Diagnosis of Opisthorchiasis by Enzyme Linked Immunosorbent Assay Using Partially Purified Antigens.

Transcription

1 Asan Pacfc Journal of Allergy and mmunology (199) 8 : Dagnoss of Opshorchass by Enzyme Lnked mmunosorben Assay Usng Parally Purfed Angens. Nlaya Poopyruchpong 1, Vlhoon Vlyananl, E.S. Upaham 1 and Pecharln Srlvaanakul 2 Opshorchass s one of he dseases of publc healh and economc mporance n Thaland. The prevalence of Opshorchs vverrn nfecon among he populaon n he norheasern par of he counry has rsen from 3.5 mllon cases n 1965 o 5.4 mllon cases n Recenly, has been esmaed ha a leas 7 mllon people are nfeced wh hs parase. 2 Curren mehods for he dagnoss of O. vverrn nfecon are based.:.. he demonsraon of Opshorchs eggs n eher sool, duodenal flud or ble. However, he echnques are edous and unrelable n some cases, especally n cases of lgh nfecon and n cases of ble duc obsrucon. Several aemps have been made o develop mmunologcal mehods for he dagnoss of he lver fluke nfecon. 3-7 The resuls are sll no sasfacory due o he low sensvy and cross-reacvy of he echnques. has been knowp. ha a more refned angen would provde a more specfc serologcal es for he <;\agnoss of parasc nfecon, ncludng he caused by lver flukes. Therefore, he followng sudy s an aemp o solae several purfed angenc maerals from O. vverrn SUMMARY Opshorchs vverrn angens were parally purfed from adul worms colleced from lver and exrahepac blary sysem of nfeced hamsers. Tegumen fracon was obaned by chemcal exracon, whereas oher fracons were purfed by Sephadex gel flraon chromaography. Fve fracons of O. vverrn angens were obaned, namely egumen exrac, somac exrac, fracon 1 (P)' fracon 2 (P2) and fracon 3 (P3>, respecvely. The enzym.llnked mmunosorben assay echnque was used o compare he reacvy of he fve parally purfed angens. The sensvy and specfcy of all fve angens were compared by esng agans he sera of 78 O. vverrlnl n'eced ndvduals from o. vverrn endemc areas and 7 ndvduals from non endemlc areas nfeced wh hookworm, Trchurs and Ascars ncludng 49 ndvduals wh negave sool examl naon. The assays performed wh egumen exrac, somac exrac and P 1 frac lon were found o have 1% sensvy, whereas he sensves of hose wh P2 and P 3 were 98.1% and 83.3%, respecvely. The egumen exrac had he hghes specfcy as demonsraed by he lowes croaa.reaclvly wh oher para ales. Our resula ndcaed ha surface egumen la he mos aulable angen for use n mmunologcal dlagnoala of opshorchass. and o assess he mmunodagnosc one hour. Afer surface egumen poenal of hese parally purfed exracon, all adul worms were angens. ground and homogenzed n a glass ssue grnder and soncaed fve MATERALS AND METHODS mes for 3 seconds each a 6 was. f Opshorchs vverrn angens Adul worms were colleced from he lver of nfeced hamsers afer four weeks of nfecon. The surface egumen of adul worms was prepared by exracon wh non onc deergen (Tron X-OO soluon conanng 5 mm Trs HC ph 7.4, 15 mm NaC, 1 mm EDTApH 7.) a 5 C wh connuous shakng for From he 1Cener for Appled Malacology and Enomology, Deparmen of Bology, Faculy of Scence, Mahdol Unversy, Rama 6 Road. Bangkok 14, Thaland and 2Research Dvson, Naonal Cancer nsue, Mnsry of Publc Healh. Bangkok 14, Thaland. Correspondence: Dr. Vhoon Vyanan f f,f f.

2 28 POOPYRUCHPONG. ET AL. The homogenae was cenrfuged a 5, rpm for 3 mn a 4" C. An alquo of he supernaan desgnaed as somac O. vverrn angens was furher purfed by Sephadex G- gel flraon chromaography, usng phosphae buffered salne soluon (PBS,pH 7.2,.15 M) as eluen. The fracons belongng o he same peaks were pooled, lyophlzed, reconsued wh dslled waer and sored a -7" C unl use. The proen conens of each fracon of O. vverrn angens were deermned by Foln Cocaleau mehod. 8 Human sera Serum samples were obaned from hree groups of nhabans. Group 1 consss of he sera from 78 ndvduals from he endemc area and has been found posve for O. vverrn nfecon by sool examnaon. Group 2 consss of he sera from 7 ndvduals resde n Pang-Nga Provnce whch s a non-endemc area for O. vverrn. Sool exam naon for hs group of people revealed nfecon wh oher parases, such as hookworm, Trchurs rchura or Ascars lumbrcodes. Group 3 consss of he sera from 49 ndvduals resde n boh non-endemc and endemc areas of O. vverrn whch has been found negave for parasc nfecon by sool examnaon. Sool examnaons were performed by he mehod of Formaln-eher concenraon echnque 9 and Soll's egg counng echnque. 1 Enzyme-Lnked Assay (ELSA) mmunosorben The ndrec echnque of enzyme-lnked mmunosorben assay for anbody o O. vverrn followed he mehod of Srvaanakul e al. 5 All fracons of he angen were esed agans he sera of groups 1, 2 and 3 ndvduals. A reference of he negave conrol was obaned by usng a pool of sera from a group of ndvduals who resde n non-endemc areas, who never consumed raw fsh and were found negave for O. vverrn; nfecon by repeaed sool examnaon. Pool sera from he groups of ndvduals wh proved posve for O. vverrn nfecon by sool examnaon were used as he posve conrol. RESULTS The somac angen of O. vverrn was separaed by gel fraon chromaography no hree fracons, namely P, P2 and P3, respecvely (Fg. ). Therefore, O. vverrn angens used n hs sudy were composed of egumen exrac, somac exrac, and P, P2 and P3 fracons of he somac exrac. The relaonshp beween he egg coun and he anbody er o each of fve angens of O. vverrn n 197 ndvduals wh and whou O. vverrn eggs n faeces are shown n Fgs. 2 o 6. When he cu off for ELSA readng was made a he serum er of 1:4, P, somac exrac ',.. " - O.lS '" z Q ;;J. U " -Q,. Fg FRACTON and he egumen fracons gave 1; posve for all of he ndvduals wh O. vverrn nfecon regardless of he number of parase eggs n he faeces (Fg. 2,5,6). A raher poor sensvy was obaned wh P2 and P3 fracons (Fgs. 3,4). When he relaonshp beween he number of parase eggs per gram of faeces and he recprocal of he anbody ers were compared, he bes correlaon was observed wh he egumen fracon (Fg. 6). Table 1 shows he resuls of ELSA assay among he group of pe ple wh confrmed O. vverrnnfecon, a group of people wh oher nesnal parasc nfecons (hookworm, Trchurs and Ascars) and a group of people wh negave sool examnaon. Alhough P fracon was found o have 1% sensvy, also reaced wh 52.8% of he sera from paens wh oher nesnal parases and 28.5% wh sera from NUMBER Gel flraon chromaography of O. vverrn somac angens on Sephadex G- column (5.3x75. em). Fracons of 3. ml wh a connuous flow rae of 21 ml/h were colleced. Fracon 1 (P1) fracon number Fracon 2 (P2) fracon number 64-1 Fracon 3 (P3) fracon number

3 ELSA FOR DAGNOSS OF OPSTHORCHASS < w v w 1. 'f <:) 5, : C w w'" a) 1. z 5 r : Vl. 1. u 5. C g. a:. 1, a) z: 5 J 29,, 1. J f 1 r r =.329 Negave 4 BO 16, BO L L -' Negave ,28 1 RECPROCAl OF THE ANTBODY TTER RECPROCAL OF THE ANTBODY TTER Fg. 2 o Pl fracon of O. vverrn angens and he number of egg/g of faeces. Fg. 3 o P2 fracon of O. vverrn angens and he number of eggs/g of faeces. Table 1. Resuls of ELSA assay among he group of ndvduals wh confrmed opshorchass, he group wh oher parasc nfecons and he group wh negave sool exam. Group of ndvdual Toal number No. Pos. ELSA wh varous esed angens esed (%) Pl P2 P3 Somac Tegumen Confrmed opshorchass (1) (96.1 ) (83.3) (1) (1) Oher parasc nfecons (52.8) (3.) (48.5) (24.2) (2.8) Negave sool exam (28.5) (14.2) (22.4) (4.) (8.1 )

4 3 POOPYRUCHPONG, ET AL. 1,, lo,ooo 5. 5, 1 V, 1, ụ, 5. loa. <:> (J) m loa. <:> co:: z: 1. 5.,. '" u '".. 1, r1: 5,. co 1, '" Ị. 5 1 r o r =.89 Nega 1 ve Fg. 5., eg.he ,24 RECPROCAl OF THE ANTlBOOY TTER of somac exrac of O. vverrn angens and he number of eggs/g of faeces. Fg. 4 RECPROCAL OF THE ANTBODY TTER o P3 fracon of O. vverrn angens and he number of eggs/g of faeces. 1, 5, negave ndvduals. Somac exrac also exhbed hgh sensvy, bu s specfcy was relavely low snce reaced wh relavely large number of he sera from paens wh oher parases. The negumen fracon was he only fracon found o have he hghes sensvy and specfcy. Among he fve angens shown o have posve correlaon beween he er of anbodes and he nensy of O. vverrn nfecon based on Soll's egg counng echnque, he egumen exrac was found o have he hghes correlaon (correlaon coeffcen, r =.399) (Fgs. 2 o 6). DSCUSSON Anbodes o O. vverrn nfecon have been deeced by varous V 1,. :rl f 5., p 1.. '" 5 z: Fg. 6 1 r :.399 Negave RECPROCAL OF THE ANTBODY TTER o he egumen fracon of O. vverrn angens and he number of eggs/g faeces.

5 ELSA FOR DAGNOSS OF OPSTHORCHASS 31 mmunologcal echnques, such as a very low parase nfecon whch medae hoss, ransmsson o man and mmunoelecrophoress,2,3,7 and canno be deeced by sool examna geographcal dsrbuon n Thaland. ELSA.4-6 Wh regard o ELSA on. Drug Res 1984; 34: Janechawa J, Tharavanj S, Vajrashra assay, Feldhem and Knobloch 4 used a crude exrac of O. vverrn and repored ha hgh sensvy was obaned bu low levels of crossreacvy wh varous speces of remaodes and nemaodes sll occurred. Smlar resuls were also obaned by Srvaanakul e al. 5, hey repored ha ELSA assay deeced of confrmed opshorchass ndvduals bu 46.5% of non-opshorchass ndvduals also were found o gve posve resuls. n our sudy, we showed ha by usng parally purfed angens of O. vverrn, sensvy and specfcy of he ELSA assay can be mproved. Among he fve fracons of parally purfed angens used n hs sudy, he egumen exrac, P angen and somac exrac rendered 1% sensvy. However, when specfcy of he fve angens were esed, he egumen exrac was found o have he hghes specfcy as ndcaed by he lowes crossreacvy wh anbody o oher nesnal parases (Table ). he occurrence of false posve resuls of ELSA assay n he group of ndvduals wh negave sool examnaon was probably due o he fac ha he sera were obaned from heerogeneous groups of people from boh non-endemc and endemc areas. Some of hese ndvduals may have s apparen ha whle he egumen exrac, P fracon and somac exrac have smlar sensvy, he P fracon and somac exrac are less specfc. The low specfcy of P fracon and somac exrac as compared o he egumen exrac s probably due o he presence of non-specfc epopes n he frs wo angens. Snce hookworm, Trchurs and Ascars are helmnh parases ha have been found hroughob Thaland, he es ha shows less cross-reacon wh hese parases would be he mos preferable. Based on our resuls, can be concluded ha he egumen exrac s he mos suable angen o be used for mmunologcal assay for he dagnoss of opshorchass. ACKNOWLEDGEMENT We are graeful o Mrs. Pssanee Ardsungnoen for her echncal asssance n examnng he sool specmens for parasc nfecons. REFERENCES. Preuksaraj S, Jerad C, Sahayaa A, Kjvanee S, Srdonsrasm T. Sudes on prevalence and nensy of nesnal nfecon n rural populaon of Thaland Comm Ds J 1982; 8 : Harnasua, C, Harnasua, T. Opshorchs vverrn: Lfe cycle, ner S, Chacumpa W. The mmunologcal dagnoss of human opshorchass and he humoral mmune response o opshorchass nfecon n he hamser. J Med Assoc Tha 198; 63: Feldhem W, Knobloch J. Serodagnoss of Opshorchs vverrn nfesaon by, an enzyme mmunoassay. Tropenmed Parasol 1982; 33 : Srvaanakul P, Vyanan V, Kurahong S, Twawech D. Enzyme-lnked mmuf [ nosorben assay for deecon of Ops horchs vverrn nfecon. Souheas, Asan J Trop Med Publc Healh 1985;, \ 16: Wongraanacheewn S, Bunnag D, Srsnha S. Enzyme-lnked mmunosorben assay for deecon of anbodes o Opshorchs vverrn. Asan Pac J Allergy lmmunol1985; 3: Vyanan V, Vvaanasesh P,Upaham E.S, Sornman S, Sreramongko1 S,, lmlarp S. Anbodes o opshorchass afer reamen wh prazquanel. J Parasol Trop Med Ass Thaland 1985; 8: Lowry OH, Rosenbrough NJ, Farr SL, Randal RJ. Proen measuremen wh he Foln phenol reagen. J Bo Chern 1951; 193: , 9. Rche, LS. An eher sedmenaon echnque for roune sool examnaon. Bull. Uned Saes Army Med Dep 1948; 8 : Soll NR. An effecve mehod of counng hookworm eggs n faeces. Amer J Hyg 1923; 3 : 59.