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Home , Gnathostoma spinigerum, Opisthorchiasis

[ ASIAN PACIFIC JOURNAL OF ALLERGY AND IMMUNOLOGY (2000) 18: 37-45 j

Trichine//a spira/is-Specific Monoclonal Antibodies and Affinity-Purified Antigen­ Based Diagnosis

Potjanee Srimanote\ Wannaporn Ittiprasert\ Banguorn Sermsart\ Urai Chaisri\ Pakpimol Mahannop2, 3 Yuwaporn Sakolvaree\ Pramuan Tapchaisri\ Wanchai Maleewong , Hisao Kurazono\ Hideo Hayashi4 and Wanpen Chaicumpa1

Both excretory-secretory SUMMARY Hybridomas secreting monoclonal antibodies (MAbs) to Trichi­ (E-S) and crude somatic (CE) anti­ nella spiralis were produced. Myeloma cells were fused with splenocytes gens have been used for the immu­ of a mouse immunized with excretory-secretory (E-S) antigen of infective nodiagnosis of trichinellosis. These larvae. A large percentage of growing hybrids secreted antibodies cross­ reactive to many of 23 heterologous parasites tested. Only 6 monoclones antigens can be obtained from (designated 3F2, 501, 10F6, 11E4, 1306 and 14011) secreted MAbs specific either adult worms or infective lar­ to the E-S antigen and/or a crude extract (CE) of T. spiralis infective larvae. vae of T. spiralis. Larval antigens The 6 monoclones secreted IgM, IgG3, IgM, IgG3, IgG3 and IgG3, respec­ are more often used because large tively. Clone 501 was selected to mass produce MAbs which were then coupled to CNBr-activated Sepharose CL-4B to prepare an affinity-purified numbers of parasites can be recov­ antigen. Dot-blot ELISA with either purified antigen or CE was evaluated. ered from the muscles of There were 17 patients with acute trichinellosis and 76 individuals con­ such as laboratory mice. Adult valescing from T. spiralis infection (group 1). Controls were 170 patients worms, however, must be detached with parasitic infections other than trichinellosis (group 2) and 35 healthy individually from the mucosa of parasite-free controls (group 3). CE-ELISA was positive in all group 1 patients. However, sera from many group 2 patients also were reactive the small intestine. E-S antigen (-44.2%, -44%, -30%, para­ from adult parasites is sometimes gonimiasis-28.6%, -27.3%, -23.1% and poorly immunogenic. l An antibody infections-20%). Affinity-purified antigen was 100% specific, all sera from detection assay using E-S antigens group 2 and group 3 individuals tested negative. Although 74 of 76 patients (97.4%) with convalescing trichinellosis tested positive, sera from only 3 of of infective larvae is not only use­ 17 patients (17.6%) with acute T. spiralis were reactive. Thus, CE antigen is ful for diagnosis but may also serve appropriate when sensitivity is needed, while purified antigen should be as a test of cure.2 used when specificity is required. Dot-blot ELISA is easier to perform, more rapid and less expensive than indirect ELISA. Many samples can be assayed simultaneously, special equipment is not required, and results can E-S antigen is more specific be preserved for retrospective analysis. Dot-blot ELISA is therefore the than CE antigen for the immuno­ method of choice for the rapid diagnosis of trichinellosis, particularly when diagnosis of human and porcine more complex laboratory tests are unavailable. trichinellosis.3-7 CE cross-reacts with antibodies elicited by other no assay like Western blot9 or by 'Department of Microbiology and Immunol­ ogy, Faculty of Tropical Medicine, Mahidol parasites, including using purified antigen. University, Bangkok, 10400 Thailand, 2De­ spp.,8 spinigerum, partment of Parasitology, Faculty of Public viverrini, Capillaria Purified antigen can be Health, Mahidol University, Bangkok, Thai­ land. JDepartment of ParaSitology, Faculty philippinensis, Strongyloides ster­ prepared by passing crude antigen of Medicine. Khon Kaen University, Khon coralis and others.9,lO CE-ELISA through a specific monoclonal anti­ Kaen, Thailand, 'Department of Microbiol­ cross-reactivity can be reduced by body-affinity column and then ogy, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Japan either using a more specific immu- eluting out the bound antigen. In Correspondence: Wan pen Chaicumpa 38 SRIMANOTE, ET AL.

this study, specific monoclonal district, when 117 villagers ate and larvae were collected by the antibodies (MAbs) to T. spira/is under-cooked pork from a wild pig. Baerman's technique I I with slight E-S antigen were produced and then The second outbreak was in 1992 modifications. used to prepare an affinity column at Ban Pasak, Chiang Saen district. for antigen purification. Affinity­ Muscle biopsies taken the same Larvae were then washed purified antigen specific to T. day that convalescent sera were ob­ thoroughly with sterile RPMI-1640 spiralis was then used in a dot-blot tained revealed T. spiralis encysted medium and cultivated in serum­ ELISA for the immunodiagnosis of larvae. The 170 patients in a group free RPMI-1640 medium contain­ human trichinellosis. 2 had a variety of non-trichinella ing 2 mM L-glutamine, 10 mM parasitic infections (Table 1). HEPES, 50 J.lglml gentamycin and MATERIALS AND METHODS Group 3 sera were from 35 healthy protease inhibitors: 0.1 mM phenyl­ individuals with no detectable methyl sulfonyl fluoride (PM SF) Serum samples parasitic infection. and 0.1 mM N-tosyl-L-phenylala­ nine-chloromethyl ketone (TPCK). Sera were obtained from larvae and There were 5,000 larvae/ml of three groups of individuals. Group antigens medium and cultivation was per­ 1 patient sera were obtained on formed for 18 hours at 37°C in a admission from 17 individuals with E-S antigen from a Thai 5% CO2 incubator.2 More than acute trichinellosis. Sera from 76 isolate of Trichinella spiralis was 95% of the larvae remained alive convalescing patiehts were also prepared as previously described.2 after culture, as shown by motility. tested. Group 1 patients were in­ Individual mice were given 350 The culture fluid was collected, fected during 2 outbreaks of trichi­ infective larvae orally and were centrifuged at 200 x g at 4°C for 5 nellosis in Chiang Rai province, sacrificed one month after infec­ minutes and dialyzed thoroughly Northern Thailand. The first oc­ tion. Muscle digestion was per­ against excess phosphate buffered curred in May 1989 in Mae Chan formed using 1% HCI-2% pepsin saline (PBS), pH 7.2 at 4°C. The

Table 1 Diagnoses of patients of group 2 by parasitology and/or serological method

Amount of patients Infection(s)/disease Diagnostic method(s)

16 Schistosomiasis mekongi eggs in stool

9 Schistosomiasis japonicum eggs in stool

20 Gnathostomiasis Western blot analysis with the presence of band at 24 kDal o

7 heterotremus heteratremus eggs in stool andlor sputum and positive bands in Western blot analysis21

43 Opisthorchiasis viverrini Opisthorchis vivemni eggs in stool andlor adult worms in stool after treatment and purgation

26 Strongyloidiasis Stool cultures were positive for filariform larvae of Strongyloides stercoraliSJ2

11 Taeniasis Mature segments of Taenia spp. in stool

10 (s) Hookworm eggs in stool

18 Malaria Plasmodium falciparum in blood smears SERODIAGNOSIS OF TRICHINELLOSIS 39

preparation was concentrated by hybridoma production. Sera of the immune BALB/C mice as feeder Amicon ultrafiltration through a remaining mice were pooled to be cells. Culture supernatants from PM I 0 membrane and protein con­ used as positive control serum (PS). these clones (hybridomas) were re­ tent was determined) 2 using bovine tested against the homologous anti­ serum albumin as standard. E-S Hybridoma and MAb production gen as well as against the panel of antigen was obtained. . the heterologous antigens listed in The donor mouse was relm- Table 2 for cross-reactivity by in­ CE antigens was prepared munized with 50 /J.g of the immu- direct ELISA. MAb antigenic speci­ from larvae as previously des­ nogen m 0.2 ml of NSS mtra- ficities were determined by Western cribed.6 Larvae were homogenized venously 3 days bef~re cell fusion. blotting against SDS-PAGE-sep­ by a glass tissue grinder in PBS On the day of cell fUSIOn, serum was arated homologous antigen and T containing protease inhibitors at collected for use as Immune serum spira/is CE antigen.9 Immunoglobu. pH 7.2. The homogenate was sub­ (IS). The mouse was sacnfice.d, the lin isotyping was performed with jected to a MSE sonicator at 20 spleen was dissected aseptically, Bio-Rad mouse immunoglobulin iso· kHz in an ice bath for 10 minut~s washed several times with serum- typing kits.14 twice and the preparation then cen­ free RPMI-1640 medium and placed trifuged at 10,000 x g at 4°C for 30 on a fine nylon mesh in a small petri Immuno-alkaline phosphatase minutes. The supernatant was col­ dish containing RPMI-1640 medium. staining of T. spiralis tissue sec­ lected and its protein content deter­ The spleen was homogenized with a tions mined. IZ E-S and CE antigens were sterile glass syringe plunger. Single kept in small aliquots at -70°C until spleen cells were collected from the Frozen sections of T use. medium outside the mesh into a spiralis larvae were reacted with sterile plastic centrifuge tube and monoclonal antibodies from hy­ The heterologous antigens washed once with the same medium bridoma clone 5Dl (Fig. 2) and used for cross-reactivity testing of by centrifugation at 200 x g for 10 stained by immuno-alkaline phos­ trichinella MAbs are shown in minutes at room temperature. Via­ phatase (Dakopatt, Denmark) to Table 2. bility of the cells was checked by determine the anatomical localiza­ trypan blue exclusion. Immune tion of worm tissue recognized by splenocytes with more than 98% MAbs. Mouse immunization viability were fused with P3x-63­ Ag8.653 myeloma cells non-immu- Preparation of affinity-purified Five 7 week-old BALB/c noglobulin secreters with more than T. spiralis antigen mice were immunized intra peri­ 98% viability, using polyethylene toneally with 50 /J.g of T spira/is glycol 4,000 as a fusogen at a ratio Hybr i do ma- sec reti ng E-S antigen in 0.1 ml of normal of 10 spleen cells to 1 myeloma cell. MAbs to T spira/is (clone 5Dl) saline solution (NSS) mixed with Fused cells were suspended in hypo­ with IgG3 isotype were grown in equal volume of Freund's complete xanthine-azaserine selective medium large scale for bulk production of adjuvant. The mice were reimmu­ to I x 106 cells/ml and aIiquots of MAb 5D 1. MAbs were attached to nized intraperitoneally twice with 200 /J.I were distributed into 96-well Sepharose CL-4B (Pharmacia, Upp­ 100 /J.g of the same Immunogen tissue culture plates. All plates were sala, Sweden) to prepare an immu­ mixed with Freund's incomplete placed in a humidified 5% COz nosorbent affinity column. Amicon adjuvant. Each mouse was bled incubator at 37°C and media was concentrated MAb was repeatedly from the retro-orbital plexus 14 changed periodically. Supernatants precipitated with saturated am­ days after the third immunization. from wells containing growing hy­ monium sulfate and MAb IgG was Serum antibody titers were deter­ brids were collected and screened for then coupled to CNBr-activated mined by indirect ELISA against antibodies against the homologous Sepharose CL-4B according to the homologous antigen 10 /J.g/ml antigen by indirect ELISA. Cells manufacturer's instructions. T of coating buffer. 13 The mouse from the antibody positive wells spira/is larvae CE was applied to with the highest ELISA titer was were cloned by limiting dilution the affinity column and adsorbed selected as a splenocyte donor for usmg spleen cells of normal non- antigen was eluted out. 15 Affinity­ 40 SRIMANOTE, ET Al.

Table 2 Parasites used for preparing heterologous antigens

Name Developmental stage and source

Angiostrongylus cantonensis Male and female adult worms from lungs of infected rats

Ascaris lumbricoides Adult worms from stool of a patient after purgation

Ascaris suum Kind gift from Dr. P. Setasuban, Department of Helminthology, Faculty of Tropical Cysticercus cellulosae Medicine, Mahidol University

Dirofilaria immitis Adult worms collected from hearts of infected dogs

Echinococcus granulosus Hydatid fluid

Echinostoma ilocanum Echinostoma malayanum Echinostoma revolutum Adult worms from stools of infected individuals after Praziquantel treatment and Haplorchis taichui purgation 17 Opisthorchis viverrini

Opisthorchis felineus Adult worm extract, a gift from Dr. Grenady, Martsinovski Institute, Moscow, Russia

Gnathostoma spinigerum Infective larvae from livers of naturally infected eels22

Entamoeba histolytica Trophozoites from axenic culture

Paragonimus heterotremus Adult worms collected from cats infected two months previously with meta cercariae collected from crabs21

Paragonimus westermani A gift from Department of Social Medicine and Environment, Faculty of Tropical Medicine, Mahidol University

Plasmodium falciparum Blood stages from in vitro culture

Schistosoma mansoni Male and female adult worms were kind gift from Dr. H. Sugiyama, National Institute of Infectious Diseases, Tokyo, Japan

Schistosoma japonicum Schistosoma mekongi Adult worms from mice infected with the respective cercariae

Strongyloides stercoralis Filariform larvae2l

Toxoplasma gondii Tachyzoites; a kind gift from Dr. Vanna Mahakittikun, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok SERODIAGNOSIS OF TRICHINELLOSIS 41

purified antigen was used in the dot-blot ELISA to detect specific kOa antibodies in serum samples from the 3 groups of individuals. 107 Dot-blot ELISA 76 Fifty microliter aliquots of either CE or affinity-purified anti­ 52 gen at a concentration of 1 )lg/ml in PBS, pH 7.4 were dotted onto a nitrocellulose membrane (NCM) using a 96-well slot-blot device (Bio-Rad, USA) and air dried. The 36 NCM was submerged in 3% bovine serum albumin (BSA) for I hour to block unoccupied sites. The NCM was cut into individual squares; each square accommodated one spot of the dotted antigen; all squares were then labeled and placed into ELISA wells con­ 19 taining individually diluted serum samples (I :200). The antigen-anti­ body reaction was allowed to take ABC 0 E F place for 30 minutes; the NCM pieces were washed individually Fig. 1. Western blot patterns of MAbs from the six specific mono­ clones against SDS-PAGE-separated crude extract (CE) of with PBS at pH 7.4 and then placed Trichinella spira/is: Lanes A through F, Western blot pat­ in a container of goat anti-human terns of immune mouse serum and MAbs from clones 3F2, IgG-horseradish peroxidase conju­ 501, 10F6, 11 E4, 1006 and 1401, respectively. Numbers gate (Dakopatt, Denmark; diluted at left are relative molecular masses x 10-3. 1: 1 ,000 in substrate buffer) fol-

Fig. 2. Specificity of monoclonal antibodies from clone 501 to tissue of infective larvae of Trichinella spira/is as revealed by indirect immuno-alkaline phosphatase staining : A =negative control; B = MAbs reacted to the intestinal contents of the . 42 SRIMANOTE, ET AL.

lowed by substrate, respectively. (19.4%). Supernatants from 50 of affinity-puri fied antigen. Tissue Positive and negative controls (a 513 wells (1.9%) contained T. specificity of clone 5DI MAb as pool of five sera from patients with spiralis E-S antigen by indirect determined by immuno-alkaline convalescing trichinellosis [diluted ELISA. Antibodies from these 50 phosphatase staining is shown in I :200] and PBS, respectively) were wells were tested against crude Fig. 2. The MAb reacted to in­ included in the test. A positive extract of the infective stage of testinal content of the parasite. dot-blot ELISA was seen as a obtained Fig. 3 illustrates SDS-PAGE-sep­ brownish-red spot on the NCM from the livers of naturally infected arated patterns of purified antigen square, where the antigen had been eels.17 Culture supernatants of 28 stained by silver and Coomassie dotted and was clearly distinguish­ wells (56%) were positive by in­ brilliant blue dye. No sera from able from the negative control direct ELISA, and only the cells group 2 or 3 had positive dot-blot (NCM square to which PBS but no from the 22 wells which did not ELISA results with purified anti­ antibody had been added). Sen­ cross react with Gnathostoma gen. Three of 17 acute group I sera sitivity, specificity and accuracy of spinigerum antigen were cloned. (17.6%) and 74 (including the 14 the dot-blot ELISA for trichinel­ samples which were negative at the losis diagnosis were calculated Six hybridomas whose cul­ acute phase of illness) of 76 con­ according to Galen's method. 16 ture supernatant MAbs did not valescent samples (97.4%) were react to any of the heterologous positive using purified antigen. RESULTS antigens listed in Table 2 were The sensitivity, specificity and ac­ finally obtained. These monoclones curacy of diagnostic assays using Indirect ELISA antibody were designated 3F2, 5DI, IOF6, purified antigen were 17.6%, 100% titers against homologous antigen IIE4, 13D6 and 14DII. All 6 and 92.1 %, respectively, in acute were measured 14 days after mice clones secreted kappa light chains. trichinellosis and 97.4%, 100% and received the last intraperitoneal Clones 3F2 and IOF6 produced 99.2%, respectively, in convales­ injection. Mice I, 2 and 3 had titers IgM heavy chains. 5D 1, II E4, cent trichinellosis. CE antigen­ of I: 102,400. The fourth mouse 13D6 and 14DI produced IgG3 based dot-blot ELISA was positive had a titer of 1:51,200 and the fifth (Table 3). Supernatant reciprocal in 100% of group I sera but was mouse had a titer of 1 :25,600. indirect ELISA titers ranged from also positive in sera from as many Pooled sera from mice 2-5 were PS 256 to 1,024 (Table 3). Fig. 1 as 44% of patients infected by and mouse I was chosen as the illustrates the antigenic specifici­ parasites other than T. spira/is splenocyte donor. Five hundred ties of MAbs to SDS-PAGE-sep­ (Table 4). and thirteen of 2,640 wells arated T. spiralis CEo Clone 5Dl contained growing hybrid cells IgG3 MAbs were used to prepare

Table 3 Specific hybridomas, their secreted immunoglobulins and the reciprocal indirect ELISA titers of their culture supernatants at stationary phase of growth

Ig isotype Hybridoma Reciprocal Indirect H chain L chain ELISA titer

3F2 11 K 512 501' YJ K 1,024 10F6 11 K 512 11E4 YJ K 256 1306 YJ K 512 14011 YJ K 1,024 "The hybridoma the culture supematant of which was used in the affinity chromatography for purification of T. spira/is specific antigen

------.. SERODIAGNOSIS OF TRICHINELLOSIS 43

Table 4 Results of dot-blot ELISA using affinity-purified antigen and crude somatic extract (CE) of T. spira/is infective larvae to detect antibodies in serum samples of all groups of individuals

No. positive cases/total (%) when the antigen used Group infection/disease in the dot-blot ELISA was

Purified antigen CE

1. Acute trichinellosis 3/17 (17.6) 17/17 (100) Convalescing trichinellosis 74/76 (97.4) 76/76 (100) 2 Opisthorchiasis viverrini 0/43 (0) 19/43 (44.2) Schistosomiasis 0/25 (0) 11/25(44) Schistosomiasis japonicum 0/9 (0) 7/9 (77.8) Schistosomiasis mekongi 0/16 (0) 4/16 (25) Gnathostomiasis 0/20 (0) 6/20 (30) Paragonimiasis heterotremus 0/7 (0) 2/7 (28.6) Taeniasis 0/11 (0) 3/11 (273) Strongyloidiasis 0/26 (0) 6/26 (231) Hookworm infections 0/20 (0) 2/10(20) Malaria 0/18 (0) 0/18 (0) 3. Normal controls 0/35 (0) 0/35 (0)

DISCUSSION kDa kDa Potential applications of 210 210 hybridoma technology to parasitic 127 127 disease were outlined by Nabholz 84 84 and Lambert in 1979.8 MAbs of the desired immunoglobulin isotype 49.5 49.5 directed against a specific epitope can be used to detect specific para­ site antigen in clinical specimens. 35.3 35.3 IgG isotype MAbs are appropriate for high antigen binding affinity 28 .1 while IgM isotypes provide strong 28.1 antigen agglutination. Specific antibodies avoid the problems of 20.5 cross-reactivity between parasite 20.5 antigens and the non-specificity conventional polyclonal antisera. A B C D E F The detection of parasite antigen(s) Fig. 3. SDS-PAGE-separated patterns of CE, E-S and affinity-puri­ in the host can be used to diagnose fied antigen of T. spiralis stained with Coomassie brilliant current infection, to test treatment blue (left; lanes A, Band C, respectively) and Silver stain efficacy, and can provide preva­ (right; lanes D. E and F, respectively). lence data for epidemiological surveys. Monoclonal antibodies 44 SRIMANOTE, ET AL.

are ideal reagents for diagnosis, for components shared by other para­ simpler and allows testing of mul­ strain typing, and for the detection sites. In this study, this suggestion tiple samples at a single time. of parasite variants such as African is confinned. Twenty-eight of 50 Results can be seen without a trypanosomes, Trichinella spp., hybrids produced antibodies cross­ microscope and no special equip­ Gnathostoma spp. and Schistosoma reactive with somatic extract of ment is required. We therefore spp. Established hybridoma clones infective larvae of Gnathostoma recommend the CE-dot-blot ELISA can produce an unlimited supply, spznzgerum. Clones of the re­ during acute trichinellosis and stable, defined monoclonal anti­ maining 22 hybrids still yielded affinity-purified antigen for con­ bodies. MAbs can be used to stand­ MAbs which cross-reacted with finnation during convalescence. ardize parasite antigens and for the parasites such as Angiostrongylus quality control of vaccine prepara­ cantonensis, Ascaris suum. cys­ tions. Monoclonal antibodies are ticercus, Dirofilaria immitis, Echi­ ACKNOWLEDGEMENTS excellent tools for purification of nostoma ilocanum, Echinostoma parasite antigens by affinity chro­ malayanum, Echinostoma revolu­ The work was financially matography. Purified antigens, in tum, , Haplor­ co-supported by the Thailand tum, can be used in immunodiag­ chis taichui, Opisthorchis viverrini, Research Fund (TRF), the Faculty nosis and in characterizing the host Paragonimus heterotremus, Para­ of Tropical Medicine, Mahidol response to infection as antibody or gonimus westermani, Plasmodium University and the National Centre cell-mediated or both. Jalciparum and Schistosoma spp. of Genetic Engineering and Bio­ (data not shown). Only 6 mono­ Trichinellosis has been technology, National Science and clones secreted MAbs which were diagnosed in both humans and pigs Technology Development Agency, specifically directed against T. by the detection of serum anti­ Ministry of Science, Technology spiralis E-S and CE antigens. bodies.5,19 The sensitivity and spec­ and Environment, Thailand. The authors thank Dr. Montip Gettaya­ ificity of these assays depends on Preparing crude somatic camin, Department of Veterinary the nature of the antigen and the parasite extracts is less laborious Medicine, AFRIMS, U.S. Compo­ timing of serum samples.2,9, 10 than preparing E-S antigen and CE Dunng symptomatic, acute illness, yields are higher. Specific T. nent, Bangkok, for the supply of indirect ELISA using T. spiralis spiralis hybridomas allowed their BALB/C mice; Dr. George Watt, larval CE antigen was 100% sen­ secreted MAbs to capture. T. Department of Retrovirology for sitive in biopsy-confinned trichi­ spiralis-specific antigens from CE providing some serum samples of nellosis.6 However, cross-reactivity by affinity chromatography. Eluted trichi-nellosis patients and for was observed with sera from specific antigen can then be used to revlewmg the manuscript; Dr. patients infected with other para­ detect diagnostic antibody. We used Hiromu Sugiyama, Department of sites.6 Dot-blot ELISA using CE as MAbs secreted by clone 50 I for Parasitology, National Institute of the antigen in this study con finned affinity chromatography of CE ex­ Infectious Diseases, Japan; Dr. that CE contains antigenic com­ Wanchai Maleewong, Khon Kaen tract; these MAbs were the IgG3 ponents shared by a broad range of isotype, which has high binding University; Dr. Prasert Setasuban, parasites (see Table 4). E-S antigen affinity. Dot-blot ELISA using this Faculty of Tropical Medicine, prepared from infective larvae, on pure antigen was 100% specific for Mahidol University, Bangkok and the other hand, yielded test results both acute and convalescing trichi­ Dr. Vanna Mahakittik~on, Depart­ which could differentiate patients nellosis. It is sensitive (93.4%) for ment of Parasitology, Faculty of with trichinellosis from those in­ diagnosis of convalescing trichinel­ Medicine Siriraj Hospital, Mahidol fected with other parasites.2 How­ losis but less so (17.7%) during University, Bangkok, for the sup­ ever, indirect ELISA results using acute illness. The dot-blot ELISA ply of some heterologous antigens. E-S antigen require a high cut-off offers several advantages over the optical density to differentiate indirect ELISA and Western blot trichinellosis from other parasitic assays previously described for REFERENCES infections.2 This suggests that E-S serodiagnosis of trichinellosis.2,6,9 I. Ko Re, Yeung, H.F. Specificity of ES antigen preparations contains some The dot-blot ELISA is quicker, antigens in detection of Trichinella SERODIAGNOSIS OF TRICHINELLOSIS 45

spira lis antibodies in Chinese pig. Chaicumpa W. Immunodiagnosis of Tapchaisri P, Setasuban P. Mono­ Trop Biomed 1989; 6: 99-111. trichinellosis: efficacy of somatic anti­ clonal antibody to a diagnostic Mr 2. Mahannop P, Chaicumpa W, Setasuban gen in early detection of human trichi­ 24,000 antigen of Gnathostoma spini­ P, Morakote N, Tapchaisri P. Im­ nellosis. Asian Pac J Allerg Immunol gerum. Int J Parasitol 1991; 2 I: 735­ munodiagnosis of human trichinellosis 1994; 12: 39-42. 8. using excretory-secretory (ES) antigen. 10. Sakolvaree Y, Ybanez L, Chaicumpa 18. Nabholz M, Lambert PH. Introduc­ J Helmithol 1992; 66: 297-304. W. Parasites elicited cross-reacting tion. In: Hybridoma Technology with 3. Zarlenga OS, Gamble SR. Molecular antibodies to Opisthorchis viverrini. Special Reference to Parasitic Dis­ cloning and expression of immuno­ Asian Pac J Allerg Immunol 1997; 15: eases. UNDP/World Bank WHO dominant 53-kDa excretory-secretory 115-22 Special Programme for Research and antigen from Trichinella spiralis mus­ II. Justus DE, Morakote N. Mast cell Training in Tropical Diseases 1997; cle larvae. Mol Biochem Parasitol degranulation associated with seques­ IX-X III. 1990; 42: 165-74. tration and removal of Trichinella 19. Ko RC. A brief update on the diag­ 4. Su X, Prestwood AK. A dot-ELISA spiralis antigen. lnt Arch Appl Immu­ nosis of trichinellosis. Southeast Asian mimicry Western blot test for detec­ no11981; 64: 371-84. J Trop Med Public Health 1997; 28 tion of swine trichinellosis. J Parasitol 12. Lowry OH, Rosebrough NJ, Farr AL, (Suppl.): S91-S98. 1991; 77: 76-82. Randall RJ. Protein measurement with 20. Tapchaisri P, Nopparatana C, Chai­ 5. Ko RC, Yeung MH. Enhanced chem­ the Folin phenol reagent. J Biochem cumpa W, Setasuban P. Specific iluminescent enzyme immunoassay for 1951; 193 265-75. antigen of Gnathostoma spinigerum for the detection of trichinellosis anti­ 13. Chaicumpa W, Thin-inta W, Khusmith imrnunodiagnosis of human gnathos­ bodies in pigs. Vet Parasitol 1992; 42: S, Tapchaisri P; Echeverria P, Kalam­ tomiasis. Int J Parasitol 199 I; 21: 315­ 101-10. baheti T, Chongsa-Nguan M. Detec­ 9. 6. Mahannop P, Setasuban P, Morakote tion of Salmonella typhi antigen 9 in 21. Indrawati I, Chaicumpa W, Setasuban N, Tapchaisri P, Cliaicumpa W. Immu­ clinical specimens with monoclonal P, Ruangkunaporn Y. Studies on im­ nodiagnosis of human trichinellosis and antibody. J Clin Microbiol 1988; 26: munodiagnosis of human paragoni­ identification of specific antigen. Int J 1824-30. miasis and specific antigen of Para­ Parasitol 1995; 25: 87-94 14. Chaicumpa W, Thattiyaphong A, Su­ gonimus heterotremus. Int J Parasitol 7. Gamble HR, Anderson WR, Graham pawat K, Chongsa-nguan M, Kalam­ 1991; 21: 395-401. CW, Murrell KD. Diagnosis of swine baheti T, Eampokalap B, Ruangku­ 22. Nopparatana C, Setasuban P, Chai­ trichinellosis by enzyme-linked immu­ naporn Y. Rapid detection of Vibrio cumpa W, Tapchaisri P. Purification of nosorbent assay (ELISA) using an cholerae 0 I. Serodiagn Immunother Gnathostoma spinigerum specific anti­ excretory-secretory antigen. Vet Para­ Infect Dis 1994; 13: 161-72. gen and immunodiagnosis of human sitol 1993; 13: 349-61. 15. Coico R. Antibody detection and gnathostomiasis. Int J Parasitol 1991; 8 Linder E, Thors C, Lundin L, preparation. In: Current Protocols in 21: 677-81. LJungstrom I, Farah S, Hagi H, Dias F. Immunology 1995; Volume I, New 23. Mangali A, Chaicl!mpa W, Nontasut P, Schistosome antigen gp-50 is respon­ York, John Wiley and Sons, Inc. Chanthzvanich P, Tapchaisri P, De­ sible for serological cross-reactivity 16. Galen RS. Predictive value and ef­ kumyoy P, Viravan C. Immunodiag­ with Trichinella spiralis. J Parasitol ficiency of laboratory testing. Pediat nosis uf human strongyloidiasis and 1992; 8: 999-1005. Clin North Am 1980; 27: 861-9. specific antigen of Strongyloides ster­ 9. Ruangkunaporn Y, Watt G, Harinasuta 17. Chaicumpa W, Ruangkunaporn Y, coralis. Southeast Asian J Trop Med C, Jongsakul K, Chongsa-nguan M, Nopparatana C, Chongsa-nguan M, Public Health 1991; 22: 88-92.