Parasite Immunology 1990, 12,545-557
Monoclonal antibodies against Opisthorchis viverrini antigens P.B.BILLINGS, N . U T S A K H I T & S.SIRISINHA Department of Microbiology, Faculty of Science, Mahidol University. Bangkok, Thailand Accepted for publication 21 November 1989 Summary Monoclonal antibodies (MoAb) were produced against somatic antigens of adult human liver fluke Opisthorchis cicerrini. Earlier studies attached diagnostic potential to an 89-90 kD antigen present in both somatic extracts and in uitro culture supernatants as well as to the abundant 16-17 kD tegumental protein doublet. Mice made excellent immune responses to low dose somatic extract adsorbed onto nitrocellulose or to the 80-95 kD region of SDS gel Western blots. The antigen specificities of hybridomas reactive with somatic antigen by ELISA were determined by radioimmunoprecipitation or immunoblotting. Six MoAb reacted with the desired 16 kD tegumental protein. A 90 kD somatic protein was identified by 9 clones. By indirect immunofluotescence, monoclonals reactive with the 16 kD polypeptide identified the outermost surface of the tegument. The 90 kD antigen was associated with all major muscle systems, most strikingly the crossed subtegumental layers, oral and ventral suckers, pharynx and a thin layer surrounding caeca. The biochemical identity of this muscle-associated antigen is unknown, but it is clearly distinct from the previously identified speciesspecific 89 kD exoantigen. The 16 kD tegumental protein shares epitopes with a number ofrelated flukes. However, 2 MoAb which react with this protein show no crossreaction.
Keywords: monoclonal antibody. Opisthorchis ricerrini, trematode, serodiagnosis
Introduction Liver fluke infection is still an important public health problem in many countries throughout the world, including East and Southeast Asia a n d Eastern Europe. In Thailand, roughly 15% of the population as a whole are infected with Opisthorchis uiuerrini. Surveys made in the highly endemic area have reported prevalences as high a s 85-90%. Current diagnosis based on stool examination for characteristic eggs is variable a n d undependable, particularly in cases of light infection, demands highly trained technicians and is invariably time-consuming. All of which render the method certainly not amenable to even moderate-scale mass screening. One might expect that the Correspondence: Dr Stitaya Sirisinhn, Department of Microbiology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400. Thailand.
P.B.Billings, N . Utsakhit & S.Sirisinha
availability of effective anthelminthic treatment might lessen the demand for a more acceptable and practical diagnostic test. However, it is precisely this ability to intervene with individual or mass chemotherapy which demands more practical evaluation of the effectiveness of mass therapy and some means of assessing the extent of reinfection and identifying persons requiring further treatment. A large number of serodiagnostic methods have been attempted in the expectation of finding a more simple and practical diagnostic technique for this infection. However, none has been found entirely satisfactory in terms of specificity and sensitivity. For the most part, this is attributable to insufficient knowledge of individual antigens, abundancies, and immunogenicities and consequent use of poorly characterized and unrefined complex mixtures ofantigens. Several years previously, our laboratory initiated a detailed study of the immune response in opisthorchiasis and individual antigens recognized in natural infection, in the hope that this could serve as a more focused starting point for the development of improved immunodiagnostic reagents (Wongratanacheewin & Sirisinha 1987, Wongratanacheewin er al. 1988a, b). These earlier studies using opisthorchiasis sera and polyclonal antibodies against various fluke antigens identified 2 antigens sufficiently abundant and species-specific to warrant further detailed study. In attempting to produce monoclonal antibodies (MoAb) which might be of use in isolating and characterizing antigens of Opisrkorchis and perhaps lead to an improved immunodiagnostic assay, we hoped to recover at least some MoAb with specificities for these known antigens. The first of these antigens is the 16-17 k D tegumental proteins, which, by some estimates together comprise almost half of the total saline-soluble protein in worm homogenate. The second antigen is an 89-90 kD protein present in relatively small amount in somatic extracts, but comprising a high proportion of the metabolic products or excretory-secretory (ES)exo-antigens recovered from worm culture supernatants. All opisthorchiasis sera react strongly with an antigen of this size in both fractions, demonstrating that it is highly immunogenic in natural infection. However, we could not be sure of the proportional response to this protein when animals were immunized with the complex total somatic extract. A special immunization scheme using somatic antigen was undertaken in the hope of shifting more of the response to this less abundant but more highly immunogenic protein. A number of hloAb against both 16 kD and 90 kD somatic antigens were recovered and characterized in terms of specificities and immunofluorescence localization of the corresponding antigen.
Materials and methods ANTIGEN PREPARATION
Syrian hamsters were experimentally infected with 0. ~~irerriiii mstacercaria recovered from naturally infected cyprinoid fish and both somatic antigen extracts and ES antigens were prepared as previously described (Wongratanacheewin ei al. 1988a). Somatic antigens from other flukes were prepared and made available by Dr S. Wongratanacheewin (Khon Kaen University, Thailand). Protein concentrations were determined by standard Folin phenol procedures or a modified Coomassie blue G binding assay (Read & Northcote 1981) To prepare tegumental fraction, the worms were subjected to ;I freeze-thaw, vortex
Monoclonal antibodies against Opisthorchis
procedure to strip the tegumental surface component (Roberts et 01. 1983) followed by a 30 min extraction with 1% deoxycholate in 20 mM Tris-buffered saline, pH 7.4 and 10 mM EDTA. Extracts were concentrated by cold 10%trichloroacetic acid precipitation; pellets were washed with 70% ethanol and dissolved in sample buffer for electrophoresis (Laemmli 1970) containing 2% sodium dodecyl sulphate (SDS) and 4 M urea. I M M U N I Z A T I O N OF M I C E FOR HYBRIDOMA CONSTRUCTION
Two fusions were made with BALB/c mice immunized with 25-35 pg of total adult worm somatic extract directly spotted and adsorbed onto nitrocellulose. For two additional fusions, total somatic antigen extract was fractionated by preparative SDS gel electrophoresis and electroblotted onto 0.1 pm pore nitrocellulose. The region of the blot containing the 89-90 kD somatic antigen was identified by an indirect enzyme immunoassay using reference sera reactive with this polypeptide, and the entire 80-95 k D region was excised for use as immunogen. Because of poor transfer of high molecular weight (MW) proteins, mice received no more than 1-2 pg of the 89-90 kD antigen. The nitrocellulose in each case was solubilized in dimethylsulphoxide (DMSO), mixed with 3 volumes of Freund's adjuvant ( I part complete:2 parts incomplete) and 0.1 ml was injected into mice subcutaneously on the back with an equal volume intraperitoneally. The response was monitored by ELISA against plates coated with somatic antigen. Regardless of antigen in primary immunization. mice received intraperitoneally a single 100 pg dose of total somatic antigen in saline 3 days prior to fusion. SOMATIC C E L L FUSION A N D SELECTION O F ANTIBODY-SECRETING H Y B R I D S
Washed spleen cells from selected immunized mice were fused with 1 x lo7myeloma cells (X63Ag8.653) in 5: 1 ratio using40-50% polyethyleneglycol MW 3000-3700 (Sigma, cell culture tested) in RPMI 1640 or phosphate-buffered saline (PBS) pH 7.2 with 10 mM glucose containing 7-10% DMSO (Billings et al. 1985).The class/subclass determination was made after cloning by spotting supernatants onto replicate nitrocellulose strips, which after blocking in 5% non-fat powdered milk, were reacted independently with different (goat) anti-mouse Ig class/subclass sera (Sigma). A (rabbit) anti-goat Ig peroxidase conjugate was used with H202 and Cchloro- I-naphthol substrate for detection (Hawkes, Niday & Gordon 1982). RADIOIMMUNOPREClPlTATlON
Freshly prepared somatic antigen extracts were used for radioimmunoprecipitation to minimize degradation. Bolton-Hunter reagent was iodinated by the chloramine-T reaction (Bolton & Hunter 1973). A 100 pl of antigen at 1 mg/ml in saline containing 50 mM sodium phosphate pH 8.2 was added directly to the dry labelled reagent and kept on ice for 30 min. The reaction was stopped with an equal volume of 2% glycine, 10 mM KI and after 10 min the mixture was passed through column of Sephadex G-25 in PBS containing 0.25% gelatin. Radioactivity peak fractions were pooled, adjusted to I'X bovine serum albumin (BSA) and frozen. Protein A-agarose (Sigma) was rehydrated and washed in 0-35 11 NaCl, 5 mM EDTA, 20 mM Tris-HC1 pH 8.2 containing 1% BSA and 0.2% Nonidet P-40.Two packed
P.B. Billings, N . Vtsakhit & S.Sirisinha
volumes of protein A-agarose was mixed with 1 volume of normal mouse serum in the above solution. After 3 washes, the normal mouse Ig-protein A-agarose was resuspended in iodinated antigen also in the same solution using 2-6 x lo5c.p.m. per 2 pl agarose. This ‘pre-absorbed’ labelled antigen was then used in the same proportions to resuspend 2 pl aliquots of protein A-agarose coated with test sera or hybridoma supernatants ( 1 pI serum or 50-500 p1 supernatant reacted with 2 pl agarose). After 2 h incubation at room temperature with end-over-end mixing, test immunosorbents were washed 3 times in the same buffer and twice with buffer without detergent or BSA diluted 1 : 5 in water. Elution was effected by boiling in SDS and proteins were separated by electrophoresis through SDS-containing 12.5% polyacrylamide gels. Dried gels were exposed to X-ray film backed with an intensifying screen for 1-4 days at -70°C. I M M U N O F L U O R E S C E N C E MICROSCOPY
Cryostat sections 6 pm in thickness were cut from freshly frozen adult flukes. Sections were air-dried onto gelatin-coated slides and fixed in acetone. After washing in PBS containing 1 Yo BSA (PBS-BSA), sections were incubated with 1 drop of test antibody for 30 min. Test sera were appropriately diluted in PBS-BSA and hybridoma supernatants were concentrated roughly 20-fold by half-saturated ammonium sulphate precipitation, followed by dialysis and addition of BSA. After washing, slides were incubated in the dark with fluorescein-conjugated (rabbit) anti-mouse Ig (Dakopatts, Denmark), again washed and counterstained for 5 min with 0.1% Evan’s blue. Sections were mounted in 90% glycerol, 10% PBS pH 8.6 containing 5 mM 1,4-diaminobenzene (Johnson er al. 1982)and examined under a fluorescence microscope.
Results Our earlier studies had singled out 2 antigens tentatively showing diagnostic potential: a highly abundant 16-1 7 kD tegumental protein and an 89-90 kD polypeptide figuring prominently in the ES fraction from cultured worms but also present in small quantity in the somatic extracts. For hybridoma production, a method of immunization was selected which would stimulate a strong antibody response with a very small amount of antigen. and yet lend itself if necessary to use of these selected antigen fractions partially purified by SDS gel electrophoresis in order to ensure recovery of these specificities. The method chosen was to immunize animals with antigen adsorbed onto nitrocellulose membrane, either total antigen directly spotted or a pre-selected region from a Western blot. The first mouse which received primary and booster immunizations with total somatic antigen adsorbed onto nitrocellulose gave a high antibody response. and 6 hybridomas were recovered-all with anti- 16 kD tegumental polypeptide specificity as determined by radioimmunoprecipitation (Figure I, clones 3, 5, 16,54,60 and 77). The response of this mouse to individual somatic proteins, as analysed in the pre-fusion serum by immunoprecipitation, showed roughly equivalent label in the 3 predominant bands of M, 90, 17 and 16 kD, yet only the latter was recovered in monoclonals. The mouse used in a second fusion was ‘rested’ for a long period after a single low-dose primary immunization with nitrocellulose-adsorbed total somatic extract to allow antibody against components other than the highly immunogenic 89-90 kD polypeptide
Monoclonal antibodies against Opisthorchis M W ( ~ O - ~Ag' I IMS
Figure 1. Autoradiographic patterns of IzJIlabelled 0. uiuerrini somatic extract before (Ag*) and after being immunoprecipitated with monoclonal antibodies which selectively reacted with 90 kD (clones 12,18,20,21,22 and 29) and 16 kD (clones 3,5,16,54,60 and 77) polypeptides. Immune mouse serum (IMS)served as positive control and myeloma condition medium (CM) served as negative control. Molecular weight markers are shown on the left.
to decline and administered a pre-fusion booster of a total somatic extract which by electrophoresis appeared markedly depleted of the major tegumental proteins, i.e., the worms had been frozen, thawed and allowed to sediment before homogenization. This 'rested' mouse, given a prefusion booster depleted of the major 16-1 7 kD antigen, gave 5 hybridomas, each reactive by immunoprecipitation with a 90 kD polypeptide in the somatic antigen extract (Figure I , clones 12, 18, 20,21 and 22). Two additional fusions were made using mice immunized with the 80-95 kD region of an SDS-gel Western blot of total somatic antigen, to increase further our chance of recovering monoclonals with specificity against the 89-90 kD polypeptide. These mice were prefusion boosted with total somatic antigen but gave only 1 clone (#29) which immunoprecipitatedthe90 kD somatic antigen, and 3 additional IgM monoclonals which failed to immunoprccipitate but reacted with this component on immunoblots (results not shown). With several additional clones obtained from immunizations with the Western blot band, the antigen could not be identified by any of the procedures here including SDS-gel immunoblot reaction and immunofluorescence.Of perhaps some significancein terms of the suitability of immunization with solubilized SDS-gel Western blot bands on nitrocellulose, 7 of 8 clones from these fusions were IgM, and of only moderate ELISA titre; the single immunoprecipitating IgG exhibited high antibody titre. The classes/ subclasses of the various MoAb are listed in Table 1. The MoAb were screened by ELISA against plates coated with antigen extracts prepared from various related parasites in order to assess the species-specificities of the antigens/epitopes recognized. The somatic antigen extracts tested included the closely
P.B.BiNings, N . Utsakhit & S.Sirisinha Table 1. Crossreaction of monoclonal antibodies with somatic antigen extracts from other trematodes determined by ELISA
An ti body Polyclonal
Anti-Ov(som) Anti-Ov (ES) Monoclonal Anti80 kD Som 12 IgA 18 IgGl 20 IgG1 21 IgGl 22 IgM 29 IgGl Anti-16 kD Som 3 n.d. 5 IgG2b 16 IgM 54 IgG1 60 IgA 77 IgM
++++ +++ + + + + ++++ + ++
+ + +++ +++ +++ ++++
++ + ++
++ ++ ++
++ ++++ -
+++ +++ - ++++ - ++++ - ++++ - ++++ -
++++ ++++ ++++ - ++++ + +++ ++++ - ++++ -
Somatic antigens used in coating plate were: Cs, Clonorchis sinensis; Fg, Fasciola gigantica; Int FI, an unidentified bovine intestinal fluke; Sm,Schistosorna mansoni; Ov, Opisthorchis viverrini. related Clonorchis sinensis, as well as Fasciola gigantica, Schistosoma mansoni and an unidentified bovine intestinal fluke. The results summarized in Table 1 showed that most MoAb which immunoprecipitated the 90 kD polypeptide component of somatic extract with the exception of clone 22 appeared highly specific for 0. viverrini, with little or no reactivity against extracts of other somewhat distantly related parasites. However, they all showed some degree of crossreactivity with the close relative C. sinensis. Two clones (clones 12 and 18) of this group, however, exhibited a fairly low crossreactivity with C. sinensis in this assay. The 3 IgM monoclonals raised against SDS-gel Western blot bands and which failed to immunoprecipitate the 90 kD polypeptide, but did react on immunoblots from SDS-gels, recognized presumably sequence-determined epitopes common to Opisthorchis, Clonorchis and Fasciola (data not shown). Of the MoAb reactive with the 16 kD polypeptide, 4 of 6 showed moderate cross reaction with Fasciola antigen. On the'other hand, 2 anti-16 k D MoAb (clones 5 and 16), appeared to recognize epitopes expressed exclusively by 0. oiverrini with essentially no crossreaction with other parasites, including C. sinensis. The antigens identified by the MoAb were localized in thin cryostat sections of adult worms by indirect immunotluorescence. All the MoAb reactive with the 90 k D
Figure 2. Indirect immunofluorescence localization of antigens on fresh-frozen 0.uiuerrinisections stained with monoclonal antibody clone 20 (A to G) and with polyclonal prefusion serum (H). (A) ( x 100) identified caeca (c), pharynx (p) and oral sucker (0s); (B) ( x 250) shows enlarged pharynx and part of oral sucker; (C) ( x 400) shows staining of thin muscles underlying caeca; (D) ( x 250) shows unidentified structure at anterior-lateral surface close to the oral sucker (see inset in (A)); (E) ( x 250) and (F)( x 400) show bright staining of circular and longitudinal muscular (m) layers underneath unstained tegument (t); (G) ( x 400) shows a transverse section through subtegumental muscles; and (H) ( x 400) shows bright staining of subtegumental muscles and unidentified uniform spots distributed throughout the body of the worm including areas between the unstained vitteline follicles (v).
P.B.Billings. N.Utsakhit & S.Sirisinha
polypeptide identified components intimately associated with all major muscular systems of the worm (Figure 2). These monoclonals gave extremely bright fluorescence over the circular and longitudinal muscular layers lyingjust beneath the tegument (Figure 2D-2G) as well as the strong muscles associated with pharynx and oral and ventral suckers (Figure 2A-2B), and the comparatively thinner muscles underlying the intestinal caeca (Figure 2C). In addition, all anti-90 kD polypeptide MoAb reacted strongly with some small uniformly-stainingstructures widely distributed throughout the body of the worm which often appeared paired or in close apposition (Figure2E). The uniform stain density across these structures, similar to transversely cut subtegumental muscles, suggests that they are also muscle-associated. It is unlikely that these represent some second minor crossreacting antigen, since all 6 monoclonals exhibited this same dual reaction pattern, yet none immunoprecipitated even a trace amount of any second labelled antigen. The eggs, uterus, vitteline follicles and ducts, ovary and testes all failed to react with these MoAb. The MoAb which immunoprecipitated the 16 kD somatic antigen all gave a bright immunofluorescence reaction over the very surface of the tegument (Figure 3) with decreased intensity through the bulk of the tegument. In addition, these antibodies identified an irregular, presumably proteinaceous, coating over and around the eggs (results not shown). These antibodies stained neither muscle fibres nor subtegumental cells. In contrast to the monoclonal antibodies, the prefusion sera from mice immunized with total worm somatic antigen used as positive control identified almost all structures of the worms (Figure 2H). Particularly strong reaction was observed with tegument, subtegumental muscles and the smaller ubiquitous probably muscle-associated structures. In addition to a more uniform fluorescence over all of the internal tissues of the worm, caecal and uterine contents, caeca and eggs were strongly reactive. On the other hand, normal mouse serum and medium in which the myeloma cell line had been cultured, so-called 'myeloma conditioned medium', used at comparable dilution as the negative control, failed to react. A test was set up to determine whether the present 90 kD somatic antigen corresponds
Figure 3. Indirect immunofluorescence staining of the tegument (t) with monoclonal antibody clone 5. Subtegumental muscular (m) layers were unstained (A x 250; B x 400).
Monoclonal antibodies against 0pis t h orch i s
E S Agl
Som ~ 9 1
9 O h O Sam21 29
Ab I6 hD Som-
Figure 4. Antigenic difference between 89 kD exoantigen and 90 kD muscle-associated somatic antigen. Separate wells of a plate were coated with the ES fraction or somatic extract and then the reaction of polyclonal mouse anti-ES (PoAb ES) and anti-somatic extract (PoAb Som), monoclonals to 90 kD somatic antigen (MoAb 90 kD Som: 12,18,20,21 and 29) and MoAb to 16 kD somatic antigen (MoAb 16 kD Som:3,5,54,60 and 77)were compared by ELISA against these two antigens.
to the previously identified 0. uiverrini specific 89-90 kD exoantigen recoverable from worm culture supernatants, where it comprises the single most abundant polypeptide. Reactivities of the antisomatic 90 kD MoAb were compared against immunoassay plates coated with either total somatic antigen or with the worm culture supernatant. An antiserum raised by immunization of a BALB/c mouse with the ES fraction (kindly provided by S.Amompunt, Mahidol University) showed higher ELISA reaction with the homologous ES antigen than with total somatic antigen (Figure 4). Similarly, sera from BALB/c mice immunized with total somatic antigen reacted more strongly with the complex homologous somatic extract than with the ES products. It was expected that the MoAb against 90 kD somatic antigen would behave somewhat similarly to the anti-ES with a stronger reaction against the more refined and concentrated ES antigen. However, all of our monoclonals reactive against the muscle-associated 90 kD polypeptide exhibited no or only trace reaction against the ES fraction. Thus, the 89 kD exoantigen is totally distinct from the muscle-associated 90 kD somatic antigen reactive with our MoAb.
Discussion Exactly how and where cells of the immune system come in contact with 0. viverrini antigens, and exactly which antigens are stimulatory and in what proportions remain
P .B.Billings, N . Utsakhit & SSirisinha
matters of some speculation. That infected humans and experimental animals normally mount an intense humoral response to various antigenic components present in egg, juvenile and mature adult stages (Flavell 1981, Wongratanacheewin et al. 1988a, b) indicates that the integrity of the biliary mucosal barrier has been interrupted and that localized epithelial lesions have allowed direct access or diffusion of soluble antigens to underlying infiltrating inflammatory cells. Two antigens of the adult worm figure prominently in this humoral response, and from previous studies based upon polyclonal data, appear species-specific and of potential diagnostic value (Wongratanacheewin & Sirisinha 1987, Wongratanacheewin et al. 1988a). Thus, in the present attempt to produce MoAb for the purpose of extending these earlier studies by analysing individual specific antigenic components of 0. oiverrini, we hoped to ensure recovery of at least some hybridomas of these specificities. One of these, a metabolic exoantigen with M, 89 kD, is of foremost importance and great interest because it is recognized by essentially all opisthorchiasis sera; conversely, it does not react with sera from individuals or animals with other parasitic infections (Wongratanacheewin et al. 1988a). This exceptional immunogenicity is no doubt due in large part to the fact that it is released or secreted in soluble form during the course of normal metabolic activity of living worms, and this secreted product is likely to be the form encountered by the immune system in natural infection. In fact, this polypeptide represents the single most abundant protein in the operationally-defined excretory/ secretory fraction recovered from parasite culture supernatants. As might be expected, the same sera reactive with this exoantigen also immunoprecipitated a similarly-sized labelled antigen from adult worm somatic extracts (Wongratanacheewin & Sirisinha 1987, Wongratanacheewin e t a ) . 1988a). Although previous data were consistent with the antigenic identity or precursor-product relationship between the 89 k D polypeptides in these two fractions, this could not be definitively established using polyclonal sera. Addressing this question was one of our goals in producing monospecific reagents in the present study. Insofar as adult somatic extracts are more readily obtained and in greater quantity, we elected to approach the problem from this side, using this source as immunogen for the production of hybridomas secreting MoAb reactive against this exoantigen. The 5 MoAb which reacted by radioimmunoprecipitation with a somatic antigen of M, 90 kD were produced from fusions involving immunization with total somatic antigen. Another clone which immunoprecipitated a radiolabelled somatic antigen at this position was obtained from a mouse immunized with the high MW region of a Western blot of somatic antigen from an SDS gel. Only one of these monoclonals crossreacted by ELISA with several distantly related flukes, but all 6 clones reacted to some extent with antigen from the very close relative C. sinensis. The previous data based upon the immunofluorescence pattern of experimental polyclonal anti-ES sera, which could be shown by immunoprecipitation to be mainly against the 89 kD exoantigen, suggested this polypeptide to be localized predominantly within the female reproductive tract where it was associated with eggs, the secretions around eggs and the lining of the uterus (Wongratanacheewin & Sirisinha 1987. Wongratanacheewin et al. 1988a). In light of this, it was surprising therefore to discover that by indirect immunofluorescence localization, this entire panel of MoAb reacting with the 90 kD somatic polypeptide, identified an antigen associated with all of the recognizable muscles of the parasite, and not with the reproductive system nor any other
Monoclonal antibodies against Opisthorchis
organ more readily reconciled with a secretory function. This then prompted a more careful reinvestigation of the nature of the muscle-associated 90 kD somatic antigen and its exact relationship to the 89 kD exoantigen. Results of this evaluation allow us to conclude that the 2 antigens are quite distinct. The muscle-associated antigen is not present to any appreciable extent in the ES fraction, while as would be expected, the 89 kD exoantigen is present in both fractions, although comprising a correspondingly higher proportion of the metabolic product fraction. The muscle-associated 90 kD antigen reported in this study resembles the paramyosin described for S.mansoni (Pearce er al. 1986, Matsumoto el al. 1988) with regard to their nonsurface localization. However, the distribution of these two antigens, as judged from the immunofluorescent patterns, is not similar to one another. The staining pattern of the S. mansoni paramyosin appears to more closely resemble that obtained when the 0. uicerrini was stained with polyclonal rabbit antibody to the 89 k D exoantigen (unpublished observations). We can only speculate at this point as to why the two somewhat similar immunization schemes using nitrocellulose-adsorbed antigen focused the humoral response predominantly toward one of two co-migrating antigens present in the same somatic extract, resulting in the exclusive recovery of monoclonals of this single specificity. The most likely explanation, supported by the ELISA reactivity against the two antigen fractions (Figure 4), is simply that in the somatic extract the muscle-associated polypeptide is far more abundant than the pre-secreted form of the exoantigen. The response to nitrocelluloseadsorbed immunogen should reflect the proportional abundance and the inherent relative immunogenicities. On the other hand, antibodies stimulated in natural infection, just as in experimental immunization with the ES fraction, are limited to the soluble, secreted components which alone have access to the immune system. In support of this, in an indirect competition ELISA, we were unable to block binding of these monoclonals to plates coated with somatic antigen, using human opisthorchiasis sera (unpublished observations). The epitopes borne by this muscle-associated antigen and probably the entire antigen itself are poorly immunogenic in natural infection. A second antigen thought to have some diagnostic value is the lower MW component of a closely-spaced stoichiornetric doublet of tegumental proteins migrating at 16-1 7 kD position. However, the 16 kD antigen is not as universally recognized by opisthorchiasis sera, as only about 75% of all infected humans have antibodies reactive by immunofluorescence against surface antigen (Boonpucknavig, Kurathong & Thamavit 1986). However, this protein itself, and not the antibodies against it, may prove to be of important diagnostic value because of this unusually high abundance. This polypeptide together with the 17 kD molecule comprise almost half the saline-soluble component of adult worm extracts (Ruppel, Boonpucknavig & Hempelmann 1985, Wongratanacheewin et al. 1988a), and hence both tegumental proteins command further careful study. One suggestion based upon the unusual abundance, the surface location and the low molecular weight is that these proteins, and particularly the more species-restricted 16 kD component, might be exploited in an antigen-capture diagnostic assay. However, it is not known to what extent this protein is released from living worms as part of normal tegumental turnover or shedding. It is detectable but not highly abundant in the ES fraction recovered from cultured worms, but in the absence of specific antibodies. this identification has been based upon size of the intact protein. The proteolytic stability of the released polypeptide in culture medium from which other ES antigens have been recovered is unknown, perhaps resulting in a tremendous underestimate of its true
P.B.Billings, N . Utsakhit & SSirisinha
presence. If this antigen, or even only epitopes from it. can be identified in s e r u m - o r better, in urine, by transglomerular passage facilitated by the low MW-this would provide a significant advance toward a diagnostic test. Clearly, a search should be made to determine whether a suitable and practical source of this antigen can be identified in clinical specimens. Six monoclonals reactive with the 16 kD tegumental protein were obtained from a single fusion. By immunofluorescence, all showed the expected localization to the surface membrane of the tegument, consistent with earlier reports based upon Triton and deoxycholate surface extraction together with immunoprecipitation and immunofluorescenct using polyclonal antisera raised against surface extracts (Wongratanacheewin & Sirisinha 1987, Wongratanacheewin et al. 1988a). In addition, this antigen was largely stripped from the surface by the simple freeze-thaw technique and the protein released from denuded worms gave a strong immunoblot reaction with these 6 monoclonals. While most showed moderate crossreaction with other flukes, 2 showed a very high degree of species-specificity,exhibiting no detectable ELISA reaction even against the Clonorchis somatic extract. Used in an antigen-capture ELISA, these 2 monoclonals could confer the required species-specificity to an assay where the antigen shares a number of common epitopes. The apparent relatively low immunogenicity of the 16 kD polypeptide would be of little concern, and might in fact be advantageous, since the presence of immune complexes could be detrimental in a capture assay.
This investigation received financial support from the U.S.Agency for International Development (Grant No. 936-5542-G-00-6027-00) and National Centre for Genetic Engineering and Biotechnology, Thailand. The authors are grateful to Professor H.J.Rim (Korea University, Seoul, Korea) who kindly made Clonorchis sinensis extract available for this investigation.
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Monoclonal antibodies against Opisthorchis
LAEMMLI U.K.(1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680 MATSUMOTO Y . ,PERRYG., LEVINER.J.C., BLANTONR., MAHMOLPA.A.F. & AIKAWAM. (1988) Paramyosin and actin in schistosomal teguments. Nature 333, 76 PEARCE E.J., JAMES S.L., DALTON J., BARRALLA., RAMOSC., STRANDM. & SHERA. (1986) Immunochemical characterization and purification of Sm-97, a Schisrosoma mansoni antigen monospecifically recognized by antibodies from mice protectively immunized with a nonliving vaccine. Journal of Immunology 137, 3593 READ S.M. & NORTHCOTE D.H. (1981) Minimization of variation in the response to different proteins of the Coomassie blue G dye-binding assay for protein. Analyrical Biochemistry 116, 53 ROBERTS S.M., MACGREGOR A.N., VOJVODIC M., WELLSE., CRABTREE J.E. & WILSONR . A . (1983) Tegument surface membranes of adult Schisrosoma mansoni: development of a method for their isolation. Molecular and Biochemical Parasitology 9, 105 RUPPELA., BOONPUCKNAVIG S. & HEMPELMANN E. (1985) Unusual protein pattern of Opisthorchis viverrini. Journal of Helmin rhology 59, 349 WONGRATANACHEEWIN S. & SIRISINHA S. (1 987) Analysis of Opisthorchis viverrini antigens: physicochemical characterization and antigen localization. Sourheasr Asian Journal of Tropical Medicine and Public Health 18, 5 1 1 WONGRATANACHEEWIN S., CHAWENGKIRTTIKUL R., BUNNAGD. & SIRISINHA S. (1988a) Analysis of Opisthorchis viuerrini antigens by imrnunoprecipitation and polyacrylamide gel electrophoresis. Parasirology 96, 119 WONGRATANACHEEWIN S., BUNNAGD., VAEUSORN N. & SIRISINHA S. (1988b) Characterization of humoral immune response in the serum and bile of patients with opisthorchiasis and its application in immunodiagnosis. American Journal of Tropical Medicine and Hygiene 38,356