Development of Hybrid IgG-Aptamer Sandwich Immunoassay Platform for Aflatoxin B1 Detection and Its Evaluation Onto Various Field Samples.

The present study was aimed to develop a novel antibody-aptamer based hybrid detection strategy for specific and sensitive detection of aflatoxin B1 (AFB1) from contaminated food grains. The study comprises generation of ssDNA aptamers and anti-AFB1 IgG against AFB1 toxin. The generated bio-probes (aptamers and antibodies) were further characterized for their specificity and sensitivity using indirect ELISA. The generated aptamers namely AFB1a and AFB1b showed prominent reactivity and selectivity against AFB1 toxin. These aptamers were further characterized for their secondary structures and dG values were determined as -4.6 and -2.75 Kcal/mol, respectively. The detection limit (LOD) of AFB1a and anti-AFB1 IgG was determined as 5 and 10 ng/mL, respectively. The characterized aptamers and antibodies against AFB1 were used to develop the sandwich immunoassay. Anti AFB1 IgG was used as a capturing antibody whereas anti-AFB1a aptamer was used as its revealing partner in the assay. The limit of detection (LOD) of the immunoassay was determined to be 5 ng/mL of AFB1 standard toxin and showed no cross-reactivity with closely related mycotoxins. To assess the reliability of the developed method, several field samples contaminated with aflatoxin B1 was included in the study and results were validated with commercial AFB1-ELISA Kit. Additionally, the spiking studies were also carried out to demonstrate the consistency and dependability of the developed hybrid sandwich immunoassay wherein the toxins recovered were found to be ranging between 73 and 98.80% with the LOD at 5 ng/mL. In conclusion, the developed method may find the better utility in routine food testing laboratories for assessment of AFB1.

Development of a FRET-based fluorescence aptasensor for the detection of aflatoxin B1 in contaminated food grain samples.

The present study aimed to develop an aptamer-based FRET detection strategy for the specific and sensitive detection of AFB1 in contaminated food grains. The study comprises generation of ssDNA aptamers against AFB1 by whole-cell SELEX and their application in a FRET-based platform utilizing graphene oxide (GO) and quantum dots (QDs). The generated aptamers were characterized to determine their specificity and sensitivity using indirect ELISA where AFB1-OVA was used as a coating antigen. Among the aptamers generated, the ATB1 aptamer showed good reactivity and selectivity against AFB1. This aptamer was further characterized to determine its secondary structure and KD value, which was found to be 5.9 kcal mol-1. The characterized aptamers were conjugated onto Cd/Se quantum dots to develop a fluorimetric system for the detection of aflatoxin B1 using a graphene oxide platform. The presence of graphene oxide quenches the fluorescence ability of the quantum dots due to π-π stacking interactions between the aptamer and GO. Upon target addition, the aptamer forms a complex with aflatoxin B1 thereby restoring the fluorescence intensity. The developed assay shows a linear response from 0.002 µg µl-1 to 0.2 µg µl-1 with a detection limit of 0.004 µg µl-1 for the AFB1 standard toxin and showed no cross-reactivity with other closely related mycotoxins. To validate the reliability of the developed method, several field samples spiked with AFB1 were included in this study and the results obtained were cross verified using a standard commercial AFB1 kit. In conclusion, the developed method may find good utility in routine food testing laboratories for risk assessment of AFB1.

Microdevice for plasma separation from whole human blood using bio-physical and geometrical effects.

In this research work, we present a simple and efficient passive microfluidic device for plasma separation from pure blood. The microdevice has been fabricated using conventional photolithography technique on a single layer of polydimethylsiloxane, and has been extensively tested on whole blood and enhanced (upto 62%) hematocrit levels of human blood. The microdevice employs elevated dimensions of about 100 µm; such elevated dimensions ensure clog-free operation of the microdevice and is relatively easy to fabricate. We show that our microdevice achieves almost 100% separation efficiency on undiluted blood in the flow rate range of 0.3 to 0.5 ml/min. Detailed biological characterization of the plasma obtained from the microdevice is carried out by testing: proteins by ultra-violet spectrophotometric method, hCG (human chorionic gonadotropin) hormone, and conducting random blood glucose test. Additionally, flow cytometry study has also been carried on the separated plasma. These tests attest to the high quality of plasma recovered. The microdevice developed in this work is an outcome of extensive experimental research on understanding the flow behavior and separation phenomenon of blood in microchannels. The microdevice is compact, economical and effective, and is particularly suited in continuous flow operations.

Sheep uterus dual lipoxygenase in the synthesis of 14,15-leukotrienes.

Lipoxygenase was purified to homogeneity from sheep uterus cytosol using a combination of ion exchangers, ammonium sulfate fractionation, and gel filtration. The purified enzyme was found to be a homodimeric protein with monomer molecular weight of 66 kDa. When incubated with arachidonic acid, the enzyme showed two lipoxygenase activities producing both 12- and 15-HPETEs at the optimum pH of 5.5. The relative concentration of 12- and 15-HETEs, however, changed with the pH of the reaction, 12-HETE being higher in the alkaline range and 15-HETE being higher in the acidic range. Furthermore the enzyme showed the expected dual lipoxygenase based 14,15-LTA4 synthase activity as evidenced by the formation of 8,15-diHETEs, the hydrolysis products of 14,15-LTA4. Isolation of 14,15-LTC4 from the homogenates of sheep uterus gave further evidence on the formation of leukotrienes. This is the first report of the formation of 14,15-series leukotrienes in mammalian reproductive tissue.

Arachidonic acid metabolites as intratesticular factors controlling androgen production.

The effect of inhibitors and products of arachidonic acid metabolism on rat testicular steroidogenesis has been investigated. In the presence of indomethacin (inhibitor of cyclooxygenase) and nordihydroguaiaretic acid (NDGA) (inhibitor of lipoxygenase), the activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) were both inhibited. The LH-stimulated increase in secretion of testosterone and progesterone was also inhibited by indomethacin and NDGA. On the other hand, vitamin E (antioxidant and inhibitor of lipoxygenase), stimulated the activity of both 3 beta-HSD and 17 beta-HSD and enhanced LH-stimulated androgen production. The metabolites of lipoxygenase (15-HPETE, 15-HETE, 5-HPETE and 5-HETE) and cyclooxygenase (PGF2 alpha) pathways stimulated 3 beta-HSD and 17 beta-HSD activity and enhanced the secretion of progesterone and testosterone. It is concluded that arachidonic acid metabolites are intratesticular factors which can regulate LH-stimulated testicular steroidogenesis.

Differential effects of 15-HPETE and 15-HETE on BHK-21 cell proliferation and macromolecular composition.

Arachidonate and/or linoleate metabolites have been implicated in modulating cell growth, replication and cell transformations. In studies with BHK-21 cells, we found lipoxygenase and cyclooxygenase inhibitors (NDGA and indomethacin, respectively) to be antiproliferative. Studies on the metabolism of arachidonic acid in BHK-21 cells have demonstrated that prostaglandin D2 is the major cyclooxygenase product, and 15-hydroxyeicosatetraenoic acid (15-HETE) is the major lipoxygenase product. Addition of D2 showed a significant decrease in the BHK-21 cell number showing antiproliferative action. Addition of lipoxygenase products, on the other hand, showed differential effects in that 15-HPETE decreased the cell number while 15-HETE increased. NDGA and 15-HPETE decreased DNA, RNA and protein contents, while 15-HETE significantly increased them. 5-HPETE and 5-HETE also showed similar results but were less potent than 15-H(P)ETEs. The differential effects of 15-HPETE and 15-HETE could be due to the generation of free radicals by the hydroperoxide and mitogenic response by hydroxide.

The production of arachidonic acid metabolites in rat testis.

There is growing evidence that arachidonic acid is oxygenated enzymatically in every cell type and that the oxygenated metabolites regulate a variety of pathological and physiological processes including reproduction. In the present study, the metabolism of arachidonic acid in the testis via cyclooxygenase and lipoxygenase pathways was analyzed. Testicular microsomes showed substantial cyclooxygenase activity as measured by the polarographic method. Analysis of the products on TLC revealed PGF2 alpha (79.5%) as the main product followed by PGE2 (20.3%) and PGD2 (0.17%). At higher substrate concentrations (150 microM), however, 6-keto-PGF1 alpha, the stable metabolite of prostacyclin, was observed in substantial quantities. Maximum activity of lipoxygenase was observed at pH 6.4 in both microsomes and cytosol, the activity being higher in cytosol. Analysis of lipoxygenase pathway products with arachidonic acid as the substrate, revealed the presence of 12-HPETE as the major product both in cytosol and in microsomes. Besides this, 15- and 5-HPETEs were also observed in substantial quantities.

Lithium neurotoxicity at 'therapeutic' levels a case report.

A case of a young manic patient who developed severe neurotoxicity when on lithium alone has been presented. Investigations did not reveal presence of any infection, electrolyte imbalance or rise in lithium level.The possibility of lithium producing neurotoxicity at therapeutic levels for as yet unknown reasons is pointed out. It is suggested that this element of risk be considered when starting lithium for therapy or prophylaxis of affective disorders.

Alcoholic hallucinosis and paranoid schizophrenia-a comparative (clinical and follow up) study.

In a Study Of 90 patients of Alcoholic Hallucinosis and 30 patients of Paranoid Schizophrenia, it was found that delusions, delusions of infidelity, third person and running commentary auditory hallucinations and insight were not different in the two groups.Delusions of grandeur, passivity, thought echo and thought broadcast were significantly more frequent in paranoid schizophrenic patients. Anxiety, visual iiafracinatians and hallucinations in more than one modality at the same time were commoner in alcoholic hallucionsis. Recovery from acute symptoms was much earlier in alcoholic hallucinosis.Number of first degree relatives with schizophrenia was much higher in the paranoid schizophrenic group.In a mean follow up period of 18 months, it was found that patients with alcoholic hallucinosis did much better than patients with paranoid schizophrenia.