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Application of hybridoma technology to the diagnosis of parasitic disease
Veterinary Parasitology, 14 (1984) 251--261 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
251
APPLICATION OF HYBRIDOMA TECHNOLOGY TO THE DIAGNOSIS OF PARASITIC DISEASE
H.R. GAMBLE
U.S. Department of Agriculture, Agricultural Research Service, Animal Parasitology Institute, Beltsville, MD 20705 (U.S.A.)
ABSTRACT
Gamble, H.R., 1984. Application of hybridoma technology to the diagnosis of parasitic disease. Vet. Parasitol., 14: 251--261. Recent applications of hybridoma-derived monoclonal antibodies to the diagnosis of parasitic disease are reviewed. Diagnostic tests, utilizing monoclonal antibodies, have included radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) techniques to detect parasite antigen in host tissues and body fluids and circulating host antiparasite antibody. In general, the use of monoclonal-derived reagents has greatly increased the specificity of diagnosis by eliminating cross-reactions between closely related parasite species, without suffering a significant loss of sensitivity. INTRODUCTION
A variety of procedures for the diagnosis of parasitic diseases have been employed, ranging from the direct detection of parasites or parasite eggs to serologic assays for host antibodies directed against the parasite. Serologic tests such as radioimmunoassay (RIA) or the enzyme-linked immunosorbent assay (ELISA) have become very popular because of their high sensitivity. RIA and ELISA procedures have been utilized widely to detect both parasite antigens in host body fluids and parasite-specific host antibodies (Bidwell et al., 1976; Voller and deSavigny, 1981; Huldt, 1981). Generally, these tests have used crude parasite antigen preparations and/or polyclonal antisera as test reagents. However, such reagents have often produced poor specificity and lack uniformity. The introduction of monoclonal antibody technology has offered a means for overcoming these limitations and has greatly increased the usefulness of diagnostic tests. Monoclonal antibodies may be used to: (1) detect parasite antigens in body fluids; (2) identify and isolate antigens to be used in antibody detection. The advantages of monoclonal antibodies in diagnostic tests are several. First, each monoclonal antibody recognizes only one antigenic determinant or epitope. Therefore, a monoclonal antibody may be selected which has specificity for an antigenic determinant that is unique to the parasite of in-
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252 terest. Because antigens shared with other parasites or antibodies directed against interspecific antigens are not detected in the diagnostic test, falsepositive reactions due to cross-reactivity are minimized or eliminated. Second, while diagnostic reagents may vary from laboratory to laboratory (Kagan, 1982), monoclonal antibodies provide great uniformity. The parasite antigen recognized or isolated using a monoclonal antibody is invariant, thus permitting reproducibility and standardization in diagnostic tests. Finally, monoclonal antibodies permit the production of large quantities of a specific reagent, up to 10 mg m1-1 of antibody, from murine ascites fluid. One potential problem associated with the use of monoclonal antibodies, however, is decreased assay sensitivity. The quantities of parasite antigen or specific host antibody to be detected will be considerably less than in tests for total parasite antigen or polyclonal host antibody, because the antigen specificity of the diagnostic test has been reduced to one epitope. To help overcome this, an antibody specific for a prominent or immunodominant parasite antigen should be selected for use in the development of a diagnostic test. For maximal sensitivity, most immunodiagnostic tests using hybridomaderived reagents have used highly sensitive RIA or ELISA methods to quantify parasite antigen or host antibodies. DISCUSSION
Types of immunodiagnostic tests Monoclonal antibodies may be used in several variations of the classical RIA and ELISA procedures. Frequently, the monoclonal antibody is used directly in combination with a crude parasite extract. Alternatively, monoclonal antibody affinity-isolated antigen is used. For most applications, the immunodiagnostic test utilizes the format of a solid phase assay. Antibody (generally a monoclonal) or antigen (crude or affinity-isolated) will be attached to any of a variety of solid phases. Most popular supports are 96-well polystyrene or polyvinyl microtiter plates, although plastic tubes and Sepharose beads have also been used. Diagnostic tests can be divided into two categories, one which detects parasite antigen and one which detects host anti-parasite antibodies. The former is a more definitive test, a positive result indicating a current parasitic infection and perhaps also the level of parasitemia. In m a n y cases, however, parasite antigen is not present in detectable quantities, hence an indirect test for host anti-parasite antibody is conducted. When testing for anti-parasite antibody, immunological amplification due to serum antibody production aids in the sensitivity of the diagnosis. However it should be recognized when performing such a test that host ~antiparasite antibodies often persist after the parasite has died or been cleared by the host; this m a y then result in false-positive reactions. The principles involved in the performance of RIA and ELISA are the
253 (O)
SOLID-PHASE IMMUNOASSAYS FOR PARASITE ANTIGEN
$ $ DOUBLE ANTIBODY SANDWICH
(b)
COMPETITION
SOLID--PHASE IMMUNOASSA YS FOR ANTI-PARASITE HOST ANTIBODIES
INHIBITION Legend
IANT,BOD,Esl ANTI-PARASITE MONOCLONAL ANTIBODY A N T I - P A R A S I T E HOST SERUM ANTIBODY P O L Y C L O N A L SECOND A N T I B O D Y TO HOST SERUM Ig's
IANTIGENS I
M O N O C L O N A L A N T I B O D Y AFFINITY ISOLATED P A R A S I T E ANTIGEN P A R A S I T E ANTIGEN IN SAMPLE FROM HOST SERUM/TISSUE X COMPETITION
INDIRECT
BINDING INDICATOR
(ENZYME OR RADIOLABEL )
Fig. 1.(a) Three general methods (ELISA or RIA) for the detection of parasite antigen in host body fluids using monoclonal antibodies. (b) Two general methods (ELISA or RIA) for the detection of host antibodies directed against parasite antigen using monoclonal antibodies (see text for description).
same, the major difference being the final m o d e of detection. Tests for the detection of antigen m a y be divided into 3 types (Fig. 1).
Double-antibody sandwich Monoclonal antibody is bound to a solid phase. A sample containing putative antigen is added, incubated, removed and followed by a labeled antib o d y (monoclonal or polyclonal). The amount of b o u n d label is directly proportional to the amount of antigen present. The choice of labeled antib o d y depends on the antigen being detected. If repeating epitopes are present on one protein, the monoclonal antibody bound to the plate m a y also be used as the labeled antibody. If this is not the case, a labeled polyclonal anti-serum or labeled monoclonal antibodies with specificity for different epitopes on the same protein m a y be used. A further modification of the double-antibody sandwich procedure is the use of an unlabeled second antibody, followed b y a labeled third antibody. However, the first and second antibody must be from different species so that only the second antibody is recognized by the labeled antibody-conjugate.
Competitive (labeled ant~en method) Monoclonal antibody is b o u n d to the solid phase, and reacted with a mix-
254 ture of labeled antigen and a serum or tissue sample containing putative antigen. A reduction in bound label as compared to negative controls indicates the presence of antigen in the sample.
Inhibition (labeled antibody method) Antigen (crude or purified by immuno-affinity chromatography) is bound to the solid phase and reacted with a mixture of labeled monoclonal antibody and a serum or tissue sample to be tested for the presence of antigen. A reduction in bound label compared to negative controls indicates the presence of antigen (or antibody, see below). This procedure m a y be amplified by using unlabeled monoclonal antibody in the inhibition step, followed by a labeled second antibody or labeled staphylococcal Protein A (which binds specifically to the Fc portion of mouse IgG). There are 2 basic types of RIA or ELISA procedures utilizing monoclonal antibodies for the detection of host antibodies directed against parasite antigens (Fig. 1).
Competitive (labeled antibody method) This assay is performed exactly as in inhibition. However, in this case anti-parasite antibodies compete for binding to solid phase antigen. When this assay is modified for amplification, by using an unlabeled primary (monoclonal) antibody and a labeled second antibody, the species of the competing antibody (serum sample) must not be bound by the labeled second antibody.
Indirect method Antigen, immuno-affinity purified using monoclonal antibodies, is bound to the solid phase and reacted with a serum sample containing putative antiparasite antibodies. A second labeled antibody, with species specificity for antibodies in the serum sample, is added and the amount of bound labeled antibody determined. This assay may be amplified by using an unlabeled second antibody and a labeled third antibody.
Important considerations in the application of monoclonal antibodies for the development of parasite immunodiagnostic tests When preparing monoclonal antibodies for use in an immunodiagnostic test, several aspects should be considered. First, an attempt should be made to develop antibodies against a prominent protein, or an i m m u n d o m i n a n t antigen specific to the life-cycle stage to be diagnosed. This m a y best be accomplished by directly infecting the rodent spleen cell donor with the parasite. This approach is limited, however, by the species specificity of the parasite (i.e. requires infectivity for mice or rats, for which m y e l o m a fusion partners are available). An alternative approach is to immunize the spleen cell donor with antigens from the parasite life-cycle stage of interest. Second,
255 a screening procedure should be devised so that only hybridomas relevant for diagnosis are selected. This screening process might include assays for crossreactions with heterologous parasite antigens to exclude unwanted clones. It has been shown, however, that direct binding assays, indicating unique parasite antigens, may be contradicted by competitive binding assays involving polyclonal sera (Craig et at., 1981). Finally, characteristics of the monoclonal antibody, such as affinity or isotope, may be selected for optimal development of a particular type of diagnostic test. A number of monoclonal antibody-based diagnostic tests have been developed and have demonstrated, in general, significant improvement over previously described procedures. Some specific examples are discussed below.
Pro tozoa Monoclonal antibodies to Toxoplasma gondii were produced by Araujo et al. (1980) and used to detect antigen in the serum of infected humans. Using a double-antibody sandwich (ELISA), antigen was detected in sera from all of 4 acutely infected individuals. The sensitivity of the assay was approximated at 4 #g of antigen m1-1 of serum. No positive reactions were obtained with sera from uninfected or chronically infected individuals or from patients with rheumatoid factor titers. However, 2 of 10 sera from patients with anti-nuclear antibodies did react with the anti-T, gondii monoclonal antibody. A similar approach with monoclonal antibodies was utilized for the detection of Trypanosoma cruzi antigenemia in mice (Araujo et al., 1982). The success of this assay m a y lead to the development of a similar type of diagnostic test for Chagas' disease in humans. Several RIA procedures have been reported for the detection of parasite circumsporozoite proteins in Plasmodium-infected mosquitos. In one system, mosquito extracts were b o u n d to microtiter wells and reacted directly with 12SI-labeled monoclonal antibody (Zavala et al., 1982). By this method, the species of Plasmodium in infected mosquitos was identified accurately with no cross-reactions among species. The same authors designed another immunoassay, a double-antibody sandwich RIA, for the same purpose. Microtiter wells were coated with one monoclonal antibody, reacted with mosquito extracts and then with an l~SI-labeled monoclonal antibody with different epitope specificity on the same protein. Using this procedure, the authors were able to detect as few as 1 infected mosquito in a group of 25. Potocnjak et al. (1982) demonstrated that a circumsporozoite protein (Pb44) from Plasmodium berghei could be detected in infected mosquitos using an inhibitory RIA. Monoclonal antibodies directed against Pb44 were b o u n d to microtiter wells, exposed to a mosquito extract and then to an 12SI-labeled anti-idiotype monoclonal antibody specific for the idiotype of the b o u n d monoclonal antibody. When Pb44 was present in the mosquito extract, binding of labeled antibody was inhibited. This assay system eliminated the need for purified parasite antigen (except as a control).
256
Helminths A series of studies, using monoclonal antibodies in immunodiagnostic tests for helminth infection, has come from the Walter and Eliza Hall Institute of Medical Research in Australia. A model system was first developed using Mesocestoides corti infections in mice (Mitchell et al., 1979). The authors employed a competitive RIA with crude parasite antigen and l~SI-labeled monoclonal antibody to detect anti-parasite antibodies in serum. No false-positive or false-negative reactions were obtained, and the test was found to be highly species-specific. Attempts to substitute a potyclonal anti-idiotype antisera for the parasite antigen were unsuccessful. A competitive RIA for diagnosis of Taenia hydatigena infection was developed by Craig et al. (1980). A monoclonal antibody was selected based on direct binding studies which indicated specificity for T. hydatigena but not for larval extracts of other taeniid species. Binding of 12SI-labeled monoclonal antibody in the RIA was inhibited by all the sera from T. hydatigena-infected sheep with no false-positive reactions with uninfected sera. However, 2 of 6 sera from T. ov/s-infected sheep inhibited binding of the anti-T, hydatigena monoclonal antibody, contradicting the species specificity indicated by direct binding assays. In a different approach, Craig et al. (1981) used monoclonal antibodies produced against Echinococcus granulosus, but shown to cross-react with sera from T. ovis-, T. hydatigena and Fasciola hepatica-infected sheep, to deplete a crude antigen extract of the responsible antigens. The partially purified E. granulosus antigen showed improved specificity in an indirect ELISA for the diagnosis of infected sheep. Monoclonal antibodies from a single hybridoma line have been shown to be useful for the diagnosis of Schistosoma japonicum infections in humans. Mitchell et al. (1981) described a monoclonal antibody, IPH.134, with specificity unique to S. ]aponicum adult worms as demonstrated by direct binding studies. When used in a competitive RIA with crude worm antigen, IPH.134 identified > 90% of serum samples from individuals who were positive for S. japonicum by fecal examination. No false-positive reactions were obtained from normal h u m a n sera or sera from humans with a variety of other protozoan or helminth infections. In addition, there was some indication that the RIA titer in the competitive assay might be correlated to fecal egg o u t p u t or prominant clinical disease. The diagnostic potential of monoclonal antibody IPH.134 was further validated by Cruise et al. (1981), who confirmed the serum a n t i b o d y basis of competitive interaction in the RIA; no evidence of anti-idiotype or immune complex interaction was found. The same authors demonstrated that as little as 4 ng of specific antibody would inhibit 50% of m a x i m u m binding of IPH.134 in the competitive RIA. The antigen to which IPH.134 was directed was considered i m m u n o d o m i n a n t , because up to 1 mg m1-1 of specific antibody was detected in human serum, and because even low responder or partially resistant inbred mice could be diagnosed.
257 As a further modification, Mitchell et al. (1983) reported a competitive ELISA using hybridoma IPH.134. Using a peniciUinase-conjugated monoclonal antibody, no false-positives and approximately 10% false-negatives in human serum samples were obtained. A monoclonal antibody recognizing a circulating antigen of adult Onchocerca volvulus was used by des Moutis et al. (1983) in a radio-immune precipitation (RIP) assay. Radio-labeled monoclonal antibody was reacted with human sera containing soluble O. volvulus antigen and the antigen--antibody complexes were precipitated with polyethylene glycol. Circulating antigen was detected in 80% of African onchocerciasis patients and 76% of Venezuelan onchocerciasis patients tested. Cross-reactivity was minimized in this test. However, several sera from patients with Brugia malayi, Loa loa or Schistosoma mansoni infections gave false-positive results. No correlation between the RIP and microfilaradermia (as determined by the skin snip test) was found, suggesting the need for using these tests in tandem. Swine trichinosis The efficacy of diagnostic tests for trichinosis has been enhanced by the identification and isolation of more refined antigen preparations. For example, Trichinella spiralis excretory--secretory (ES) products and an immunopurified fraction from stichocyte particles (PAW) have been shown to have good diagnostic properties (Gamble et al., 1983; Seawright et al., 1983, respectively), while crude extracts of T. spiralis muscle larvae have been shown to produce 10--15% false-positive reactions (Ruitenberg et al., 1976; Saunders et al., 1977; Clinard, 1978; Gamble et al., 1983). Monoclonal antibodies directed against T. spiralis were produced by cell fusion using spleen cells from Balb/c mice orally inoculated with infective muscle larvae (Gamble and Graham, 1983a). Hybridomas producing antibodies directed against a crude extract of muscle larvae were cloned and were screened by ELISA against T. spiralis adults, larvae and larval ES antigens, as well as a panel of antigens from other helminth parasites of swine (Table I). Most monoclonal antibodies recognized antigens shared by larval and adult stages of T. spiralis and by other swine parasites, particularly Trichuris suis. However, several monoclonal antibodies were specific for Trichinella spiralis larvae and adult antigens or larval ES antigens. Because of the demonstrated diagnostic value of assays utilizing larval ES products (Gamble et al., 1983), one hybridoma with specificity for an ES antigen of muscle larvae (7C2C5) was selected for further study. Hybridoma 7C2C5 secreted an IgM monoclonal antibody with specificity for antigenic determinants present on proteins of molecular weight 45 000, 49 000 and 53 000 (Ts.45, Ts.49, Ts.53). Target antigens of 7C2C5 are secreted orally by muscle larvae, as indicated by immune precipitation reactions, which occurred when larvae were incubated with ascites fluid. Additionally, immunoperoxidase staining demonstrated that these antigens are located in the stichocyte cells. Antigens Ts.45, Ts.49 and Ts.53 were isolated by affinity-chromatography
258
and used in a triple-antibody indirect ELISA for the detection of antibodies to T. spiralis as described by Gamble et al. (1983). Using this test, no falsepositives were obtained with sera from farm-raised hogs at slaughter, or serum from hogs infected experimentally with swine nematodes other than T. spiralis (Fig. 2). In addition, 100% of the experimentally-infected hogs were detected at a 1:100 dilution of serum. Subsequent studies have shown TABLE I R e a c t i v i t y o f s e l e c t e d m o n o c l o n a l a n t i b o d i e s w i t h a n t i g e n s f r o m Trichinella spiralis l i f e c y c l e s t a g e s a n d o t h e r p a r a s i t e s p e c i e s (Ascaris suum, Strongyloides ransomi, Stephanurus dentatus, Trichurus suis) in a n E L I S A . S e e G a m b l e a n d G r a h a m ( 1 9 8 3 a ) f o r d e t a i l s o f t h e
assay Clone
T. spiralis Larvae
A. suum
Adult
ES*
Larvae
1A5C 3 + 1D3 C 3 + 7A5C 1 +
+ + +
. _
. + _
7A6C 1 + 7B1 C 3 + 7B4C s + 7C2 C 5 + 7C5 C = +
+ + + +
_ . + -
+ + . . +
Adult .
Egg
.
S. ransomi
S. dentatus
T. suis
Larvae
Adult
Adult + + +
.
.
+ _
+ +
-
+ +
+ _
+ +
+ +
+ +
.
.
.
.
. +
+ + +
. .
+
.
. +
+
+
*ES = excretory--secretory products.
o
T..s..piralis neg o
o I
T, soiralis neg o
T. {~oiralis pos
•
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I
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I
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•
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o
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'
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o'.1
012 ELISA
0!3
0!4
o'.s
0.6
O. D.
Fig. 2. E n z y m e - l i n k e d i m m u n o s o r b e n t a s s a y f o r t h e d e t e c t i o n o f s w i n e a n t i b o d i e s dir e c t e d a g a i n s t Trichinella spiralis i n f e c t i o n . A n t i g e n s w e r e c r u d e w o r m e x t r a c t ( C W E ) o r an excretory--secretory antigen (McAff-Ag) isolated by affinity-chromatography using h y b r i d o m a 7C2C 5. S w i n e s e r a i n c l u d e d 3 5 s e r a f r o m f a r m - r a i s e d s l a u g h t e r h o g s ( c ) , 3 0 s e r a f r o m h o g s i n o c u l a t e d w i t h 1 0 000 T. spiralis m u s c l e l a r v a e a n d b l e d a f t e r 6 m o n t h s ( e ) , a n d p o o l e d s a m p l e s f r o m a n i m a l s e x p e r i m e n t a l l y i n f e c t e d w i t h Ascaris suum ( a ) , Strongyloides ransomi (A) o r Stephanurus dentatus ( o ) . S e e G a m b l e a n d G r a h a m ( 1 9 8 3 a ) for assay details.
259
that similar results are obtained with hogs experimentally-infected with lower infecting doses of T. spiralis muscle larvae (500 instead of 10 000). Because of the time and expense involved in affinity-isolation of antigen, the potential for antigen variation, and the presence of additional antigen epitopes on affinity-isolated proteins, we sought to develop a competitive immunoassay using hybridoma 7C2C5 (Gamble and Graham, 1983b). Microtiter plates were coated with crude ES antigen, serum samples and biotinlabeled monoclonal antibody 7C2C5 were added, and the plates were incubated. Bound biotinylated monoclonal antibody was detected by the addition of an avidin-peroxidase conjugate followed by peroxidase substrate; a reduction in enzyme activity as compared to normal serum controls indicated the presence of competing host serum antibodies. The presence of circulating worm antigen or anti-idiotype antibodies could n o t be detected in serum samples from heavily-infected animals by direct binding assay. Therefore, it was concluded that competition was due exclusively to serum antibodies. With the competitive ELISA, no false-positive reactions were discovered in a group of sera from non-infected farm-raised hogs and all serum samples from hogs experimentally-infected with T. spiralis were positive (Fig. 3). Additionally, no cross-reactions were obtained with serum pools from hogs infected with Ascaris suum, Strongyloides ransomi or Stephanurus dentatus or from hogs with serum antibody titers against Trichuris suis. Therefore, this assay appears to offer a more simple, b u t still sensitive and specific test for swine trichinosis. % Inhibition 0 I:. soiralis neg o T
=gpiralis pos . T. 8uis pos •
20
oooo
40
60
•
80
100
• =.!i-=.=1t=-'-
i| °°
A, s u u m pool A & ransomi pool • S. dentatus pool o
Fig. 3. Competitive enzyme-linked immunosorbent assay for the detection of swine antibodies directed against Trichinella spiralis infection. Antigen was a crude excretory-secretory preparation. Swine sara included 35 sera from farm-raised slaughter hogs (o), 30 sera from hogs inoculated with 10 000 T. spiralis muscle larvae and bled after 6 months (e), 13 sera from hogs with natural infections of Trichuris suis (=), and pooled samples from animals experimentally infected with Ascaris suum (~), Strongyloides ransomi (4) or Stephanurus dentatus (o). See Gamble and Graham (1983b) for assay details.
260 PROSPECTS FOR THE FUTURE
Hybridoma technology is just beginning to be applied in the field of parasite immunodiagnostics. However, the recent success in research employing these techniques should encourage greater interest. Preliminary reports indicate that monoclonal antibodies with immunodiagnostic value have been produced against Entamoeba histolytica (Lopez et al., 1982), Toxocara canis (Nicholas et al., 1982), Trichomonas vaginalis (Desmond et al., 1983), Leismania spp. (Williams et al., 1983), Schistosoma mansoni (Abdel-Hafiz et al., 1983; Zodda et al., 1983) and Dirofilaria immitis (Scott et al., 1983). By the proper selection of monoclonal antibodies and/or their use in immuno-affinity isolation of specific antigens, useful diagnostic tests should eventually be developed for all important parasitic diseases.
REFERENCES Abdel-Hafiz, S.K., Phillips, S.M. and Zodda, D.M., 1983. Schistosoma mansoni: Detection and characterization of antigens and antigenemia by inhibition enzyme-linked immunosorbent assay (IELISA). Exp. Parasitol., 55 : 219--232. Araujo, F.G., Handman, E. and Remington, J.S., 1980. Use of monoclonal antibodies to detect antigens of Toxoplasma gondii in serum and other body fluids. Infect. Immun., 30: 12--16. Araujo, F.G., Sharma, S.D., Tsai, V., Cox, P. and Remington, J.S., 1982. Monoclonal antibodies to stages of Trypanosoma cruzi: Characterization and use for antigen detection. Infect. Immun., 37: 344--349. Bidwell, D.E., Buck, A.A., Diesfeld, H.J., Enders, B., Haworth, J., Huldt, G., Kent, N.H., Kirsten, C., Mattern, P., Ruitenberg, E.J. and Voller, A., 1976. The enzyme-linked immunosorbent assay (ELISA). Bull. W.H.O., 54: 129--139. Clinard, E.H., 1978. Serum fractions associated with positive and false-positive reactions in the ELA test for trichinellosis in swine. In: C.W. Kim and Z.S. Pawlowski (Editors), Trichinellosis. 4th Int. Conf., University Press of New England, Hannover, NH, pp. 509--517. Craig, P.S., Mitchell, G.F., Cruise, K.M. and Rickard, M.D., 1980. Hybridoma antibody immunoassays for the detection of parasitic infection: Attempts to produce an immunodiagnostic reagent for a larval taeniid cestode infection. Aust. J. Exp. Biol. Med. Sci., 58: 339--350. Craig, P.S., Hocking, R.E., Mitchell, G.F. and Rickard, M.D., 1981. Murine hybridomaderived antibodies in the processing of antigens for the immunodiagnosis of hydatid (Echinococcus granulosus) infection in sheep. Parasitology, 83: 303--317. Cruise, K.M., Mitchell, G.F., Garcia, E.G. and Anders, R.F., 1981. Hybridoma antibody immunoassays for the detection of parasitic infection: Further studies on a monoclonal antibody with immunodiagnostic potential for schistosomiasis japonica. Acta Trop., 38.' 437--447. DesMoutis, I., Ouaissi, A., Grzych, J.M., Yarzabal, L., Haque, A. and Capron, A., 1983. Onchocerca volvulus: detection of circulating antigen by monoclonal antibodies in human onchocerciasis. Am. J. Trop. Med. Hyg., 32: 533--542. Desmond, W., McEvoy, S., Bowley, K. and Leung, J., 1983. An ELISA assay with a monoclonal antibody to all geographic and drug resistant strains of Trichomonas vaginalis. Proc. Fed. Am. Soc. Exp. Biol., 42: 855.
261 Gamble, H.R. and Graham, C.E., 1983a. A monoclonal antibody-purified antigen for the immunodiagnosis of trichinosis. Am. J. Vet. Res., 45: 67--73. Gamble, H.R. and Graham, C.E., 1983b. Comparison of monoclonal antibody-based competitive and indirect enzyme-linked immunosorbent assays for the diagnosis of swine trichinosis. Vet. Immunol. Immunopathol., in press. Gamble, H.R., Anderson, W.R., Graham, C.E. and Murrell, K.D., 1983. Diagnosis of swine trichinosis by enzyme-linked immunosorbent assay (ELISA) using an excretory--secretory antigen, vet. Parasitol., 13: 349--361. Huldt, G., 1981. Serodiagnosis of parasitic infections. Parasitology, 82: 49--50. Kagan, I.G., 1982. Standardization and quality control of immunodiagnostic methods. In: D.F. Mettrick and S.S. Desser (Editors), Parasites -- Their World and OurS. Elsevier Biomedical Press, Amsterdam, pp. 387--390. Lopez, J.S., Jensen, F.L., Ximenez, C. and Ortiz-Ortiz, L., 1982. Monoclonal antibody specific for Entamoeba histolytica. Proc. Fed. Am. Soc. Exp. Biol., 41 : 484. Mitchell, G.F., Cruise, K.M., Chapman, C.B., Anders, R.F. and Howard, M.C., 1979. Hybridoma antibody immunoassays for the detection of parasitic infection: development of a model system using a larval cestode infection in mice. Aust. J. Exp. Biol. Med. Sci., 57: 287--302. Mitchell, G.F., Cruise, K.M., Garcia, E.G. and Anders, R.F., 1981. Hybridoma-derived antibody with immunodiagnostic potential for schistosomiasis japonica. Proc. Natl. Acad. Sci., 78: 3165--3169. Mitchell, G.F., Premier, R.R., Garcia, E.G., Hurrell, J.G.R., Chandler, H.M., Cruise, K.M., Tapales, F.P. and Tiu, W.O., 1983. Hybridoma antibody-based competitive ELISA in Schistosoma japonicum infection. Am. J. Trop. Med. Hyg., 32: 114--117. Nicholas, W.L., Mitchell, G.F. and Stewart, A.C., 1982. Monoclonal antibodies to larval Toxocara canis (Nematoda). Mol. Biochem. Parasitol. Suppl., Parasites -- Their world and Ours, Elsevier Biomedical Press, Amsterdam, p. 96. Potocnjak, P., Zavala, F., Nussenzweig, R. and Nussenzweig, V., 1982. Inhibition of idiotype--anti-idiotype interaction for detection of a parasite antigen: a new immunoassay. Science, 215 : 1637--1639. Ruitenberg, E.J., Lungstrom, I., Steerenberg, P.A. and Buys, J., 1976. Reliability of the enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Trichinella spiralis infections in conventionally raised pigs. J. Immunol. Methods, 10: 67--83. Saunders, G.C., Clinard, E.H., Bartlett, M.L. and Sanders, W.M., 1977. Application of the indirect enzyme-labelled antibody microtest to the detection and surveillance of animal diseases. J. Infect. Dis., 136: $258--$266. Scott, A.L., Showalter, S. and Levy, D.A., 1983. Monoclonal antibodies against somatic antigens of Dirofilaria immitis. Proc. Fed. Am. Soc. Exp. Biol., 42: 1089. Seawright, G.L., Zimmerman, W.J., Isenstein, R.A. and Despommier, D.D., 1983. Enzyme immunoassay for swine trichinellosis using antigens purified by immunoaffinity chromatography. Am. J. Trop. Med. Hyg., in press. Voller, A. and deSavigny, D., 1981. Diagnostic serology of tropical parasitic diseases. J. Immunol. Methods, 46: 1--29. Williams, K.M., Constantine, N.T. and Anthony, R.L., 1983. Differentiation of new world Leishmania species by means of their reactivity with monoclonal antibodies in immunofluorescent assays. Proc. Fed. Am. Soc. Exp. Biol., 42: 854. Zavala, F., Gwadz, R.W., Collins, F.H., Nussenzweig, R.S. and Nussenzweig, V., 1982. Monoclonal antibodies to circumsporozoite proteins identify the species of malaria parasite in infected mosquitos. Nature (London), 299: 737--738. Zodda, D.M., Abdel-Hafez, K.D. and Phillips, S.M., 1983. Characterization of monoclonal antibodies against Schistosoma mansoni. Am. J. Trop. Med. Hyg., 32: 69--77.
251
APPLICATION OF HYBRIDOMA TECHNOLOGY TO THE DIAGNOSIS OF PARASITIC DISEASE
H.R. GAMBLE
U.S. Department of Agriculture, Agricultural Research Service, Animal Parasitology Institute, Beltsville, MD 20705 (U.S.A.)
ABSTRACT
Gamble, H.R., 1984. Application of hybridoma technology to the diagnosis of parasitic disease. Vet. Parasitol., 14: 251--261. Recent applications of hybridoma-derived monoclonal antibodies to the diagnosis of parasitic disease are reviewed. Diagnostic tests, utilizing monoclonal antibodies, have included radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) techniques to detect parasite antigen in host tissues and body fluids and circulating host antiparasite antibody. In general, the use of monoclonal-derived reagents has greatly increased the specificity of diagnosis by eliminating cross-reactions between closely related parasite species, without suffering a significant loss of sensitivity. INTRODUCTION
A variety of procedures for the diagnosis of parasitic diseases have been employed, ranging from the direct detection of parasites or parasite eggs to serologic assays for host antibodies directed against the parasite. Serologic tests such as radioimmunoassay (RIA) or the enzyme-linked immunosorbent assay (ELISA) have become very popular because of their high sensitivity. RIA and ELISA procedures have been utilized widely to detect both parasite antigens in host body fluids and parasite-specific host antibodies (Bidwell et al., 1976; Voller and deSavigny, 1981; Huldt, 1981). Generally, these tests have used crude parasite antigen preparations and/or polyclonal antisera as test reagents. However, such reagents have often produced poor specificity and lack uniformity. The introduction of monoclonal antibody technology has offered a means for overcoming these limitations and has greatly increased the usefulness of diagnostic tests. Monoclonal antibodies may be used to: (1) detect parasite antigens in body fluids; (2) identify and isolate antigens to be used in antibody detection. The advantages of monoclonal antibodies in diagnostic tests are several. First, each monoclonal antibody recognizes only one antigenic determinant or epitope. Therefore, a monoclonal antibody may be selected which has specificity for an antigenic determinant that is unique to the parasite of in-
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252 terest. Because antigens shared with other parasites or antibodies directed against interspecific antigens are not detected in the diagnostic test, falsepositive reactions due to cross-reactivity are minimized or eliminated. Second, while diagnostic reagents may vary from laboratory to laboratory (Kagan, 1982), monoclonal antibodies provide great uniformity. The parasite antigen recognized or isolated using a monoclonal antibody is invariant, thus permitting reproducibility and standardization in diagnostic tests. Finally, monoclonal antibodies permit the production of large quantities of a specific reagent, up to 10 mg m1-1 of antibody, from murine ascites fluid. One potential problem associated with the use of monoclonal antibodies, however, is decreased assay sensitivity. The quantities of parasite antigen or specific host antibody to be detected will be considerably less than in tests for total parasite antigen or polyclonal host antibody, because the antigen specificity of the diagnostic test has been reduced to one epitope. To help overcome this, an antibody specific for a prominent or immunodominant parasite antigen should be selected for use in the development of a diagnostic test. For maximal sensitivity, most immunodiagnostic tests using hybridomaderived reagents have used highly sensitive RIA or ELISA methods to quantify parasite antigen or host antibodies. DISCUSSION
Types of immunodiagnostic tests Monoclonal antibodies may be used in several variations of the classical RIA and ELISA procedures. Frequently, the monoclonal antibody is used directly in combination with a crude parasite extract. Alternatively, monoclonal antibody affinity-isolated antigen is used. For most applications, the immunodiagnostic test utilizes the format of a solid phase assay. Antibody (generally a monoclonal) or antigen (crude or affinity-isolated) will be attached to any of a variety of solid phases. Most popular supports are 96-well polystyrene or polyvinyl microtiter plates, although plastic tubes and Sepharose beads have also been used. Diagnostic tests can be divided into two categories, one which detects parasite antigen and one which detects host anti-parasite antibodies. The former is a more definitive test, a positive result indicating a current parasitic infection and perhaps also the level of parasitemia. In m a n y cases, however, parasite antigen is not present in detectable quantities, hence an indirect test for host anti-parasite antibody is conducted. When testing for anti-parasite antibody, immunological amplification due to serum antibody production aids in the sensitivity of the diagnosis. However it should be recognized when performing such a test that host ~antiparasite antibodies often persist after the parasite has died or been cleared by the host; this m a y then result in false-positive reactions. The principles involved in the performance of RIA and ELISA are the
253 (O)
SOLID-PHASE IMMUNOASSAYS FOR PARASITE ANTIGEN
$ $ DOUBLE ANTIBODY SANDWICH
(b)
COMPETITION
SOLID--PHASE IMMUNOASSA YS FOR ANTI-PARASITE HOST ANTIBODIES
INHIBITION Legend
IANT,BOD,Esl ANTI-PARASITE MONOCLONAL ANTIBODY A N T I - P A R A S I T E HOST SERUM ANTIBODY P O L Y C L O N A L SECOND A N T I B O D Y TO HOST SERUM Ig's
IANTIGENS I
M O N O C L O N A L A N T I B O D Y AFFINITY ISOLATED P A R A S I T E ANTIGEN P A R A S I T E ANTIGEN IN SAMPLE FROM HOST SERUM/TISSUE X COMPETITION
INDIRECT
BINDING INDICATOR
(ENZYME OR RADIOLABEL )
Fig. 1.(a) Three general methods (ELISA or RIA) for the detection of parasite antigen in host body fluids using monoclonal antibodies. (b) Two general methods (ELISA or RIA) for the detection of host antibodies directed against parasite antigen using monoclonal antibodies (see text for description).
same, the major difference being the final m o d e of detection. Tests for the detection of antigen m a y be divided into 3 types (Fig. 1).
Double-antibody sandwich Monoclonal antibody is bound to a solid phase. A sample containing putative antigen is added, incubated, removed and followed by a labeled antib o d y (monoclonal or polyclonal). The amount of b o u n d label is directly proportional to the amount of antigen present. The choice of labeled antib o d y depends on the antigen being detected. If repeating epitopes are present on one protein, the monoclonal antibody bound to the plate m a y also be used as the labeled antibody. If this is not the case, a labeled polyclonal anti-serum or labeled monoclonal antibodies with specificity for different epitopes on the same protein m a y be used. A further modification of the double-antibody sandwich procedure is the use of an unlabeled second antibody, followed b y a labeled third antibody. However, the first and second antibody must be from different species so that only the second antibody is recognized by the labeled antibody-conjugate.
Competitive (labeled ant~en method) Monoclonal antibody is b o u n d to the solid phase, and reacted with a mix-
254 ture of labeled antigen and a serum or tissue sample containing putative antigen. A reduction in bound label as compared to negative controls indicates the presence of antigen in the sample.
Inhibition (labeled antibody method) Antigen (crude or purified by immuno-affinity chromatography) is bound to the solid phase and reacted with a mixture of labeled monoclonal antibody and a serum or tissue sample to be tested for the presence of antigen. A reduction in bound label compared to negative controls indicates the presence of antigen (or antibody, see below). This procedure m a y be amplified by using unlabeled monoclonal antibody in the inhibition step, followed by a labeled second antibody or labeled staphylococcal Protein A (which binds specifically to the Fc portion of mouse IgG). There are 2 basic types of RIA or ELISA procedures utilizing monoclonal antibodies for the detection of host antibodies directed against parasite antigens (Fig. 1).
Competitive (labeled antibody method) This assay is performed exactly as in inhibition. However, in this case anti-parasite antibodies compete for binding to solid phase antigen. When this assay is modified for amplification, by using an unlabeled primary (monoclonal) antibody and a labeled second antibody, the species of the competing antibody (serum sample) must not be bound by the labeled second antibody.
Indirect method Antigen, immuno-affinity purified using monoclonal antibodies, is bound to the solid phase and reacted with a serum sample containing putative antiparasite antibodies. A second labeled antibody, with species specificity for antibodies in the serum sample, is added and the amount of bound labeled antibody determined. This assay may be amplified by using an unlabeled second antibody and a labeled third antibody.
Important considerations in the application of monoclonal antibodies for the development of parasite immunodiagnostic tests When preparing monoclonal antibodies for use in an immunodiagnostic test, several aspects should be considered. First, an attempt should be made to develop antibodies against a prominent protein, or an i m m u n d o m i n a n t antigen specific to the life-cycle stage to be diagnosed. This m a y best be accomplished by directly infecting the rodent spleen cell donor with the parasite. This approach is limited, however, by the species specificity of the parasite (i.e. requires infectivity for mice or rats, for which m y e l o m a fusion partners are available). An alternative approach is to immunize the spleen cell donor with antigens from the parasite life-cycle stage of interest. Second,
255 a screening procedure should be devised so that only hybridomas relevant for diagnosis are selected. This screening process might include assays for crossreactions with heterologous parasite antigens to exclude unwanted clones. It has been shown, however, that direct binding assays, indicating unique parasite antigens, may be contradicted by competitive binding assays involving polyclonal sera (Craig et at., 1981). Finally, characteristics of the monoclonal antibody, such as affinity or isotope, may be selected for optimal development of a particular type of diagnostic test. A number of monoclonal antibody-based diagnostic tests have been developed and have demonstrated, in general, significant improvement over previously described procedures. Some specific examples are discussed below.
Pro tozoa Monoclonal antibodies to Toxoplasma gondii were produced by Araujo et al. (1980) and used to detect antigen in the serum of infected humans. Using a double-antibody sandwich (ELISA), antigen was detected in sera from all of 4 acutely infected individuals. The sensitivity of the assay was approximated at 4 #g of antigen m1-1 of serum. No positive reactions were obtained with sera from uninfected or chronically infected individuals or from patients with rheumatoid factor titers. However, 2 of 10 sera from patients with anti-nuclear antibodies did react with the anti-T, gondii monoclonal antibody. A similar approach with monoclonal antibodies was utilized for the detection of Trypanosoma cruzi antigenemia in mice (Araujo et al., 1982). The success of this assay m a y lead to the development of a similar type of diagnostic test for Chagas' disease in humans. Several RIA procedures have been reported for the detection of parasite circumsporozoite proteins in Plasmodium-infected mosquitos. In one system, mosquito extracts were b o u n d to microtiter wells and reacted directly with 12SI-labeled monoclonal antibody (Zavala et al., 1982). By this method, the species of Plasmodium in infected mosquitos was identified accurately with no cross-reactions among species. The same authors designed another immunoassay, a double-antibody sandwich RIA, for the same purpose. Microtiter wells were coated with one monoclonal antibody, reacted with mosquito extracts and then with an l~SI-labeled monoclonal antibody with different epitope specificity on the same protein. Using this procedure, the authors were able to detect as few as 1 infected mosquito in a group of 25. Potocnjak et al. (1982) demonstrated that a circumsporozoite protein (Pb44) from Plasmodium berghei could be detected in infected mosquitos using an inhibitory RIA. Monoclonal antibodies directed against Pb44 were b o u n d to microtiter wells, exposed to a mosquito extract and then to an 12SI-labeled anti-idiotype monoclonal antibody specific for the idiotype of the b o u n d monoclonal antibody. When Pb44 was present in the mosquito extract, binding of labeled antibody was inhibited. This assay system eliminated the need for purified parasite antigen (except as a control).
256
Helminths A series of studies, using monoclonal antibodies in immunodiagnostic tests for helminth infection, has come from the Walter and Eliza Hall Institute of Medical Research in Australia. A model system was first developed using Mesocestoides corti infections in mice (Mitchell et al., 1979). The authors employed a competitive RIA with crude parasite antigen and l~SI-labeled monoclonal antibody to detect anti-parasite antibodies in serum. No false-positive or false-negative reactions were obtained, and the test was found to be highly species-specific. Attempts to substitute a potyclonal anti-idiotype antisera for the parasite antigen were unsuccessful. A competitive RIA for diagnosis of Taenia hydatigena infection was developed by Craig et al. (1980). A monoclonal antibody was selected based on direct binding studies which indicated specificity for T. hydatigena but not for larval extracts of other taeniid species. Binding of 12SI-labeled monoclonal antibody in the RIA was inhibited by all the sera from T. hydatigena-infected sheep with no false-positive reactions with uninfected sera. However, 2 of 6 sera from T. ov/s-infected sheep inhibited binding of the anti-T, hydatigena monoclonal antibody, contradicting the species specificity indicated by direct binding assays. In a different approach, Craig et al. (1981) used monoclonal antibodies produced against Echinococcus granulosus, but shown to cross-react with sera from T. ovis-, T. hydatigena and Fasciola hepatica-infected sheep, to deplete a crude antigen extract of the responsible antigens. The partially purified E. granulosus antigen showed improved specificity in an indirect ELISA for the diagnosis of infected sheep. Monoclonal antibodies from a single hybridoma line have been shown to be useful for the diagnosis of Schistosoma japonicum infections in humans. Mitchell et al. (1981) described a monoclonal antibody, IPH.134, with specificity unique to S. ]aponicum adult worms as demonstrated by direct binding studies. When used in a competitive RIA with crude worm antigen, IPH.134 identified > 90% of serum samples from individuals who were positive for S. japonicum by fecal examination. No false-positive reactions were obtained from normal h u m a n sera or sera from humans with a variety of other protozoan or helminth infections. In addition, there was some indication that the RIA titer in the competitive assay might be correlated to fecal egg o u t p u t or prominant clinical disease. The diagnostic potential of monoclonal antibody IPH.134 was further validated by Cruise et al. (1981), who confirmed the serum a n t i b o d y basis of competitive interaction in the RIA; no evidence of anti-idiotype or immune complex interaction was found. The same authors demonstrated that as little as 4 ng of specific antibody would inhibit 50% of m a x i m u m binding of IPH.134 in the competitive RIA. The antigen to which IPH.134 was directed was considered i m m u n o d o m i n a n t , because up to 1 mg m1-1 of specific antibody was detected in human serum, and because even low responder or partially resistant inbred mice could be diagnosed.
257 As a further modification, Mitchell et al. (1983) reported a competitive ELISA using hybridoma IPH.134. Using a peniciUinase-conjugated monoclonal antibody, no false-positives and approximately 10% false-negatives in human serum samples were obtained. A monoclonal antibody recognizing a circulating antigen of adult Onchocerca volvulus was used by des Moutis et al. (1983) in a radio-immune precipitation (RIP) assay. Radio-labeled monoclonal antibody was reacted with human sera containing soluble O. volvulus antigen and the antigen--antibody complexes were precipitated with polyethylene glycol. Circulating antigen was detected in 80% of African onchocerciasis patients and 76% of Venezuelan onchocerciasis patients tested. Cross-reactivity was minimized in this test. However, several sera from patients with Brugia malayi, Loa loa or Schistosoma mansoni infections gave false-positive results. No correlation between the RIP and microfilaradermia (as determined by the skin snip test) was found, suggesting the need for using these tests in tandem. Swine trichinosis The efficacy of diagnostic tests for trichinosis has been enhanced by the identification and isolation of more refined antigen preparations. For example, Trichinella spiralis excretory--secretory (ES) products and an immunopurified fraction from stichocyte particles (PAW) have been shown to have good diagnostic properties (Gamble et al., 1983; Seawright et al., 1983, respectively), while crude extracts of T. spiralis muscle larvae have been shown to produce 10--15% false-positive reactions (Ruitenberg et al., 1976; Saunders et al., 1977; Clinard, 1978; Gamble et al., 1983). Monoclonal antibodies directed against T. spiralis were produced by cell fusion using spleen cells from Balb/c mice orally inoculated with infective muscle larvae (Gamble and Graham, 1983a). Hybridomas producing antibodies directed against a crude extract of muscle larvae were cloned and were screened by ELISA against T. spiralis adults, larvae and larval ES antigens, as well as a panel of antigens from other helminth parasites of swine (Table I). Most monoclonal antibodies recognized antigens shared by larval and adult stages of T. spiralis and by other swine parasites, particularly Trichuris suis. However, several monoclonal antibodies were specific for Trichinella spiralis larvae and adult antigens or larval ES antigens. Because of the demonstrated diagnostic value of assays utilizing larval ES products (Gamble et al., 1983), one hybridoma with specificity for an ES antigen of muscle larvae (7C2C5) was selected for further study. Hybridoma 7C2C5 secreted an IgM monoclonal antibody with specificity for antigenic determinants present on proteins of molecular weight 45 000, 49 000 and 53 000 (Ts.45, Ts.49, Ts.53). Target antigens of 7C2C5 are secreted orally by muscle larvae, as indicated by immune precipitation reactions, which occurred when larvae were incubated with ascites fluid. Additionally, immunoperoxidase staining demonstrated that these antigens are located in the stichocyte cells. Antigens Ts.45, Ts.49 and Ts.53 were isolated by affinity-chromatography
258
and used in a triple-antibody indirect ELISA for the detection of antibodies to T. spiralis as described by Gamble et al. (1983). Using this test, no falsepositives were obtained with sera from farm-raised hogs at slaughter, or serum from hogs infected experimentally with swine nematodes other than T. spiralis (Fig. 2). In addition, 100% of the experimentally-infected hogs were detected at a 1:100 dilution of serum. Subsequent studies have shown TABLE I R e a c t i v i t y o f s e l e c t e d m o n o c l o n a l a n t i b o d i e s w i t h a n t i g e n s f r o m Trichinella spiralis l i f e c y c l e s t a g e s a n d o t h e r p a r a s i t e s p e c i e s (Ascaris suum, Strongyloides ransomi, Stephanurus dentatus, Trichurus suis) in a n E L I S A . S e e G a m b l e a n d G r a h a m ( 1 9 8 3 a ) f o r d e t a i l s o f t h e
assay Clone
T. spiralis Larvae
A. suum
Adult
ES*
Larvae
1A5C 3 + 1D3 C 3 + 7A5C 1 +
+ + +
. _
. + _
7A6C 1 + 7B1 C 3 + 7B4C s + 7C2 C 5 + 7C5 C = +
+ + + +
_ . + -
+ + . . +
Adult .
Egg
.
S. ransomi
S. dentatus
T. suis
Larvae
Adult
Adult + + +
.
.
+ _
+ +
-
+ +
+ _
+ +
+ +
+ +
.
.
.
.
. +
+ + +
. .
+
.
. +
+
+
*ES = excretory--secretory products.
o
T..s..piralis neg o
o I
T, soiralis neg o
T. {~oiralis pos
•
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I
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•
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)>
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'
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o'.1
012 ELISA
0!3
0!4
o'.s
0.6
O. D.
Fig. 2. E n z y m e - l i n k e d i m m u n o s o r b e n t a s s a y f o r t h e d e t e c t i o n o f s w i n e a n t i b o d i e s dir e c t e d a g a i n s t Trichinella spiralis i n f e c t i o n . A n t i g e n s w e r e c r u d e w o r m e x t r a c t ( C W E ) o r an excretory--secretory antigen (McAff-Ag) isolated by affinity-chromatography using h y b r i d o m a 7C2C 5. S w i n e s e r a i n c l u d e d 3 5 s e r a f r o m f a r m - r a i s e d s l a u g h t e r h o g s ( c ) , 3 0 s e r a f r o m h o g s i n o c u l a t e d w i t h 1 0 000 T. spiralis m u s c l e l a r v a e a n d b l e d a f t e r 6 m o n t h s ( e ) , a n d p o o l e d s a m p l e s f r o m a n i m a l s e x p e r i m e n t a l l y i n f e c t e d w i t h Ascaris suum ( a ) , Strongyloides ransomi (A) o r Stephanurus dentatus ( o ) . S e e G a m b l e a n d G r a h a m ( 1 9 8 3 a ) for assay details.
259
that similar results are obtained with hogs experimentally-infected with lower infecting doses of T. spiralis muscle larvae (500 instead of 10 000). Because of the time and expense involved in affinity-isolation of antigen, the potential for antigen variation, and the presence of additional antigen epitopes on affinity-isolated proteins, we sought to develop a competitive immunoassay using hybridoma 7C2C5 (Gamble and Graham, 1983b). Microtiter plates were coated with crude ES antigen, serum samples and biotinlabeled monoclonal antibody 7C2C5 were added, and the plates were incubated. Bound biotinylated monoclonal antibody was detected by the addition of an avidin-peroxidase conjugate followed by peroxidase substrate; a reduction in enzyme activity as compared to normal serum controls indicated the presence of competing host serum antibodies. The presence of circulating worm antigen or anti-idiotype antibodies could n o t be detected in serum samples from heavily-infected animals by direct binding assay. Therefore, it was concluded that competition was due exclusively to serum antibodies. With the competitive ELISA, no false-positive reactions were discovered in a group of sera from non-infected farm-raised hogs and all serum samples from hogs experimentally-infected with T. spiralis were positive (Fig. 3). Additionally, no cross-reactions were obtained with serum pools from hogs infected with Ascaris suum, Strongyloides ransomi or Stephanurus dentatus or from hogs with serum antibody titers against Trichuris suis. Therefore, this assay appears to offer a more simple, b u t still sensitive and specific test for swine trichinosis. % Inhibition 0 I:. soiralis neg o T
=gpiralis pos . T. 8uis pos •
20
oooo
40
60
•
80
100
• =.!i-=.=1t=-'-
i| °°
A, s u u m pool A & ransomi pool • S. dentatus pool o
Fig. 3. Competitive enzyme-linked immunosorbent assay for the detection of swine antibodies directed against Trichinella spiralis infection. Antigen was a crude excretory-secretory preparation. Swine sara included 35 sera from farm-raised slaughter hogs (o), 30 sera from hogs inoculated with 10 000 T. spiralis muscle larvae and bled after 6 months (e), 13 sera from hogs with natural infections of Trichuris suis (=), and pooled samples from animals experimentally infected with Ascaris suum (~), Strongyloides ransomi (4) or Stephanurus dentatus (o). See Gamble and Graham (1983b) for assay details.
260 PROSPECTS FOR THE FUTURE
Hybridoma technology is just beginning to be applied in the field of parasite immunodiagnostics. However, the recent success in research employing these techniques should encourage greater interest. Preliminary reports indicate that monoclonal antibodies with immunodiagnostic value have been produced against Entamoeba histolytica (Lopez et al., 1982), Toxocara canis (Nicholas et al., 1982), Trichomonas vaginalis (Desmond et al., 1983), Leismania spp. (Williams et al., 1983), Schistosoma mansoni (Abdel-Hafiz et al., 1983; Zodda et al., 1983) and Dirofilaria immitis (Scott et al., 1983). By the proper selection of monoclonal antibodies and/or their use in immuno-affinity isolation of specific antigens, useful diagnostic tests should eventually be developed for all important parasitic diseases.
REFERENCES Abdel-Hafiz, S.K., Phillips, S.M. and Zodda, D.M., 1983. Schistosoma mansoni: Detection and characterization of antigens and antigenemia by inhibition enzyme-linked immunosorbent assay (IELISA). Exp. Parasitol., 55 : 219--232. Araujo, F.G., Handman, E. and Remington, J.S., 1980. Use of monoclonal antibodies to detect antigens of Toxoplasma gondii in serum and other body fluids. Infect. Immun., 30: 12--16. Araujo, F.G., Sharma, S.D., Tsai, V., Cox, P. and Remington, J.S., 1982. Monoclonal antibodies to stages of Trypanosoma cruzi: Characterization and use for antigen detection. Infect. Immun., 37: 344--349. Bidwell, D.E., Buck, A.A., Diesfeld, H.J., Enders, B., Haworth, J., Huldt, G., Kent, N.H., Kirsten, C., Mattern, P., Ruitenberg, E.J. and Voller, A., 1976. The enzyme-linked immunosorbent assay (ELISA). Bull. W.H.O., 54: 129--139. Clinard, E.H., 1978. Serum fractions associated with positive and false-positive reactions in the ELA test for trichinellosis in swine. In: C.W. Kim and Z.S. Pawlowski (Editors), Trichinellosis. 4th Int. Conf., University Press of New England, Hannover, NH, pp. 509--517. Craig, P.S., Mitchell, G.F., Cruise, K.M. and Rickard, M.D., 1980. Hybridoma antibody immunoassays for the detection of parasitic infection: Attempts to produce an immunodiagnostic reagent for a larval taeniid cestode infection. Aust. J. Exp. Biol. Med. Sci., 58: 339--350. Craig, P.S., Hocking, R.E., Mitchell, G.F. and Rickard, M.D., 1981. Murine hybridomaderived antibodies in the processing of antigens for the immunodiagnosis of hydatid (Echinococcus granulosus) infection in sheep. Parasitology, 83: 303--317. Cruise, K.M., Mitchell, G.F., Garcia, E.G. and Anders, R.F., 1981. Hybridoma antibody immunoassays for the detection of parasitic infection: Further studies on a monoclonal antibody with immunodiagnostic potential for schistosomiasis japonica. Acta Trop., 38.' 437--447. DesMoutis, I., Ouaissi, A., Grzych, J.M., Yarzabal, L., Haque, A. and Capron, A., 1983. Onchocerca volvulus: detection of circulating antigen by monoclonal antibodies in human onchocerciasis. Am. J. Trop. Med. Hyg., 32: 533--542. Desmond, W., McEvoy, S., Bowley, K. and Leung, J., 1983. An ELISA assay with a monoclonal antibody to all geographic and drug resistant strains of Trichomonas vaginalis. Proc. Fed. Am. Soc. Exp. Biol., 42: 855.
261 Gamble, H.R. and Graham, C.E., 1983a. A monoclonal antibody-purified antigen for the immunodiagnosis of trichinosis. Am. J. Vet. Res., 45: 67--73. Gamble, H.R. and Graham, C.E., 1983b. Comparison of monoclonal antibody-based competitive and indirect enzyme-linked immunosorbent assays for the diagnosis of swine trichinosis. Vet. Immunol. Immunopathol., in press. Gamble, H.R., Anderson, W.R., Graham, C.E. and Murrell, K.D., 1983. Diagnosis of swine trichinosis by enzyme-linked immunosorbent assay (ELISA) using an excretory--secretory antigen, vet. Parasitol., 13: 349--361. Huldt, G., 1981. Serodiagnosis of parasitic infections. Parasitology, 82: 49--50. Kagan, I.G., 1982. Standardization and quality control of immunodiagnostic methods. In: D.F. Mettrick and S.S. Desser (Editors), Parasites -- Their World and OurS. Elsevier Biomedical Press, Amsterdam, pp. 387--390. Lopez, J.S., Jensen, F.L., Ximenez, C. and Ortiz-Ortiz, L., 1982. Monoclonal antibody specific for Entamoeba histolytica. Proc. Fed. Am. Soc. Exp. Biol., 41 : 484. Mitchell, G.F., Cruise, K.M., Chapman, C.B., Anders, R.F. and Howard, M.C., 1979. Hybridoma antibody immunoassays for the detection of parasitic infection: development of a model system using a larval cestode infection in mice. Aust. J. Exp. Biol. Med. Sci., 57: 287--302. Mitchell, G.F., Cruise, K.M., Garcia, E.G. and Anders, R.F., 1981. Hybridoma-derived antibody with immunodiagnostic potential for schistosomiasis japonica. Proc. Natl. Acad. Sci., 78: 3165--3169. Mitchell, G.F., Premier, R.R., Garcia, E.G., Hurrell, J.G.R., Chandler, H.M., Cruise, K.M., Tapales, F.P. and Tiu, W.O., 1983. Hybridoma antibody-based competitive ELISA in Schistosoma japonicum infection. Am. J. Trop. Med. Hyg., 32: 114--117. Nicholas, W.L., Mitchell, G.F. and Stewart, A.C., 1982. Monoclonal antibodies to larval Toxocara canis (Nematoda). Mol. Biochem. Parasitol. Suppl., Parasites -- Their world and Ours, Elsevier Biomedical Press, Amsterdam, p. 96. Potocnjak, P., Zavala, F., Nussenzweig, R. and Nussenzweig, V., 1982. Inhibition of idiotype--anti-idiotype interaction for detection of a parasite antigen: a new immunoassay. Science, 215 : 1637--1639. Ruitenberg, E.J., Lungstrom, I., Steerenberg, P.A. and Buys, J., 1976. Reliability of the enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Trichinella spiralis infections in conventionally raised pigs. J. Immunol. Methods, 10: 67--83. Saunders, G.C., Clinard, E.H., Bartlett, M.L. and Sanders, W.M., 1977. Application of the indirect enzyme-labelled antibody microtest to the detection and surveillance of animal diseases. J. Infect. Dis., 136: $258--$266. Scott, A.L., Showalter, S. and Levy, D.A., 1983. Monoclonal antibodies against somatic antigens of Dirofilaria immitis. Proc. Fed. Am. Soc. Exp. Biol., 42: 1089. Seawright, G.L., Zimmerman, W.J., Isenstein, R.A. and Despommier, D.D., 1983. Enzyme immunoassay for swine trichinellosis using antigens purified by immunoaffinity chromatography. Am. J. Trop. Med. Hyg., in press. Voller, A. and deSavigny, D., 1981. Diagnostic serology of tropical parasitic diseases. J. Immunol. Methods, 46: 1--29. Williams, K.M., Constantine, N.T. and Anthony, R.L., 1983. Differentiation of new world Leishmania species by means of their reactivity with monoclonal antibodies in immunofluorescent assays. Proc. Fed. Am. Soc. Exp. Biol., 42: 854. Zavala, F., Gwadz, R.W., Collins, F.H., Nussenzweig, R.S. and Nussenzweig, V., 1982. Monoclonal antibodies to circumsporozoite proteins identify the species of malaria parasite in infected mosquitos. Nature (London), 299: 737--738. Zodda, D.M., Abdel-Hafez, K.D. and Phillips, S.M., 1983. Characterization of monoclonal antibodies against Schistosoma mansoni. Am. J. Trop. Med. Hyg., 32: 69--77.