Management of Snakebite and Research Management of Snakebite ...

Study on improvement of toxoidation methods and its stability
Many attempts have been made by the DMR working group on RVV toxoid research on
the toxidation methods and stability of the formal toxoid regarding both safety and
immunogenicity. Khin-Maung-Maung et al (1996) fractionate of crude RVV with sephadex
G 75 super fine gel filtration chromatography and pooled the first four fractions which
were further treated under slow and step wise formalinization. The fractionated RVV toxoid
obtained showed no reversion in toxicity and also preserved immunogenicity upto six
months storage at room temperature as well as storage at 4°C. However, electrophoretic
study of the toxoid revealed that the low molecular weight protein bands reappeared in
the toxoid stored at room temperature for three months and above, probably indicating
the re- dissociation of polymerized protein during storage at room temperature.
San-Aye et al (1997) studied sodium bisulfite for preservation of the formaldehyde
linkage in the toxoid. Thirty-five percent sodium bisulfite was added into the formalinized
crude RVV toxoid during its preparation. In another experiment, RVV toxoid was prepared
by adding formalin upto 2% concentration and sodium bisulfite was added upto 35%.
Both of these two preparations of toxoid were stored at 4°C and 37°C for six months and
tested for safety and immunogenicity. Both the experiments showed that toxoid stored at
37°C and 4°C were safe and immunogenic. However, the imunogenicity of toxoid with
sodium bisulfite stored at 37°C is less than the toxoid at 4°C.
In 1999 Win-Aung et al lyophilized the liquid form of RVV toxoid and stored at 4°C
and room temperature. It was apparent that lyophilized toxoid stored at both conditions
showed minimal changes in activities of enzymes, less reversion in formalin linkages, more
immunogenic and no change in toxicity when compared with those of liquid form of RVV
toxoid. However, there was considerable amount of protein loss in lyophilized toxoid
during the process of freeze drying. This will be obstacle in producing large amount of
lyophilized toxoid for human immunization.
Recently, five major enzymatic components of RVV, namely phospholipase A2,
phosphodiesterase, arginase esterase, coagulase and phosphomonoesterase were purified
from crude RVV by using an appropriate gel filtration and or ion-exchange chromatographic
columns and the formalinized RVV toxoid against these enzymes were then developed.
This toxoid also showed no toxicity and also induced satisfactory immune responses in
experimental mice compared to those of a conventional toxoid. However, when the fully
immunized mice were challenged with the 3 LD50 i.e. lethal dose RVV, they could not
totally withstand and died just after challenging (San-Aye, personal communication).
CONCLUSION
Development of Russell’s Viper Toxoid
Generally, snake venoms are highly complex mixtures of various enzymes and toxic
elements. Most of the constituents are proteins but low molecular weight compounds
such as peptides, nucleotides and metal ions are also present (Karlsson, 1973). Therefore,
toxoidation of the snake venoms is very difficult as compared to that of single toxin like a
bacterial toxin. In addition, because of the antigenic complexity of the venom proteins
and their great local and systemic effects, active immunization against venom for the
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