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Papers by Tanuja Kosuri

TLC-Digital Image-Based Fluorometric Analysis of Ergosterol and Chitin Content in Food Grains Artificially Infested with Aspergillus flavus and Fusarium verticillioides

Food Analytical Methods

Immuno‐analytical Method Development for Detection of Transgenic Cry1Ac Protein and Its Validation

Journal of the Science of Food and Agriculture

Biochemistry & Analytical Biochemistry, 2016

A commercial fabric fluorescent optical brightener Ranipal  (F-OB), has been successfully employ... more A commercial fabric fluorescent optical brightener Ranipal  (F-OB), has been successfully employed to stain proteins on native and SDS-1D-PAGE. The FOB was purified by using a biphasic solvent system of dichloromethane and water. The R f value (0.63) of purified and crude FOB were comparable on TLC. The mass spectrometry of purified FOB indicated base peak at 414 (m/z). Absorption and emission maxima of FOB were found to be 350 nm and 430 nm, respectively. The FOB could stain the proteins both pre-and post-electrophoretic run, on native gel. Postelectrophoretic staining was rapid and required 20 min to visualize the stained proteins. On the other hand, in SDS gels, an additional 20 min was required for the extraction of SDS before staining the proteins with FOB. SDS was found to interfere with binding of FOB to proteins. Varying concentrations of molecular weight markers were loaded and their fluorescent intensity was plotted against the concentration of the proteins. The r 2 values ranged from 0.965 to 0.997 indicating excellent linearity. The detection for carbonic anhydrase was in the range of 8.0-800 ng. Unlike most of the dyes used for protein staining, staining with FOB could be carried out in tank buffer (2 mg/100 mL) and was also reversible. The FOB , perhaps would be the most cost-effective fluorescent dye to stain the proteins (US $ 0.04/25.0 g). The FOB was found to be simple, safe, sensitive, less time consuming and economical fluorescent dye as an alternative, for staining proteins on polyacrylamide gels.

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

Journal of Environmental Radioactivity, 2015

An ecofriendly green chemistry method using a natural biopolymer, Gum Kondagogu (GK) for the remo... more 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.

Mycotoxin Research, 2010

The effect of surfactants (two cationic, one anionic and three non-ionic) at 0.001, 0.01, 0.1 and... more The effect of surfactants (two cationic, one anionic and three non-ionic) at 0.001, 0.01, 0.1 and 1.0 % concentrations on aflatoxin production, ergosterol content and sugar consumption by Aspergillus parasiticus (NRRL 2999) in YES liquid culture medium is reported. At 0.01% concentration, the cationic surfactants, cetyl dimethyl ammonium bromide (CDAB) and dodecyl trimethyl ammonium bromide (DTAB), and the anionic surfactant, sodium dodecyl sulfate (SDS), completely inhibited spore germination, while DTAB also inhibited the production of ergosterol and toxin (p p-tert-octylphenol (Triton X-100) delayed the spore germination up to day 5 at all concentrations and inhibited toxin and ergosterol production at 0.001% concentration. The affect was found to be dose-dependent from 0.001% to 1%, for Triton X-100 only. Positive correlation between ergosterol content and toxin production in the presence of different surfactants at various time periods (3, 5, 7, 9 and 12 days) was found. Tween-20 was most effective in inhibiting toxin production on day 7, when aflatoxin production was found to be maximal in control group. Sugar consumption was directly proportional to the ergosterol content, showing a significant correlation with aflatoxin production.

A simple thin-layer chromatography-digital image-based analytical method has been developed for t... more A simple thin-layer chromatography-digital image-based analytical method has been developed for the quantitation of the botanical pesticide, azadirachtin. The method was validated by analyzing azadirachtin in the spiked food matrixes and processed commercial pesticide formulations, using acidified vanillin reagent as a postchromatographic derivatizing agent. The separated azadirachtin was clearly identified as a green spot. The R f value was found to be 0.55, which was similar to that of a reference standard. A standard calibration plot was established using a reference standard, based on the linear regression analysis [r 2 = 0.996; y = 371.43 + (634.82)x]. The sensitivity of the method was found to be 0.875 mg azadirachtin. Spiking studies conducted at the 1 ppm (mg/g) level in various agricultural matrixes, such as brinjal, tomato, coffee, and cotton seeds, revealed the recoveries of azadirachtin in the range of 67-92%. Azadirachtin content of commercial neem formulations analyzed by the method was in the range of 190-1825 ppm (mg/mL). Further, the present method was compared with an immunoanalytical method enzyme-linked immonosorbent assay developed earlier in our laboratory. Statistical comparison of the 2 methods, using Fischer's F-test, indicated no significant difference in variance, suggesting that both methods are comparable.

The effect of surfactants (two cationic, one anionic and three non-ionic) at 0.001, 0.01, 0.1 and... more The effect of surfactants (two cationic, one anionic and three non-ionic) at 0.001, 0.01, 0.1 and 1.0 % concentrations on aflatoxin production, ergosterol content and sugar consumption by Aspergillus parasiticus (NRRL 2999) in YES liquid culture medium is reported. At 0.01% concentration, the cationic surfactants, cetyl dimethyl ammonium bromide (CDAB) and dodecyl trimethyl ammonium bromide (DTAB), and the anionic surfactant, sodium dodecyl sulfate (SDS), completely inhibited spore germination, while DTAB also inhibited the production of ergosterol and toxin (p<0.05). At a concentration of 0.001%, CDAB was found to enhance toxin production, while SDS was found to inhibit it when compared with other surfactants. Non-ionic surfactants, polyoxyethylene sorbitan monolaurate (Tween-20), polyoxyethylene lauryl ether (Brij-35) and ethoxylated p-tert-octylphenol (Triton X-100) delayed the spore germination up to day 5 at all concentrations and inhibited toxin and ergosterol production at 0.001% concentration. The affect was found to be dose-dependent from 0.001% to 1%, for Triton X-100 only. Positive correlation between ergosterol content and toxin production in the presence of different surfactants at various time periods (3, 5, 7, 9 and 12 days) was found. Tween-20 was most effective in inhibiting toxin production on day 7, when aflatoxin production was found to be maximal in control group. Sugar consumption was directly proportional to the ergosterol content, showing a significant correlation with aflatoxin production.

A simple and sensitive indirect noncompetitive enzyme immunoassay to quantitate mercapturic acid-... more A simple and sensitive indirect noncompetitive enzyme immunoassay to quantitate mercapturic acid-aflatoxin B 1 (AFB 1 ) adduct in rat urine is reported. A novel procedure was developed for in vitro synthesis of an immunogen, bovine serum albumen-glutathione-aflatoxin B 1 (BSA-GSH-AFB 1 ) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. Sulphydryl group's analysis confirmed the conjugation of -SH groups to AFB 1 . Thin-layer chromatography and spectral analysis (absorption, fluorescence, and Fourier transform infrared) of the conjugates further confirmed the formation of the adducts. Polyclonal antibodies specific to mercapturic acid-AFB 1 adduct were produced against BSA-GSH-AFB 1 . The assay was found to be linear in the range of 100 pg-100 ng of the analyte (y = a + bx). A 50% displacement of BSA-GSH-AFB 1 antibodies was achieved at an inhibitory concentration (IC 50 ) of 11.9 ng GSH-AFB 1 (r 2 = 0.98) and 1.22 ng N-acetyl-L-cysteine (NAC)-AFB 1 (r 2 = 0.98). Spiking 5 mg/mL of reference standard to the control rat urine showed a recovery of 98 ± 2%. The immunoassay was validated in a rodent model exposed to a single oral dose of 1 mg/kg body mass of pure AFB 1 . The excretion of NAC-AFB 1 adduct was quantitated at the end of 24 h. The concentration of the NAC-AFB 1 adduct excreted in urine as determined by the immunoassay was found to be in the range of 3.22-5.97 mg/mg creatinine. The present method may find wide application as a biochemical tool in molecular epidemiological and intervention studies with respect to human exposure to dietary aflatoxins.

Neem seed kernels artificially infested with Aspergillus parasiticus (NRRL 2999) was evaluated fo... more Neem seed kernels artificially infested with Aspergillus parasiticus (NRRL 2999) was evaluated for aflatoxin elaboration and fungal growth, and compared with groundnut, a high risk commodity for aflatoxin contamination. At optimal moisture content (10-12%) the total, individual toxins (AFB 1 , AFB 2 , AFG 1 and AFG 2 ) and ergosterol content increased and showed maximum levels on day 9. Crude protein and polyphenols increased while, fat and total sugar content decreased during the period of infection. The protein content correlated positively (r = 0.734) with total toxin levels, whereas fat content (r = -0.761) and total sugars (r = -0.891) showed negative correlation and they were all statistically significant (p<0.01). The polyphenols showed negative and non-significant correlation with total toxin levels. Azadirachtin one of the major active principles of neem seed kernel showed significant decrease on day 3 (P<0.05) and day 6 (P<0.01). Neem seed kernel has shown 54 and 74% less aflatoxin production on day 9 and 12, respectively in comparison to groundnut seeds. Ergosterol content also showed 60% decrease on day 9, conferring it a poor substrate for fungal growth and aflatoxin elaboration.

Purpose: The aim of this investigation was to exploit lens-specific glycated crystallins as an im... more 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]+α, HMW+α-glycated, β, β-glycated, γ, and γ-glycated) as antigens. The cross-reactivity of these lens specific antibodies against rat β-, β-glycated, γ-, and γ-glycated lens crystallins was also analyzed. A noncompetitive enzyme linked immunosorbent assay (ELISA) methodology was developed for the detection of circulating lens crystallins in human sera using HMW+α, HMW+α-glycated, β-, and β-glycated crystallins from humans and γ-and γ-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 γ-and β-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+α (16.45 ng), (iii) human HMW+α-glycated (273 ng), (iv) human β-(37.82 ng), (v) human β-glycated (260 ng), (vi) rat γ-(105.34 ng), and (vii) rat γ-glycated (313 ng). The immunochemical analysis of human serum indicated a significant change (p<0.001) in the levels of circulating β-glycated and γ-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 +α-glycated crystallins in the age group of 40-80 years as compared to their age-matched controls. Notably, the levels of serum γ-glycated crystallins were found to be threefold higher than that of HMW+α-glycated and β-glycated crystallins in the age group of 70-80 years. Circulating autoantibodies to HMW+α-glycated, β-glycated, and γ-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 γ-glycated crystallins were found to be twofold and 3.2 fold higher as compared to the levels of autoantibodies to β-glycated and HMW+α-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+α, HMW+α-glycated, β, β-glycated, γ, and γ-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 lensspecific 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.

TLC-Digital Image-Based Fluorometric Analysis of Ergosterol and Chitin Content in Food Grains Artificially Infested with Aspergillus flavus and Fusarium verticillioides

Food Analytical Methods

Immuno‐analytical Method Development for Detection of Transgenic Cry1Ac Protein and Its Validation

Journal of the Science of Food and Agriculture

Biochemistry & Analytical Biochemistry, 2016

A commercial fabric fluorescent optical brightener Ranipal  (F-OB), has been successfully employ... more A commercial fabric fluorescent optical brightener Ranipal  (F-OB), has been successfully employed to stain proteins on native and SDS-1D-PAGE. The FOB was purified by using a biphasic solvent system of dichloromethane and water. The R f value (0.63) of purified and crude FOB were comparable on TLC. The mass spectrometry of purified FOB indicated base peak at 414 (m/z). Absorption and emission maxima of FOB were found to be 350 nm and 430 nm, respectively. The FOB could stain the proteins both pre-and post-electrophoretic run, on native gel. Postelectrophoretic staining was rapid and required 20 min to visualize the stained proteins. On the other hand, in SDS gels, an additional 20 min was required for the extraction of SDS before staining the proteins with FOB. SDS was found to interfere with binding of FOB to proteins. Varying concentrations of molecular weight markers were loaded and their fluorescent intensity was plotted against the concentration of the proteins. The r 2 values ranged from 0.965 to 0.997 indicating excellent linearity. The detection for carbonic anhydrase was in the range of 8.0-800 ng. Unlike most of the dyes used for protein staining, staining with FOB could be carried out in tank buffer (2 mg/100 mL) and was also reversible. The FOB , perhaps would be the most cost-effective fluorescent dye to stain the proteins (US $ 0.04/25.0 g). The FOB was found to be simple, safe, sensitive, less time consuming and economical fluorescent dye as an alternative, for staining proteins on polyacrylamide gels.

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

Journal of Environmental Radioactivity, 2015

An ecofriendly green chemistry method using a natural biopolymer, Gum Kondagogu (GK) for the remo... more 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.

Mycotoxin Research, 2010

The effect of surfactants (two cationic, one anionic and three non-ionic) at 0.001, 0.01, 0.1 and... more The effect of surfactants (two cationic, one anionic and three non-ionic) at 0.001, 0.01, 0.1 and 1.0 % concentrations on aflatoxin production, ergosterol content and sugar consumption by Aspergillus parasiticus (NRRL 2999) in YES liquid culture medium is reported. At 0.01% concentration, the cationic surfactants, cetyl dimethyl ammonium bromide (CDAB) and dodecyl trimethyl ammonium bromide (DTAB), and the anionic surfactant, sodium dodecyl sulfate (SDS), completely inhibited spore germination, while DTAB also inhibited the production of ergosterol and toxin (p p-tert-octylphenol (Triton X-100) delayed the spore germination up to day 5 at all concentrations and inhibited toxin and ergosterol production at 0.001% concentration. The affect was found to be dose-dependent from 0.001% to 1%, for Triton X-100 only. Positive correlation between ergosterol content and toxin production in the presence of different surfactants at various time periods (3, 5, 7, 9 and 12 days) was found. Tween-20 was most effective in inhibiting toxin production on day 7, when aflatoxin production was found to be maximal in control group. Sugar consumption was directly proportional to the ergosterol content, showing a significant correlation with aflatoxin production.

A simple thin-layer chromatography-digital image-based analytical method has been developed for t... more A simple thin-layer chromatography-digital image-based analytical method has been developed for the quantitation of the botanical pesticide, azadirachtin. The method was validated by analyzing azadirachtin in the spiked food matrixes and processed commercial pesticide formulations, using acidified vanillin reagent as a postchromatographic derivatizing agent. The separated azadirachtin was clearly identified as a green spot. The R f value was found to be 0.55, which was similar to that of a reference standard. A standard calibration plot was established using a reference standard, based on the linear regression analysis [r 2 = 0.996; y = 371.43 + (634.82)x]. The sensitivity of the method was found to be 0.875 mg azadirachtin. Spiking studies conducted at the 1 ppm (mg/g) level in various agricultural matrixes, such as brinjal, tomato, coffee, and cotton seeds, revealed the recoveries of azadirachtin in the range of 67-92%. Azadirachtin content of commercial neem formulations analyzed by the method was in the range of 190-1825 ppm (mg/mL). Further, the present method was compared with an immunoanalytical method enzyme-linked immonosorbent assay developed earlier in our laboratory. Statistical comparison of the 2 methods, using Fischer's F-test, indicated no significant difference in variance, suggesting that both methods are comparable.

The effect of surfactants (two cationic, one anionic and three non-ionic) at 0.001, 0.01, 0.1 and... more The effect of surfactants (two cationic, one anionic and three non-ionic) at 0.001, 0.01, 0.1 and 1.0 % concentrations on aflatoxin production, ergosterol content and sugar consumption by Aspergillus parasiticus (NRRL 2999) in YES liquid culture medium is reported. At 0.01% concentration, the cationic surfactants, cetyl dimethyl ammonium bromide (CDAB) and dodecyl trimethyl ammonium bromide (DTAB), and the anionic surfactant, sodium dodecyl sulfate (SDS), completely inhibited spore germination, while DTAB also inhibited the production of ergosterol and toxin (p<0.05). At a concentration of 0.001%, CDAB was found to enhance toxin production, while SDS was found to inhibit it when compared with other surfactants. Non-ionic surfactants, polyoxyethylene sorbitan monolaurate (Tween-20), polyoxyethylene lauryl ether (Brij-35) and ethoxylated p-tert-octylphenol (Triton X-100) delayed the spore germination up to day 5 at all concentrations and inhibited toxin and ergosterol production at 0.001% concentration. The affect was found to be dose-dependent from 0.001% to 1%, for Triton X-100 only. Positive correlation between ergosterol content and toxin production in the presence of different surfactants at various time periods (3, 5, 7, 9 and 12 days) was found. Tween-20 was most effective in inhibiting toxin production on day 7, when aflatoxin production was found to be maximal in control group. Sugar consumption was directly proportional to the ergosterol content, showing a significant correlation with aflatoxin production.

A simple and sensitive indirect noncompetitive enzyme immunoassay to quantitate mercapturic acid-... more A simple and sensitive indirect noncompetitive enzyme immunoassay to quantitate mercapturic acid-aflatoxin B 1 (AFB 1 ) adduct in rat urine is reported. A novel procedure was developed for in vitro synthesis of an immunogen, bovine serum albumen-glutathione-aflatoxin B 1 (BSA-GSH-AFB 1 ) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. Sulphydryl group's analysis confirmed the conjugation of -SH groups to AFB 1 . Thin-layer chromatography and spectral analysis (absorption, fluorescence, and Fourier transform infrared) of the conjugates further confirmed the formation of the adducts. Polyclonal antibodies specific to mercapturic acid-AFB 1 adduct were produced against BSA-GSH-AFB 1 . The assay was found to be linear in the range of 100 pg-100 ng of the analyte (y = a + bx). A 50% displacement of BSA-GSH-AFB 1 antibodies was achieved at an inhibitory concentration (IC 50 ) of 11.9 ng GSH-AFB 1 (r 2 = 0.98) and 1.22 ng N-acetyl-L-cysteine (NAC)-AFB 1 (r 2 = 0.98). Spiking 5 mg/mL of reference standard to the control rat urine showed a recovery of 98 ± 2%. The immunoassay was validated in a rodent model exposed to a single oral dose of 1 mg/kg body mass of pure AFB 1 . The excretion of NAC-AFB 1 adduct was quantitated at the end of 24 h. The concentration of the NAC-AFB 1 adduct excreted in urine as determined by the immunoassay was found to be in the range of 3.22-5.97 mg/mg creatinine. The present method may find wide application as a biochemical tool in molecular epidemiological and intervention studies with respect to human exposure to dietary aflatoxins.

Neem seed kernels artificially infested with Aspergillus parasiticus (NRRL 2999) was evaluated fo... more Neem seed kernels artificially infested with Aspergillus parasiticus (NRRL 2999) was evaluated for aflatoxin elaboration and fungal growth, and compared with groundnut, a high risk commodity for aflatoxin contamination. At optimal moisture content (10-12%) the total, individual toxins (AFB 1 , AFB 2 , AFG 1 and AFG 2 ) and ergosterol content increased and showed maximum levels on day 9. Crude protein and polyphenols increased while, fat and total sugar content decreased during the period of infection. The protein content correlated positively (r = 0.734) with total toxin levels, whereas fat content (r = -0.761) and total sugars (r = -0.891) showed negative correlation and they were all statistically significant (p<0.01). The polyphenols showed negative and non-significant correlation with total toxin levels. Azadirachtin one of the major active principles of neem seed kernel showed significant decrease on day 3 (P<0.05) and day 6 (P<0.01). Neem seed kernel has shown 54 and 74% less aflatoxin production on day 9 and 12, respectively in comparison to groundnut seeds. Ergosterol content also showed 60% decrease on day 9, conferring it a poor substrate for fungal growth and aflatoxin elaboration.

Purpose: The aim of this investigation was to exploit lens-specific glycated crystallins as an im... more 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]+α, HMW+α-glycated, β, β-glycated, γ, and γ-glycated) as antigens. The cross-reactivity of these lens specific antibodies against rat β-, β-glycated, γ-, and γ-glycated lens crystallins was also analyzed. A noncompetitive enzyme linked immunosorbent assay (ELISA) methodology was developed for the detection of circulating lens crystallins in human sera using HMW+α, HMW+α-glycated, β-, and β-glycated crystallins from humans and γ-and γ-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 γ-and β-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+α (16.45 ng), (iii) human HMW+α-glycated (273 ng), (iv) human β-(37.82 ng), (v) human β-glycated (260 ng), (vi) rat γ-(105.34 ng), and (vii) rat γ-glycated (313 ng). The immunochemical analysis of human serum indicated a significant change (p<0.001) in the levels of circulating β-glycated and γ-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 +α-glycated crystallins in the age group of 40-80 years as compared to their age-matched controls. Notably, the levels of serum γ-glycated crystallins were found to be threefold higher than that of HMW+α-glycated and β-glycated crystallins in the age group of 70-80 years. Circulating autoantibodies to HMW+α-glycated, β-glycated, and γ-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 γ-glycated crystallins were found to be twofold and 3.2 fold higher as compared to the levels of autoantibodies to β-glycated and HMW+α-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+α, HMW+α-glycated, β, β-glycated, γ, and γ-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 lensspecific 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.