Title: Monoclonal antibodies to Sarcocystis neurona and
United States Patent: 6,891,024
Issued: May 10, 2005
Inventors: Marsh; Antoinette (Columbia, MO)
Assignee: The Curators of the University of Missouri
Appl. No.: 140754
Filed: May 7, 2002
The present invention is directed to particular monoclonal antibodies
that find use in the identification and purification of Sarcocystis
neurona and related antigens. In particular, these antibodies permit the
diagnosis of Sarcocystis related diseases such as equine protozoal
Description of the Invention
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of microbiology,
immunology and pathology. More particularly, it concerns the development of
particular monoclonal antibodies for use in the diagnosis and therapy of
disease caused by Sarcocystis neurona infections.
2. Description of Related Art
Equine protozoal myeloencephalitis (EPM) is a widespread neurological
disease in horses. Similar syndromes have been recognized in other species,
such as marine mammals. It is progressive and in advanced stages, the horse
will suffer from spinal cord and brain stem damage resulting in ataxia of
the limbs and other signs of muscular incoordination, loss of response to
certain sensory stimuli and muscle atrophy. In severe cases of recurrent
neurological signs that do not respond to therapy, horses must be euthanized,
which is very costly to owners.
Most cases of EPM are caused by a protozoan parasite, Sarcocystis neurona.
This organism has been identified in other species and has been associated
with encephalitis as well. The horse is thought to become infected with this
parasite by ingestion of sporocysts shed by the opossum (Didelphis
virginiana) or closely related species that are found in the Americas.
This would suggest that horses shipped to other parts of the world could
develop EPM later; therefore, EPM is not just a disease found in the
To date, there has been only one group—the inventors—that have disclosed a
monoclonal antibody directed Sarcocystis neurona. Marsh et al.
(2000). However, this report did not disclose the target for the antibody or
how it was made. In addition, the antibody failed to react with certain
strains of S. neurona, suggesting limited suitability for use in
diagnostic screens. Thus, there clearly remains a need to identify
additional antibodies that can be used in both diagnosis and therapy of
S. neurona disease.
SUMMARY OF THE INVENTION
Thus, in accordance with the present invention, there is provided a
monoclonal antibody that binds immunologically to a Sarcocystis neurona
organism or antigen, designated 2A7-18 or 2G5-2. The antibody may
comprise a label, for example, a radioisotope, bead, a ligand, a
chemilluminescent molecule, a fluorescent molecule, or an enzyme. It may
also comprise a therapeutic compound, such as a radioisotope or a
In another embodiment, there is provided a method of identifying a
Sarcocystis neurona organism or antigen in a sample comprising (a)
contacting the sample with a monoclonal antibody that binds immunologically
to a Sarcocystis neurona organism or antigen, designated 2G5-2 or
2A7-18; and (b) determining binding of the monoclonal antibody to a
Sarcocystis neurona organism or antigen in the sample, whereby binding
of the monoclonal antibody indicates the presence of Sarcocystis neurona
organism or antigen. The sample may be obtained from a warm-blooded
animal, such as a horse, cow, dog, cat, mink, raccoon, skunk, harbor seal,
sea otter, mouse, armadillo or human. The sample may be a tissue sample, a
fluid sample or a fecal sample. The tissue sample may be from brain, spinal
cord, placenta, lung, liver, muscle, connective tissue, vascular endothelium
or gastrointestinal tract. The fluid sample may be from blood, serum,
plasma, urine, milk, ascites, cerebrospinal fluid or fetal fluid.
The assay format may be a Western blot, a radioimmunoprecipitation, RIA, or
an ELISA, including a sandwich ELISA. The method may employ a solid support
such as a column, a dipstick, a filter or a microtiter dish. The ELISA may
comprise detection of bound 2G5-2 or 2A7-18 using a labeled anti-Ig
antibody. The ELISA also may be is a competitive assay. The assay also may
involve quantification. The assay may further comprise determining antigenic
profile of the Sarcocystis neurona organism.
In yet another embodiment, there is provided a kit comprising at least one a
monoclonal antibody that binds immunologically to a Sarcocystis neurona
organism or antigen, designated 2A7-18 or 2G5-2, in suitable container.
The kit may comprise both 2A7-18 and 2G5-2. The antibody may comprise a
label, for example, a bead, a radioisotope, a ligand, a chemilluminescent
molecule, a fluorescent molecule, or an enzyme.
In still yet another embodiment, there is provided a method of detecting the
presence, in a sample, of antibodies that bind immunologically to a
Sarcocystis neurona organism or antigen, comprising (a) providing a test
composition comprising a Sarcocystis neurona organism or antigen; (b)
contacting the test composition with a known amount of a monoclonal antibody
that binds immunologically to a Sarcocystis neurona organism or
antigen, designated 2G5-2 or 2A7-18, the monoclonal antibody comprising a
detectable label; (c) contacting the product of step (b) with the sample;
and (d) measuring a change in the amount of label associated with the test
composition, as compared to the amount observed in step (b), wherein a
decrease in the amount of label associated with the test composition
indicates the presence of antibodies that bind immunologically to a
Sarcocystis neurona organism or antigen in the sample. The measuring of
change may be quantitative. The label may be an enzyme label, a radiolabel,
a chemilluminescent label or a fluorescent label.
In yet a further embodiment, there is provided a method of isolating a
Sarcocystis neurona organism or antigen comprising (a) providing a
monoclonal antibody that binds immunologically to a Sarcocystis neurona
organism or antigen, designated 2G5-2 or 2A7-18; (b) contacting the
antibody with a sample containing a Sarcocystis neurona organism or
antigen; and (c) isolating the antibody from the sample, whereby isolation
of the antibody also isolates the Sarcocystis neurona organism or
antigen. The antibody may be bound to a support, for example, a column, a
dipstick, a filter or a plate. The antibody also may comprise a label that
permits isolation thereof.
The method may further comprise isolating the Sarcocystis neurona
organism or antigen away from the monoclonal antibody. The monoclonal
antibody may further comprise a label that permit isolation of the antibody,
such as a bead, a chemilluminescent or fluorescent tag or a ligand. The
isolating step may comprise affinity chromatography, fluorescence activated
cell sorting, precipitation or centrifugation.
In still yet a further embodiment, there is provided a method of treating a
horse for equine protozoal myeloencephalitis (EPM) comprising (a) obtaining
a tissue or fluid sample from the horse; (b) contacting the sample with a
monoclonal antibody that binds immunologically to a Sarcocystis neurona
organism or antigen, the antibody designated 2G5-2 or 2A7-18; (c)
determining binding of the monoclonal antibody to a Sarcocystis neurona
organism or antigen in the sample; and (d) in the event that a
Sarcocystis neurona organism or antigen is identified in the sample,
treating the horse for EPM.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
I. The Present Invention
The present invention is directed to the identification of two particular
antibodies against Sarcocystis neurona and antigens thereof. This
application provides the first enabling disclosure for antibodies to
Sarcocystis neurona. In addition is provides for the use of these
antibodies, both individually and in combination, in the screening of
samples for a Sarcocystis neurona organism, antigen or antibodies.
This is particularly important as Sarcocystis neurona is a
significant pathogen for horses, where it is the cause of equine protozoal
myeloencephalitis, or "EPM." The details of the invention are provided
II. Sarcocystis Neurona and Equine Protozoal Myeloencephalitis (EPM)
Sarcocystis neurona is a coccidian protozoan that exhibits a wide
geographic distribution, including North and South America, as well as parts
of the Old World. It has a number of hosts including opossum (definitive
host), skunks, armadillos, cats (intermediate host) and horses (accidental
host). Sites of infection include the intestinal lining (carnivore host),
skeletal muscle (intermediate hosts) and brain & spinal cord (horses).
Horses are infected by ingesting infective S. neurona sporocysts.
Sarcocystis neurona has an obligate 2-host species life cycle, including
the natural intermediate host which is a prey species of some kind, and a
definitive host, the opossum. Until recently, S. neurona was
considered likely to be S. falcatula, a species of Sarcocystis that
utilizes birds of passerine (grackles, cowbirds, starlings), psittacine
(budgerigars) or columborid (pigeons) birds as intermediate hosts, but
recent research conducted at the University of Florida, the University of
California, the University of Missouri and Cornell University has revealed
that S. neurona and S. falcatula are not the same species.
However, the opossum (Didelphis virginiana) is the definitive host
for both species, and is the source of the infective sporocysts to horses.
The parasite encysts in the muscle tissue of the intermediate host. When
this tissue is eaten by opossums, the organism undergoes sexual reproduction
in intestinal epithelium, and forms infective sporocysts contained within an
oocyst. Oocysts and sporocysts are found in the intestinal contents but the
fragile oocyst is commonly disrupted by the time feces are passed. The
intermediate host becomes infected by ingesting sporocysts which presumably
contaminate the feed or water. Sporozoites emerge from the sporocysts,
penetrate the intestines, and become tachyzoites (merozoites), which undergo
a series of replicative cycles in the vascular endothelial cells, and
possibly white blood cells. The protozoa enter host cells, and become the
intracellular stage, the schizont, or meront. This schizont is a "mother
cell" that divides asexually into many tachyzoite offspring.
In the natural intermediate hosts, later generations of tachyzoites migrate
to muscle, and encyst in myocytes, forming sarcocysts. These later
generations of tachyzoites develop into a slowly dividing stage, called
bradyzoites. Bradyzoites divide slowly over the course of the host's
lifetime. Some species of Sarcocystis cause myositis in the intermediate
host (e.g., eosinophilic myositis caused by S. cruzi in cattle) while
others evade the host immune system, and remain asymptomatic (e.g., S.
fayeri in horses is rarely associated with myositis). When the natural
intermediate host is killed, or dies and is eaten, these cysts are activated
in the gastrointestinal tract of the definitive host, and the cycle starts
again. Most Sarcocystis spp. are found encysted in the muscle tissue
of the intermediate host, but some Sarcocystis spp., as well as the
closely related protozoa, Toxoplasma gondii, have been found in cysts
in the central nervous system.
Horses are an aberrant intermediate host of S. neurona. Sporocysts
are eaten, pass into the small intestines and excyst in the horse. From
there, the infective stage of the organism, the sporozoites, enter the
horse's blood stream. In some horses, they undergo several replicative
cycles in endothelial cells (in blood vessels), becoming tachyzoites, and
migrate to the central nervous system. They replicate asexually within
neurons and microglial cells, without forming tissue cysts. In the central
nervous system of the horse, they slowly divide and grow, gradually
destroying the nervous tissue, causing incoordination and the other clinical
signs that result from EPM. The stage of the organism found horses cannot be
transmitted to other horses. Because the organism does not encyst in the
tissues, it cannot be transmitted to opossums, even if the opossum were to
eat the tissue. Therefore, the horse is a dead end host for the protozoan.
Diagnosis is limited, relying on unexplained neurologic symptoms, and
testing spinal fluid for antibodies. Treatment recommendations vary;
however, the trends if toward: (a) trimethoprim-sulfadiazine, 15-25 mg/kg of
body weight, orally, every 12 hours for at least 12 weeks; (b) pyrimethamine
(Daraprim), 1.0 mg/kg orally, every 24 hours for at least 12 weeks; (c) rest
and minimizing of stress; and (d) anti-inflammatories. While most horses
respond to treatment, few make a complete recovery.
III. Producing Monoclonal Antibodies
It will be understood that monoclonal antibodies binding to S. neurona
and related proteins will have utilities in several applications. These
include the production of diagnostic kits for use in detecting and
diagnosing disease. In these contexts, one may to link such antibodies to
diagnostic or therapeutic agents, or use them as capture agents or
competitors in competitive assays. Means for preparing and characterizing
antibodies are well known in the art (see, e.g., Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory, 1988; U.S. Pat. No. 4,196,265).
The methods for generating monoclonal antibodies (MAbs) generally begin
along the same lines as those for preparing polyclonal antibodies. The first
step for both these methods is immunization of an appropriate host. As is
well known in the art, a given composition may vary in its immunogenicity.
It is often necessary therefore to boost the host immune system, as may be
achieved by coupling a peptide or polypeptide immunogen to a carrier.
Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and
bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum
albumin or rabbit serum albumin can also be used as carriers. Means for
conjugating a polypeptide to a carrier protein are well known in the art and
include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester,
carbodiimyde and bis-biazotized benzidine.
As also is well known in the art, the immunogenicity of a particular
immunogen composition can be enhanced by the use of non-specific stimulators
of the immune response, known as adjuvants. Exemplary and preferred
adjuvants include complete Freund's adjuvant (a non-specific stimulator of
the immune response containing killed Mycobacterium tuberculosis),
incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
The amount of immunogen composition used in the production of polyclonal
antibodies varies upon the nature of the immunogen as well as the animal
used for immunization. A variety of routes can be used to administer the
immunogen (subcutaneous, intramuscular, intradermal, intravenous and
intraperitoneal). The production of polyclonal antibodies may be monitored
by sampling blood of the immunized animal at various points following
A second, booster injection, also may be given. The process of boosting and
titering is repeated until a suitable titer is achieved. When a desired
level of immunogenicity is obtained, the immunized animal can be bled and
the serum isolated and stored, and/or the animal can be used to generate
Following immunization, somatic cells with the potential for producing
antibodies, specifically B lymphocytes (B cells), are selected for use in
the MAb generating protocol. These cells may be obtained from biopsied
spleens or lymph nodes. Spleen cells and lymph node cells are preferred, the
former because they are a rich source of antibody-producing cells that are
in the dividing plasmablast stage. Often, a panel of animals will have been
immunized and the spleen of animal with the highest antibody titer will be
removed and the spleen lymphocytes obtained by homogenizing the spleen with
a syringe. Typically, a spleen from an immunized mouse contains
approximately 5×107 to 2×108 lymphocytes.
The antibody-producing B lymphocytes from the immunized animal are then
fused with cells of an immortal myeloma cell, generally one of the same
species as the animal that was immunized. Myeloma cell lines suited for use
in hybridoma-producing fusion procedures preferably are
non-antibody-producing, have high fusion efficiency, and enzyme deficiencies
that render then incapable of growing in certain selective media which
support the growth of only the desired fused cells (hybridomas).
Any one of a number of myeloma cells may be used, as are known to those of
skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984). For
example, where the immunized animal is a mouse, one may use P3-X63/Ag8,
X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC,-11, MPC11-X45-GTG 1.7
and S194/5XXO Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and
4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in
connection with human cell fusions.
One preferred murine myeloma cell is the NS-1 myeloma cell line (also termed
P3-NS-1-Ag4-1), which is readily available from the NIGMS Human Genetic
Mutant Cell Repository by requesting cell line repository number GM3573.
Another mouse myeloma cell line that may be used is the
8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.
Methods for generating hybrids of antibody-producing spleen or lymph node
cells and myeloma cells usually comprise mixing somatic cells with myeloma
cells in a 2:1 proportion, though the proportion may vary from about 20:1 to
about 1:1, respectively, in the presence of an agent or agents (chemical or
electrical) that promote the fusion of cell membranes. Fusion methods using
Sendai virus have been described by Kohler and Milstein (1975; 1976), and
those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et
al. (1977). The use of electrically induced fusion methods also is
appropriate (Goding, pp. 71-74, 1986).
Fusion procedures usually produce viable hybrids at low frequencies, about
1×10-6 to 1×10-8. However, this does not pose a
problem, as the viable, fused hybrids are differentiated from the parental,
infused cells (particularly the infused myeloma cells that would normally
continue to divide indefinitely) by culturing in a selective medium. The
selective medium is generally one that contains an agent that blocks the de
novo synthesis of nucleotides in the tissue culture media. Exemplary and
preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin
and methotrexate block de novo synthesis of both purines and pyrimidines,
whereas azaserine blocks only purine synthesis. Where aminopterin or
methotrexate is used, the media is supplemented with hypoxanthine and
thymidine as a source of nucleotides (HAT medium). Where azaserine is used,
the media is supplemented with hypoxanthine.
The preferred selection medium is HAT. Only cells capable of operating
nucleotide salvage pathways are able to survive in HAT medium. The myeloma
cells are defective in key enzymes of the salvage pathway, e.g.,
hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The
B cells can operate this pathway, but they have a limited life span in
culture and generally die within about two weeks. Therefore, the only cells
that can survive in the selective media are those hybrids formed from
myeloma and B cells.
This culturing provides a population of hybridomas from which specific
hybridomas are selected. Typically, selection of hybridomas is performed by
culturing the cells by single-clone dilution in microtiter plates, followed
by testing the individual clonal supernatants (after about two to three
weeks) for the desired reactivity. The assay should be sensitive, simple and
rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays,
plaque assays, dot immunobinding assays, and the like.
The selected hybridomas are then serially diluted and cloned into individual
antibody-producing cell lines, which clones can then be propagated
indefinitely to provide MAbs. The cell lines may be exploited for MAb
production in two basic ways. A sample of the hybridoma can be injected
(often into the peritoneal cavity) into a histocompatible animal of the type
that was used to provide the somatic and myeloma cells for the original
fusion (e.g., a syngeneic mouse). Optionally, the animals are primed with a
hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior
to injection. The injected animal develops tumors secreting the specific
monoclonal antibody produced by the fused cell hybrid. The body fluids of
the animal, such as serum or ascites fluid, can then be tapped to provide
MAbs in high concentration. The individual cell lines could also be cultured
in vitro, where the MAbs are naturally secreted into the culture medium from
which they can be readily obtained in high concentrations.
MAbs produced by either means may be further purified, if desired, using
filtration, centrifugation and various chromatographic methods such as HPLC
or affinity chromatography. Fragments of the monoclonal antibodies of the
invention can be obtained from the purified monoclonal antibodies by methods
which include digestion with enzymes, such as pepsin or papain, and/or by
cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal
antibody fragments encompassed by the present invention can be synthesized
using an automated peptide synthesizer.
It also is contemplated that a molecular cloning approach may be used to
generate monoclonals. For this, combinatorial immunoglobulin phagemid
libraries are prepared from RNA isolated from the spleen of the immunized
animal, and phagemids expressing appropriate antibodies are selected by
panning using cells expressing the antigen and control cells e.g.,
normal-versus-tumor cells. The advantages of this approach over conventional
hybridoma techniques are that approximately 104 times as many
antibodies can be produced and screened in a single round, and that new
specificities are generated by H and L chain combination which further
increases the chance of finding appropriate antibodies.
Other U.S. patents, each incorporated herein by reference, that teach the
production of antibodies useful in the present invention include U.S. Pat.
No. 5,565,332, which describes the production of chimeric antibodies using a
combinatorial approach; U.S. Pat. No. 4,816,567 which describes recombinant
immunoglobin preparations; and U.S. Pat. No. 4,867,973 which describes
antibody-therapeutic agent conjugates.
IV. Antibody Conjugates
Antibodies of the present invention may be linked to at least one agent to
form an antibody conjugate. In order to increase the efficacy of antibody
molecules as diagnostic or therapeutic agents, it is conventional to link or
covalently bind or complex at least one desired molecule or moiety. Such a
molecule or moiety may be, but is not limited to, at least one effector or
reporter molecule. Effector molecules comprise molecules having a desired
activity, e.g., cytotoxic activity. Non-limiting examples of effector
molecules which have been attached to antibodies include toxins, anti-tumor
agents, therapeutic enzymes, radionuclides, antiviral agents, chelating
agents, cytokines, growth factors, and oligo- or polynucleotides. By
contrast, a reporter molecule is defined as any moiety which may be detected
using an assay. Non-limiting examples of reporter molecules which have been
conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent
labels, phosphorescent molecules, chemilluminescent molecules, chromophores,
photoaffinity molecules, colored particles or ligands, such as biotin.
Antibody conjugates are generally preferred for use as diagnostic agents.
Antibody diagnostics generally fall within two classes, those for use in in
vitro diagnostics, such as in a variety of immunoassays, and those for use
in vivo diagnostic protocols, generally known as "antibody-directed
imaging." Many appropriate imaging agents are known in the art, as are
methods for their attachment to antibodies (see, for e.g., U.S. Pat. Nos.
5,021,236, 4,938,948, and 4,472,509). The imaging moieties used can be
paramagnetic ions, radioactive isotopes, fluorochromes, NMR-detectable
substances, and X-ray imaging agents.
In the case of paramagnetic ions, one might mention by way of example ions
such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II),
nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III),
gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium
(III) and/or erbium (III), with gadolinium being particularly preferred.
Ions useful in other contexts, such as X-ray imaging, include but are not
limited to lanthanum (III), gold (III), lead (II), and especially bismuth
In the case of radioactive isotopes for therapeutic and/or diagnostic
application, one might mention astatine211, 14carbon,
51chromium, 36chlorine, 57cobalt, 58cobalt,
copper67, 152Eu, gallium67, 3hydrogen,
iodine123, iodine125, iodine131, indium111,
59iron, =phosphorus, rhenium186, rhenium188, 75selenium,
35sulphur, technicium99m and/or yttrium90.
125I is often being preferred for use in certain embodiments, and
technicium99m and/or indium111 are also often
preferred due to their low energy and suitability for long range detection.
Radioactively labeled monoclonal antibodies of the present invention may be
produced according to well-known methods in the art. For instance,
monoclonal antibodies can be iodinated by contact with sodium and/or
potassium iodide and a chemical oxidizing agent such as sodium hypochlorite,
or an enzymatic oxidizing agent, such as lactoperoxidase. Monoclonal
antibodies according to the invention may be labeled with technetium99m
by ligand exchange process, for example, by reducing pertechnate with
stannous solution, chelating the reduced technetium onto a Sephadex column
and applying the antibody to this column. Alternatively, direct labeling
techniques may be used, e.g., by incubating pertechnate, a reducing agent
such as SNCl2, a buffer solution such as sodium-potassium
phthalate solution, and the antibody. Intermediary functional groups which
are often used to bind radioisotopes which exist as metallic ions to
antibody are diethylenetriaminepentaacetic acid (DTPA) or ethylene
diaminetetracetic acid (EDTA).
Among the fluorescent labels contemplated for use as conjugates include
Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL,
BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM,
Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500,
Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red,
Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or Texas Red.
Another type of antibody conjugates contemplated in the present invention
are those intended primarily for use in vitro, where the antibody is linked
to a secondary binding ligand and/or to an enzyme (an enzyme tag) that will
generate a colored product upon contact with a chromogenic substrate.
Examples of suitable enzymes include urease, alkaline phosphatase,
(horseradish) hydrogen peroxidase or glucose oxidase. Preferred secondary
binding ligands are biotin and avidin and streptavidin compounds. The use of
such labels is well known to those of skill in the art and are described,
for example, in U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345,
4,277,437, 4,275,149 and 4,366,241.
Yet another known method of site-specific attachment of molecules to
antibodies comprises the reaction of antibodies with hapten-based affinity
labels. Essentially, hapten-based affinity labels react with amino acids in
the antigen binding site, thereby destroying this site and blocking specific
antigen reaction. However, this may not be advantageous since it results in
loss of antigen binding by the antibody conjugate.
Molecules containing azido groups may also be used to form covalent bonds to
proteins through reactive nitrene intermediates that are generated by low
intensity ultraviolet light (Potter and Haley, 1983). In particular, 2- and
8-azido analogues of purine nucleotides have been used as site-directed
photoprobes to identify nucleotide binding proteins in crude cell extracts
(Owens & Haley, 1987; Atherton et al., 1985). The 2- and 8-azido nucleotides
have also been used to map nucleotide binding domains of purified proteins (Khatoon
et al., 1989; King et al, 1989; Dholakia et al., 1989) and may be used as
antibody binding agents.
Several methods are known in the art for the attachment or conjugation of an
antibody to its conjugate moiety. Some attachment methods involve the use of
a metal chelate complex employing, for example, an organic chelating agent
such a diethylenetriaminepentaacetic acid anhydride (DTPA);
ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/or
tetrachloro-3α-6α-diphenylglycouril-3 attached to the antibody (U.S. Pat.
Nos. 4,472,509 and 4,938,948). Monoclonal antibodies may also be reacted
with an enzyme in the presence of a coupling agent such as glutaraldehyde or
periodate. Conjugates with fluorescein markers are prepared in the presence
of these coupling agents or by reaction with an isothiocyanate. In U.S. Pat.
No. 4,938,948, imaging of breast tumors is achieved using monoclonal
antibodies and the detectable imaging moieties are bound to the antibody
using linkers such as methyl-p-hydroxybenzimidate or
In other embodiments, derivatization of immunoglobulins by selectively
introducing sulfhydryl groups in the Fe region of an immunoglobulin, using
reaction conditions that do not alter the antibody combining site are
contemplated. Antibody conjugates produced according to this methodology are
disclosed to exhibit improved longevity, specificity and sensitivity (U.S.
Pat. No. 5,196,066, incorporated herein by reference). Site-specific
attachment of effector or reporter molecules, wherein the reporter or
effector molecule is conjugated to a carbohydrate residue in the Fe region
have also been disclosed in the literature (O'Shannessy et al., 1987). This
approach has been reported to produce diagnostically and therapeutically
promising antibodies which are currently in clinical evaluation.
V. Immunodetection Methods
In still further embodiments, the present invention concerns immunodetection
methods for binding, purifying, removing, quantifying and otherwise
generally detecting Sarcocystis neurona and its associated antigens.
Some immunodetection methods include enzyme linked immunosorbent assay
(ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay,
chemiluminescent assay, bioluminescent assay, and Western blot to mention a
few. In particular, a competitive assay for the detection and quantitation
of S. neurona antibodies directed to specific parasite epitopes in
samples also is provided. The steps of various useful immunodetection
methods have been described in the scientific literature, such as, e.g.,
Doolittle and Ben-Zeev (1999), Gulbis and Galand (1993), De Jager et al.
(1993), and Nakamura et al. (1987). In general, the immunobinding methods
include obtaining a sample suspected of containing S. neurona, and
contacting the sample with a first antibody in accordance with the present
invention, as the case may be, under conditions effective to allow the
formation of immunocomplexes.
These methods include methods for purifying S. neurona or related
antigens from a sample. The antibody will preferably be linked to a solid
support, such as in the form of a column matrix, and the sample suspected of
containing the S. neurona or antigenic component will be applied to
the immobilized antibody. The unwanted components will be washed from the
column, leaving the S. neurona antigen immunocomplexed to the
immobilized antibody, which is then collected by removing the organism or
antigen from the column.
The immunobinding methods also include methods for detecting and quantifying
the amount of S. neurona or related components in a sample and the
detection and quantification of any immune complexes formed during the
binding process. Here, one would obtain a sample suspected of containing
S. neurona or its antigens, and contact the sample with an antibody that
binds S. neurona or components thereof, followed by detecting and
quantifying the amount of immune complexes formed under the specific
conditions. In terms of antigen detection, the biological sample analyzed
may be any sample that is suspected of containing S. neurona or S.
neurona antigen, such as a tissue section or specimen, a homogenized tissue
extract, a biological fluid, including blood and serum, or a secretion, such
as feces or urine.
Contacting the chosen biological sample with the antibody under effective
conditions and for a period of time sufficient to allow the formation of
immune complexes (primary immune complexes) is generally a matter of simply
adding the antibody composition to the sample and incubating the mixture for
a period of time long enough for the antibodies to form immune complexes
with, i.e., to bind to S. neurona or antigens present. After this
time, the sample-antibody antibody composition, such as a tissue section,
ELISA plate, dot blot or Western blot, will generally be washed to remove
any non-specifically bound antibody species, allowing only those antibodies
specifically bound within the primary immune complexes to be detected.
In general, the detection of immunocomplex formation is well known in the
art and may be achieved through the application of numerous approaches.
These methods are generally based upon the detection of a label or marker,
such as any of those radioactive, fluorescent, biological and enzymatic
tags. Patents concerning the use of such labels include U.S. Pat. Nos.
3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and
4,366,241. Of course, one may find additional advantages through the use of
a secondary binding ligand such as a second antibody and/or a biotin/avidin
ligand binding arrangement, as is known in the art.
The antibody employed in the detection may itself be linked to a detectable
label, wherein one would then simply detect this label, thereby allowing the
amount of the primary immune complexes in the composition to be determined.
Alternatively, the first antibody that becomes bound within the primary
immune complexes may be detected by means of a second binding ligand that
has binding affinity for the antibody. In these cases, the second binding
ligand may be linked to a detectable label. The second binding ligand is
itself often an antibody, which may thus be termed a "secondary" antibody.
The primary immune complexes are contacted with the labeled, secondary
binding ligand, or antibody, under effective conditions and for a period of
time sufficient to allow the formation of secondary immune complexes. The
secondary immune complexes are then generally washed to remove any
non-specifically bound labeled secondary antibodies or ligands, and the
remaining label in the secondary immune complexes is then detected.
Further methods include the detection of primary immune complexes by a two
step approach. A second binding ligand, such as an antibody that has binding
affinity for the antibody, is used to form secondary immune complexes, as
described above. After washing, the secondary immune complexes are contacted
with a third binding ligand or antibody that has binding affinity for the
second antibody, again under effective conditions and for a period of time
sufficient to allow the formation of immune complexes (tertiary immune
complexes). The third ligand or antibody is linked to a detectable label,
allowing detection of the tertiary immune complexes thus formed. This system
may provide for signal amplification if this is desired.
One method of immunodetection uses two different antibodies. A first
biotinylated antibody is used to detect the target antigen, and a second
antibody is then used to detect the biotin attached to the complexed biotin.
In that method, the sample to be tested is first incubated in a solution
containing the first step antibody. If the target antigen is present, some
of the antibody binds to the antigen to form a biotinylated antibody/antigen
complex. The antibody/antigen complex is then amplified by incubation in
successive solutions of streptavidin (or avidin), biotinylated DNA, and/or
complementary biotinylated DNA, with each step adding additional biotin
sites to the antibody/antigen complex. The amplification steps are repeated
until a suitable level of amplification is achieved, at which point the
sample is incubated in a solution containing the second step antibody
against biotin. This second step antibody is labeled, as for example with an
enzyme that can be used to detect the presence of the antibody/antigen
complex by histoenzymology using a chromogen substrate. With suitable
amplification, a conjugate can be produced which is macroscopically visible.
Another known method of immunodetection takes advantage of the immuno-PCR
(Polymerase Chain Reaction) methodology. The PCR method is similar to the
Cantor method up to the incubation with biotinylated DNA, however, instead
of using multiple rounds of streptavidin and biotinylated DNA incubation,
the DNA/biotin/streptavidin/antibody complex is washed out with a low pH or
high salt buffer that releases the antibody. The resulting wash solution is
then used to carry out a PCR reaction with suitable primers with appropriate
controls. At least in theory, the enormous amplification capability and
specificity of PCR can be utilized to detect a single antigen molecule.
Immunoassays, in their most simple and direct sense, are binding assays.
Certain preferred immunoassays are the various types of enzyme linked
immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art.
Immunohistochemical detection using tissue sections is also particularly
useful. However, it will be readily appreciated that detection is not
limited to such techniques, and western blotting, dot blotting, FACS
analyses, and the like may also be used.
In one exemplary ELISA, the antibodies of the invention are immobilized onto
a selected surface exhibiting protein affinity, such as a well in a
polystyrene microtiter plate. Then, a test composition suspected of
containing the S. neurona or S. neurona antigen is added to
the wells. After binding and washing to remove non-specifically bound immune
complexes, the bound antigen may be detected. Detection may be achieved by
the addition of another anti-S. neurona antibody that is linked to a
detectable label. This type of ELISA is a simple "sandwich ELISA." Detection
may also be achieved by the addition of a second anti-S. neurona
antibody, followed by the addition of a third antibody that has binding
affinity for the second antibody, with the third antibody being linked to a
In another exemplary ELISA, the samples suspected of containing the S.
neurona or S. neurona antigen are immobilized onto the well
surface and then contacted with the anti-S. neurona antibodies of the
invention. After binding and washing to remove non-specifically bound immune
complexes, the bound anti-S. neurona antibodies are detected. Where
the initial anti-S. neurona antibodies are linked to a detectable
label, the immune complexes may be detected directly. Again, the immune
complexes may be detected using a second antibody that has binding affinity
for the first anti-S. neurona antibody, with the second antibody
being linked to a detectable label.
Irrespective of the format employed, ELISAs have certain features in common,
such as coating, incubating and binding, washing to remove non-specifically
bound species, and detecting the bound immune complexes. These are described
In coating a plate with either antigen or antibody, one will generally
incubate the wells of the plate with a solution of the antigen or antibody,
either overnight or for a specified period of hours. The wells of the plate
will then be washed to remove incompletely adsorbed material. Any remaining
available surfaces of the wells are then "coated" with a nonspecific protein
that is antigenically neutral with regard to the test antisera. These
include bovine serum albumin (BSA), casein or solutions of milk powder. The
coating allows for blocking of nonspecific adsorption sites on the
immobilizing surface and thus reduces the background caused by nonspecific
binding of antisera onto the surface.
In ELISAs, it is probably more customary to use a secondary or tertiary
detection means rather than a direct procedure. Thus, after binding of a
protein or antibody to the well, coating with a non-reactive material to
reduce background, and washing to remove unbound material, the immobilizing
surface is contacted with the biological sample to be tested under
conditions effective to allow immune complex (antigen/antibody) formation.
Detection of the immune complex then requires a labeled secondary binding
ligand or antibody, and a secondary binding ligand or antibody in
conjunction with a labeled tertiary antibody or a third binding ligand.
"Under conditions effective to allow immune complex (antigen/antibody)
formation" means that the conditions preferably include diluting the
antigens and/or antibodies with solutions such as BSA, bovine gamma globulin
(BGG) or phosphate buffered saline (PBS)/Tween. These added agents also tend
to assist in the reduction of nonspecific background.
The "suitable" conditions also mean that the incubation is at a temperature
or for a period of time sufficient to allow effective binding. Incubation
steps are typically from about 1 to 2 to 4 hours or so, at temperatures
preferably on the order of 25° C. to 27° C., or may be overnight at about 4°
C. or so.
Following all incubation steps in an ELISA, the contacted surface is washed
so as to remove non-complexed material. A preferred washing procedure
includes washing with a solution such as PBS/Tween, or borate buffer.
Following the formation of specific immune complexes between the test sample
and the originally bound material, and subsequent washing, the occurrence of
even minute amounts of immune complexes may be determined.
To provide a detecting means, the second or third antibody will have an
associated label to allow detection. Preferably, this will be an enzyme that
will generate color development upon incubating with an appropriate
chromogenic substrate. Thus, for example, one will desire to contact or
incubate the first and second immune complex with a urease, glucose oxidase,
alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period
of time and under conditions that favor the development of further immune
complex formation (e.g., incubation for 2 hours at room temperature in a
PBS-containing solution such as PBS-Tween).
After incubation with the labeled antibody, and subsequent to washing to
remove unbound material, the amount of label is quantified, e.g., by
incubation with a chromogenic substrate such as urea, or bromocresol purple,
or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS), or H2O2,
in the case of peroxidase as the enzyme label. Quantification is then
achieved by measuring the degree of color generated, e.g., using a visible
In another embodiment, the present invention contemplates the use of
competitive formats. This is particularly useful in the detection of S.
neurona antibodies in sample. In competition based assays, an unknown
amount of analyte or antibody is determined by its ability to displace a
known amount of labeled antibody or analyte. Thus, the quantifiable loss of
a signal is an indication of the amount of unknown antibody or analyte in a
Here, the inventor proposes the use of labeled S. neurona monoclonal
antibodies to determine the amount of S. neurona antibodies in a
sample. The basic format would include contacting a known amount of S.
neurona monoclonal antibody (linked to a detectable label) with S.
neurona antigen or organism. The S. neurona antigen or organism
is preferably attached to a support. After binding of the labeled monoclonal
antibody to the support, the sample is added and incubated under conditions
permitting any unlabeled antibody in the sample to compete with, and hence
displace, the labeled monoclonal antibody. By measuring either the lost
label or the label remaining (and subtracting that from the original amount
of bound label), one can determine how much non-labeled antibody is bound to
the support, and thus how much antibody was present in the sample.
The antibodies of the present invention may also be used in conjunction with
both fresh-frozen and/or formalin-fixed, paraffin-embedded tissue blocks
prepared. for study by immunohistochemistry (IHC). The method of preparing
tissue blocks from these particulate specimens has been successfully used in
previous IHC studies of various prognostic factors, and is well known to
those of skill in the art (Brown et al., 1990; Abbondanzo et al., 1990;
Allred et al., 1990).
Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen
"pulverized" tissue at room temperature in phosphate buffered saline (PBS)
in small plastic capsules; pelleting the particles by centrifugation;
resuspending them in a viscous embedding medium (OCT); inverting the capsule
and/or pelleting again by centrifugation; snap-freezing in -70° C.
isopentane; cutting the plastic capsule and/or removing the frozen cylinder
of tissue; securing the tissue cylinder on a cryostat microtome chuck;
and/or cutting 25-50 serial sections from the capsule. Alternatively, whole
frozen tissue samples may be used for serial section cuttings.
Permanent-sections may be prepared by a similar method involving rehydration
of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in
10% formalin for 4 hours fixation; washing/pelleting; resuspending in warm
2.5% agar; pelleting; cooling in ice water to harden the agar; removing the
tissue/agar block from the tube; infiltrating and/or embedding the block in
paraffin; and/or cutting up to 50 serial permanent sections. Again, whole
tissue samples may be substituted.
3. Immunodetection Kits
In still further embodiments, the present invention concerns immunodetection
kits for use with the immunodetection methods described above. As the S.
neurona antibodies are generally used to detect S. neurona or
S. neurona antigens, the antibodies will be included in the kit. The
immunodetection kits will thus comprise, in suitable container means, a
first antibody that binds to S. neurona or S. neurona antigen,
and optionally an immunodetection reagent.
In certain embodiments, the S. neurona antibody may be pre-bound to a
solid support, such as a column matrix and/or well of a microtitre plate.
The immunodetection reagents of the kit may take any one of a variety of
forms, including those detectable labels that are associated with or linked
to the given antibody. Detectable labels that are associated with or
attached to a secondary binding ligand are also contemplated. Exemplary
secondary ligands are those secondary antibodies that have binding affinity
for the first antibody.
Further suitable immunodetection reagents for use in the present kits
include the two-component reagent that comprises a secondary antibody that
has binding affinity for the first antibody, along with a third antibody
that has binding affinity for the second antibody, the third antibody being
linked to a detectable label. As noted above, a number of exemplary labels
are known in the art and all such labels may be employed in connection with
the present invention.
The kits may further comprise a suitably aliquoted composition of the S.
neurona or S. neurona antigens, whether labeled or unlabeled, as
may be used to prepare a standard curve for a detection assay. The kits may
contain antibody-label conjugates either in fully conjugated form, in the
form of intermediates, or as separate moieties to be conjugated by the user
of the kit. The components of the kits may be packaged either in aqueous
media or in lyophilized form.
The container means of the kits will generally include at least one vial,
test tube, flask, bottle, syringe or other container means, into which the
antibody may be placed, or preferably, suitably aliquoted. The kits of the
present invention will also typically include a means for containing the
antibody, antigen, and any other reagent containers in close confinement for
commercial sale. Such containers may include injection or blow-molded
plastic containers into which the desired vials are retained.
Claim 1 of 11 Claims
1. A monoclonal antibody that binds immunologically to a Sarcocystis
neurona organism or antigen, designated 2A7-18, deposited with the
ATCC as PTA-3418, or 2G5-2, deposited with the ATCC as PTA-3419.
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