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.

Purification and properties of three new phospholipase A2 isoenzymes from Micropechis ikaheka venom.

Three new phospholipase A2 (PLA2) isoenzymes were purified from the Micropechis ikaheka venom by successive chromatographies. The homogeneity of them was accessed by capillary zone electrophoresis and mass spectrometry. Their N-terminal sequences showed high identity (94, 88 and 90, respectively) with MiPLA-1, a group IB PLA2 also from this venom. In addition, strong immuno-cross-reaction with anti-MiPLA-1 serum was observed. These results suggested that three newly purified PLA2 belonged to group IB. Beside enzymatic activity, they induced various pharmacological effects, including myotoxic, anticoagulant effects and insulin secretion stimulating effects. Our results indicated that enzymatic activity is essential for their myotoxic and anticoagulant effects. On the other hand, no direct correlation between their insulin secretion stimulating effect and enzymatic activity was observed, suggesting that they may stimulate insulin secretion through a non-enzymatic mechanism.

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.

Tests for detection of snake venoms, toxins and venom antibodies: review on recent trends (1987-1997).

Various methods developed for the detection of snake venoms, toxins and venom antibodies, during the last decade is reviewed. Radioimmunoassay, agglutination assay, enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay etc. have been used for detection of venoms and toxins. Important contributions have been made to improve the specificity, sensitivity, rapidity and simplicity of the ELISA method. Monoclonal antibodies and affinity-purified venom-specific antibodies were used to achieve species specificity of ELISA and the latter seems to be the ideal for venom detection. Incorporation of avidin-biotin system as well as the fluorogenic substrate in the enzyme immunoassay sufficiently increased the sensitivity of the assay to detect venom concentrations to picogram levels. The ability to use undiluted blood and other whole biological fluids reduce the assay time considerably. Although there have been several reports were on venom detection, so far only a few field kits have been developed. This implies that the experiments and design were only at the laboratory levels and still more work has to be carried out before it could be used in the field. Concerning the venom antibody detection, ELISA has been used extensively and the humoral response of patients envenomed by snake has been investigated in detail. Non-specific reactivity along with cross-reactivity still limits the use of ELISA for species identification in epidemiological studies. Overall, ELISA remains the suitable method for the detection of snake venoms, toxins and venom antibodies in body fluids. The possible use of a biosensor approach to solve some of the problems associated with the ELISA method are also discussed.

Ehretianone, a novel quinonoid xanthene from Ehretia buxifolia with antisnake venom activity.

Ehretianone (1), a new quinonoid xanthene, together with known sterols, was isolated from a MeOH extract of the root bark of Ehretia buxifolia. The structure of ehretianone was elucidated as 7-hydroxy-9a alpha-(3-methylbut-2-enyl)-4a alpha,9 alpha-(2-methylprop-2-enyl)-4a, 9a-dihydro-1,4-dioxoxanthene on the basis of spectroscopic data and X-ray crystallographic analysis. The antisnake venom activity of ehretianone against Echis carinatus venom in mice is also reported.