[0059] 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.
[0060] 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.
[0061] 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.
[0062] 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 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 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.
[0063] 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.
[0064] The invention will be further illustrated in the following
examples, which do not limit the scope of the appended claims.
EXAMPLE 1
Glycomolecule Analysis Using Antibodies as First and Second
Sequence-Specific Agents
[0065] 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.
[0066] The structure of the sugars is as follows: [0067] MFLNH-II
(HS):
[0067] ##STR00001## [0068] NS:
##STR00002##
[0069] 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.sup.x
(Le.sup.x), or Lewis.sup.b antigen (Le.sup.b). 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 rifle-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-US-00002 TABLE 2 On the matrix anti T-antigen Anti-Le.sup.x
anti-Le.sup.b Saccharide bound HS HS Second mAb nile-red
anti-Le.sup.x Signal nile-red, reducing Reducing end none end
TABLE-US-00003 TABLE 3 On the matrix anti T-antigen Anti-Le.sup.x
anti-Le.sup.b Saccharide bound NS NS Second mAb Green anti-Le.sup.b
nile-red anti-Le.sup.x Signal Green, reducing nile-red, reducing
end end
[0070] 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-US-00004 TABLE 4 On the matrix anti T-antigen Anti-Le.sup.x
anti-Le.sup.b HS nile-red, reducing Reducing end end NS Green,
reducing nile-red, reducing end end NS Green, reducing nile red,
reducing end end
[0071] 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 .alpha.1-2 Fucosidase or Exo
.beta. 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-US-00005 TABLE 5 reactions after applying .alpha.1-3,4
Fucosidase: On the matrix anti T-antigen Anti-Le.sup.x
anti-Le.sup.b HS reducing end NS
TABLE-US-00006 TABLE 6 reaction after applying Exo .beta.
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.sup.x anti-Le.sup.b HS nile-red NS
Green nile-red
TABLE-US-00007 TABLE 7 reactions after applying .alpha.1-2
Fucosidase: On the matrix anti T-antigen Anti-Le.sup.x
anti-Le.sup.b HS nile-red, Reducing end reducing end NS Reducing
end
[0072] 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.
TABLE-US-00008 TABLE 8 On the matrix anti T-antigen Anti-Le.sup.x
anti-Le.sup.b 0 nile-red, reducing Reducing end end 1 reducing end
-- -- 2 nile-red 3 nile-red, reducing Reducing end end
TABLE-US-00009 TABLE 9 On the matrix anti T-antigen Anti-Le.sup.x
anti-Le.sup.b 0 Green, reducing nile red, reducing end end 1 -- --
-- 2 Green nile red 3 Reducing end
[0073] 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
[0074] 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 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.
[0075] The method of sequential labeling of reducing ends is now
described in more detail in the following steps:
1. Blocking:
[0076] A polysaccharide having a reducing end is incubated in a
solution containing NaBH.sub.4/NaOH at pH 11.5.
[0077] This treatment blocks the reducing end, so that the
polysaccharide is now devoid of a reducing end (RE).
2. Exposing:
[0078] 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
[0079] 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.
[0080] 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.dbd.N double bond is then reduced to a CH.sub.2--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, Calif.,
USA, as detailed e.g., in the Glyko, Inc. catalog, p. 8-13, which
is incorporated herein by reference.
4. Reaction With a Second Endoglycosidase
[0081] 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
[0082] 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.
[0083] 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.
[0084] 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 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.
[0085] 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-.beta.(1,4)[Fuc-.alpha.(1,3)]-GlcNAc-.beta.(1,3)-Gal.beta.(1,4)-Glc.
The pentasaccharide is branched at The GlcNAc 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-.beta.-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.
[0086] 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.
[0087] In order to ascertain that the enzymes had digested the
saccharide, the various reactions are size-separated using
thin-layer chromatography (TLC).
[0088] 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.
##STR00003##
[0089] 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 1 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).
[0090] 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.
[0091] The reaction buffer is phosphate-buffered saline (PBS) with
1 mM CaCl and 1 mM MgCl. After binding of the lectins, the filter
was blocked with 1% BSA in reaction buffer. As controls, reactions
without lectin and with 10 .mu.g BSA as immobilized protein were
used.
[0092] 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-US-00010 TABLE 10 AAA PNA LCA DSA RCA I 1 ++ 2 ++ ++ ++ 3 ++
4 ++ ++ ++ ++
[0093] 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)GlcNAc, GlcNAc, and
Galactose/GalNAc 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-.beta.-Galactosidase recognize
cleavage sequences in the saccharide. These sequences are
Fuc(1-3/1-4)GlcNAc and GlcNAc.beta.(1-3)Gal.beta.(1-3/4)Glc/GlcNAc,
respectively.
[0094] 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 GlcNAc 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.
[0095] Moreover, the reaction with PNA as immobilized agent shows
that the reducing end is cleaved only if Endo-.beta.Galactosidase
is used (reactions 1 and 3). This indicates that the
Endo-.beta.Galactosidase 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.
[0096] When taking into account the above data, it is now possible
to propose a sequence of the saccharide as follows:
Fuc.alpha.(1-3,1-4)GlcNAc(1-3)Gal(1-4)Glc/GlcNAcreducing end
[0097] 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 GlcNAc 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.
EXAMPLE 4
Derivation of Partial or Complete Sequence Information
[0098] 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.
[0099] 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".
[0100] It is now possible to create a second list of triplets of
recognition sites of the above type (type 1 triplets):
(first recognition site)-(glycosidase)-(second recognition
site).
[0101] 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)
[0102] 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.
[0103] The process of obtaining sequence information, i.e., of
collapsing the triplets into a map of recognition sites, is
described below.
[0104] 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.
[0105] 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>
[0106] 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
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.
[0107] 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.
[0108] 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.
[0109] 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 recognition site 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.
[0110] 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.
[0111] 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 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 S J, MacKenzie C R, Sadowska J,
Miehniewicz J, Young N M, Bundle D R, Narang; Selection of antibody
single-chain variable fragments with improved carbohydrate binding
by phage display. J. Biol. Chem. 269, 9533-38, 1994).
[0112] 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.
[0113] 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 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 and the respective recognition site (a or b).
[0114] 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.
[0115] 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: [0116] 1.
collecting triplets of type 1 and type 2 [0117] 2. sorting said
triplets according to similarity [0118] 3. comparing triplets with
different glycosidase recognition sites [0119] 4. arranging the
triplets in the order of occurrence on the saccharide [0120] 5.
arranging the glycosidase recognition sites [0121] 6. checking the
compatibility to the triplets [0122] 7. arranging recognition
sequences of glycosidases and of first and second essentially
sequence-specific agents in a single file order [0123] 8.
translating the recognition sequences (sites) into polysaccharide
sequence [0124] 9. correcting "overlap" problems [0125] 10.
outputting a sequence [0126] 11. checking against all available
data
[0127] 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.
[0128] 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.
[0129] 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 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
[0130] The aim of this example is to demonstrate the application of
the GMID technique to the analysis and comparison of milk
samples.
[0131] A. Membranes and layer lectins:
[0132] The supporting surface used in the experiments described
hereinbelow is a nitrocellulose membrane. The membranes were
prepared as follows: [0133] 1. Nitrocellulose membranes were cut
out and their top surface marked out into an array of 9.times.6
squares (3 mm.sup.2 each square). The membranes were then placed on
absorbent paper and the top left square of each one marked with a
pen. [0134] 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
.mu.l 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:
TABLE-US-00011 ##STR00004##
[0135] The lectins used in this experiment are listed in Table
11.
TABLE-US-00012 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
[0136] 3. The prepared blots were placed in 90 mm petri dishes.
[0137] 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). [0138] 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.degree. C., without rotation). [0139] 6. The blots
were then washed by addition of 10 ml washing solution to each
petri dish. 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:
[0140] The milk samples used were as follows: [0141] 1. Bovine UHT
long-life milk (3% fat) obtained from Ramat haGolan dairies, Israel
(lot 522104); [0142] 2. Pasteurized goat's milk, obtained from
Mechek dairies, Israel (lots 1 and 2); [0143] 3. Non-pasteurized
goat's milked obtained as in 2. (lots 3 and 4).
[0144] The milk samples were diluted to 10% v/v and approximately 5
ml of each sample applied to separate blots.
[0145] 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).
[0146] The blots were then incubated at room temperature with
agitation for one hour.
C. Colored lectins:
[0147] 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.
[0148] A mixture of these two lectins was prepared in washing
solution, such that the concentration of each colored lectin was 2
mg/ml.
[0149] 500 .mu.l 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
[0150] 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.
[0151] 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:
[0152] The bovine milk sample yielded a GMID indicating that the
polysaccharide in the sample contains saccharides that yield
positive results for lectins specific for: [0153] a.
glucose/mannose (ConA, PSA and LCA); [0154] b. GlcNac (WGA and
DSA).
[0155] The pasteurized goat milk samples yielded positive results
for: [0156] a. glucose/mannose (conA, PSA and LCA); [0157] b.
GlcNac (DSA).
[0158] No difference in lectin reactivity between the lots tested
was observed. The non-pasteurized goat milk sample gave a positive
reaction for: [0159] a. glucose/mannose (ConA, PSA and LCA); [0160]
b. GlcNac (DSA).
[0161] 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
[0162] A GMID analysis was performed on five different bacterial
lipopolysaccharides obtained from Sigma Chemical Co. (St. Louis,
Mo., 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.
[0163] 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.
EXAMPLE 7
Method for Selecting Colored Lectins
[0164] 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.
[0165] 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.
[0166] 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.
[0167] In step 105, 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.
[0168] 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.
[0169] 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 therefore, 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
[0170] 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. FIG. 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.
[0171] 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.
[0172] 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.
[0173] 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 for 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.
[0174] 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, Hydra.TM. (Robbins
Inc., USA), MicroGrid II/TAS/Pro.TM. (BioRobotics Ltd., United
Kingdom) and GMS417.TM. (Genetic Microsystems Affymetrix Inc.,
USA).
[0175] 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.
[0176] 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.
[0177] 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. Optionally, a control solution
is also incubated with at least a portion of the solid support, as
a measurement for non-specific binding.
[0178] 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.
[0179] 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.
[0180] 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).
[0181] 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.
[0182] 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.
[0183] 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, GeneTools.TM.
(BioRobotics Ltd., United Kingdom); GenePix Pro 3.0.TM. (Axon
Instruments Inc.) and QuantArray.TM. (GSI Lumonics Inc.).
Alternatively, these steps could be optionally performed with
firmware and/or hardware, or some combination thereof.
[0184] 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.
[0185] 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.
[0186] 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.
* * * * *