[ 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 animals 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 (opisthorchiasis-44.2%, schistosomiasis-44%, gnathostomiasis-30%, para­ from adult parasites is sometimes gonimiasis-28.6%, taeniasis-27.3%, strongyloidiasis-23.1% and hookworm 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 Schistosoma using purified antigen. University, Bangkok, 10400 Thailand, 2De­ spp.,8 Gnathostoma spinigerum, partment of Parasitology, Faculty of Public Opisthorchis 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 Trichinella spiralis 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 Schistosoma mekongi eggs in stool 9 Schistosomiasis japonicum Schistosoma japonicum eggs in stool 20 Gnathostomiasis Western blot analysis with the presence of band at 24 kDal o 7 Paragonimiasis heterotremus Paragonimus heteratremus eggs in stool andlor sputum and positive bands in Western blot analysis21 43 Opisthorchiasis viverrini Opisthorchis vivemni eggs in stool andlor Opisthorchis viverrini adult worms in stool after PraziQuantel 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 Hookworm infection(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.