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Claims  |
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What is claimed is:
1. A method of obtaining peptide molecules which selectively bind to
antibodies uniquely associated with a disease of a patient infected with a
pathogenic bacterium or virus, comprising:
(a) isolating antibodies from the serum of a first patient having
antibodies uniquely associated with the disease;
(b) binding the antibodies to a support surface;
(c) contacting the antibodies on the support surface with a library of
peptide molecules in a manner so as to allow peptide molecules in the
library to bind to the antibodies;
(d) obtaining peptide molecules in step (c) which bind to an antibody from
the first patient;
(e) isolating antibodies from the serum of a second patient having
antibodies uniquely associated with the same disease as the first patient;
(f) labeling the antibodies from the second patient with a detectable
label;
(g) contacting the labeled antibodies of step (f) with the peptide
molecules of step (d) in a manner so as to allow binding of the labeled
antibodies to the molecules of step (d); and
(h) obtaining peptide molecules which bind to antibodies of the first
patient and to antibodies of the second patient.
2. The method of claim 1, further comprising:
(i) isolating antibodies from the serum of a third patient having
antibodies uniquely associated with the same disease as the first patient;
(j) labeling the antibodies from the third patient with a detectable label;
(k) contacting the labeled antibodies of step (j) with the peptide
molecules of step (h) in a manner so as to allow binding of the labeled
antibodies to the peptide molecules of step (j); and
(l) obtaining peptide molecules which bind to antibodies of the first,
second and third patient.
3. The method of claim 2, further comprising:
(m) isolating antibodies from the serum of an additional patient having
antibodies uniquely associated with the same disease as the first patient
wherein the additional patient is different from the first patient, the
second patient, and the third patient;
(n) labelling the antibodies from the additional patient with a detectable
label;
(o) contacting the labelled antibodies of the additional patient with the
peptide molecules of step (l) in a manner so as to allow binding of the
labelled antibodies to the peptide molecules of step (l); and
(p) obtaining peptide molecules which bind to antibodies of the first
patient, second patient, third patient, and additional patient.
4. The method of claim 3, further comprising:
(q) repeating steps (m)-(p) a plurality of times thereby obtaining peptide
molecules which bind to disease specific antibodies which antibodies are
specific to the disease of the first patient, second patient, third
patient, and each additional patient.
5. The method of claim 1, further comprising:
isolating the peptide molecules of step (h).
6. The method of claim 5, further comprising:
identifying the isolated peptide molecules.
7. The method of claim 1, further comprising:
obtaining peptide molecules which bind to disease specific antibodies which
antibodies are specific to the disease of the first patient and the second
patient by observing antibodies which bind to the peptide molecules of
step (h).
8. The method of claim 1, further comprising:
repeating step (c) under more stringent binding conditions in order to
determine antibodies with a higher binding affinity for the library of
peptide molecules than the antibodies determined by carrying out step (c)
a first time.
9. The method of claim 1, wherein the antibodies of step (f) are labelled
with a label selected from the group consisting of a radioactive label,
biotin, and fluorescein.
10. The method of claim 1, wherein the disease is caused by a virus
selected from the group consisting of HIV, HCV and HBV.
11. The method of claim 1, where the support surface is the surface of
polystyrene beads.
12. The method of claim 1, wherein the library of peptides is a library
produced on the surface of a bacteriophage.
13. The method of claim 12, wherein the library of peptides contains more
than 400 different bacteriophage with each having on its surface a peptide
with a distinct, unique and different amino acid sequence.
14. The method of claim 13, wherein the library of peptides contains more
than 8,000 different bacteriophage with each having on its surface a
peptide with a distinct, unique and different amino acid sequence.
15. The method of claim 14, wherein the library of peptides contains more
than 160,000 different bacteriophage with each having on its surface a
distinct, unique and different amino acid sequence.
16. The method of claim 15, wherein the library of peptides contains more
than 3,200,000 different bacteriophage with each having on its surface a
distinct, unique and different amino acid sequence.
17. The method of claim 16, wherein the library of peptides contains more
than 64,000,000 different bacteriophage with each having on its surface a
distinct, unique and different amino acid sequence.
18. The method of claim 1, wherein the library of peptides is a library of
peptides on the surface of bacteria.
19. A method of obtaining peptides which selectively bind to an antibodies
uniquely associated with a disease of a patient infected with a pathogenic
bacterium or virus, comprising:
(a) isolating antibodies from the serum of a first patient having
antibodies uniquely associated with the disease;
(b) binding the antibodies to a support surface;
(c) contacting the antibodies on the support surface with a library of
peptides expressed on bacteriophage in a manner so as to allow peptides to
bind to the antibodies;
(d) obtaining the peptides expressed on bacteriophage in step (c) which
bind to antibodies from the first patient;
(e) diluting the bacteriophage of step (d) and contacting the diluted
bacteriophage with bacteria under conditions which allow the bacteriophage
to infect the bacteria, reproduce and produce plaques;
(f) isolating antibodies from the serum of a second patient having
antibodies uniquely associated with the same disease as the first patient;
(g) labeling the antibodies from the second patient having antibodies
uniquely associated with a detectable label;
(h) contacting the labeled antibodies of step (g) with the plaques of step
(e) in a manner so as to allow binding of the labeled antibodies to the
plaques; and
(i) obtaining peptides on the bacteriophage in the plaques which bind to
antibodies of the first patient and to antibodies of the second patient
and isolating these peptides.
20. A method of obtaining antibodies uniquely associated with a disease of
a patient infected with a pathogenic bacterium or virus, comprising:
(a) isolating antibodies from the serum of a first patient having
antibodies uniquely associated with the disease;
(b) binding the antibodies to a support surface;
(c) contacting the antibodies on the support surface with a library of
peptides expressed on bacteriophage in a manner so as to allow peptides to
bind to the antibodies;
(d) obtaining the peptides expressed on bacteriophage in step (c) which
bind to an antibody from the first patient;
(e) diluting the bacteriophage of step (d) and contacting the diluted
bacteriophage with bacteria under conditions which allow the bacteriophage
to infect the bacteria, reproduce and produce plaques;
(f) isolating antibodies from the serum of a second patient having
antibodies uniquely associated with the same disease as the first patient;
(g) labeling the antibodies from the second patient with a detectable
label;
(h) contacting the labeled antibodies of step (g) with the plaques of step
(e) in a manner so as to allow binding of the labeled antibodies to the
plaques; and
(i) obtaining antibodies of the second patient which bind to the same
peptides as the antibodies of the first patient and isolating those
antibodies. |
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Claims |
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Description |
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FIELD OF THE INVENTION
This invention relates generally to the field of technology involved in the
isolation, recovery, formulation and use of various compounds including
peptides which selectively bind specific antibodies or other specific
binding proteins that are characteristic of a disease state.
BACKGROUND OF THE INVENTION
The present invention is based, in part, on conventional knowledge of
antibodies, the antigens which bind them, and known procedures for making
large mixtures of peptides and other molecules which may bind to naturally
occurring antibodies. Antibodies are proteins produced by lymphoid cells
(plasma cells). The antibodies are produced by the cells in response to
"foreign" substances (antigens), either exogenous or endogenous, and are
capable of binding specifically with the antigen which stimulated the
immune response. The antibodies can also bind with substances which are
structurally similar to that same antigen. An antigen can be any foreign
substance that, upon introduction into a vertebrate animal, stimulates the
production of antibodies. An antigen may also be of endogenous source,
such as antigens in auto-immune diseases or from tissue destruction. A
complex antigen-like molecule may carry several antigenically distinct
sites which are referred to as determinants. A substance which is
structurally similar to certain parts of an immunogen (in general the
determinants) can react specifically with its antibody. However, such
smaller substances are generally too small to stimulate antibody synthesis
by themselves and can be referred to as an incomplete antigen or hapten.
Throughout the life of vertebrate animals, such as humans, foreign
substances are continually introduced. These foreign substances include
viruses and bacteria which cause disease. Each time an antigenic substance
is introduced it may result in the generation of a different antibody or
several antibodies. Since many foreign substances are introduced over the
life of the animal, and since many antibodies may be generated in response
to a single foreign substance, each individual animal will have a large
numbers of different antibodies. With present technology, it is very
difficult to isolate and recover a specific antibody associated with a
specific antigen or disease especially if nothing is known about the
characteristics of the antibody or antigens involved. More specifically,
if one or more individuals is known to have a given disease (wherein the
agent responsible for the disease has not been characterized), it is not a
simple matter to isolate the antibodies in that individual which have been
generated and amplified as a result of that disease, nor is it a simple
matter to isolate and recover antigens which bind to those antibodies. The
present invention endeavors to provide approaches which make it possible
to isolate and recover antibodies which are characteristic of a specific
disease and to simultaneously isolate and recover antigens and/or related
molecules which specifically bind to those antibodies.
In order to carry out the methodology of the present invention it is
necessary to produce large numbers of peptides and/or other molecules
which can be tested for their ability to bind to antibodies. These large
mixtures of peptides and/or other molecules can be produced using
technology described in the literature. One of the initial methods of
producing multiple peptides more rapidly than the standard Merrifield
method is disclosed by Houghten, R. A., Proc Natl Acad Sci USA (1985)
82:5131-5135. The Houghten method involves a modification of the
Merrifield method but uses many individual polyethylene bags resulting in
a method wherein each bag will contain a different peptide. An alternative
method was devised by Geysen, H. M., et al., Proc Natl Acad Sci USA (1984)
81:3998-4002. (See also, W086/06487 and W086/00991). In accordance with
the Geysen method, C-terminal amino acid residues are bound to solid
supports in the form of multiple polyethylene pins and the pins treated in
parallel to attach additional amino acid residues.
More recently, machines have been introduced which produce many peptides by
parallel synthesis (e.g. Advanced Chem Tech, Gilson). An advancement in
the ability to produce extremely large numbers of peptides as mixtures was
disclosed within U.S. Pat. No. 5,010,175 issued Apr. 23, 1991 to Rutter
and Santi. The Rutter and Santi method makes it possible to generate large
numbers of peptides in either equimolar amounts or in predictable amounts.
The methodology makes it possible to quickly synthesize large numbers of
peptides such as mixtures containing 64 million or more different and
distinct peptides. Another method for producing mixtures of peptides
including large numbers of different peptides is disclosed within issued
U.S. Pat. No. 5,182,366 issued Jan. 26, 1993 to Huebner et al. and in
publications by Lam et al., Nature, (1991) 354, 82-84, and Prague paper.
The procedures described in the patents listed above allow for the
production of peptides and/or modified peptides using chemical synthesis
technology i.e. one compound is reacted with another in a chemical
reaction in order to obtain a reaction product. Although these methods can
be used in connection with the present invention, other recent technology
involves the biological synthesis of peptides in large numbers. More
specifically, the genetic material of bacteria or phage can be modified so
that each bacterium or phage produce an individual peptide on their
surface. By randomly producing large numbers of different pieces of
altered genetic material it is possible to produce a mixture of bacteria
or phage wherein the different bacteria or phage in the mixture include a
different peptide expressed on the surface. One method of producing
peptides on phage is taught by Devlin et al., Science (1990) 249:404-406
which discloses a method for the production and rapid evaluation of random
libraries of millions of peptides on the surface of phage. A similar
method was published by Scott and Smith, Science (1990) 249: 386-390.
Scott and Smith disclose a method wherein peptides are produced on the
surface of bacteriophage and the phage expressing a particular peptide tag
can be selected from a mixture of tens of millions of clones expressing
oligopeptides of random sequences using affinity purification with a
protein ligand. Christian et al., J. Mol. Biol., (1992) 227:711-718
discloses simplified methods for the construction, assessment and rapid
screening of peptide libraries in bacteriophage. Another related method
involves the generation of libraries by insertion of peptides into the
external domain of bacterial outer-membrane proteins, such as lam B, using
recombinant technology Brown, Proc. Natl. Acad. Sci. USA (1992) 89:
8651-8655. Still another method of producing large numbers of peptides is
taught in U.S. Pat. No. 5,223,409 issued Jun. 29, 1993 to Ladner et al.
All the above discussed methods for producing libraries of peptides and
modified peptides can be used in connection with the present invention.
Although the methods are extremely useful for producing large mixtures of
peptides and modified peptides, the methodologies do not allow one to
identify molecules which bind to antibodies in sera when neither the
antibody nor the antigen is known, or to identify antibodies specific to a
given disease. The present invention endeavors to provide technology which
makes such possible.
SUMMARY OF THE INVENTION
The methodology of the present invention makes it possible to isolate
peptides and/or other molecules which specifically bind to antibodies in
sera which antibodies are specific to a given disease while simultaneously
isolating those antibodies. In order to carry out the methodology,
antibodies are isolated from the sera of a number "n" of patients, each
with a history of having had the same given disease. The antibodies of a
first patient are isolated and immobilized on a support surface in a
manner which does not interfere with specific binding of antigens. To
carry out the initial selection, libraries of peptides displayed on the
surface of bacteriophage (or bacteria, or synthetic peptides on a solid
support) are brought into contact with the immobilized antibodies of the
first patient under conditions where binding will occur. (For descriptive
purposes procedures using peptides displayed on phage are described below.
However, similar approaches can be performed with bacteria, synthetic
peptides, etc., by persons skilled in the art.). Unbound phage-peptides
are washed away and antibody specific phage-peptides are removed,
isolated, and recovered as plaques grown on a lawn of bacteria. Secondary
screening is then carried out by isolating and labeling (radioisotopically
or other method) the antibodies of a second patient and using the labeled
antibodies to probe the peptides of the isolated bacteriophage binders
identified in the initial selection (e.g. using methods similar to that
described with pure monoclonal antibodies by Christian et al., J. Mol.
Biol., (1992) 227:711-718 and Castagnoliet al., J. Mol. Biol. (1991) 222:
301-310. In that many of the antibodies of the first patient will differ
from the antibodies of the second patient, many or most of the non-disease
specific antibodies (of the second patient) will not bind to the peptides
of the isolated bacteriophage which bound to the antibodies of the first
patient. The peptides which are not bound by antibodies of the second
patient are identified as not binding to antibodies of the disease of
interest and are eliminated from the screening process. The process may be
repeated with labeled antibodies from a third, fourth, etc. patient to
obtain and identify those peptides having the highest affinity to the
antibodies specific to the disease of interest. Those peptides found to
have the greatest binding affinity are characterized--e.g. their amino
acid sequences are determined by deducing such from the DNA sequences if
the peptides are on phage or bacteria. The predicted peptides can then be
chemically synthesized and bound to a support for use as a diagnostic
assay to detect the presence of antibodies, or formulated in a
pharmaceutical preparation which can be used to deactivate or neutralize
antibodies in a living being.
A primary object of the invention is to provide a method for determining
specific peptides which selectively bind to disease specific antibodies
and isolating antibodies associated with a disease.
An important advantage of the present invention is that the methodology
requires no prior knowledge of the molecular entities which cause a given
disease or the antibodies which are generated by infection with the
disease.
Another advantage of the present invention is that the isolated molecules
which specifically bind to antibodies characteristic of a disease can be
used to assay the sera of an individual for the presence of the disease
and can, at times, be used to diagnose the progress of the disease within
the individual by quantitative or qualitative differences in disease
specific antibodies.
A feature of the invention is that the diagnostic reagents isolated, using
the methodology of the present invention, are isolated and defined solely
by the presence of common antibodies generated during a disease state of a
number of individuals who have had or who are suffering from the disease.
As such these antibodies define the disease by clinical description and
are thus termed "disease-specific."
Another advantage and feature of the present invention is that the
antibodies of a number of different patients (all with a given disease in
common) are comparatively analyzed to eliminate non-common antibodies and
thereby isolate common antibodies specific to a given disease of interest.
Another advantage of the invention is that very large numbers of peptides
and other molecules can be quickly screened for their ability to bind to
antibodies and thereafter isolated and characterized.
A feature of the invention is that it can be used in connection with
libraries of synthetic peptides or cell surface peptides displayed on
bacteriophage or bacteria.
Another object of the invention is to provide a diagnostic assay device
comprised of a substrate having single or multiple antibody-binding
peptides bound to the surface of the substrate.
Another advantage of the invention is that the methodology can be used to
produce assays for a variety of different diseases which can be used to
assay human and animal sera to determine the presence of antibodies
associated with specific diseases and, perhaps associated with a specific
state of a disease.
Another feature of the invention is that it is useful in developing not
only assays but pharmaceutical formulations used in neutralizing the
antibodies and thus, in some cases, the treatment of patients..
These and other objects, advantages and features of the present invention
will become apparent to those persons skilled in the art upon reading the
details of the methodology and usage as more fully set forth below.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Before the present method of obtaining peptide diagnostic reagents, and
assays and formulations using such are described, it is to be understood
that this invention is not limited to the particular methodologies, assays
and formulations described as such and may, of course, vary. It is also to
be understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to be limiting
since the scope of the present invention will be limited only by the
appended claims.
It must be noted that as used in this specification and the appended
claims, the singular "a", "and" and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example, reference to "a
peptide" includes mixtures of and large numbers of peptides, reference to
"an antibody" includes large numbers of antibodies and reference to "the
method of synthesis" includes one or more methods of synthesis known to
those skilled in the art or understood by those skilled in the art upon
reading the present application and so forth.
Unless defined otherwise 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 any methods and
materials similar or equivalent to those described herein can be used in
the practice or testing of the present invention, the preferred methods
and materials are now described. All publications mentioned herein are
incorporated herein by reference in order to describe and disclose the
specific information for which the reference has been cited.
Definitions
The term "antibody" is defined in its broadest sense to encompass specific
binding moieties, i.e., chemical moieties which specifically bind to other
chemical moieties within a biological system. The term includes a protein
molecule formed within the body of an animal in order to neutralize the
effect of a foreign invading protein (called an antigen). The term further
includes antibodies produced by lymphocytes in response to the presence of
antigens wherein each antibody has a molecular structure that exactly fits
the structure of one particular antigen molecule so that the antigen and
antibody fit specifically to each other like a lock and key. The term
encompasses antibody molecules which attach themselves to invading antigen
molecules (which antigen molecules are generally on the surface of a
pathogenic bacteria or virus) and thereby renders the bacteria or virus
inactive.
The terms "peptide library" and "library of peptides" are used
interchangeably herein. The terms are intended to encompass a mixture of
peptides, preferably in the form of linear chains of amino acids
containing four to 20 amino acids. A peptide library is intended to
encompass mixtures which include 400 or more distinct and unique peptides,
each present in the mixture in retrievable and analyzable amounts.
Preferred mixtures include peptides which include the same number of amino
acids e.g. mixtures containing only hexapeptides. A particularly preferred
mixture includes peptides wherein each of the 20 naturally occurring amino
acids is positioned at all possible positions in one of the peptides in
the mixture. For example, a particularly preferred mixture of hexapeptides
includes 206 hexapeptides (64 million) with each of the 64 million
hexapeptides being present in retrievable and analyzable amounts.
The term "peptide analog library" and "library of peptide analogs" are used
interchangeably herein. The terms are intended to encompass a library of
molecules which are preferably polymers and are preferably in the form of
a linear chain of monomer units containing four to 20 monomer units. A
peptide analog library includes large numbers of molecules such as defined
above with respect to a "peptide library." Peptide analogs include
peptides which incorporate Doamino acids, amino acid analogs and other
organic molecules which can be linked together as monomer units in order
to form a polymer.
The term "disease specific antibody" (DSA) is intended to encompass any
antibody produced by a living being having an immune system which is
uniquely associated with a given disease and, as such, is an antibody or
antibodies specifically generated as a direct or indirect result of the
disease.
The term "non-disease specific antibody" (NDSA) is intended to encompass
all antibodies in an organism other than the disease specific antibodies.
Non-disease specific antibodies may have been generated as the result of a
wide range of different antigenic materials including prior diseases which
are not the specific disease of interest.
The term "antibody-binding peptides" is intended to encompass linear
peptides (preferably containing four to 20 amino acids) which will bind to
an antibody under conventional antibody/antigen binding conditions.
Preferred antibody-binding peptides are linear peptides which selectively
bind to disease specific antibodies and which do not bind to non-disease
specific antibodies.
The term "antibody-binding molecule" is intended to encompass
antibody-binding peptides and other molecules such as peptide analogs or
organic molecules which selectively bind to an antibody under conventional
antibody-antigen binding conditions. Preferred antibody-binding molecules
are linear polymeric molecules or organic molecules containing four or
more monomer units which selectively bind to disease specific antibodies
and which do not bind, or bind less tightly, to non-disease specific
antibodies.
General Methodology
The methodology of the present invention makes it possible to isolate
molecules, such as peptides, which selectively bind to the disease
specific antibodies with no prior knowledge of the immune response and
subsequently to isolate and identify these antibody molecules. Having
isolated antibody-binding molecules such as peptides they can be
chemically synthesized and used in assays or formulated for use as
therapeutics. Having isolated and recovered pure antibodies they might in
some cases be sequenced and copies can be produced.
The methodology is carried out by extracting the antibodies from sera from
a number of patients all of whom have the same disease of interest but who
are otherwise immunologically heterogeneous. The antibodies from different
patients are compared to ascertain which antibodies are the disease
specific antibodies (DSA) common to patients who have the disease of
interest. The antibody-binding peptides or other molecules of interest can
then be identified using the DSA. Thereafter, the antibody-binding
molecules can be used in assays to determine the presence of disease
specific antibodies within the sera of other patients being tested, or can
be formulated for use as therapeutics.
The "Background of the Invention" section put forth above describes a
number of methodologies for producing peptide libraries and/or peptide
analog libraries. Any of these methodologies and others can be used in
connection with the general methodology of the present invention. In
general, chemical synthesis methodologies such as disclosed within U.S.
Pat. No. 5,010,175 to Rutter and Santi or Huber and Santi, or Lam et al.,
can be used to produce large libraries of peptides synthetically. The
amino acid monomer units can be modified so that this technology can be
used to produce peptide analog libraries synthetically. Alternatively,
methods such as disclosed within the above cited Devlin et al. and Scott
and Smith articles can be used to produce large libraries of peptides
expressed on the surface of bacteriophage. Alternately, organic-diversity
libraries as described (Pauwels, et al. Proc. Natl. Acad. Sci. USA (1993)
90: 1711-1715; Bunin, B. A. and Ellman, J. A. (1992) 114, 10997-10998;
DeWitt, S. H. et al., (1993) 90: 6909-6913) might be used to identify
novel organic-molecule diagnostic reagents. Although any of these methods
can be used it is, of course, necessary to use chemical synthesis
methodology if the antibody binding molecules are different from peptides.
More specifically, chemical synthesis methodology must be used to produce
peptide analogs and related organic compounds which might bind antigens.
However, for purposes of simplicity and efficiency it is preferable to use
peptide libraries where the peptides are produced on the surface of
bacteriophage or bacteria in accordance with the nonproprietary methods
described in Devlin et al., Science (1990) 249: 404-406; Scott and Smith,
Science (1990) 249: 386-390; Christian et al., J. Mol. Biol., (1992) 227:
711-718; Castagnoliet al., J. Mol. Biol. (1991) 222: 301-310 and Brown,
Proc. Natl. Acad. Sci. USA (1992) 89: 8651-8655. For this reason, the
following specific description refers to the use of a peptide library
produced on bacteriophage. However, it should be noted that, having read
the present disclosure, those skilled in the art could readily modify the
following description so that the methodology could be carried out using
various libraries including libraries on the surface of bacteria,
synthetic peptide analog libraries and libraries of organic compounds
produced using chemical synthesis technology.
Initial Sera/Antibody Cleanup
To carry out the methodology of the invention the sera of a number "n" of
patients (e.g. 10 patients), each diagnosed with the same disease of
interest, are extracted. It is critical that each of the patients be
definitively diagnosed as having the disease of interest. It is desirable
that the patients are otherwise different with respect to their
immunological background so that most antibodies in common are disease
specific. This could be accomplished, to a certain extent, by choosing
patients of diverse genetic backgrounds and from diverse locations around
the world.
Having extracted the sera from the different patients, the serum from each
of the patients is subjected to an initial cleanup procedure which
involves the removal of interfering substances in order to simplify the
search for the antibodies of interest. For example, the sera are brought
into contact with the proteins of E. coli, and the antibodies which
specifically bind to the E. coli proteins (due to previous exposures) are
removed. Other common high titer antibodies can also be removed from the
sera in an analogous fashion. Techniques for carrying out such cleanup
procedures involve contacting the antibodies with E. coli K-12 lysate
using procedures such as those described in laboratory handbooks such as
Sambrook, Fritsch and Maniatis (1989), Cold Spring Harbor Laboratory
Press. The antibodies are then purified by column separation techniques
such as a protein A column in order to isolate IgG antibodies. The
isolated antibodies can then be subjected to elimination methodology in an
attempt to remove as many non-disease specific antibodies as possible.
The serum extracted from each of the "n" patients is subjected to the
initial antibody extraction and cleanup procedures of the type described
above. Having done so, one will obtain individual pools of antibodies
(e.g. 10 pools of antibodies from 10 individuals) which include the
disease specific antibodies from each of the 10 individuals along with
large numbers of non-disease specific antibodies. It is these 10 pools
which are to be comparatively sorted per the present invention, one
against the other, in order to discern the disease specific antibodies
common to all of the 10 pools. Any antibodies common to all 10 pools is
almost certainly a disease specific antibody and the present invention is
directed towards obtaining such disease specific antibodies.
It should be pointed out that the broadest possible interpretation of the
present invention would not require the use of the initial clean-up
procedure. In accordance with the broadest interpretation of the present
invention, the antibodies of a first patient would be comparatively sorted
against the antibodies of a second patient. If the first and second
patient did not share any antibodies except for the disease specific
antibodies of interest, antibody-binding molecules could be readily
identified along with the binding antibodies. However, in practice the use
of the initial clean-up procedure could be important in reducing the
number of antibodies which would need to be subjected to further
comparative sorting per the present invention.
A variety of initial antibody clean-up procedures can be used. Those
skilled in the art will recognize that different procedures might be more
useful in connection with the treatment of the sera from particular
populations than would other techniques. For example, patients from a
given population may be very likely to have generated antibodies with
respect to a given disease, whereas patients from another population would
be very unlikely to have generated antibodies specific to that disease.
For example, malaria is more common in some populations than in others.
Accordingly it might be more efficient to screen out malaria specific
antibodies from some populations routinely whereas screening for such
antibodies in a patient population unlikely to have been infected with
malaria would not be efficient.
The initial clean-up procedure described above does not require the use of
any peptide library or peptide analog library and thus can be carried out
in the same manner regardless of what type of library is being used for
subsequent screening. However, with respect to the next section referred
to as "Initial Selection" it is necessary to generate some type of
library. As indicated above, for purposes of simplicity, the methodology
is described with respect to the use of a peptide library and specifically
a peptide library which has been generated on the surface of bacteriophage
as described in the Scott and Smith, 1990, article cited above. However,
all types of libraries (including organic compounds which are not
peptides, but which mimic peptide or other molecules (e.g. carbohydrates,
nucleic acids, etc.) in their ability to bind antibodies) could be used in
the methodology of the present invention.
Initial Selection
Even after the initial extraction and cleanup procedures it would be
expected that each of the 10 pools of extracted antibodies would include a
number of disease specific antibodies and disease non-specific antibodies,
e.g. 10 disease specific antibodies mixed with a much larger number of
non-disease specific antibodies, e.g. 1,000 non-disease specific
antibodies. As indicated, the numbers used here are chosen merely for
example. The exact number of disease specific antibodies and non-disease
specific antibodies present in each pool are unknown, and will vary based
on a number of factors such as the disease and the individual from whom
the antibodies were extracted. However, in general, each pool will include
several antibodies specific to the disease and a much larger number of
antibodies which are not specific to the disease. The fact that a small
number of disease-specific antibodies are hidden within a much larger
number of non-disease-specific antibodies is a natural phenomenon which
emphasizes the importance of the present invention. More specifically, the
methodology of the present invention makes it possible to select out the
disease-specific antibodies from among a much larger number of
non-disease-specific antibodies.
A pool of antibodies from a first patient (patient A) are immobilized to a
given support surface. The antibodies are bound in a manner so as to not
hinder binding of antigens to them. A library of peptides (phage,
bacteria, synthetic peptides, or other molecules) is then brought into
contact with the immobilized antibodies bound to the support surface. The
library of peptides is preferably created on the surface of bacteriophage
wherein each of the phage will express a different peptide, e.g., a
hexapeptide. Such a phage library can be produced in accordance with
procedures known to those skilled in the art e.g. as described in the
references cited above. In essence, the procedures involve the random
synthesis of nucleotides which will encode 64 or more million hexapeptides
and randomly inserting the oligonucleotides into the genome of
bacteriophage. It is preferable to use a peptide library created on the
surface of bacteriophage in that any given peptide in the library which is
found to be of interest can be readily purified by plaque purification and
amplified by using the bacteriophage to infect bacteria and undergo
replication.
The peptide library on the bacteriophage is brought into contact with the
immobilized antibodies from patient A which were bound to a support. In
order to reduce the background of non-disease-specific antibodies, an
excess of blocking antibodies from disease-free individuals may be added
to the bacteriophage library in advance and/or simultaneously
co-incubated. Conventional antibody/peptide binding conditions can be
used. After sufficient time has been allotted for the binding of the
peptides (on the bacteriophage) to the immobilized antibodies bound to the
support, conventional procedures are utilized to wash unbound peptides
(bacteriophage) and bacteriophage-"blocking antibody" complexes from the
area leaving only those peptides (bacteriophage) which bind to the
immobilized antibodies.
The antibody-binding peptides (phage) are then removed and isolated. The
isolated peptides (phage) can be used to infect bacteria and create
unique, purified plaques of the bacteriophage. At this point, the initial
selection step of the present invention has been completed. Specifically,
a library of molecules has been generated and those molecules have been
demonstrated to bind to the antibodies from a first patient. At this
point, the "initial selection" step of the invention has been completed.
However, the initial selection step can be further elaborated on in order
to determine which of the antibody-binding peptides bind most strongly to
the antibodies of the first patient. This procedure is described below.
Initial Selection (Increased Binding Affinity)
Copies of the antibody-binding peptides can be brought into contact with
the antibodies from patient A bound to a support in order to determine
which of the peptides bind to the antibodies with greatest affinity. The
binding conditions can be changed (such as to make conditions for binding
more difficult) in order to determine those antibody binding peptides
which have the highest affinity for the antibodies. Although the
procedures need not be repeated, it can be repeated any number of times
and can be used to identify not only peptides which have higher affinity
for antibodies but used to deduce which antibodies are present in high
titer and therefore more likely to be related to the specific disease of
interest.
Secondary Screening--Identifying Overlapping Epitopes in Different Sera
To a large extent the present invention is characterized by the secondary
subsequent screening step. In the second step antibodies which have been
isolated and subjected to initial "cleanup" procedures from a second
patient (patient B) are labeled with an appropriate detectable label such
as biotin, fluorescein or appropriate radioactivity. The labeled
antibodies of patient B are then used as probes by bringing the labeled
antibodies into contact with the bacteriophage colonies created by the
antibody-binding peptides from patient A. In one method, the
antibody-binding peptides are bound to a membrane and plaque hybridization
procedures are used Christian et al., J. Mol. Biol., (1992) 227: 711-718.
A representative sample of phage are used to infect male E. coli (phage:E.
coli::1:100). Approximately 50,000 colonies are plated on Luria broth (LB)
plates with tetracycline (20 .mu.g/ml; 1000 colonies/plate) and incubated
overnight at 37.degree. C. Phage are transferred to nitrocellulose filters
by simple overlay. The filters are immediately washed twice (30
minutes/wash) with TNT buffer (10 mMTris-HCl, pH 8.0, 150 nM NaCl, 0.05%
Tween 20) and soaked for 30 minutes at room temperature in a blocking
solution containin | | |
