140

3 Sigal, N. H. and Klinman, N. K. (1978) A&ni. I~ru~~uw/, 26, 255 4 Karush, F. (1970) /!nn.~V.?! Acad SLZ. 169, 56 5 Steward, M. W. (1978) in Handbook I!/ E.x~erznientnl Immirnoiogy I). M. Weir, ed.) 3rd ed, p 16, 1 Blackwell, Oxford 6 Eisen, H. N. and Siskind, G. W. (1964) ~zoch~mzvtry 3, 966 7 Pressman, D., Roholt, D. A., Grossberg, A. I,. (1970) Ann. Y.?“. Acad. St. 169, 65 8 Werblin, T. and Siskind, G. W. (1972) Zmmunoc/zem~stry 9, 987 9 Morgan, A. G. and Soothill, J. F. (1975) &x~aluw jlonrlon/ 254, 711 10 Dekruyff, R. and Siskind, G. W. (1979) C:rll. fvurwzol. 47, 134 II Dekruyff, R. and Siskind, G. W. (1980) (XI. Zrrimurrol. 49, 90 12 Petty, R. E. and Steward, M. W. (1977) Zrrrrrrunoiog 32, 49 I3 Soothill, J, F. and Steward, M. W. (1971) clin. Exp. Zmmmoi. 9, 193 14 Katz, F. E. and Steward, M. W. (1975) Irruw,uu~lugy 29, 543 15 Ruscetti, S. K., Kunz, M. W. and Gill, T. J. (1974) ,j’. Irrrmurrnl. 133, 1468 16 Steward, M. W. and Petty, R. E. (1976) Imrnunolqy 30, 789 17 Kim, Y. T. and Siskind, G. W. (1978) Irrurwu&~y 34, 669 I8 Reinhardt, M. C. and Steward, IM. W. (1979) Irr,mw&gy, 38, 735 19 Steward, M. W., Reinhardt, M. C. and Staines, N. A. (1979) Immunology 37, 697 20 Chandra, R. K. (1980) Irr~~rw~ology nj .Nu~nlzonal I~zI~&~.s, Edward Arnold, London 21 Steward, M. W., Alpcrs, J. H. and Soothill, J. F. (1973) Aoc. Roy. Sot. Med. 66, 808 22 Steward, M. W. and Voller, A. (1973) Wr.3.&//. t’&. 54, 198 23 Siskind, G. W. and Benacerraf, B. (1969) Ado. Imnmrtnol. 10, I 24 Ghose, A. C. and Karush, F. (1973) Biochernirlry 12, 2437 25 Macario, A. J. L. and Conway de Macario, E. (1973) &tnr~ (London) 254, 263 26 Herzenberg, L. A., Black, S. J., Tokuhisa, 1‘. and I lerzenberg, L. A. (198O)J. &b. Med. 151, 1071 27 Bystryn, J-C., Siskind, G. W. and Uhr, J W. (1973) J. Eup. Med. 137, 301 28 Delisi, C. (1978) in 77zheoretzcal lmn~unology (Bell, G. I., Perelson, A. S. and Pimbley, G. H. eds.) Vol. 8, p 215 Marcel Dekker, New York

29 Klaus, G. G II., Howard, J. C and Feldman, M. (1976) Hrzi. Med. Hzdi. 32, 141 30 Klinman, N. R. (1980) in &rogw\.\ irz hnmw~olugy IV (Yougereau, M. and I)ausset, J. eds.) p 122, Academic Press, London 31 Nossal, G. J. V. and Pike, B. (1980) in &~IP.T.I. uz Ivzmrrnnlqy IV (M. Fourgeau and J. Dausset, eds.) p. 136, Academic Press, London 32 Perelson, A. S. (I 978) in 7%~orrlzcm/ Irr~r2mruu~iogy (Bell, G. I., Perelson, A. S. and Pimbley, G. H. eds.) Vol. 8, p. 171. Marcel Dekker, New York 33 Petty, R. E., Steward, M. W. and Soothill, J. F. (1972) C,‘lul. Ex;h. Inmtnol. 12, 231 34 Urbain, J., van Acker, A. de vos Cloetens, C. H. and UrbainVansanten, G. (1972) Irr,munochrmz,lryy 9, 121 35 Steward, M. W. (1978) in Immunochrmutry: An Adunncrd lbxlbook (Glynn, L. E. and Steward, M. W. eds.) p. 233, Wiley, Chichester 36 Theis, G. A. and Siskind, G. W. (1968) j’. Irruwnt~l. 100, 138 37 Germuth, F. G. and Rodriguez, K. (I 973) Zrnrnlin~~~~~lh~j~fj~yo/lhP Xenczl &rrrerulu.~ Little, Brown and Co. Boston 38 Dixon, F. C., Feldman, J. D. and Vasquez, J. J. (196l)J. &/I. Med. 133, 899 39 Pincus, 1‘., Haberkern, R. and Christian, C. I,. (1968),7. Ex#. Med. 127, 819 40 Kuriyama, T. (1973) Lab. Znw~l. 28, 224 41 Soothill, J. F., Smith, M. I). and Morgan, A. G. (1975) in Pafhogvnzr &XPIIP.\ zn Pcuuilu fn@lwn.! (Taylor, A. E. R. ANd Miller, R. eds.) Vol. 13, p 59, Blackwell, Oxford 42 Steward, M. W. (1979) c/in. I&fi. Zmmunol. 38, 414 43 Devey, M. E. and Steward, M. W. (1980) Irrururwlogy 41, 303 44 Koyama, A., Niwa, Y., Shigematsu, II , Taniguchi, M. and Tada, ‘r. (1978) I&J Inwl. 38, 253 45 Germuth, F. G., Rodriguez, E., I,orelle, C. A., Trump, E. J., Micano, L. and Wise, O’L. (1979) Lab. Inwst. 41, 360 46 Steward, M. W. (1976) in I+rtion rind Imrrwnology vi thu Rheumotzi- /)z.tunsur (Dumonde, D. C. ed.) p 439, Blackwell, Oxford 47 Winfield, J. B., Fairferman, I. and Koffler, D. (1977) j’ Clm. huesi. 59, 90 48 Causer, W. G. and Salant, D. J. (1979) Kzdwy Inl. 17, 1

Hybridoma antibodies in immunodiagnosis of parasitic infection Graham Laboratory

of Immunoparasitology,

‘The

Walter

and

F. Mitchell

Eliza

Hall

Institute

of rCledica1 Research, Melbourne,

Victoria

3050,

Australia Immunological methods for the indirect detection of parasites have found wide use in epidemiological investigations of parasitic infections and at the level of the infected individual’-/. Many parasites ake simply a tlseu,er,No,th-ll”lllirrd Ummedlcai PressIL)“, o,G,~4n,n,a,,o,,r,rrllrlllll/S112 ill

not amenable to direct detection in the living host and detection of parasite products (eggs in faeces, antigcns or metabolites in serum or urine) may be impossible, impractical, time consuming or unreliable. The

old adversaries of the serologist, low sensitivity, inadequate specificity, and difficulties in standardization, are those which plague most available tests used by the parasite immunodiagnostician or epidemiologist. It is entirely predictable that the use of a mixture of a large number of antigens extracted from any life cycle stage of any complex parasite will lead to false positive reactions in serology or skin testing. On the other hand, the greater the restriction in the number of antibodies or sensitized cell specificities detected, the greater the probability of lowering the sensitivity of the immunodiagnostic test (IDT). In light infestations with various parasites there is no reason to expect the immune system to be engaged in antiparasite immune responses to an exaggerated extent. This is particularly so with those parasites which are relatively sequestered or excluded (e.g. at mucous membranes) from the immune system. Interpretation problems will always be experienced when using methods which rely on the detection of long-lived antiparasite antibody responses or heightened sensitivity (memory) to parasite antigens. However, such methods are at least capable of exploiting the potential amplification power of the immune response. The value of an IDT is improved if it is quantitative, readily applied in the field, and capable of providing some information on (1) level of infection, (2) duration of infection, (3) potential immunopathologic consequences of the immune responses being detected, and (4) relative host protection to infection; detection of a potent immune response which is protecting the host against infection is no test for the presence of a resident parasite (c.f. exposure). All this is a lot to ask of an IDT. With the advent of hybridoma technologyK,9, it was predicted that the .~,&:c$cily of IDTs would be improved. By using carefully selected hybbidomaderived antibodies with specificity for a particular parasite or parasite life cycle stage, ELBA tests”’ could be designed which were based on detection of antibodies directed against antigens purified by hybridoma antibody affinity chromatography. The property of high specificity is precisely that which is required of a parasite typing reagent, for example. However, it was suspected that sensitivity in immunodiagnosis would be lost - the IDT would be based on the detection of an immune response to a single determinant (e.g. in a competitive radio immunoassay (RIA), or a limited number of antigenic determinants (after use of the antibody to isolate antigens containing the determinant). For the hybridoma-based IDT to be useful all infected individuals would be required to respond to the limited array of antigens. These valid concerns about sensitivity have led to two distinct approaches in the use of hybridoma antibodies in the development of new IDTs: (1) the identification of hybridoma antibodies directed against immunodominant antigens specific for a parasite and to be used for antigen isolation; (2) the use of hybridoma

antibodies directed against shared antigens (and which are responsible for cross reactions and false positives) for the depletion of these antigens from crude antigen mixtures. The first demonstration that high specificity and adequate sensitivity could be obtained by using ‘single specificity’ hybridoma antibodies for immunodiagnosis of a parasite infection was made in a model system”. A hybridoma (designated McH. 105), secreting an IgC, antibody directed against the murinc larval cestode, Mmcatoich cod, and produced by fusion of spleen cells from chronically infected mice, was labelled with rzj1 and used in a competitive solid-phase RIA with the crudest of parasite extracts. The test had absolute specificity, in that sera from mice infected with various other parasites did not inhibit the binding of [rziI] McH. 105 to its target determinant in the crude antigen mixture. The test also had high sensitivity in that sera from all M. corti-infected mice of various inbred strains, some of which had been infected for only 2 weeks, were highly inhibitory. Infected hypothymic nudes were the only mice not detected in this model ID?‘. Attempts to substitute the crude antigen mixture with a large pool 01 anti-idiotypic antibody directed against McH. 105 were not successful since sensitivity was lost’ I. The M. corti parasite, though a natural larval cestode of mice, does not really provide a fair test of the sensitivi?y of hybridoma antibody-based IDTs. This parasite is an unusual helminth in that it proliferates in the host (liver and peritoneal cavity) and mice develop huge worm burdens with prominent and hypergammaglobulinaemia’z~r3. l’he disease McH hybridoma antibody with the above-mentioned properties was identified with a selection method based on the binding of antibodies in culture supernatants to living parasites denuded of surface immunoglobulin. We have found in other systems that apparent high specificity of a hybridoma antibody cannot be inferred from a demonstration of high binding activity for the homologous parasite (or antigen preparation) and no binding to heterologous parasite antigens in solid-phase RIAs. A large battery of sera from clinically delined ‘mono-infected’ patients or animals in competitive assays is far more useful for testing the parasite specificity of a hybridoma-derived antibody than are binding assaysr4. Presumably, the amplification provided by the host immune response unmasks the presence of minority antigenic determinants in parasites (and which may not bind to plates in the assay). The appropriate life cycle stage which shares the determinant in question may also not be available for preparation of antigen to be used in the RIAIS. This being so, it is imperative that large banks of sera from clinically defined patient groups, and centres to collect and distribute this sera, be formed. This approach to the development of new hybridoma-based IDTs with high sensitivitv and

umm.mol~~gy today, j’uly

142 specificity for parasitic infection has recently been extended to ,Wzislosomn ja,hrzicum in mani6,i7. Numerous 1DTs for schistosomiasis are available (or at a highly advanced stage of development), one of the most useful and simplest being the circumoval precipitin (COP) test described many years ago by Oliver-GonzaleziX. The availability of COP-positive hybridomas directed against egg antigens of s. manwzi’9 and S&wnicum2” will aid in the analysis of this extraordinary reaction and in the isolation of immunodiagnostic (and immunopathologic) antigens. A hybridoma-derived antibody (designated IPH.134) and directed against S.p,bonicum adult worms has been shown to have high immunodiagnostic potential for schistosomiasis japonica in the Philippines’“,“. The binding of [i251]IPH. 134 to a crude extract of S$fionicum worms is inhibited by >90% of sera from approximately 50 known infected individuals and, most importantly, the majority of patients with high faecal egg counts or prominent hepatosplenomegaly have high inhibitory titres of antibody in their sera. Thus, an IDT based on detection of serum antibody to the antigen to which IPH.134 is directed may give clues to infection level and disease status. This remains to be determined under field or laboratory conditions using large numbers of sera and an ELISA test with isolated antigen (or anti-idiotypic antibodies) coated to plates. The alternative approach of using cross-reactive hybridoma-derived antibodies to deplrte crude antigen mixtures of antigenic determinants shared between parasites has been successfully employed in the case of echinococcosis (hydatids) in sheep2’. Although also in its infancy and far from being a proven approach, this strategy may be particularly useful in those parasitic infections where there is a sensitivity problem in using crude antigen mixtures and where there is concern that genetically based unresponsiveness (or low responsiveness resulting from neonatal infection) will dictate that multiple antigenic determinants be included in the IDT. In some situations it is already known that a limited number of other parasite infections leads to false positives. The use of cells from mice injected or infected with the homologous parasite for fusion and selection for antibodies directed against the hcterologous parasite(s) should lead to the development of useful reagents to be used in solid phase for antigen processing (depletion).

l&S’1

These examples demonstrate the power of the hybridoma technology in the development of new immunodiagnostic reagents for parasitic infection. It must be emphasized, however, that the only valid test for a new reagent is in the field or routine immunodiagnostic laboratory, and hybridoma antibody-based IDTs have not yet reached that stage. One restriction to progress in this type of applied immunoparasitology is the lack of modified myeloma cells in species other than the mouse for fusing with lymphoid cells from naturally infected natural hosts. Although murine cell lines have proven, and will continue to prove, their worth in this area, the opportunity to produce families of hybridomas within the species (e.g. sheep, cattle, man, etc.) will have obvious advantages. References 1 Cohen, S. and Sadun, E. H. (Eds.) (1976) Inzmunology qf Parn.rzi~c In/&on Blackwell Scientific, Oxford 2 Fife Ir., E. I-i. (1971) E&I. PuvL~I~~. 30, 132 3 Fifejr., E. H. (1972) &xi. I’araszlol. 31, 136 4 Kacan, I. G. (1974) 7. Para~i~mkd. 45. 163 5 K&n, I. G. (1979j z&n.]. Tro,b. Mcd Hyg. 28,429 6 klouba, V. (Ed.) (1980) Inzwzol~~gzcal Inue\~rg&n o/ Trqcal Pnm.rilic I1iwn1p.s. Churchill Livingstone, Edinburgh 7 Soltys, M. A. and Woo, P. ‘1’. K. (1972) z, 7-ro,benmeci. Paraszt. 23, 172 8 Kijhler, G. and Milstcin, C. (I 975) Nature jLw~&n) 256, 495 9 KGhler, G. and Milstein, C. (1976) Eur.3. hmund. 6, 511 IO Vollcr, A., Bartlett, A. and Bidwell, D. E. (1976) Tran~. R. Sot. Tryb. Mrd. Ilyg. 70, 98 11 Mitchell, G. F., Cruise, K. M., Chapman, C. B., Anders, R. F. and Howard, M. C. (1979) An.ll.3. ix/~. Bid. MP~. Scz. 57, 287 12 Mitchell, G. F., Marchalonis, I. I.. Smith, P. M., Nicholas, W. L. and Warner, N. L. (1977) A?(\y.J. Exp. Binl. Mud. Scz. 55,‘187 13 Chapman, C. B., Knopf, P. M., Hicks, J. D. and Mitchell, G. F. (1979) Axit. j’ Exp. Bzol. Med. Sk. 57, 369 14 Craig, P. S., Mitchcil, G. F., Cruise, K. M and Kickard, M. D. (1980) Ausl. j’. E.x{I. Bzrd. Mrd. Sri. 58, 339 15 Mitchell, CT‘: F. (1781) in ~Monoclonni An~~bod~e~ ancf T-C& EIybrrdwnn.~ (Himmerling, G. J., Hgmmerling, U. and Kearney, ,J. F. eds.) Elsevier, Amsterdam (in press) 16 Mitchell, G. F., Cruise, K. M., Garcia, E. G. and Andcrs, R. F. (1981) Pm. Nd. A~ad. Sz. I/SA (in press) 17 Cruise, K. M., Mitchell, G. F., Garcia, E. G. and Anders, R. F. (1981) Ach hpzrci (in press) 18 Oliver-Gonzalez,,J. (1954)3. hjkt. ,015. 95, 86 19 Hillycr, G. V. and Pelley, R. P. (1980) Am. 3. 7;op. Mrd. ffyg. 29, 582 20 Cruise, K. M.. Mitchell, G. F., ‘I‘apalcs, Y. P., Garcia, E. G. and Huang, S-R. (1981) Au51.3. Exp, Bud. Mrd. Sci. (in press) 21 Craig, P. S., Hocking, R. E., Mitchell, G. F. and Rickard, M. D. (1981) Pnra.szlo/ogy (in press)