Comparison of Antivenom potential of chicken Egg yolk Antibodies Generated against Bentonite and Adjuvant coated Echis carinatus venom
S Meenatchisundaram, R Selvakumaran, G Parameswari, A Michael
S Meenatchisundaram, R Selvakumaran, G Parameswari, A Michael. Comparison of Antivenom potential of chicken Egg yolk Antibodies Generated against Bentonite and Adjuvant coated Echis carinatus venom. The Internet Journal of Pharmacology. 2008 Volume 7 Number 1.
The Chicken egg yolk antibodies generated against Bentonite and Adjuvant (FCA) coated
Snake envenoming is a major public health issue in the rural tropics with large numbers of envenoming and deaths. There are nearly 3000 different species of snakes found in the world of which approximately 300 are venomous. In India there are about 216 different species are found, of which 53 species are reported to be poisonous. The common poisonous snakes found in India are Cobra, Krait, Russell’s viper and Saw Scaled Viper (Bawaskar, 2004). About 35,000 to 50,000 people die of snakebite every year in India. Antivenom immunotherapy is the only specific treatment against snake venom envenomation. In India polyvalent antisnake venom effective against venoms of Cobras, Krait, Russell's viper and Saw-scaled viper is available. Each ml of polyvalant Antisnake venom can neutralise 0.6mg of Cobra, 0.6mg of Russell’s viper, 0.45mg of Krait and 0.45mg of Saw Scaled Viper venom. Commercially available Horse antivenom contained high concentrations of non-immunoglobulins which frequently caused complement mediated side effects, serum sickness and renal failure which may be reduced by using sufficiently pure antibodies (Mayadevi
The present study involves the comparison of the effectiveness of antivenom antibodies generated in chicken immunized with Freund’s complete adjuvant and bentonite coated venoms of
Materials and Methods
Venom and Experimental animals
The free-dried snake venom powder of
Development of antivenom antibodies in chicken
The lyophilized snake venom powder of
Purification and Characterization of antivenom antibodies from egg yolk
The antibodies were extracted from egg yolk by the method of Polson
Determination of antibody titer by Indirect ELISA
The antibody titer of the antibodies generated against
Specificity of antibodies by Western Blot
The western blot analysis to determine the specificity of IgY antibodies was carried out according to the method described by Towbin
Tests for anti-snake venom activities
The median lethal dose (LD50) of
Edema- forming Activity
The Minimum edema-forming dose (MED) of
The minimum haemorrhagic dose (MHD) of venom was determined by the method described by Theakston and Reid, 1983. The minimum haemorrhagic dose was defined as the least amount of venom which when injected intradermaly (i.d.) into mice results in a haemorrhagic lesion of 10mm diameter in 24 hours. Neutralization of the haemorrhagic activity was estimated by mixing a fixed amount of chicken egg yolk antibodies with different amounts of venom. The IgY–venom mixture was incubated at 37 ◦C for 1 h and 0.1 ml of the mixture was injected intradermaly into mice. The haemorrhagic lesion was estimated after 24 h.
Phospholipase A2 activity was measured using an indirect hemolytic assay on agarose–erythrocyte–egg yolk gel plate by the methods described by Gutierrez et al., 1988. Increasing concentrations of
The procoagulant activity was done according to the method described by Theakston and Reid, 1983 modified by Laing
Statistical evaluation was performed using XL stat 2007 and SPSS 10 Softwares. P< 0.005 was considered statistically significant.
Generation of anti- antivenom antibodies in Chicken
White leghorn Chickens were immunized intramuscularly with sublethal dose of
Adjuvant Effects on Antivenom Titers
The ELISA there was a gradual increase in the antibody titer in both egg yolk antibodies (FCA and Bentonite) and reached a pleatue and remained stable till 180th day of observation. The booster doses administered at regular time intervals increased and maintained the antivenom level in yolk. The birds immunized with freunds adjuvant coated venoms showed very high titer at dilutions of more than 1:10000 detecting even less than 0.080µg of specific antivenom (Fig 1).
The birds immunized with bentonite venoms also showed good titre value but lesser than FCA. All the birds show a significant titer as compared with the unimmunized control. The chickens immunized with FCA coated venoms did not produce any side effects in birds but Bentonite caused a decrease in the laying frequency of the bird. These side affects caused by bentonite may lead to decrease in antivenom titres in egg yolk of immunized hens.
The antivenom potential of chicken egg yolk antibodies generated against bentonite and Adjuvant(FCA) coated
Determination of lethal toxicity of and their Neutralization by using chicken egg yolk antibodies
When comparing to IgY generated against bentonite coated venom, IgY generated against F CA coated
In edema forming activity, the mice immunized with
In the case of hemorrhagic activity,
Snakebite is a common medical emergency encountered in the tropics and estimated 35,000 to 50,000 people die of snakebite every year in India (Sharma
The present investigation was carried out to raise specific polyclonal hyper-immune antibodies in Chicken against Bentonite and Adjuvant (FCA) coated