Proc. Natl. Acad. Sci. USAVol. 84, pp. 6914-6918, October 1987Medical Sciences

Monoclonal antibody to a unique surface epitope of the humanfilaria Brugia malayi identifies infective larvae in mosquito vectors

(surface antigen/diagnosis/speciation)

CLOTILDE K. S. CARLOW*t, EILEEN D. FRANKEt, ROBERT C. LOWRIE, JR.§, FELIX PARTONO¶,AND MARIO PHILIPP**Molecular Parasitology Group, New England Biolabs, 32 Tozer Road, Beverly, MA 01915; tDepartment of Parasitology, U.S. Naval Medical Research Unit 2Detachment, c/o American Embassy, JI. Medan Merdeka Salatan 5, Jakarta 10110, Indonesia; §Delta Regional Primate Research Center, Tulane University,Covington, LA 70433; and IDepartment of Parasitology, University of Indonesia, Salemba 6, Jakarta, Indonesia

Communicated by Charles C. Richardson, June 8, 1987

ABSTRACT We describe properties ofan IgM monoclonalantibody (NEB-DEs) raised against the human filarial parasiteBrugia malayi. The antibody reacts with a stage- and species-specific determinant located on the surface of the infective-stage larva, as determined by indirect immunofluorescence. Touse this reagent in epidemiological field studies, we developedan enzyme-linked immunoassay with which B. malayi larvaecan be differentiated from other filarial parasites in mosquitovectors, including the morphologically indistinguishable par-asite of animalsBrugiapahangi. The immunoenzyme assay was91-94% specific and 90-97% sensitive when performed oninfected mosquitoes. In the absence of mosquito tissue, thelevels of specificity and sensitivity increased to 100% and97.5-100%, respectively. Binding of antibody to the surface ofliving larvae was abrogated by treatment of the worms with theenzymes pronase and proteinase K and with the detergentsTriton X-100, ocyl fl-D-glucopyranoside, and 3-[(3-cholami-dopropyl)dimethylammonio]-1-propanesulphonate (CHAPS).In contrast, treatment with trypsin, endoglycosidase-F, 0-Glycanase, N-Glycanase, lipase, various phospholipases, boil-ing, 2-mercaptoethanol at 370C, or periodate did not reduce theantigenicity of the larval surface to antibody NEB-DjE5. Theseresults suggest that the species-specific epitope is a peptidedomain attached to a hydrophobic anchoring residue.

Filarial nematodes have a biphasic life cycle comprising aperiod of larval development in a blood-sucking arthropodvector and one of maturation and sexual reproduction withina vertebrate host. Of the filariae that infect man, the mostprevalent are those that parasitize the lymphatics, and amongthem are Wuchereria bancrofti and Brugia malayi (1). Infec-tion with either species causes both acute and chronic clinicalmanifestations-namely, fevers, lymphadenitis, and adeno-lymphangitis during the acute phase, followed byelephantiasis in later stages of both Brugian and Bancroftianfilariasis, often compounded with hydrocele and chyluria ininfections with W. bancrofti (2). This parasite can be foundthroughout the equatorial belt of the world, whereas B.malayi is confined to regions of South and Southeast Asia (1).A number of mosquito genera transmit lymphatic filariasis,including Mansonia, Culex, Aedes, and Anopheles. Theseare also vectors of animal filariae, which are often sympatricwith the filariae of man (1, 3). It is this fact that makes theentomological assessment of disease control measures sodifficult, for its accuracy depends on the precise evaluationof the larval species that infect the prevailing mosquitoes.Skilled personnel is required to discriminate morphologicallythe various filariae. In some cases, however, such as theinfective larvae of B. malayi and the animal filariae Brugia

pahangi, their morphological distinction is virtually impos-sible (4, 5). Here we describe an IgM monoclonal antibody(NEB-D1E5) with which this distinction can be easilyachieved. It is directed against a surface epitope expressed bythe infective larvae of all geographic isolates and strains ofB.malayi examined. We also report the development ofa highlysensitive diagnostic test utilizing antibody NEB-D1E5. Theprocedure is suitable for stage- and species-specific detectionof infective larvae in mosquitoes under conditions prevailingin field stations of endemic areas.

MATERIALS AND METHODSMonoclonal Antibody. Monoclonal antibody NEB-D1E5

was obtained by a method described elsewhere (6) from miceimmunized by an intrasplenic injection of living B. malayiinfective larvae of the subperiodic strain.

Parasites. Six different geographic isolates of B. malayiwere used. These included subperiodic strains from Malay-sia, Sumatra, and Sulawesi, Indonesia; periodic strains fromKerala, India, and Sumatra; and an aperiodic strain from EastKalimantan, Indonesia. Brugia timori larvae were harvestedfrom Aedes togoi mosquitoes that fed on blood collected froman infected individual from Timor, Indonesia. Aedes aegyptimosquitoes infected with B. malayi (subperiodic strain fromMalaysia) or B. pahangi or infective larvae of Dirofilariaimmitis, Litomosoides carinii, or Acanthocheilonema viteaewere purchased from TRS Laboratories (Athens, GA). In-fective larvae of Brugia patei and Onchocerca lienalis weregifts from Ann Vickery (University of South Florida) andP. J. Ham (London School of Hygiene and Tropical Medi-cine), respectively. Dirofilaria corynodes was obtained as anatural infection in a patas monkey Erythrocebus patas andmaintained in patas monkeys by using Ae. aegypti mosqui-toes. Mansonella ozzardi third-stage larvae were derivedfrom Culicoides furens midges that fed on infected bloodfrom Haitians and subsequently were maintained in patasmonkeys by using Culicoides hollensis and Culicoidesvariipennis midges. Loa loa obtained from Chrysops deerfliesfrom Cameroon were maintained in patas monkeys usinglocally collected Chrysops spp.; W. bancrofti larvae wereisolated from Ae. aegypti mosquitoes after feeding on a patasmonkey infected with a Haitian isolate. Wuchereria kali-mantani was obtained from a naturally infected leaf monkeyPresbytis cristatus using Ae. togoi mosquitoes.

Immunofluorescence. Larvae were incubated for 1 hr at 40Cin ascitic fluid containing the anti-surface-epitope monoclo-nal antibody appropriately diluted in phosphate-bufferedsaline (PBS; GIBCO). Control samples consisted of parasites

Abbreviation: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate.tTo whom reprint requests should be addressed.

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incubated in PBS, in normal mouse serum, or in ascitic fluidcontaining an IgM monoclonal antibody (clone NEB-C6C6)against internal antigens of B. malayi. The samples werewashed in PBS and incubated for a further 45 min in a 1:20dilution of fluorescein-conjugated goat anti-mouse immuno-globulin antibody (Cooper Biomedical, Malvern, PA). Para-sites were washed and examined for bound antibody underultraviolet light by using a Nikon DIAPHOT TMD invertedmicroscope with an epifluorescence attachment "TMD-EF"at x100-200 magnification. All determinations were per-formed in duplicate.Immunoenzyme Assay. Previously harvested larvae or

individual infected mosquitoes with wings and legs removedwere placed in the wells of a 96-well microfiltration plate(Millipore) containing 100 ,1 ofPBS/0.2%Tween 20 (Sigma).Each mosquito was gently crushed with a wooden applicatorto liberate parasites, and the plate was left for 30 min at roomtemperature. Fluid was removed from the wells by filtration,and 100 gI of PBS, normal mouse serum, or ascitic fluidcontaining either monoclonal antibody NEB-D1E5 or mono-clonal antibody NEB-C6C6 (all appropriately diluted inPBS/0.2%Tween 20) was added; the plate was incubated for1 hr. After three 3-min washes in PBS/0.2%Tween 20, 100 1.d

a

of biotinylated anti-mouse antibody (Amersham) diluted 1:50was added to each well, and the plate was incubated foranother hour. The plate was washed as before, and 100 A.l ofa 1:500 dilution of horseradish peroxidase linked tostreptavidin (Amersham) was then added. After 30 min theplate was washed, and 100 pl of enzyme substrate solution[4-chloro-1-naphthol, Bio-Rad; at 3 mg/ml ofmethanol/PBS,1:5 (vol/vol), containing 0.075% hydrogen peroxide] wasplaced in each well. B. malayi parasites were stainedblue/black within 5 min only in the presence of boundantibody. Otherwise, worms remained invisible against thewhite nitrocellulose background.Enzymatic Treatment of Living B. malayi Infective Larvae.

Intact B. malayi infective larvae were exposed to variousenzyme treatments: endoglycosidase F (endo F, New En-gland Nuclear), 2 units (1 unit = 15 ,g of deglycosidation perhour) in 0.1 M sodium phosphate, pH 6.1/0.05 M Na2EDTA/1% bovine serum albumin for 16 hr at 37°C; trypsin(Sigma; T-8642, L-1-tosylamido-2-phenylethyl chloromethylketone-treated) at 25 ,g/ml for 1 hr at 37°C, with soybeantrypsin inhibitor (Sigma; T-9003) subsequently added to aconcentration of 25 ,g/ml; Pronase (Boehringer Mannheim)at 10 ,Ag/ml for 1 hr at 37°C, with phenylmethylsulphonyl

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FIG. 1. Anti-surface-epitope reactivity of monoclonal antibody NEB-DIE5 with infective third-stage larvae of B. malayi as determined byindirect immunofluorescence. B. malayi (a and b), B. pahangi (c and d), or W. bancrofti (e and I) infective larvae were incubated withanti-surface-epitope monoclonal antibody and viewed under visible (a, c, and e), or ultraviolet light (b, d, and f). Larvae were incubated for1 hr at 4°C in ascitic fluid containing the anti-surface-epitope monoclonal antibody appropriately diluted in PBS. The samples were washed inPBS and incubated for a further 45 min in a 1:20 dilution of fluorescein-conjugated goat anti-mouse immunoglobulin antibody. Parasites werewashed and examined for bound antibody as described. (x 150-300.)

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fluoride (Sigma) then added to 2.5 mM; proteinase K(Boehringer Mannheim) at 0.01 jig/ml for 1 hr at 370C;O-Glycanase (endo-a-N-acetylgalactosaminidase from Dip-lococcus pneumoniae; Genzyme, Boston, MA) at 120 milli-units/ml overnight at 370C; N-Glycanase (peptide:N-glyco-sidase F; peptide-W4[N-acetyl-(3-glucosaminyl]asparagineamidase from Flavobacterium meningosepticum; Genzyme)at 90 units per ml overnight at 370C; and lipase (Chromobac-terium viscosum; catalyzes the hydrolysis of triglycerides toglycerol and fatty acids), phospholipase C (Bacillus cereus),phospholipase D (Streptomyces chromofuscus), or phospho-lipase A2 (porcine pancreas phospholipase) at 500 units per mlfor 2 hr at 370C (all from Calbiochem-Behring). All solutionswere prepared in PBS unless otherwise stated. Treatedworms were washed extensively with PBS prior to im-munofluorescence analysis. When fluorescence was abrogat-ed by the treatment, the integrity of the unbound monoclonalantibody used was confirmed by resubmitting the sample toa test with untreated worms.

Antigen Solubilization and Chemical Sensitivity Studies.Epitope loss from the epicuticle was monitored by im-munofluorescence after the following treatments: (i) incuba-tion of intact B. malayi third-stage larvae in 0.1 M sodiummetaperiodate (Sigma)/50 mM sodium acetate, pH 4.5, for 1hr at room temperature in the dark, followed by 1% glycinefor 30 min to block aldehyde groups; (ii) 1% 2-mercaptoeth-anol (Kodak) for 1 hr at 37°C; (iii) 2% 3-[(3-cholamido-propyl)dimethylammonio]-1-propane-sulphonate (CHAPS;Sigma)/2% Triton X-100 (Pierce)/2% octyl f3-D-glucopy-ranoside (Calbiochem) for 15 min at 80°C; or boiling for 10min in PBS.

RESULTS

Surface Reactivity of Monoclonal Antibody NEB-DEs. Thesurface reactivity of monoclonal antibody NEB-D1E5 wasdetermined by using an indirect immunofluorescent antibodytest and living B. malayi infective larvae within or dissectedfrom crushed mosquitoes (Ae. aegypti). An intense uniformstaining ofthe parasites was observed (Fig. 1), while exposedinternal tissues of any damaged larvae did not fluoresce,implying that the epitope is surface-restricted. Parasitesincubated in normal mouse serum, PBS, or ascitic fluidcontaining monoclonal antibody NEB-C6C6 did not displaysurface fluorescence. Freeze-killed or fixed (3% formolsaline) B. malayi infective larvae were still able to bindantibody NEB-DE5, which retained activity after freezing orstorage in a lyophilized state for at least 9 months.

Specificity of Monoclonal Antibody NEB-DEs. To evaluatethe specificity of the monoclonal antibody and its potential as

a diagnostic reagent, a rather extensive range of infectivelarvae of various filarial species was screened by indirectimmunofluorescence (Table 1). Notably included were lym-phatic filariae of genera other than Brugia-namely, W.bancrofti and W. kalimantani; larvae ofthe genus Brugia, butdifferent species such as B. pahangi, B. patei, and B. timori;and also different geographic isolates of B. malayi. Inaddition, infective larvae of two other human filariae, M.ozzardi and L. loa; the monkey parasite D. corynodes; tworodent filariae, L. carinii and A. viteae; and the dog parasiteD. immitis were also analyzed. With the exception of B.timori, the target epitope was species-specific, since nobinding of antibody to other species was observed (Fig. 1 andTable 1). Furthermore, the antibody reacted equally well withisolates of B. malayi from regions throughout South andSoutheast Asia. These included a subperiodic strain fromMalaysia; subperiodic, aperiodic, and periodic strains of thezoophilic type from Indonesia; and a periodic strain fromKerala, India (Table 1).

Table 1. Reactivity of the anti-surface-epitope monoclonalantibody with infective larvae of various filarial species

Species/isolate Vector Fluorescence

B. malayiMalaysiat Mansonia spp.* +IndonesiatO§ Mansonia spp.* +India§ Mansonia spp.* +

B. timori Anopheles barbirostris* +B. pahangi Mansonia spp.*B. patei Ae. aegypti¶W. bancrofti Culex, Aedes, spp.*W. kalimantani Ae. togoilL. loa Chrysops spp.*M. ozzardi Culicoides, Simulium spp.*D. immitis Aedes, Culex, Anopheles spp.* -

D. corynodes Ae. aegyptlr0. lienalis Simulium spp.*L. carinii Ornithonyssus bacoti*A. viteae Ornithodorus tartakovskPyi**Most common genus transmitting infection.tSubperiodic strain.tAperiodic strain.§Periodic strain.lUsed for transmission in the laboratory. Natural vector unknown.

Identification ofB. malayi Larvae in Mosquito Vectors Usingan Enzyme-Linked Assay. Considering the conditions inwhich a specific diagnostic tool of this nature would be used,we applied a more practical enzyme-based detection systemas described. Previously extracted larvae or individuallycrushed mosquitoes were used (Fig. 2). B. malayi infectivelarvae stained blue/black in the presence of bound antibody.No worms were colored in appropriate control wells con-taining B. malayi larvae incubated in normal mouse serum,PBS, or ascitic fluid containing monoclonal antibody NEB-C6C6, or in wells with B. pahangi larvae incubated withantibody NEB-D1E5. The performance of the test in a fieldtrial simulation was also determined. A double-blind trial wascarried out using 96 freeze-killed mosquitoes harboring B.malayi or B. pahangi infective larvae. The test scored 94%specificity and 90%o sensitivity based on the actual number ofinfected mosquitoes determined by dissection. A wormburden of 2.5 larvae per mosquito was observed, whereas afigure of 3.7 was obtained by dissection. In a repeat trial withmosquitoes carrying an average of 2.2 larvae per mosquito (aburden perhaps more akin to that found in natural transmis-sion), the levels of specificity and sensitivity were still 91%and 97%, respectively, and the worm burden detected by theassay was 1.8. Higher levels of sensitivity and specificitywere obtained when larvae cleared of mosquito tissues wereused. On two separate occasions using five worms per wellin a total of 220 wells, sensitivity was 97.5% and 100%o.Specificity was 100% when 110 B. pahangi larvae dissectedfrom mosquitoes were distributed among 20 wells and ana-lyzed.No parasites were detectable when the enzyme test was

performed on 96 mosquitoes harboring B. malayi larvae thathad been allowed to develop for 7 days only and that, at thatpoint, were not infective to man.

Attempts to Determine the Nature of the Antigen(s) Bearingthe Target Epitope. The sensitivity of the antigen(s) bearingthe target epitope to enzymatic hydrolysis and to variouschemical and physical treatments and its solubility in deter-gents were determined. The effect of the various treatmentswas monitored by indirect immunofluorescence. As indicat-ed in Table 2, the epitope was no longer detectable in situafter incubation in pronase or proteinase K or in the deter-gents Triton X-100, CHAPS, or octyl 3-D-glucopyranoside.

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FIG. 2. Identification of B. malayi infection in individual mosquitoes (Ae. aegyph) by an enzyme-based detection assay. B. malayi larvaedissected from mosquitoes (a) or crushed mosquitoes harboring B. malayi infection (b) were treated as described. B. malayi parasites werestained blue/black only in the presence of bound antibody. Otherwise worms remained invisible against the white nitrocellulose background.(x20-90.)

The remaining treatments failed to reduce significantly thefluorescent signal.

DISCUSSIONAdvances in the epidemiology of mosquito-borne filariasishave been greatly hindered by our inability to discern whichinsects harbor human parasites. A number of mosquitogenera transmit filarial parasites of both man and animalorigin, and in some instances the filariae are morphologicallyidentical. As a consequence, it has been impossible to obtainaccurate entomological data on the effect ofmosquito controlor drug therapy. We have produced a monoclonal antibodythat can easily differentiate a major filarial parasite of man,B. malayi, from its morphologically indistinguishable coun-terpart of animals, B. pahangi. This antibody reacts with allgeographical isolates of B. malayi examined.A practical immunoenzyme assay has been developed that

enables the accurate identification of infective larvae inmosquito vectors. The assay is species-specific (91-94%), thefew false positives being attributable to cross-contaminationof the single applicator used to crush mosquito samples; it isalso sensitive (90-97%), even though a crude method was

Table 2. Effect of various treatments on the surface reactivity ofB. malayi infective larvae

Treatment

Untreated controlPronaseProteinase KTrypsinEndoglycosidase FO-GlycanaseN-GlycanaseLipasePhospholipase CPhospholipase DPhospholipase A2CHAPSTriton X-100Octyl 8-E-glucopyranosideBoiling in PBS2-MercaptoethanolPeriodate oxidation

Fluorescence

+

used to liberate worms from the vector, and is simple enoughto be used in field-station laboratories. Other importantpractical advantages are that the antibody can be lyophilizedfor easy transport and storage without any loss of surfacereactivity and that larval antigenicity is preserved by fixationwith 3% formalin. Such features further substantiate thesuitability of the assay for epidemiological surveys. Anadditional quality of the assay described is its unique abilityto discriminate between mosquitoes carrying third-stagelarvae and, therefore, infective to man and those carryingearlier larval stages. Consistently, no fluorescence wasobserved on any of the larval phases ofB. malayi prior to theinfective stage, including sheathed and exsheathed micro-filariae, sausage larvae, and early second-stage larvae, whenthese worms were incubated in antibody NEB-D1E6 (7). Theexception was late second-stage larvae, which demonstrateda dim positive fluorescence signal at their anterior andposterior ends (7). However, only third-stage larvae weredetectable by the immunoenzyme assay. Recently, repeatedDNA sequences of B. malayi (8, 9) and B. pahangi (8) weredescribed that allow the identification of these parasites byDNA hybridization. These probes represent an importantbreakthrough in the quest for species-specific reagents and apromising approach for the diagnosis of infection in man.However, entomological assessment of filariasis controlmeasures requires stage-specific reagents. Indiscriminatedetection of all larval stages in infected mosquitoes may leadto an overestimation of the disease transmission potential,since it is likely that a large number of the filarial-carryinginsect vectors die before the parasites reach the infectivestage (10, 11). Similar problems may arise in areas withinsusceptible mosquito species in which parasite develop-ment does not reach completion. With the level of stage-specificity possessed by antibody NEB-D1E5, errors due toindiscriminate detection should be circumvented.The cross-reactivity found between B. malayi and B. timori

is intriguing. Worms of these two species show differences inthe Giemsa-staining pattern of microfilariae (12) and in adultworm morphology (13) that warrant their separate taxonomicclassification, but, unlike B. pahangi and B. malayi (3), theyhave not yet been subjected to the definitive test of interspe-cies mating and subsequent analysis of "hybrid" fertility.Such experiments would be particularly interesting becauseDNA probes specific for B. malayi also react with B. timoriDNA (9, 14). Fortunately, the precise phylogenetic relation-

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ship between these two species is of minor practical impor-tance, for, unlike B. pahangi, B. timori is not sympatric withB. malayi (15, 16).

Sensitivity to the proteolytic enzymes pronase and pro-teinase K but not to trypsin or to mild treatment with2-mercaptoethanol suggests that the epitope-binding anti-body NEB-D1E5 is itself, or is associated with, a polypeptidewith no available trypsin sites or conformationally importantdisulfide bridges. In agreement with this contention is ourfailure to influence antibody binding to the worms by treat-ment with endoglycosidases or by periodate oxidation, im-plying that the epitope may not be a carbohydrate. Indeedsome sugar moities seem to be scarcely expressed on thesurface of B. malayi infective larvae, as judged by the totalabsence of binding of a panel of seven different lectins (17).Worms subjected to a mild treatment (15 min at 800C) withnonionic or zwitterionic detergents no longer bound themonoclonal antibody. Although this treatment may havedenatured the epitope, we favor the interpretation that thelatter was solubilized because the harsher procedure ofboiling the parasites in PBS had no effect on antibodybinding. Solubilization of the epitope with detergents indi-cates that the antigen is anchored in a hydrophobic environ-ment. In this respect, it may be significant that binding ofmonoclonal antibody to the worms was not affected bytreatment of the larvae with lipase or phospholipases (Table2). Therefore, the interpretation that is most consistent withour results is that the species-specific epitope is part of ahydrophobically anchored peptide domain. However, using avariety of biochemical and immunochemical procedures(C.K.S.C. & M.P., unpublished data), we have been unableto identify this molecule(s) further. Detergent extracts ofworms surface-radioiodinated by the Iodo-Gen procedure(18) did not contain material immunoprecipitable by antibodyNEB-DE5. Similarly, the latter did not bind to larval anti-gens electroblotted onto nitrocellulose after NaDodSO4/polyacrylamide gel electrophoresis, even after attempts to"renature" antigens by incubating the nitrocellulose strips indecreasing concentrations of urea. Material extracted fromintact larvae with Triton X-100 was immunoprecipitable byantibody NEB-D1E5 after iodination with the Bolton-Hunterreagent (19). Unfortunately, this result is not yet reproducibleto our satisfaction. In addition, components of the sameradiolabeled material binding to antibody NEB-D1E5-Seph-arose columns were indistinguishable from those binding toSepharose columns with the control antibody NEB-C6C6.Finally, extraction of iodine-labeled larvae with Triton X-100after antibody binding to the larval surface did not yieldimmunoprecipitable immune complexes containing identifi-able radiolabeled material.The widespread crossreactivity of filarial antigens and that

ofsurface antigens ofBrugia in particular have been analyzedextensively (20-22), but to date no species or stage-specificcomponent(s) has been identified. As pointed out by Maizelset al. (20), however, the biochemical and antigenic similaritybetween Brugia surface molecules does not preclude theexistence of epitopes unique both to stage and species. With

the aid ofmonoclonal antibody NEB-D1E5, we have localizedone such epitope as part of a polypeptide domain. Positiveidentification of the molecule(s) bearing this epitope willdiscern whether these antigens are hitherto unidentifiedsurface components unique to B. malayi infective larvae or,in fact, are shared with other stages and/or species in amodified form not containing the NEB-DjE5 antibody-bind-ing epitope. The molecular basis of antigenic diversity amongfilariae could thus begin to be understood.

We gratefully acknowledge the support and advice of Dr. DonaldG. Comb. We thank Dr. Ann Vickery and Dr. Peter Ham for their giftof frozen specimens of third-stage larvae of B. patei and 0. lienalis,respectively. E.D.F. thanks Soeroto Atmosoedjono and Purnomofor their technical assistance.

1. Sasa, M. (1976) Human Filariasis: A Global Survey of Epi-demiology and Control (University Park Press, Baltimore).

2. Manson-Bahr, P. E. C. & Apted, F. I. C. (1982) Manson'sTropical Diseases (Balliere Tandell, London), 18th Ed., pp.148-180.

3. Denham, D. A. & McGreevy, P. B. (1977) Adv. Parasitol. 15,243-309.

4. Nelson, G. S. (1959) J. Helminthol. 33, 233-256.5. Schacher, J. F. (1962) J. Parasitol. 48, 679-692.6. Carlow, C. K. S., Edwards, M. K., James, E. R. & Philipp,

M. (1987) J. Parasitol., in press.7. Carlow, C. K. S., Perrone, J., Spielman, A. & Philipp, M.

(1987) UCLA Symp. Mol. Cell. Biol. New Ser. 59, in press.8. McReynolds, L. A., DeSimone, S. M. & Williams, S. A.

(1986) Proc. Natl. Acad. Sci. USA 83, 797-801.9. Sim, B. K. L., Mak, J. W., Cheong, W. H., Sutanto, I.,

Kurniawan, L., Marwoto, H. A., Franke, E., Campbell, J. R.,Wirth, D. F. & Piessens, W. F. (1986) Am. J. Trop. Med. Hyg.35, 559-564.

10. Jordan, P. & Goatly, K. D. (1962) Ann. Trop. Med. Parasitol.56, 173-187.

11. Wharton, R. H. (1957) Ann. Trop. Med. Parasitol. 51, 278-296.

12. Purnomo, Dennis, D. T. & Partono, F. (1977) J. Parasitol. 63,1001-1006.

13. Partono, F., Purnomo, Dennis, D. T., Atmosoedjono, S.,Oemijati, S. & Cross, J. H. (1977) J. Parasitol. 63, 540-546.

14. McReynolds, L. A., Piessens, W. F. & Williams, S. A. (1987)Parasitol. Today, in press.

15. David, H. L. & Edeson, J. F. B. (1965) Ann. Trop. Med.Parasitol. 59, 193-204.

16. Dennis, D. T., Partono, F., Purnomo, Atmosoedjono, S. &Saroso, J. L. (1976) Am. J. Trop. Med. Hyg. 25, 797-802.

17. Kaushal, N. A., Simpson, A. J. G., Hussain, R. & Ottesen,E. A. (1984) Exp. Parasitol. 58, 182-187.

18. Fraker, P. J. & Speck, J. C., Jr. (1984) Biochem. Biophys.Res. Commun. 80, 849-857.

19. Bolton, A. E. & Hunter, W. M. (1973) Biochem. J. 133,529-539.

20. Maizels, R. M., Partono, F., Oemijati, S., Denham, D. A. &Ogilvie, B. M. (1983) Parasitology 87, 249-263.

21. Sutanto, I., Maizels, R. M. & Denham, D. A. (1985) Mol.Biochem. Parasitol. 15, 203-214.

22. Philipp, M., Maizels, R. M., McLaren, D. J., Davies, M. W.,Suswillo, R. & Denham, D. A. (1986) Trans. R. Soc. Trop.Med. Hyg. 80, 385-393.

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