TY - JOUR
T1 - Use of immunoturbidimetry to detect venom-antivenom binding using snake venoms
AU - O'Leary, Margaret A
AU - Maduwage, Kalana
AU - Isbister, G. K.
PY - 2013/5
Y1 - 2013/5
N2 - Introduction: Immunoturbidimetry studies the phenomenon of immunoprecipitation of antigens and antibodies in solution, where there is the formation of large, polymeric insoluble immunocomplexes that increase the turbidity of the solution. We used immunoturbidimetry to investigate the interaction between commercial snake antivenoms and snake venoms, as well as cross-reactivity between different snake venoms. Methods: Serial dilutions of commercial snake antivenoms (100 μl) in water were placed in the wells of a microtitre plate and 100 μl of a venom solution (50 μg/ml in water) was added. Absorbance readings were taken at 340. nm every minute on a BioTek ELx808 plate reader at 37 °C. Limits imposed were a 30. minute cut-off and 0.004 as the lowest significant maximum increase. Reactions with rabbit antibodies were carried out similarly, except that antibody dilutions were in PBS. Results: Mixing venom and antivenom/antibodies resulted in an immediate increase in turbidity, which either reached a maximum or continued to increase until a 30. minute cut-off. There was a peak in absorbance readings for most Australian snake venoms mixed with the corresponding commercial antivenom, except for Pseudonaja textilis venom and brown snake antivenom. There was cross-reactivity between Naja naja venom from Sri Lanka and tiger snake antivenom indicated by turbidity when they were mixed. Mixing rabbit anti-snake antibodies with snake venoms resulted in increasing turbidity, but there was not a peak suggesting the antibodies were not sufficiently concentrated. The absorbance reading at pre-determined concentrations of rabbit antibodies mixed with different venoms was able to quantify the cross-reactivity between venoms. Indian antivenoms from two manufacturers were tested against four Sri Lankan snake venoms (Daboia russelli, N. naja, Echis carinatus and Bungarus caeruleus) and showed limited formation of immunocomplexes with antivenom from one manufacturer. Discussion: The turbidity test provides an easy and rapid way to compare and characterise interactions between antivenoms and snake venoms.
AB - Introduction: Immunoturbidimetry studies the phenomenon of immunoprecipitation of antigens and antibodies in solution, where there is the formation of large, polymeric insoluble immunocomplexes that increase the turbidity of the solution. We used immunoturbidimetry to investigate the interaction between commercial snake antivenoms and snake venoms, as well as cross-reactivity between different snake venoms. Methods: Serial dilutions of commercial snake antivenoms (100 μl) in water were placed in the wells of a microtitre plate and 100 μl of a venom solution (50 μg/ml in water) was added. Absorbance readings were taken at 340. nm every minute on a BioTek ELx808 plate reader at 37 °C. Limits imposed were a 30. minute cut-off and 0.004 as the lowest significant maximum increase. Reactions with rabbit antibodies were carried out similarly, except that antibody dilutions were in PBS. Results: Mixing venom and antivenom/antibodies resulted in an immediate increase in turbidity, which either reached a maximum or continued to increase until a 30. minute cut-off. There was a peak in absorbance readings for most Australian snake venoms mixed with the corresponding commercial antivenom, except for Pseudonaja textilis venom and brown snake antivenom. There was cross-reactivity between Naja naja venom from Sri Lanka and tiger snake antivenom indicated by turbidity when they were mixed. Mixing rabbit anti-snake antibodies with snake venoms resulted in increasing turbidity, but there was not a peak suggesting the antibodies were not sufficiently concentrated. The absorbance reading at pre-determined concentrations of rabbit antibodies mixed with different venoms was able to quantify the cross-reactivity between venoms. Indian antivenoms from two manufacturers were tested against four Sri Lankan snake venoms (Daboia russelli, N. naja, Echis carinatus and Bungarus caeruleus) and showed limited formation of immunocomplexes with antivenom from one manufacturer. Discussion: The turbidity test provides an easy and rapid way to compare and characterise interactions between antivenoms and snake venoms.
KW - Antibody
KW - Antivenom
KW - Immunocomplexes
KW - Immunoturbidimetry
KW - Snake venom
UR - http://www.scopus.com/inward/record.url?scp=84875273162&partnerID=8YFLogxK
U2 - 10.1016/j.vascn.2013.02.004
DO - 10.1016/j.vascn.2013.02.004
M3 - Article
C2 - 23416032
AN - SCOPUS:84875273162
SN - 1056-8719
VL - 67
SP - 177
EP - 181
JO - Journal of Pharmacological and Toxicological Methods
JF - Journal of Pharmacological and Toxicological Methods
IS - 3
ER -