Anti-Echis carinatus venom antibodies from chicken egg yolk: isolation, purification and neutralization efficacy.

High titer antibodies (IgY) were raised in egg yolk of white leghorn chicken (Gallus gallus domesticus) by immunizing with the venom of Echis carinatus (Saw scaled viper or carpet viper), an Indian venomous snake belonging to the family Viperidae. The anti-snake venom antibodies (antivenom) were isolated from egg yolk by the water dilution method, enriched by 19% sodium sulfate precipitation and purified by immunoaffinity chromatography. A single, electrophoretically pure IgY band of 180-200 kDa was obtained on SDS-PAGE. Immunoblot analysis revealed not only the specific binding of the antivenom but also dose-dependent blocking of antivenom by venom proteins. In neutralization studies, a preincubated mixture of both affinity-purified (50 mg/kg body weight) as well as partially purified (210 mg/kg body weight) anti-E. carinatus IgY with 2 LD(50) dose of E. carinatus venom (2 x 6.65 mg/kg body weight) gave 100% protection in mice when administered subcutaneously.

Modulation of allergen-specific immune responses to the major shrimp allergen, tropomyosin, by specific targeting to scavenger receptors on macrophages.

BACKGROUND: Tropomyosin from shrimp is the major cross-reacting crustacean food allergen. Earlier studies have led to the purification and immunochemical characterization of the major IgE binding epitopes of the allergen. Maleylated proteins are known to be specifically targeted to scavenger receptors on macrophage. Since antigens processed and presented by macrophages are known to elicit Th1 type of responses and allergic responses are characterized by polarization towards Th2 phenotype, the possibility of modulation of allergen-specific immune responses by targeting of tropomyosin to macrophage via scavenger receptor was explored. METHODS: The IgG and IgE binding potential of the native maleylated form of tropomyosin was carried out by ELISA and immunoblot. The ability of the native and maleylated form of allergen to induce in vitro proliferation of splenocytes from BALB/C mice immunized with both forms of allergen was tested. The in vitro production of IL-4 and IFN-gamma by splenocytes from mice immunized with the two forms of allergen was determined from culture supernatants. The in vivo production of serum IgG1 and IgG2a antibodies following immunization with native and modified allergens was monitored by ELISA. RESULTS: The maleylated form of tropomyosin was found to have reduced antigenicity and allergenicity as compared to its native counterpart. The modified allergen was, however, found to elicit a cellular response similar to native tropomyosin in vitro. Analysis of the cytokine profiles showed a modulation from an IL-4-dominant, proallergic, Th2 phenotype to an IFN-gamma-dominant, antiallergic, Th1 phenotype that could also be correlated to a modulation in the in vivo antibody isotype. CONCLUSION: The results suggest the possible potential for modulating allergic responses in vivo by selective targeting to macrophages.

ELISA for the detection of venoms from four medically important snakes of India.

A double antibody sandwich enzyme linked immunosorbent assay (ELISA) was developed to detect Echis carinatus venom in various organs (brain, heart, lungs, liver, spleen and kidneys) as well as tissue at the site of injection of mice, at various time intervals (1, 6, 12, 18, 24 h and 12 h intervals up to 72 h) after death. The assay could detect E. carinatus venom levels up to 2.5 ng/ml of tissue homogenate and the venom was detected up to 72 h after death. A highly sensitive and species-specific avidin-biotin microtitre ELISA was also developed to detect venoms of four medically important Indian snakes (Bungarus caeruleus, Naja naja, E. carinatus and Daboia russelli russelli) in autopsy specimens of human victims of snake bite. The assay could detect venom levels as low as 100 pg/ml of tissue homogenate. Venoms were detected in brain, heart, lungs, liver, spleen, kidneys, tissue at the bite area and postmortem blood. In all 12 human victim cadavers tested the culprit species were identified. As observed in mice, tissue at the site of bite area showed the highest concentration of venom and the brain showed the least. Moderate amounts of venoms were found in liver, spleen, kidneys, heart and lungs. Development of a simple, rapid and species-specific diagnostic kit based on this ELISA technique useful to clinicians is discussed.

B- and T-cell epitopes of tropomyosin, the major shrimp allergen.

The major crustacean allergen characterized from different species of shrimp is the muscle protein tropomyosin. Two shared epitopes corresponding to 47-63 and 150-158 of the deduced amino-acid sequence of the brown shrimp, M. ensis, were identified as IgE-binding B-cell epitopes. A 21-mer peptide spanning the amino-acid residues 261-281 was identified as a putative T-cell epitope capable of reducing ongoing tropomyosin-specific IgG and IgE responses in a mouse model. These observations suggest that peptide immunotherapy may also be effective in the treatment of food hypersensitivity.