Immuno-analytical method development for detection of transgenic Cry1Ac protein and its validation.

BACKGROUND: Bacillus thuringiensis (Bt) synthesizes Cry1Ac protein, which is toxic to many lepidopteran pests, and the cry1ac gene has been expressed in several transgenic crop plants. The Cry1Ac protein has been isolated from Bt kurstaki HD73 and purified to homogeneity. Polyclonal antibodies were raised against purified Cry1Ac in rabbits and goat. Sandwich ELISA was developed for Cry1Ac using goat IgG as a coating antibody, and affinity-purified rabbit IgG as the primary antibody. RESULTS: The sensitivity of the assay was in the range of 0.47-1000 ng. It was subsequently employed in validating biological samples. Fifteen different cotton-seed samples were screened: 12 were found to be Bt positive and 3 Bt negative. The CS7 seeds showed the highest Bt content of 8.51 ± 0.45 µg g-1 , followed by CS8 (6.0 ± 0.02 µg g-1 ), CS15 (5.9 ± 0.03 µg g-1 ), CS9 (5.5 ± 0.05 µg g-1 ), and CS10 (4.83 ± 0.013 µg g-1 ). The CS5 seeds showed Bt content of 3.6 ± 0.21 µg g-1 . The F2 generation, CS6 (Kaveri seeds) showed lower Bt content (2.9 ± 0.06 µg g-1 ). The CL5 samples showed Cry1Ac content of 0.99 ± 0.009 µg g-1 . The amount of Cry1Ac protein in leaves, stem, and roots of germinated Bt cotton plants (CS10 and CS4) were 1.76 ± 0.15 µg g-1 , 1.9 ± 0.01 µg g-1 , 2.0 ± 0.1 µg g-1 , and 1.6 ± 0.15 µg g-1 , 1.9 ± 0.01 µg g-1 , and 2.0 ± 0.01 µg g-1 dry tissue, respectively. CONCLUSION: The method developed can be used for screening the expression levels of Cry1Ac in different transgenic Bt cultivars and also spurious Bt cotton seeds procured by farmers. © 2019 Society of Chemical Industry.

Bioprospecting of gum kondagogu (Cochlospermum gossypium) for bioremediation of uranium (VI) from aqueous solution and synthetic nuclear power reactor effluents.

An ecofriendly green chemistry method using a natural biopolymer, Gum Kondagogu (GK) for the removal of U (VI) from aqueous, simulated nuclear effluents was studied. The adsorption characteristic of GK towards U (VI) from aqueous solution was studied at varied pH, contact time, adsorbent dose, initial U (VI) concentration and temperature using UV-Visible spectroscopy and ICP-MS. Maximum adsorption was seen at pH 4, 0.1% GK with 60 min contact time at room temperature. The GK- U (VI) composite was characterized by FT-IR, zeta potential, TEM and SEM-EDAX. The Langmuir isotherm was found to be 487 mg of U (VI) g(-1) of GK. The adsorption capacity and (%) of U (VI) was found to be 490 ± 5.4 mg g(-1) and 98.5%. Moreover adsorption of U (VI) by GK was not influenced by other cations present in the simulated effluents. The adsorbed U (VI) was efficiently stripped from composite using 1 M HCl.

Immunochemical detection of glycated lens crystallins and their circulating autoantibodies in human serum during aging.

PURPOSE: The aim of this investigation was to exploit lens-specific glycated crystallins as an immunogen to detect human glycated crystallins and their circulating autoantibodies in human serum during aging in relation to the development of cataract. METHODS: Polyclonal antibodies were produced against human total lens proteins (40-80 years) in rabbits. The specificity of the antibodies produced were determined by antibody capture assay using purified human lens crystallins (high molecular weight fraction [HMW]+alpha, HMW+alpha-glycated, beta, beta-glycated, gamma, and gamma-glycated) as antigens. The cross-reactivity of these lens specific antibodies against rat beta-, beta-glycated, gamma-, and gamma-glycated lens crystallins was also analyzed. A non-competitive enzyme linked immunosorbent assay (ELISA) methodology was developed for the detection of circulating lens crystallins in human sera using HMW+alpha, HMW+alpha-glycated, beta-, and beta-glycated crystallins from humans and gamma- and gamma-glycated crystallins from rats as immobilized antigens. Circulating autoantibodies were also detected in human sera by antibody capture assay. The methodology was validated by evaluating 60 human serum samples collected from cataract patients and 30 human serum samples from apparently normal subjects belonging to the same age group. RESULTS: The polyclonal antibodies raised against human total lens proteins showed 90% and 65% cross-reactivity with rat gamma- and beta-crystallins, respectively, by ELISA. Further, these polyclonal antibodies were capable of detecting both native and in vitro synthesized glycated crystallins. Their IC50 values were observed to be (i) human total lens proteins (55 ng), (ii) human HMW+alpha (16.45 ng), (iii) human HMW+alpha-glycated (273 ng), (iv) human beta- (37.82 ng), (v) human beta-glycated (260 ng), (vi) rat gamma- (105.34 ng), and (vii) rat gamma-glycated (313 ng). The immunochemical analysis of human serum indicated a significant change (p<0.001) in the levels of circulating beta-glycated and gamma-glycated crystallins in the age group of 40-80 years with respect to their control groups. However, there was no statistically significant change in the levels of HMW+alpha-glycated crystallins in the age group of 40-80 years as compared to their age-matched controls. Notably, the levels of serum gamma-glycated crystallins were found to be threefold higher than that of HMW+alpha-glycated and beta-glycated crystallins in the age group of 70-80 years. Circulating autoantibodies to HMW+alpha-glycated, beta-glycated, and gamma-glycated crystallins were detected in the serum of both apparently normal and cataract patients in the age group of 40-80 years by antibody capture assay. The levels of these autoantibodies were significantly higher at every time point compared to their respective controls. Autoantibodies to gamma-glycated crystallins were found to be twofold and 3.2 fold higher as compared to the levels of autoantibodies to beta-glycated and HMW+alpha-glycated crystallins, respectively. Western blot and immunohistochemical analysis substantiated the observations made in non-competitive ELISA. CONCLUSIONS: During the course of aging, leakage of lens crystallins (HMW+alpha, HMW+alpha-glycated, beta, beta-glycated, gamma, and gamma-glycated) elicit an immune response resulting in the formation of autoantibodies in cataract patients (40-80 years) as compared to age matched controls. This is the first experimental report where polyclonal antibodies raised against lens-specific glycated crystallins were capable of detecting the early leakage of glycated crystallins in human subjects. This immunochemical approach has implications in the early detection of senile cataract.