Development of immunodiagnostic tests for snake venoms of Thailand
Issued Date
2023
Copyright Date
1988
Language
eng
File Type
application/pdf
No. of Pages/File Size
xv, 169 leaves : ill.
Access Rights
restricted access
Rights Holder(s)
Mahidol University
Bibliographic Citation
Thesis (Ph.D. (Microbiology))--Mahidol University, 1988
Suggested Citation
Leera Chinonavanig Development of immunodiagnostic tests for snake venoms of Thailand. Thesis (Ph.D. (Microbiology))--Mahidol University, 1988. Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/89699
Title
Development of immunodiagnostic tests for snake venoms of Thailand
Alternative Title(s)
การพัฒนาวิธีทางอิมมิวโนเพื่อใช้วินิจฉัยพิษงูของประเทศไทย
Author(s)
Abstract
The rational treatment of snake venom poisoning involves the administration of specific antivenom. Since horse antivenoms produced in Thailand are monovalent and cross-neutralization among them is minimal or absent, the selection of antivenom for the treatment and thus the correct identification of the culprit snake are of crucial importance. Diagnosis of snakebite based on the clinical manifestations of the victims is difficult because several venoms produce similar signs and symptoms. In order to aid the clinician in making correct diagnosis, simple immunossays for detecting venoms of the six major poisonous snakes of Thailand have been studied. The snakes were NAJA NAJA SIAMENSIS, BUNGARUS FASCIATUS, OPHIOPHAGUS HANNAH, CALLOSELASMA RHODOSTOMA, VIPERA RUSSELLI, and TRIMERESURUS ALBOLABRIS Since assays using crude venoms and their corresponding antibodies may exhibit cross-reactions between venom proteins of different species, characterization of these venoms in homologous and heterologous systems have been made. By using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, information regarding to the molecular weights, abundancies, relative immunogenicities and immunological cross-reactivities of the venom proteins was gathered. SDS-PAGE showed elapid venoms to be rich in proteins of mol. wt. 7-8 KD which are mostly postsynaptic neurotoxins and cardiotoxins. Abundant low mol. wt. proteins were also observed in the viperid venoms but their biological functions are not known. Immunoblots, stained with rabbit homologous antibody, showed high mol. wt. venom proteins were highly immunogenic while postsynaptic neurotoxins of elapids were marginally immunogenic. Two protiens of O. Hannah venom with mol. wt. of 23 and 25 KD were found to be extraordinarily immunogenic. Cross-reacting and species-specific venom proteins were readily identified by the immunoblot technique. Only a small number of venom proteins were cross-reactive among the snake species tested while the remaining appeared to be species-specific. In the development of immunoassays, four very simple tests based on latex agglutination inhibition (LAI), passive hemagglutination inhibition (HAI), reverse latex agglutination (RLA) and reverse passive hemagglutination (RPHA) have been studied. Although useful information was obtained on the LAI and HAI tests, their sensitivities in venom detection were too low to be useful clinically. In the RLA, specific rabbit antibodies purified on a Sepharose-Protein A affinity column were used to coat latex particles. The sensitized particles specifically agglutinated in the presence of homologous venom proteins with a sensitivity of detection of 0.16 to 1.2 ?g/ml. Cross-reactions were observed with heterologous venoms at concentrations about 460 to 16,000 times higher than with homologous venoms. Interference from human plasma, serum and urine on the RLA could be eliminated by adsorption with normal rabbit IgG-coated latex or by heat inactivation at 56 degree C for 30 min. The sensitized latex particles were stable to freezing, thawing, lyopthilization and to storage at 4 degree C (> 3 months) and at -20 degree C (> 3 months). The total test time was about 40 min. Vaious snake venoms were detected in 31 out of 59 (52.5%) serum samples obtained from victims of snakebites. Only one false positive case was observed. With 26 wound swabs, 10 cases (38.5%) were tested positive by the RLA. In the RPHA technique, the affinity purified rabbit antivenom IgG(s) were coupled to sheep, human or chicken erythrocytes by using chromic chloride as coupling reagent. The properties of sensitized red blood cells from these 3 species with regards to sensitivity, specificity, stability, etc. were studied and compared. The sensitivity of the test was about 2 to 635 ng/mL. Cross-reactions were observed with heterologous venoms at concentrations 60 to 500 times higher than those required by homologous venoms. Interference from clinical samples could be removed by procedures similar to those for the RLA test. The coupled red blood cells were stable at 4 degree C from 1 to 12 months depending on the antibody used. The entire test required 60 to 120 min. The RPHA test detected various venoms in 48 out of 59 (81.3%) serum samples while 16 out of 26 (61.5%) wound swabs were positive. Two serum samples were found to be false positives. The sensitivity and specificity of the RPHA test were comparable to those of ELISA but the RPHA test was more simple, faster and less expensive. Various parameters of the RLA, RPHA and ELISA, relevant to venom detection for rapid species diagnosis in developing countries, were compared and discussed.
Degree Name
Doctor of Philosophy
Degree Level
Doctoral Degree
Degree Department
Faculty of Science
Degree Discipline
Microbiology
Degree Grantor(s)
Mahidol University