Rev. sci. tech. Off. int. Epiz., 1993,12 (2), 425-433
Advances in the diagnosis of some parasitic
diseases by monoclonal antibody-based
enzyme-linked immunosorbent assays
D . P . K N O W L E S Jr and J.R. G O R H A M *
Summary: Advances in diagnostic assays for parasitic diseases include the use
of monoclonal antibodies (MAbs) in antigen capture and competitive
inhibition enzyme-linked immunosorbent assays (C-ELISA). Antigen capture
ELIS As for Anaplasma marginale and Cryptosporidium parvum provide
direct detection of these parasites during clinical disease, and the C-ELISA
format has been adapted for detection of anti-Babesia equi, anti-A. marginale
and anti-bluetongue virus antibodies. False-positive results may occur when
antigen preparations in other ELISA formats are contaminated
with
Escherichia coli, erythrocyte or cell-culture antigens. The C-ELISA format
overcomes problems of antigen purity, since the specificity of the C-ELISA
depends solely on the MAb used. For this reason, the C-ELISA format is highly
suited for use with recombinant antigens. Also, the use of recombinant protein
in diagnostic assays precludes the need to infect animals for antigen production
when the antigen cannot be produced in cell culture.
KEYWORDS: Anaplasma marginale - Antigen capture ELISA - Babesia
equi - Bluetongue virus - Competitive inhibition ELISA - Cryptosporidium
parvum - Recombinant proteins.
INTRODUCTION
New diagnostic assays are developed to improve current tests or to provide an assay
when one is not available. When a new diagnostic assay is developed as a replacement
for a current diagnostic test, methods must be found to provide accurate comparative
test performance (sensitivity and specificity) data. A common method of evaluation is
to simply compare the results of the new assay with those of the test currently in use.
This comparison results in quantification of the agreement (concordance) between the
two assays but does not address the question of differences in sensitivity or specificity.
Sensitivity is defined by the following formula:
test positives x 100
test positives + false negatives
* Animal Disease Research Unit, United States Department of Agriculture/Agricultural Research
Service, Department of Veterinary Microbiology and Pathology, 337 Bustad Hall, Washington State
University, Pullman, Washington 99164-7030, United States of America.
426
and specificity is defined as follows:
test negatives x 100
test negatives + false positives
It is clear from these formulas that sensitivity measures false-negative results and
specificity quantitates false-positive results (23).
Much of the difficulty in obtaining accurate test performance data resides in defining
a "gold standard" for disease in the above formulas. One such accepted "gold standard"
is the transmission of disease from an animal which is test positive to an animal which is
test negative. For most experimental designs, transmission data cannot be obtained for
every animal tested. Depending on the reasons for developing a new diagnostic assay,
concordance data may be a sufficient first step in test evaluation. For example, if a new
diagnostic assay is designed solely to provide a more rapid test, concordance data may
be sufficient for validation. If a test with improved performance is required, then the
best "gold standard" for comparison must be identified and utilized for validation of a
new assay. Clearly, when a diagnostic assay is the first test reported for a particular
disease, a "gold s t a n d a r d " must b e identified which can provide the criteria for test
validation. In this context, some recently-developed monoclonal antibody (MAb)based enzyme-linked immunosorbent assays (ELISAs) are reviewed.
ANTIGEN CAPTURE ENZYME-LINKED IMMUNOSORBENT ASSAY
T h e primary advantage of the antigen capture E L I S A is the ability of this test to
detect parasite antigen directly from an animal prior to or during clinical disease. The
main component of the antigen capture ELISA is a M A b . The desired characteristics of
this M A b are:
- strong binding to the parasite
- the ability to attach to an ELISA plate without loss of reactivity
- in addition, a second M A b recognizing an epitope other than the M A b which is
bound to the ELISA plate is often used as part of the indicator system.
Anaplasmosis, a vector-borne haemoparasitic rickettsial disease, is caused by
Anaplasma marginale (20) and A. centrale (21). Clinical disease is characterized by
anaemia, weight loss, abortion and death (1), and survivors are lifelong carriers of the
rickettsia (19). Recently, an antigen capture ELISA has been developed for the detection
of A. marginale rickettsaemia prior to the onset of clinical disease (22). T h e assay
sensitivity is 1.1(±0.5)% parasitized erythrocytes. This E L I S A uses two M A b s which
recognize different epitopes on a major surface protein of A. marginale (22). These
epitopes had previously been shown to be conserved among different A. marginale
isolates (17). The ELISA was validated by testing blood from twenty-four calves before
and after experimental infection with A. marginale (22). This assay was shown to be
specific for A. marginale, in that blood samples containing Babesia bovis, B. bigemina,
A. ovis and six antigenic variants of Trypanosoma brucei tested negative (22).
Since this assay discriminates between anaplasmosis and other clinically similar
haemoparasitic diseases, it permits diagnosis and treatment of cattle in a critical stage of
infection (22). A primary advantage of this assay is the detection of infected cattle early
427
in the course of disease, when tetracycline t h e r a p y is most successful in preventing
death (16).
Cryptosporidiosis (caused by the sporozoan Cryptosporidium parvum) is a common
infection of young calves (4). C. parvum is one of the major causes of n e o n a t a l
diarrhoea in calves and contributes to significant economic losses in the beef and dairy
cattle industries (9). A n antigen capture E L I S A which detects Cryptosporidium
sp.
oocysts in bovine faeces was recently described (3). F o r c a p t u r e , the assay uses an
immunoglobulin (Ig)M M A b which binds to a 40 k D a oocyst antigen, and uses
polyclonal goat anti-oocyst serum in the indicator system (3). The format of this test is
illustrated in Figure 1.
H
R
P
0
Rabbit anti-goat Ig,
HRPO conjugate
Goat anti-oocyst
C. parvum oocysts
MAb anti-oocyst
HRPO: horseradish peroxidase
Ig:
immunoglobulin
FIG.l
Schematic representation of a monoclonal antibody (MAb) capture enzyme-linked
immunosorbent assay used for Cryptosporidium parvum oocysts
Courtesy of Dr L.E. Perryman, Washington State University, Pullman, Washington,
United States of America
(3)
5
The assay detects as few as 3 x 10 C. parvum oocysts per ml of faeces and does not
detect oocysts of Eimeria auburnensis, E. bovis, E. ellipsoidalis or E. zuernii (3). T h e
assay was validated by:
a) testing faeces from naturally or experimentally-infected calves from t h r e e
locations
b) comparing the E L I S A to indirect immunofluorescence and light microscopic
analysis
c) following the kinetics of faecal shedding of C. parvum oocysts by experimentallyinfected calves (3).
428
The utility of this assay is that it is sufficiently sensitive to detect oocysts in animals
with clinical signs of disease, as well as in calves which have recovered from diarrhoea
but which continue to shed oocysts (3). F u r t h e r m o r e , this E L I S A has b e e n used to
detect C. parvum oocysts in diarrhoeic faeces from llamas and young horses with severe
combined immunodeficiency (3).
COMPETITIVE INHIBITION ELISA
In addition to the detection of parasites during clinical stages of infection, it is also
necessary to identify animals which have survived clinical disease and which are
persistent carriers. Parasite levels in carrier animals are often too low to be detected by
antigen capture ELISA; however, specific antibody is often maintained at detectable
levels. First described for use in detecting anti-bluetongue virus antibody (2), the MAbbased competitive inhibition E L I S A ( C - E L I S A ) format has b e e n used to detect
antibodies to Cowdria ruminantium, A. marginale and Babesia equi ( 1 0 , 1 1 , 24). A
C - E L I S A for detection of bovine anti-bluetongue virus antibody is commercially
available (18).
Components of the C-ELISA include a M A b and the corresponding epitope. The
M A b must possess an appropriate binding affinity such that polyclonal antibody can
replace it in the competitive reaction. Desirable epitope characteristics include a linear
peptide composition, immunodominance, and conservation among isolates. The latter
is an important characteristic in cases where production of the epitope by molecular
cloning and expression in bacteria is necessary. Since the specificity of the C-ELISA
depends entirely on the M A b used, the test is well suited for use with recombinant
antigens. If the recombinant antigen is expressed in sufficient quantity to provide an
appropriate signal:noise ratio after application to the E L I S A plate as a bacterial lysate,
additional purification of the recombinant antigen is not necessary.
T h e c o m p l e m e n t fixation (CF) test is currently the official test for a n t i - ß . equi
antibodies in many countries, including the United States of America (5,6,8). However,
the test still has a number of limitations, as follows:
a) C F cannot be used to test sera with anti-complement activity or sera which react
with the control erythrocyte antigen used in the test
b) sera containing specific IgG(T) antibody may yield false-negative results because
IgG(T) does not fix complement by the classical pathway (14)
c) the CF test has been shown to yield variable results in persistently-infected horses (7).
A MAb-based C-ELISA for detection of equine anti-B. equi antibodies has been
described recently (11). T h e format of this E L I S A is illustrated in Figure 2. This
C-ELISA uses an IgGl M A b which binds to a peptide epitope on the surface of B. equi
merozoites (12). A concordance of 94% was found when 154 sera from 19 countries
were tested by the C-ELISA and CF (11). In this study, sera yielding discrepant results
between the C F test and C - E L I S A were retested by i m m u n o p r e c i p i t a t i o n of 35Smethionine-labelled in vitro translation products of messenger ribonucleic acid from
B. equi (11,12). Immunoprecipitation results indicated that sera which tested negative
by C F and positive by C - E L I S A w e r e positive for B. equi (11). T h e C - E L I S A was
shown to be specific for anti-B. equi antibody, as horses infected with B. caballi tested
429
test serum
MAb
FIG.
2
Illustration of monoclonal antibody (MAb)-based competitive inhibition enzymelinked immunosorbent assay for Babesia equi
(12)
Courtesy of D r Elizabeth S. Visser, Onderstepoort Veterinary Institute,
Onderstepoort, South Africa
When equine test serum contains specific antibody, the specific equine antibody
competes with the mouse MAb, thus decreasing the optical density (OD) to
significantly below the level given by the negative reference serum, and the equine
serum is considered positive. When the equine test serum does not contain specific
antibody, the mouse monoclonal antibody binds to the epitope, thus yielding an
increased optical density equivalent to negative reference serum, and the equine serum
is considered negative
430
negative (11). The advantage of the C-ELISA is that the format overcomes limitations
a) and b) listed above. Furthermore, the B. equi protein bearing the epitope used in the
C - E L I S A has been molecularly cloned and expressed in Escherichia coli, and the
C-ELISA has been reformatted with this antigen (13).
Recently, a highly-conserved A. marginale major surface p r o t e i n (MSP-5) was
described (24). In preliminary testing, MSP-5 and a corresponding M A b successfully
detected anti-A marginale antibody in immune bovine sera (24). In addition, another
A. marginale major surface protein (MSP-3) has demonstrated potential as a diagnostic
antigen in the detection of anti-A marginale antibody in the sera of long-term carrier
cattle (15).
CONCLUSIONS
MAb-based ELISAs have tremendous potential for use in the diagnosis of parasitic
diseases. T h e antigen capture E L I S A can directly detect the presence of a parasite
during the clinical stages of disease when t r e a t m e n t is most efficacious, and the
C-ELISA format can be used to detect carriers of persistent parasitic infections. A final
and i m p o r t a n t attribute of the C - E L I S A is the ease with which it can be used with
recombinant antigens. This is especially important in those parasitic diseases for which
diagnostic antigen cannot yet be produced in cell culture. Furthermore, the production
of recombinant antigen in E. coli precludes the necessity of infecting animals for antigen
production.
*
PROGRÈS ACCOMPLIS DANS LE DIAGNOSTIC DE CERTAINES PARASITOSES
GRÂCE A U X TECHNIQUES IMMUNO-ENZYMATIQUES BASÉES SUR LES
ANTICORPS MONOCLONAUX. - D.P. Knowles Jr et J.R. Gorham.
Résumé : L'utilisation des anticorps monoclonaux dans les techniques
d'immunocapture
enzymatique (enzyme-linked immunosorbent assay :
ELISA) et d'ELISA «compétition/ inhibition» (C-ELISA) a permis de
progresser dans la mise au point de tests de diagnostic pour les maladies
parasitaires. Les tests d'immunocapture ELISA pour Anaplasma marginale et
Cryptosporidium parvum permettent de reconnaître directement ces parasites
pendant la phase clinique de la maladie, tandis que le test de C-ELISA a été
adapté à la détection d'anticorps anfi-Babesia equi, anti-A. marginale et antivirus de la fièvre catarrhale du mouton. Avec d'autres tests ELISA, on aboutit
parfois à des erreurs par excès, lorsque les préparations d'antigènes sont
contaminées par Escherichia coli, des érythrocytes ou des antigènes de culture
cellulaire. Le test C-ELISA résout le problème de la pureté de l'antigène,
puisque la spécificité du test ne dépend plus que de l'anticorps
monoclonal
utilisé. Aussi, est-il très recommandé d'utiliser ce test C-ELISA avec des
antigènes recombinants. De plus, grâce aux protéines recombinantes employées
dans les tests de diagnostic, il n'est plus nécessaire d'infecter des animaux pour
obtenir des antigènes lorsque ceux-ci ne peuvent être produits par culture
cellulaire.
431
MOTS-CLÉS : Anaplasma marginale - Babesia equi - Cryptosporidium
parvum - Protéines recombinantes - Test de compétition/inhibition ELISA Test d'immunocapture ELISA - Virus de la fièvre catarrhale du mouton.
*
* *
AVANCES EN EL DIAGNÓSTICO D E PARASITOSIS MEDIANTE P R U E B A S
INMUNOENZIMÁTICAS B A S A D A S E N ANTICUERPOS MONOCLONALES. D.P. Knowles Jr y J.R. Gorham.
Resumen: El uso de anticuerpos
monoclonales
en las técnicas de
inmunocaptura enzimática (ELISA) o de «competición/inhibición»
ELISA
(C-ELISA) permitió importantes avances en el establecimiento de pruebas de
diagnóstico para las parasitosis. Las pruebas de inmunocaptura ELISA para
Anaplasma marginale y Cryptosporidium parvum permiten el reconocimiento
inmediato de estos parásitos durante la fase clínica de la enfermedad; la prueba
C-ELISA se ha adaptado para la detección de anticuerpos anti-Babesia equi,
anti-A. marginale y anti-virus de la fiebre catarral ovina. Con otras pruebas
ELISA
pueden darse resultados falsamente
positivos,
cuando las
preparaciones con antígenos están contaminadas por Escherichia coli,
eritrocitos o antígenos de cultivo celular. La prueba C-ELISA resuelve el
problema de la pureza del antígeno, ya que su especificidad sólo depende del
anticuerpo monoclonal utilizado; es por lo tanto muy recomendable usar esta
prueba con antígenos recombinantes. Por otra parte, gracias a las proteínas
recombinantes que se usan en las pruebas diagnósticas, ya no es necesario
infectar animales para obtener antígenos cuando éstos no se pueden producir
por cultivo celular.
PALABRAS CLAVE: Anaplasma marginale - Babesia equi Cryptosporidium parvum - Proteínas recombinantes - Pruebas de
competición/inhibición ELISA - Pruebas de inmunocaptura ELISA - Virus
de la fiebre catarral ovina.
*
REFERENCES
1. ALDERINK F.J. & DIETRICH R. (1981). - Anaplasmosis in Texas: epidemiologic and
economic data from the questionnaire survey. In Proc. 7th National Anaplasmosis
Conference (R.J. Hidalgo & E.W. Jones, eds). Mississippi State Univ. Press, Mississippi,
27-44.
2. ANDERSON J. (1984). - Use of monoclonal antibody in a blocking ELISA to detect group
specific antibodies to bluetongue virus. J. Immunol. Meth., 74,139-149.
3. A N U S Z K.Z., M A S O N P.H., R I G G S M.W. & P E R R Y M A N L.E. (1990). - Detection of
Cryptosporidium parvum oocysts in bovine feces by monoclonal antibody capture
enzyme-linked immunosorbent assay. J. clin. Microbiol., 2 8 (12), 2770-2774.
4. FAYER R. & U N G A R L.P. (1986). - Cryptosporidium
Microbiol. Rev., 5 0 , 458-483.
spp. and cryptosporidiosis.
432
5. FRERICHS W . M . , H O L B R O O K A.A. & JOHNSON A . J . (1969). - Equine piroplasmosis:
complement fixation titers of horses infected with Babesia caballi. Am. J. vet. Res., 30,
697-702.
6. FRERICHS W . M . , H O L B R O O K A.A. & JOHNSON A . J . (1969). - Equine piroplasmosis:
production of antigens for the complement fixation test. Am. J. vet. Res., 30, 1337-1341.
7. FRIEDHOFF K.T. (1982). - The piroplasms of Equidae - significance for international
commerce. Berl. Münch. tierärztl. Wschr., 95,368-374.
8. HIRATO K . , NONOMIYA N . , UWANO Y. & KUTH T. (1945). - Studies on the complement
fixation reaction for equine piroplasmosis. Jpn. J. vet. Sci., 7,197-205.
9. HOUSE J.A. (1978). - Economic impact of rotavirus and other neonatal disease agents of
animals. J. Am. vet. med. Assoc., 173, 573-576.
10. JONGEJAN F., THIELEMANS M.J.C., D E G R O O T M., KOOTEN P.J.S. VAN & ZEIJST B.A.M.
VAN DER (1991). - Competitive enzyme-linked immunosorbent assay for heartwater
using monoclonal antibodies to a Cowdria ruminantium-specific 32-kilodalton protein.
Vet. Microbiol., 28, 199-211.
11. KNOWLES D.P. JR, P E R R Y M A N L.E., K A P P M E Y E R L.S. & H E N N A G E R S.G. (1991). -
Detection of equine antibody to Babesia equi merozoite proteins by a monoclonal
antibody-based competitive inhibition enzyme-linked immunosorbent assay. J. clin.
Microbiol., 29, 2056-2058.
12. KNOWLES D.P. JR, PERRYMAN L.E., G O F F W . L . , M I L L E R C.D., H A R R I N G T O N R.D. &
GORHAM J.R. (1991). - A monoclonal antibody defines a geographically-conserved
surface protein of Babesia equi merozoites. Infect. & Immunity, 59, 2314-2417.
13. KNOWLES D.P. JR, KAPPMEYER L.L., STILLER D., H E N N A G E R S.G. & PERRYMAN L.E.
(1992). - Antibody to a recombinant merozoite protein epitope identifies horses infected
with Babesia equi. J. clin. Microbiol., 30, 3122-3126.
14. M C G U I R E T.C., HOOSIER G. L. VAN JR & HENSON J.B. (1971). - The complement-fixation
reaction in equine infectious anemia: demonstration of inhibition by IgG(T).
J. Immunol, 107,1738-1744.
15. M C G U I R E T.C., DAVIS W . C . , BRASSFIELD A.L., MCELWAIN T.F. & PALMER G . H . (1991). -
Identification of Anaplasma marginale long-term carrier cattle by detection of serum
antibody to isolated MSP-3. J. clin. Microbiol, 29, 788-793.
16. MILLER J.G. (1956). - The prevention and treatment of anaplasmosis. Ann. N. Y. Acad.
Sci., 64, 49-55.
17. PALMER G . H . , WAGHELA S.D., B A R B E T A.F., DAVIS W . C . & M C G U I R E T.C. (1987). -
Characterization of a neutralization-sensitive epitope on the Aml05 surface protein of
Anaplasma marginale. Int. J. Parasitol, 17, 1279-1285.
18. REDDINGTON J . J . , REDDINGTON G.M. & M A C L A C H L A N N . J . (1991). - A competitive
ELISA for detection of antibodies to the group antigen of bluetongue virus. J. vet. Diagn.
I n v e s t . , 3 , 144-147.
19. SWIFT B.L. & THOMAS G.M. (1983). - Bovine anaplasmosis: elimination of the carrier
state with injectable long-acting Oxytetracycline. J. Am. vet. med. Assoc., 183, 63-65.
20. THEILER A. (1910). - Anaplasma marginale (genus nov. et species nov.). Un nouveau
protozaire du bétail. Bull. Soc. Pathol, exot.,3,135.
21. THEILER A. (1911). - Further investigations into anaplasmosis of South African cattle. In
First report of the Director of Veterinary Research, Union of South Africa, 7-76.
433
22. TRUEBLOOD E . S . , M C G U I R E T . C . & PALMER G . H . (1991). - Detection of
Anaplasma
marginale rickettsemia prior to onset of clinical signs by using an antigen capture
enzyme-linked immunosorbent assay. J. clin. Microbiol., 29 (7), 1542-1544.
23. TYLER J . W . & CULLOR J.S. (1989). - Titers, tests, and truisms: rational interpretation of
diagnostic serologic testing. J. Am. vet. med. Assoc., 194 (11), 1550-1558.
24. VISSER E . S . , M C G U I R E T . C . , PALMER G . H . , D A V I S W . C . , SHKAP V . , P I P A N O E . &
KNOWLES D . P . JR (1992). - Anaplasma marginale msp5 gene encodes a 19-kilodalton
protein conserved in all recognized Anaplasma species. Infect. & Immunity, 60,
5139-5144.