Purification and characterization of mycoferritin from Aspergillusflavus MTCC 873.

The fungus Aspergillus flavus MTCC 873, a non-toxigenic isolate demonstrated its capability to synthesize mycoferritin (MF) upon induction with iron in yeast extract sucrose (YES) medium. The molecular mass, yield, iron and carbohydrate contents of the MF were 440 kDa, 0.015 mg/g of wet mycelia, 0.8 and 30.4%, respectively. Native gel-electrophoresis revealed a band corresponding to dimeric form of equine spleen ferritin (ESF). Subunit analysis by SDS-PAGE revealed a single protein band with an apparent molecular mass of 24 kDa, suggesting similar sized subunits in the structure of apoferritin shell. Immunological cross-reactivity was observed with the anti-fish liver ferritin. Transmission electron microscopy (TEM) revealed an apparent particle size of 100 Å. N-terminal amino acid sequence of MF revealed a sequence of SLPLQDYA, which showed identities with other eukaryotic ferritin sequences. The spectral characteristics (UV/VIS, fluorescence and circular dichroic spectra) were similar to ESF. The fungus, unlike A. parasiticus 255 (non-toxigenic) was incapable of producing aflatoxins, when grown in YES media.

Epidemiological profile of snake-bite cases from Andhra Pradesh using immunoanalytical approach.

BACKGROUND & OBJECTIVE: Snake-bites are the common cause of morbidity and mortality in tropical countries. In India, there are 216 species of snakes, of which only four are venomous snakes (cobra, krait, Russell's viper and saw scaled viper). This study was undertaken to find out the epidemiological profile of snake-bite incidences in the State of Andhra Pradesh, based on the data collected from State Forensic Science Laboratory, Hyderabad. METHODS: Data from 1379 snake-bite cases were collected from case reports for a 5 yr period (1999- 2003) that included age and sex of the victim, district, month of incidence, time of incident, death of a victim and the time point of analysis. On the basis of the forensic data, specimens were collected from forensic medicine department, during rainy season and were analysed for the venom antigens (cobra and krait) by ELISA method. RESULTS: The peak number of snake-bite cases were seen during June-September. Majority of the cases were observed in the age group 21-50 yr (71%). Higher incidence of snake-bite was recorded in males (76%). Of the 22 cases analysed by the ELISA, 6 tested positive for cobra venom, while 8 cases tested positive for krait venom, the remaining specimens tested negative for both cobra and krait venom. INTERPRETATION & CONCLUSION: Evaluation of forensic specimens (autopsy & biopsy) of human snakebite victims based on specific molecular epidemiological tool like ELISA gives a true estimate of the incidence supplementing clinical and circumstantial evidence.

Development of an immunoanalytical method for the detection of beta- and gamma-crystallins and anti-crystallin antibodies. A molecular biomarker for cataract.

PURPOSE: To develop and evaluate an immunoanalytical method for the detection of beta- and gamma-crystallins and anti-crystallin antibodies. MATERIALS AND METHODS: Beta and gamma-crystallins isolated from rat lens were used as immunogens to raise polyclonal antibodies in rabbits. Antibody capture assay and western blot analysis showed that the antibodies to beta- and gamma-crystallins were specific. An indirect competitive enzyme linked immunosorbent assay (ELISA) developed to quantitate beta- and gamma-crystallin showed an IC50 value of 70 ng and 65 ng, respectively, based on regression analysis. Spiking studies with purified beta-crystallin antibodies showed that 33 ng of the purified antibody gave an absorbance of 1.1 at 450 nm, indicating the sensitivity of the method. RESULTS: Antibodies to beta- and gamma-crystallins were not detected in serum samples of the cataractous CFY/NIN rats (used as an animal model for induction of experimental cataract by feeding high galactose diet). However, the cataractous rat serum samples effectively displaced beta- and gamma-crystallin antibodies, indicating that these crystallins leak during cataract formation. The concentration of beta- and gamma-crystallins in the rat serum, as analysed by indirect competitive ELISA, was found to be in the range of 17.6-81.6 micrograms/ml [corrected] and 12.4-19.6 micrograms/ml, respectively. CONCLUSIONS: The methodology developed in the present study may find application as a biochemical tool in molecular epidemiology of cataract.

Binding characteristics of bovine serum albumin-aflatoxin B1 to polystyrene microtiter plates: importance of hapten to carrier protein molar ratio.

Binding characteristics of bovine serum albumin-aflatoxin B1 conjugates with high (1:54), medium (2:25) and low (1:9) hapten to carrier molar ratios, to the polystyrene microtiter plates are influenced by the stoichiometry of the hapten (Aflatoxin B1) to the carrier protein (bovine serum albumin). Conjugates with optimal hapten to carrier molar ratios (1:25) showed a better binding capacity to the polystyrene microtiter plate as compared to the conjugates with the high molar ratios in a non-competitive ELISA for aflatoxin B1. Denaturation of the conjugate with molar ratio of 1:54 in order to enhance its binding capacity, however, did not result in any significant improvement.