De, Anindya Kanti

etd@IISc Electronic Theses and Dissertations at Indian Institute of Science

Synopsu of the thew submztted for the PhD degree of the Ind~an Instttute of Sczence, Bangalore, Indra Title of the thesis DEVELOPMENT OF AN IMMUNODIAGNOSTIC KIT FOR SPECIES IDENTIFICATION OF SNAKE BITE AND STUDIES ON THE CROSS-REACTING VENOM ANTIGENS Name of the candidate Anindya Kanti De, M.Sc T h e  problem of snake envenomahon IS of Clin~Cal importance m many parts of the world including the developmg countries of Asla, Afnca and Lahn Amenca Throughout the world, Ind~a tops the list m the inadence of snake bite, followed by Burma, Ceylon, Venezuela and Braid m decreasing order In India too, snake blte IS a major medical and soaeeconomic problem, mostly In rural areas, espeaally among agncultunsts, plantahon workers and snakecatchers Stahshcs, reveal that annually about 200,000 persons are bitten and nearly 30,000 are fatal On an average, roughly 100 deaths occur everyday The four common Indian snakes responsible for the mortality and morbidrty are Bungarus c m l e u s  (halt), Naja naja (cobra), Echs m a t u s  (saw-scaled viper) and Vzpra russellr (Russell's wper) Administrahon of polyvalent anbsnake venom (ASV) along wth the supporhve treatment is the best way of treahng snake envenomahon Though ASV IS an effechve anhdote, ~t has mherent drawbacks Currently, m India, no tests are available to ~denhfy the b~hng spwes of snake Idenhficahon of the offendmg spwes wd1 be helpful not only to prove condusively whether or not true envenomahon has occurred but also, be useful In administering the correspondmg monovalent anhserum for effechve serotherapy Hence, an avrdin-blohn nucrotiter enzyme-hnked rmmunosorbant assay (AB-microELISA) was developed as this detection system is the most suitable, ta ktng into considerahon ~ t s  prachcal utllity and other d~stmct advantages (sensihvity, speafiaty and rap~dity) As a first step, ant~bodles were raised In rabbits aganst the venoms of the sequenhally through the indivrdual venom-speafic mlumns The affinity punfied anhbodres thus obtained were used as lmmunoreagenb in the detechon system The affini ty punfied venom-speafic antibod~es were used to coat the microh ter plates The vanous body fluids like blood, serum, unne or swab from kte area were incubated on these plates Biohnylated affinity punfied ant~body was subsequently used as the rwealing anhbody and avldin-horse radish peroxldase conjugate was finally used to detect the snake venom Venom levels were quanhtated by companng w th known concentrahons of venom The assay can detect venom levels as low as 1 ng/ml Using this assay, samples from 27 human snake bite wchms were screened Out of 27 cases 9 were found to be negahve, yet the patients underwent serotherapy The range of venom levels detected vaned from 0-479 ng/ml Mmmum bites were due to V russellz Quanhtahon of venom d l  indicate the degree of envenomahon and hence suitably mod~fy the treatment Since snake bite is medical emergency, it must be treated immediately Talung Into considerahon the hme factor mvolved and the problems encountered in the rural areas, an ELISA based ~mmunodiagnoshc lut was developed The followng are the sallent features of the lut (a) It can idenhfy the bihng speaes unambiguously wthin 30 min (b) No modem equ~pment is required (c) The test can be carned out at room temperature (d) The color reactton can be seen vrsually The test can detect venom levels upto 10 ng/ml Snake venoms have been desmbed as a "mosa~c of anhgens" and common anhgens are present In members of related and unrelated speaes The presence of common anhgens is not only important from the vlew point of taxonomy, but also forms the bass for the cross-protechon offered by heterologous venoms On the other hand the presence of common anhgens can lead to error or ambigu~ty in speaes speafic diagnosis ELISA was performed to determine the extent of cross- reachvlty among the four venoms, and the cross-reachng venom antgens were idenhfied by ~mmunoblot analysis In the ELISA method, the venoms were probed wth crude as well as affinity punfied anhbodies In general, maxmum cross- reachvity was observed between members of the same family, wz elapids and vlpends The anh-B camZeus venom anhbodles showed more cross-reacttwty wth N nala venom and we-versa (59 1 % and 621 % respechvely) Similarly, anb-E carznatus venom anhbodies showed more cro.ss-reachvlty wth V russellz venom and weversa (71 2% and 68 9%) To idenhfy the cross-reacbng antigens, the venoms of four common Indlan snakes were fiachonated on sodium dodecyl sulphate-poly acrylamide gel eledrophoresls, electroblotted to nitrocellulose membrane, and the separated anhgens were probed wth normal, crude and affinity punfied anhbod~es The affinity punfied anhbodles recognized only 1t.s homologous venom proteins while the crude sera showed varyrng degrees of mss-reachvlty wth other heterologous venoms In order to idenhb further the cross-reachng anhgens, the venoms were probed wth the bound fractions of the respechve venom speafic columns Certan bands that were not observed when probed wth the crude sera were dishnctly v ~ s u h d  when probed wth  these affinrty punfied bound frachons, mdicahng the effiaency of these columns in selectwely absorbing the cross-reachng anhbodies The man drawbacks of the currently avnlable mode of polyvalent ASV, include severe anaphylachc reachons and delayed serum adcness In addihon, since ~t has been rased agnnst four venoms, large volumes are needed for the treatment and the recovery IS slow The allewc reachons both ~mmediate and delayed, decrease with an mcrease rn the punficahon of the anhvenin Thus, administrahon of an affinity punfied monovalent anhvenin wll be the best mode of treatment for a safe and rapid recovery In order to prepare affinity punfied, monospeafic anhvenin, anhbodies against the four venoms were rased in rabbit The crude anhsnake venom anhbodies were subjected to ammonium sulphate kachonahon followed by pepsin digeshon The resulhng frachon was passed through therr respechve venom spwfic column to obtam affinity punfied F(ab), frachon In order to obtain the affinrty punfied horse monovalent ASV, the commeraally avadable polyvalent ASV was passed over the venom-speafic columns The efficacy of vanous laboratory preparahons and commeraal ASV frachons was evaluated by n v z h  neutralrzahon assay where the venom-anhvenin mixture is preincubated for 30 min at 37°C before lnjechng intravenously into mlce In each case, the neutralrzing potency of the affinity punfied frachon was the most effechve A simple, raprd, sensihve and speaes speafic immunodiagnoshc lut has been developed to detect/quanhtate the venoms of the four common Indian snakes The iut wdl be of tremendous use in both the d~agnosis and prognosis of snake bite The immunoblot technrque enables the prease identrficahon of cross-reachng venom proteins There is a future scope for the identificahon of speaes-speafic venom anhgens to prepare monoclonal antibod~es for immunodiagnoshc luts The affinity punfied horse monovalent ASV developed in the present study wtll the best akernahve and most effechve as compared to the currently used polyvalent commeraal ASV The development of affin~ty punfied antwenin m different speaes like sheep wd1 be of great therapeutic value in the case of mdividuals who are all eqp  to horse sera 

Development of an Immunodiagnostic Kit for Species Identification of Snake Bite and Studies on the Cross-Reacting Venom Antigens

http://etd.iisc.ac.in/static/etd/abstracts/3956/G14544-Abs.pdf

Development of an Immunodiagnostic Kit for Species Identification of Snake Bite and Studies on the Cross-Reacting Venom Antigens

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etd@IISc Electronic Theses and Dissertations at Indian Institute of Science