Immunoassay for equine protozoal myeloencephalitis in horses

An immunoassay for Sarcocystis neurona antibodies in equines is described. The immunoassay uses blocking of Sarcocystis antigens by antibodies to Sarcocystis sp. other than Sarcocystis neurona in connection with the immunoassay.

BACKGROUND OF THE INVENTION 
(1) Field of the Invention 
The present invention relates to an immunoassay method and test kit for 
detecting antibodies to at least one protein of Sarcocystis neurona 
produced in an equine suspected of harboring the Sarcocystis neurona which 
causes myleoencephalitis in the equine. The disease is debilitating and 
may frequently be fatal. The preferred immunoassay method uses preblocking 
of a Western blot membrane containing at least one known identifying 
antigen of Sarcocystis neurona with unlabeled antibodies of a Sarcocystis 
sp. other than Sarcocystis neurona in order to prevent false positive, 
non-specific binding of a labeled antibody to the protein (s). 
(2) Description of the Related Art 
A mammalian body relates to the presence of foreign antigens by producing 
antibody molecules from its lymphocyte cells. Antibodies have the property 
of selectively binding to certain distinctive sites, known as determinants 
on antigens, thereby rendering the antigens innocuous. The antibodies have 
a physical affinity for specific determinants or epitopes of antigenic 
material. A reaction between an antibody and a determinant on an antigen 
for which the antibody is specific results in an adduct, commonly referred 
to as an "immunocomplex". The formation of such complexes makes possible a 
wide variety of assays for antigenic material. Such assays are known 
generically as immunoassays. 
Immunoassays have replaced other procedures used for in vitro diagnostic 
methods to detect or quantitate a variety of antigens and/or antibodies in 
fluids and, particularly, body fluids such as blood serum, urine or spinal 
fluid with important biologic or pharmacologic properties. The high levels 
of sensitivity and specificity achieved with immunoassays result from the 
specific , high-affinity, reversible binding of antibodies and antigens, 
and from the existence of methods for attachment of sensitive detectable 
labels (radioactive isotopes, fluorophores, ferritin, free radicals, 
bacteriophages and enzymes) to antibodies or antigens. Enzymes are most 
commonly used today. 
Immunoassay techniques are based upon the complex binding of the antigenic 
substance being assayed (analyte) with an antibody or antibodies in which 
one or the other member of the complex may be labeled, permitting the 
detection and/or quantitative analysis of the target substance by virtue 
of the label activity associated with the labeled antigen complex or 
antibody. Immunoassays are generally classified into two groups: the 
heterogeneous immunoassay in which a labeled antigen or antibody is 
separated from the labeled antigen-antibody complex before measurement of 
label activity in either fraction; and the homogeneous immunoassay in 
which the activity of labeled antigen is measured in the presence of 
labeled antigen-antibody complex. 
Two such diagnostic assay techniques used to determine the presence or 
amount of antigen in body fluids are generally known as "competitive" 
assays and "non-competitive" or "sandwich" assays. Typically, in 
"competitive" assay techniques, an unlabeled antibody or antigen 
preparation bound to a solid support or carrier is first reacted with a 
labeled antigen or antibody reagent solution and then with the body fluid 
sample wherein the antigen or antibody in the sample competes with the 
labeled antigen for sites on the supported antibody or antigen. The amount 
of labeled antigen reagent displaced indicates the quantity of antigen 
present in the fluid sample to be detected. 
In the case of a "sandwich" or "non-competitive" assay, a quantity of 
unlabeled polyclonal or monoclonal antibody or antigen bound to a 
solid-support or carrier surface, is reacted with a body fluid sample 
being evaluated for antigens or antibodies, and then, after suitable 
incubation time and washing, the sample is further incubated with a 
solution of labeled anti-antibody. The labeled antibody bound to the solid 
phase in an antibody-antigen-antibody sandwich or the amount of unbound 
labeled antibody or antigen in the liquid phase would be determined as a 
measure of the presence of antigen or antibody in the test sample. 
Thus the analyte can be an antibody or an antigen which has produced 
antibodies in the host. 
The problem in the prior art is that some of the antibodies in samples 
recognize non-unique epitopes of proteins in the immunoassay and, thus, 
are non-specific. This can render the immunoassay non-specific. In the 
prior art this problem is referred to as "cross-reactivity". The result of 
cross-reactivity is that the immunoassay fails to accurately measure the 
specific analyte to be detected. 
Various techniques have been used to prevent non-specific reactions with 
the analyte. A polyclonal antibody (derived by exposing an animal to the 
antigen and then separation of the antibodies from the blood serum) can be 
reacted with reagents which bind non-specific proteins for removal. 
Monoclonal antibodies (from a fusion of myeloma cells and cells of the 
animal which produce the monoclonal antibody) can be used to produce more 
specific binding. The monoclonal antibodies still do not necessarily block 
non-specific reactions where the antibody recognizes a common protein 
epitope of the biological sample to be tested. Various blocking agents 
containing proteins, such as milk or gelatin, are used to block 
non-specific sites which might cause the antibody to bind 
non-specifically. These help, but do not solve the problem. 
The etiologic agent of equine protozoal myeloecephalitis (EPM) has been 
shown to be Sarcocystis neurona, (Dubey, J. P., et al., J. Parasitol. 
77:212-218 (1991)). Similar neurological disease has been described in one 
horse due to Neospora spp. (Marsh, A. E., et al., J. Am. Vet. Med. Assoc. 
190:1907-1913 (1996)). S. neurona utilizes the opossum (Didelphis 
virginiana) as its definitive host (Fenger, C. K., et. al., J. of 
Parasitology 81:916-919 (1995)); Fenger, C. K., et al., Vet. Parasitol. 
68:199-213 (1997); Conference of Research workers in Animal 
Diseases-Abstract 162, November 1997). The opossum passes infective 
sporocysts into the environment in its feces. Horses and ponies can become 
infected by ingesting sporocysts of S. neurona (Dubey, J. P, et al., J. 
Parasitol 77:212-218 (1991)), but they are dead end hosts. When the 
parasite enters the central nervous system of the horse, clinical 
neurological disease can result. No EPM cases have been reported in horses 
that have not originated from the Western Hemisphere. This is believed to 
be due to definitive host specificity of S. neurona, and therefore EPM 
does not typically occur outside the range of the opossum. 
A Western blot test was developed to detect antibodies to S. 
neurona-specific antigens (approximately 11, 13 and 10.5 kilodaltons) 
(Granstrom, D. E., et al., J. Vet Diag Invest 5:88-90 (1993)) in 
cerebrospinal fluid of horses suspected of having EPM. In 1997, a 
different criteria for a positive test (with reactivity to proteins of 
approximately 13, 11, 10.5 and 10 kDa) was used by the same laboratory in 
the only published experimental infection study of Sarcocystis from 
opossums in horses (Fenger, C. K., et al., Vet Parasitol 68:199-213 
(1997)). The Western blot test has also been used to estimate 
seroprevalence of antibodies to S. neurona in Pennsylvania, Ohio and 
Oregon (Bentz, B. G., et al., J. Am Vet Med Assoc 210:517-518 (1997); 
Saville, W. J., et al., JAVMA 210:519-518 (1997); Blythe, L. L., et al., 
JAVMA 210:525-527 (1997)). Seroprevalence estimates ranged from 22%-65% 
for various geographic regions sampled, suggesting high rates of infection 
with S. neurona. These Western blot assays have not been found to be 
reliable in predicting the presence of Sarcocystis neurona due to 
cross-reacting antibodies to other Sarcocystis sp. in the equine. 
Recent work has indicated that immunodominant proteins of approximately 12 
and 29 kDa are specific to S. neurona (Marsh, A. E., et al, J. Am Vet Med 
Assoc 190:1907-1913 (1996)), which would suggest a different criteria for 
a positive test result than that which has been used for diagnostic 
purposes and seroprevalence estimates. It was found that antibody 
cross-reactivity to other apicomplexian species can occur at or near these 
bands and therefore may cause false-positive test results. 
SUMMARY OF THE INVENTION 
The present invention relates to a method for the detection of Sarcocystis 
neurona in an immunoassay where a protein of Sarcocystis neurona is 
reacted with antibodies from an equine suspected of harboring the 
Sarcocystis neurona and the label is detected, the improvement which 
comprises: reacting the protein of the Sarcocystis neurona with a 
non-labeled antibody to proteins of a Sarcocystis sp. other than the 
Sarcocystis neurona from a species of a mammal other than equine prior to 
reaction with the antibody so that non-specific binding of the antibody is 
inhibited to thereby detect the Sarcocystis neurona. 
The present invention relates to a method for the detection of disease 
caused by Sarcocystis neurona in equines which comprises: 
(a) isolating fluid from the equine which can contain parasite induced 
antibodies to Sarcocystis neurona proteins, thus indicating the presence 
of the Sarcocystis neurona; 
(b) reacting the fluid with at least one identifying antigen of the 
Sarcocystis neurona protein bound on a substrate, wherein the substrate 
has been blocked with antibodies to Sarcocystis sp. other than Sarcocystis 
neurona so that antibodies to Sarcocystis neurona antigen in the serum are 
bound to the identifying antigen; 
(c) detecting the antibodies bound to the antigen so that the disease is 
detected. 
Further, the present invention relates to a kit for the detection of 
disease caused by Sarcocystis neurona which comprises in separate 
containers: 
(a) an identifying antibody which is specifically able to bind a first 
protein of Sarcocystis neurona; and 
(b) a non-labeled antibody which is specific for a second protein of a 
Sarcocystis sp. other than Sarcocystis neurona. 
Further still, the present invention relates to a kit for the detection of 
disease caused by Sarcocystis neurona in equines which comprises: 
(a) a substrate with at least one identifying antigen to the Sarcocystis 
neurona bound on a surface of the substrate, 
(b) antibody to a Sarcocystis sp. other than Sarcocystis neurona; and 
(c) at least one reagent for detection of an antibody in a fluid of the 
equine which antibody binds to the antigen of Sarcocystis neurona. 
OBJECTS 
It is an object of the present invention to provide a highly reliable 
immunoassay method and test kit for detecting antibodies in fluid from an 
equine which are developed as a result of infection by Sarcocystis 
neurona, the causative agent of protozoal myeloencephalitis in equines. In 
particular, it is an object of the present invention to provide a method 
and test kit which is relatively easy to perform, accurate and which is 
economical. These and other objects will become increasingly apparent by 
reference to the following description and the drawings.

DESCRIPTION OF PREFERRED EMBODIMENTS 
As used herein, the term "analyte" refers to the antibodies produced in an 
equine as a result of infection by an equine with Sarcocystis neurona. It 
is these antibodies which are to be measured. Typically the antibodies are 
measured from cerebrospinal fluid or serum. 
The antibodies used in the present assay are preferably polyclonal and 
prepared in a conventional manner. The polyclonal antibodies are raised in 
a variety of domesticated animals, preferably a goat, and isolated from 
serum. The polyclonal antibodies are purified by affinity chromatography 
and then separated. While more expensive, monoclonal antibodies raised 
from hybridomas can be obtained in a conventional manner. 
The preferred method of the present invention involves the separation of 
protein (antigens) from the Sarcocystis neurona using gel electrophoresis 
and then transferring and binding the proteins to a membrane in a Western 
blot. The serum is reacted with the proteins on the membrane which are in 
bands and then the labeled polyclonal antibody is reacted with the 
analyte-antigen complex. Certain band or bands are selected for detection 
based upon the protein(s) selected for producing the polyclonal antibody 
in the horse during infection. It has been found that the 30 and 60 kDa 
bands together provide a definitive result and thus are preferred based on 
testing statistically significant numbers of known positive and negative 
samples. 
The important aspect of the present invention is that sites of the proteins 
selected for analysis are blocked with an antibody which does not occur in 
equines to another Sarcocystis sp. This prevents non-specific binding of 
the labeled antibody with epitopes of the analyte which are not definitive 
for Sarcocystis neurona. 
It will be appreciated that other types of immunoassays are claimed where 
the antigenic protein from Sarcocystis neurona used to form the antibody 
antigen complex is reacted with an antibody from a non-equine directed 
against another Sarcocystis sp. All the available types of immunoassays 
are well known to those skilled in the art as previously discussed. 
The following Examples 1 and 2 show the preferred embodiment of the present 
invention. There are many variations which can be used. 
EXAMPLE 1 
The purpose of this Example was to develop the most stringent criteria 
possible for a positive test result, and to estimate the sensitivity and 
specifically of the preferred EPM Western blot antibody test. The Example 
shows the ability of bovine serum with antibodies to Sarcocystis cruzi, 
which is non-infective in equines, to act as a blocking agent to prevent 
false positive results in the Western blot test for S. neurona. 
Materials and Methods 
Selection of Samples 
49 equine serum samples were obtained from India, where S. neurona is not 
known to exist and where there are no opossums. These samples were defined 
as known negative controls, because the horses could not possess 
antibodies to S. neurona. Serum and cerebrospinal fluid samples from 8 
horses from which S. neurona was cultured postmortem were defined as known 
positive controls. 
Culture of Antigen 
Equine neural tissues from horses suspect for EPM were removed postmortem. 
The samples were stored in Hank's Balanced Salt Solution (HBSS) at room 
temperature. Within two hours of removal, portions of tissue were minced 
and ground in a Dounce homogenizer with cell culture medium. The slurry 
was poured onto confluent equine dermal cells and incubated at 37.degree. 
C., 5% CO.sub.2 for 24 hours. Medium was replaced after 24 hours and 
changed every two days for the first week and every week thereafter. One 
culture contaminated with an extracellular yeast resistent to amphotericin 
B at 1.25 .mu.g/ml was kept viable by washing vigorously with HBSS six 
times, refeeding with media containing 4 .mu.g/ml amphotericin B for 10 
days before replacing with normal media (penicillin and amikacin 
concentrations kept the same). Viable, replicating meroziotes of S. 
neurona were observed 7-49 days after inoculation. 
Western Blot Testing 
S. neurona meroziotes were harvested from equine dermal cell culture and 
heat denatured in sample buffer (0.5 M Tris (pH 7.4) with 10% SDS, 20% 
Glycerol and 5% B-mercaptoethanol), all by volume. Denatured proteins were 
separated by SDS-PAGE in 12%-20% linear gradient gels with a 4% stacking 
gel, both by volume. Separated proteins were electrophoretically 
transferred to Western PVDF membranes (at 100 volts for 1 1/2 hours, then 
150 volts for 1/2 hour) and blocked overnight in blocking buffer 1% by 
volume bovine serum albumen (not infected with Sarcocystis) as a blocking 
agent for the control and 0.5% Tween-Tris Buffered Saline (blocking 
buffer). Blots were air-dried and frozen for later use. 
For testing of serum samples according to the prior art, blots were wetted 
in 0.5% Tween-Tris Buffered Saline (TTBS) and clamped into a plexiglass 
press, Molecular weight markers were covered with blocking buffer in the 
first two lanes; the remaining 18 lanes were loaded with serum samples 
diluted 1:10 in blocking buffer, and incubated overnight. Subsequently, 
the blots were rinsed 3 times in TTBS, incubated in for 3 hours in 
blocking buffer with biotin labeled goat anti-horse IgG (H+L), rinsed 3 
times in TTBS, incubated in TTBS with avidin peroxidase conjugate for 45 
minutes, rinsed 3 times in TTBS and developed with aminoethyl carbazole 
(AEC) stain. 
For the bovine S. cruzi antisera treatment: The Western blot protocol was 
repeated with an additional treatment: the blots clamped in the plexiglass 
press were incubated with bovine serum with Sarcocystis cruzi antibodies 
in blocking buffer (1:10) for 2 hours, rinsed 3 times in TTBS, then loaded 
with equine samples and developed according to the prior art Western blot 
test. 
Bovine S. cruzi antisera was tested by the Western blot protocol described 
above, using biotin labeled goat anti-equine IgG (H+L) to verify that 
anti-equine IgG would not bind to bovine IgG. 
Data Analysis 
Known positive and negative samples were tested with and without the bovine 
S. cruzi antisera treatment. The protein bands detected by each sample 
were recorded and tested, alone and in combination with others, by the 
Fishers Exact test for independence (two-tailed). The combination of bands 
that was most strongly associated with a positive test was used to 
classify samples as Test Positives and Test Negatives. These totals were 
compared to the correct classification of True Positives and True 
Negatives, and test sensitivity and test specificity were calculated. 
Results 
When tested by Western block without the bovine S. cruzi antisera, no 
single band was significantly associated with S. neurona antibodies by the 
Fisher's Exact Test. This was also true of combinations of bands. 
Reactivity to proteins at 20 and 16 kDa occurred in all the 
culture-confirmed positive samples and in the majority of the known 
negative samples. No diagnostic criteria could be developed from these 
test results. 
Analysis of the proteins detected by the culture-confirmed positives and 
known negatives on the bovine S. cruzi antisera treated blots showed the 
combined presence of the 30 and 16 kDa bands to be strongly associated by 
the Fisher's Exact Test (P&gt;0.001, two-tailed) with a positive test result. 
All of the culture-confirmed positive samples exhibited both bands. 48 of 
the 49 known negatives tested negative by this criteria; one sample 
produced thin bands near enough to 30 and 16 KDa to be interpreted as a 
suspect positive. For the purposes of our analysis, that sample was 
classified as a false positive. 
Bovine IgG directed against S. cruzi was not detected by the goat 
anti-equine IgG. 
Sensitivity and Specificity Calculations 
The diagnostic criteria of simultaneous reactivity to the 30 and 16 KDa 
bands used on bovine S. cruzi antisera treated blots was compared to the 
true status of the samples. Because the culture-confirmed positive samples 
were all correctly identified, the test sensitivity is estimated to be 
approaching 100%. 48 of the 49 known negatives were correctly identified, 
thus the test specificity is estimated to be approximately 98%. Using the 
presence or absence of S. neurona-specific antibodies as the outcome, the 
sample positive predictive value was 88.9%, and the smapel negative 
predictive value was 100%. 
______________________________________ 
True (+) True (-) 
Totals 
______________________________________ 
Test (+) 8 1 9 
Test (-) 0 48 48 
Totals 8 49 57 
______________________________________ 
Example 1 supports earlier work which suggested that the best criteria for 
a positive S. neurona immunoblot test is immunodominant proteins at 
approximately 30 and 16 kDa (Marsh, A. E., et al., J. Am Vet Med Assoc 
209:1907-1913 (1996)). The culture-confirmed positive samples used in this 
analysis were highly consistent in their reactivity to those specific 
proteins. The results of this example also demonstrates that the Western 
blot S,. neurona antibody test can be subject to false positive results 
due to the antibody cross-reactivity. This is not surprising , since a 
solid-phase immunofluorescence procedure (FIAX) used until 1993 in testing 
for EPM utilized the affinity of S. neurona-directed antibodies for S. 
cruzi (Diagnostic test offered by Oklahoma State University, Dr. Carl Fox 
Laboratory--Unpublished results). Additionally, S. cruzi antisera proved 
equally effective as S. neurona antisera for detecting S. neurona by 
avidin-biotin complex immunoperoxidase immunohistochemical staining 
(Hamir, A. N., et al., J Vet Diagn Invest 5:418-422 (1993)). It is likely 
that antibodies directed against other common Sarcocystis species area 
source of false positive tests. 
Exposing S. neurona immunoblots to bovine S. cruzi antisera prior to 
testing equine samples proved to be an effective technique for reducing 
false positive test results. The bovine S. cruzi antibodies apparently 
bind to Sarcocystis genus-specific proteins, but not to S. 
neurona-specific proteins. Other Sarcocystis sp which horses may be 
exposed to include S. falcatula, S. rilei and others. By blocking the 
non-specific proteins that may be too close to discern from the specific 
proteins with the naked eye, both reading and interpretation is improved. 
The sensitivity and specificity of the Western blot test was quite high. It 
is possible that a horse from which S. neurona is cultured will not have 
antibodies directed against the immunodominant proteins at 30 and 16 kDa. 
A false negative such as this could be due to acute onset of EPM, which 
has not allowed the animal sufficient time to mount a detectable antibody 
response. Clinical EPM testing done by the Michigan East Lansing, Mich., 
has suggested that the protein at 30 kDa may be detected before the 
protein at 16 kDa during the course of serocovnersion. A horse with acute 
clinical signs consistent with EPM, whose immunoblot test detects the 30 
kDa band alone, should be retested after 7-10 days to confirm the antibody 
status of the animal. Another potential source of a false negative test 
may be a latent stage of S. neurona that does not stimulate and antibody 
response. Finally, the known negative samples from India appeared to 
provide many cross-reactive antibodies, but it is possible that further 
testing of sera from Eastern Hemisphere equids will reveal new possible 
sources of false positive test results. 
Despite these sources of potential test error, the positive criteria and 
the blocking technique described here improves the specificity of the 
Western blot antibody test for S. neurona without any concurrent loss of 
sensitivity. They represent a highly stringent "gold standard". It is 
noteworthy that 2 of the 8 horses successfully cultured were also 
diagnosed with Wobbler's syndrome, and 1 of the 8 was diagnosed with 
leukoencephalomalacia (moldy corn poisoning). Without successfully 
culturing S. neurona from these animals with dual neurological problems, 
it would have been easy to classify them as false positives for S. 
neurona. 
Finally, by classifying known positives and known negatives according to 
antibody status, rather than by clinical diagnosis of EPM or other 
neurological diseases, it is possible to evaluate the test solely by its 
ability to detect the desired antibodies. 
EXAMPLE 2 
The purpose of this experiment was to determine if pre-incubating the whole 
blots in bovine serum positive for antibodies to S. cruzi before drying 
and freezing gave the same result as blots treated as in Experiment 1. 
Western Blot Testing 
S. neurona meroziotes were harvested from equine dermal cell culture and 
heat denatured in sample buffer (0.5 M Tris (pH 7.4) with 10% SDS, 20% 
Glycerol and 5% B-mercaptoethanol). Denatured proteins and molecular 
weight markers were separated by SDS-PAGE in 12-20% linear gradient gels 
with a 4% stacking gel. Separated proteins were electophoretically 
transferred to Western PVDF membranes and blocked overnight in 1% bovine 
serum albumen and 0.5% Tween-Tris Buffered Saline (blocking buffer). Blots 
were air dried and frozen for later use. For testing of serum samples, the 
blots were wetted in 0.5% Tween-Tris Buffered Saline (TTBS) and clamped 
into a Plexiglass press. Molecular weight markers were covered in B-TTBS 
(blocking buffer and serum albumin) in the first two lanes, the remaining 
18 lanes were loaded with serum samples diluted 1:10 in blocking buffer 
and incubated overnight. Subsequently, blots were rinsed 3 times in TTBS, 
incubated for 3 hours in blocking buffer with biotin labeled goat 
anti-horse IgG H+L), rinsed 3 times in TTBS, incubated in TTBS with 
peroxidase conjugate, rinsed 3 times in TTBS and developed with aminoethyl 
carbazole (AEC) stain. 
Bovine S. cruzi Antisera Treatment 
Individual Lanes 
The Western blot protocol was repeated with an additional treatment; the 
blots were incubated with bovine serum with Sarcocystis cruzi antibodies 
in blocking buffer (1:10) for 2 hours, rinsed 3 times in TTBS, then loaded 
with equine samples and developed according to the previously described 
protocol. 
Whole Blots 
The Western blot protocol was repeated. After the blots had blocked 
overnight, the blocking buffer was poured off. Bovine S. cruzi antisera 
was diluted 1:50 in blocking buffer and the blots were blocked with 
agitation in the solution for 1-1.5 hours, then air dried and frozen for 
later use. Equine samples were loaded and developed according to the 
previously described protocol. 
The results were the same as shown in Example 1 (FIGS. 3 and 4).