BACKGROUND
in autoimmune diabetes the autoantibodies have always been important for clinical interest due to their potential role in screening, diagnosis, monitoring treatment of effectiveness and prognosis.
the gad65abs are often considered to be an epiphenomenon resulting from the autoimmune destruction of the pancreatic beta cells in t1d.
previous studies suggest that they are involved in antigen processing and presentation and thus modulate the immune response  <cit> .
because of the high diagnostic sensitivity for autoimmune diabetes, the presence of gad65ab is currently used to identify subjects at high risk for the disease  <cit> .
gad65abs are detected in about 60% of new-onset cases of type  <dig> diabetes  <cit> , and high levels of these autoantibodies were also reported in diabetic patients with secondary complications , thus leading cause of blindness and renal failure  <cit> .
the exact etiology behind these complications is not completely clear.
in our recent study; ros modified gad <dig> was found to be more immunogenic in t1d than its native form  <cit> .
gad65abs in t1d are predominantly directed at conformational epitopes located in the middle region of the molecule, whereas they also recognize linear epitopes and epitopes located in the middle, cooh- and nh2-terminuses  <cit> .
shifts in gad <dig> epitopes were detected in a subgroup of newly diagnosed children within the first  <dig> months after disease onset  <cit> .
moreover, epitope spreading has gained credence as a major driver underlying autoimmunity  <cit> .
growing evidence suggests that ros plays an important role in the initiation and progression of diabetes and its associated complications  <cit> .
these increased levels of free radicals pose a direct toxic effect on gad <dig> and increase its immunogenicity  <cit> .
specificity of autoantibodies for epitopes on gad <dig> and their levels may be a better indicator of impending or actual destruction of islet β-cells and increasing complications associated with diabetes.
in the view of the above mentioned studies we hypothesized some possible link between diabetic associated complications and presence of ros-gad65abs.
to prove this, binding characteristics of serum autoantibodies from uncomplicated and complicated  t1d patients were assessed with n-gad <dig> and ros-gad <dig> by direct binding and competitive elisa.
the avidity of modified gad <dig> was also evaluated by precipitate titration curve in different diabetic groups.
RESULTS
ros modification of gad65
ros directed modification of gad <dig> studied previously by our group showed marked structural changes  <cit> .
khan et al., demonstrated that hyperchromicity and tryptophan specific fluorescence for modified gad <dig> was found to be significantly higher than native gad <dig> and the spectral analysis also showed blue shift of  <dig> nm in modified gad <dig> over native gad <dig>  far-uv-cd spectropolarimetry of ros-gad <dig> exhibited significant changes in secondary structural elements compared to its unmodified form decrease in α-helix and an increase of in β-sheet, random coil and turns was observed  <cit> .
detection of autoantibodies against n-gad <dig> and ros-gad65
in a pilot study serum samples from uncomplicated and complicated t1d patients were screened for autoantibodies against n-gad <dig>  and ros-gad <dig>  using unmodified and ros-modified gad <dig> as antigens.
from figure  <dig>  we observed that sera from normal human  subjects showed very low level of reactivity towards n-gad <dig>  or ros-gad <dig> .
conversely, serum autoantibodies of uncomplicated t1d patients showed significant binding with ros-gad <dig>  as compared to n-gad <dig> .
moreover, sera from diabetic nephropathic  and diabetic retinopathic  patients exhibited statistically higher significant differences in the binding pattern of serum autoantibodies with ros-gad <dig> as compared to n-gad <dig> .
od was considered as a direct measure of the concentrations of autoantibodies in the serum samples.
in all the study groups there were higher levels of reactivity of modified antigen as compared to its native form.
nh samples showed 14% increased and uncomplicated serum samples exhibited  <dig> % increased reactivity towards ros-gad <dig> as compared to n-gad <dig>  whereas, nephropathic and retinopathic t1d complicated subjects showed an increase of  <dig> % and 100% respectively, in the reactivity with modified antigen when compared with unmodified antigen.
the binding specificities of serum autoantibodies from uncomplicated and complicated  t1d patients were evaluated by competitive elisa using n-gad <dig> and ros-gad <dig> as inhibitors.
significantly higher recognition of modified antigen was observed by the serum autoantibodies from nephropathic  and retinopathic patients .
n-gad <dig> exhibited no marked difference in recognition of serum igg from both complicated  and uncomplicated  subjects of t1d, table  <dig>  normal humans showed very less or negligible percent inhibition with either of the antigens .
n-gad <dig> and m-gad <dig> represents native gad <dig> and modified gad <dig>
nh represents normal humans as control.
the elisa plates were coated with n-gad <dig> and m-gad <dig> .
ros-gad <dig> and n-gad <dig> were used as inhibitor.
* p <  <dig>  vs ros-gad65abs in uncomplicated t1d.
estimation of protein bound carbonyl compounds in serum samples
in vivo carbonyl content was considered a biomarker of oxidative stress.
oxidative stress levels were estimated for every patient of each group of t1d .
data showed significant increase in serum protein bound carbonyl contents  in complicated subjects as compared to uncomplicated t1d patients.
complicated subjects, t1d who had retinopathy  exhibited higher amounts of protein bound carbonyl content as compared to nephropathic  t1d patients.
data are means ± sd or n represents number of smokers from given total respective subjects.
for blood glucose estimations, blood was collected in oxalated fluoride containers and the assays were performed immediately.
hypertension is defined as sitting systolic blood pressure ≥  <dig> mmhg and/or diastolic blood pressure ≥  <dig> mmhg or the use of antihypertensive medication.
signs * and # represents p values <  <dig>  and <  <dig>  respectively.
quantification of apparent association constant
the amount of antigen bound to antibody was also evaluated by quantitative precipitin titration curve.
igg of uncomplicated  and complicated  subjects was purified by affinity chromatography on protein a-agarose column.
the purified iggs were found to elute in a single symmetrical peak.
varying amounts of modified gad <dig>  were mixed with  <dig> μg of patient's igg and incubated for  <dig> h at 37°c and overnight at 4°c.
microsurface adsorption-spectral correction  technique showed the interaction of igg with modified protein.
langmuir equation was used to estimate aac of complicated and uncomplicated t1d samples for ros-gad <dig>  and was computed to be  <dig>  × 10- <dig> m and  <dig>  × 10- <dig> m for t1d nephropathic and retinopathic patients respectively.
uncomplicated t1d showed  <dig>  × 10- <dig> m aac.
a maximum of  <dig> μg and  <dig> μg of ros-gad <dig> was bound to  <dig> μg and  <dig> μg of igg from t1d nephropathic and retinopathic subjects respectively.
however uncomplicated t1d patients exhibited  <dig> μg of igg bound with  <dig> μg of ros-gad <dig>  no appreciable differences were observed in the aacs calculated for n-gad <dig> in same above mentioned serum samples of complicated  and uncomplicated t1d patients  as shown in figure  <dig>
discussion
the levels of ros increase many folds during t1d via formation of sorbitol by polyol pathways, regeneration of cofactors nadph and nad+ by nadh oxidase and glycation reactions  <cit> .
long term association of proteins with high concentrations of hydrogen peroxide and free radical intermediates results in protein modification both at the amino acid and protein levels  <cit> .
this statement is supported by our previous findings based on the structural characterization of native and ros-gad <dig>   <cit> .
significant recognition of ros-gad <dig> by serum autoantibodies of complicated t1d patients was estimated when compared with autoantibodies from uncomplicated t1d subjects.
n-gad <dig> did not show any marked differences in recognitions of circulatory autoantibodies from complicated and uncomplicated t1d subjects.
the affinity of gad65abs was found to be higher in subjects who had developed t1d with neuropathy  <cit>  or autoimmune polyendocrine disorders  <cit> .
in control normal humans, negligible binding was observed with either of the antigens.
from the cohort, twenty serum samples each from complicated  and uncomplicated t1d patients were selected and binding affinities of circulating autoantibodies with n-gad <dig> and ros-gad <dig> were ascertained by competitive elisa.
twenty serum samples from normal humans were taken under the same experimental condition as controls.
a characteristic difference was observed in the pattern of inhibition elisa assays obtained from complicated and uncomplicated t1d subjects in respect to ros-gad <dig> antigen.
this indicates that the ros-modified gad <dig> is an effective inhibitor showing substantial higher titres of circulating autoantibodies in complicated t1d subjects as compared to uncomplicated t1d.
moreover; amongst the complicated patients, retinopathic showed highest recognition for ros gad <dig> as compared to nephropathic and uncomplicated patients.
further the study also elucidates that with increased duration of disease and poor glycemic control leads to increased oxidative stress and hence the complications.
the oxidative stress was further ascertained by the levels of protein bound carbonyl content in patients which is a biomarker of protein oxidation  <cit> .
type  <dig> diabetes retinopathic patients had highest carbonyl content followed by nephropathic and uncomplicated respectively.
possible this heightened state of oxidative stress leads to extensive in vivo gad <dig> antigen modifications.
thus gad <dig> of complicated t1d patients presents more number of epitopes that resemble in vitro ros modified gad <dig> and conceivably generates significant number of autoantibodies.
gad65ab titers are higher and more prevalent in patients with other associated autoimmune diseases such as thyroiditis  <cit> .
the strong dependence of conformation of protein for autoantibody recognition, blocking experiments  <cit>  and recombinant fab using monoclonal antibodies  <cit>  has been useful for determining conformational gad65ab epitopes.
during immune-pathophysiology significant amounts of circulating immune complexes are formed and deposited in kidneys leading to diabetic nephropathy, retinopathy and other tissues causing severe injury  <cit> .
as we discussed in this study continuous long durations of increased levels of ros cause increase in antigenic determinants on gad <dig>  so, the avidity of gad <dig> became more complex and gain increased strength of binding because of interdependency of epitopes.
figure  <dig> and  <dig> clearly indicate better recognition of ros-gad <dig> than n-gad <dig> by igg isolated from retinopathic t1d subjects followed by nephropathic and uncomplicated t1d patient.
the enhanced recognition of ros-gad <dig> by retinopathic t1d patient igg showed the possible participation of oxidative stress and long duration of disease as given in table  <dig> that might have role in in vivo modification of gad <dig> inducing the molecule to express its cryptic epitopes.
CONCLUSIONS
in conclusion, significantly high levels of circulating ros-gad65abs were detected in complicated  as compared to uncomplicated t1d patients.
this risk of the disease may be exemplified due to acceleration in the formation of free radicals with gradual increase in duration of disease.
this leads to conformational alterations in n-gad <dig> protein which could increase or expose cryptic epitopes.
dynamic changes in the gad65abs binding pattern suggest subsequent epitopes spreading with disease progression.
this could be one of the etiologies of increased gad65ab immunogenicity that implicated in t1d complications.
measurement of these autoantibodies could be shown to be useful in assisting the prediction for the development of t1d progression/or complications.
reduction in the levels of ros may lead to decrease in in vivo gad <dig> molecules modification thus, leads to delay in the progression of complications.
hence antioxidants may play important role in the treatment.

 in autoimmune diabetes the autoantibodies have always been important for clinical interest due to their potential role in screening, diagnosis, monitoring treatment of effectiveness and prognosis. the gad65abs are often considered to be an epiphenomenon resulting from the autoimmune destruction of the pancreatic beta cells in t1d. previous studies suggest that they are involved in antigen processing and presentation and thus modulate the immune response. because of the high diagnostic sensitivity for autoimmune diabetes, the presence of gad65ab is currently used to identify subjects at high risk for the disease. gad65abs are detected in about 60% of new-onset cases of type diabetes, and high levels of these autoantibodies were also reported in diabetic patients with secondary complications, thus leading cause of blindness and renal failure. the exact etiology behind these complications is not completely clear. in our recent study; ros modified gad was found to be more immunogenic in t1d than its native form. gad65abs in t1d are predominantly directed at conformational epitopes located in the middle region of the molecule, whereas they also recognize linear epitopes and epitopes located in the middle, cooh- and nh2-terminuses. shifts in gad epitopes were detected in a subgroup of newly diagnosed children within the first months after disease onset. moreover, epitope spreading has gained credence as a major driver underlying autoimmunity. growing evidence suggests that ros plays an important role in the initiation and progression of diabetes and its associated complications. these increased levels of free radicals pose a direct toxic effect on gad and increase its immunogenicity. specificity of autoantibodies for epitopes on gad and their levels may be a better indicator of impending or actual destruction of islet β-cells and increasing complications associated with diabetes. in the view of the above mentioned studies we hypothesized some possible link between diabetic associated complications and presence of ros-gad65abs. to prove this, binding characteristics of serum autoantibodies from uncomplicated and complicated t1d patients were assessed with n-gad and ros-gad by direct binding and competitive elisa. the avidity of modified gad was also evaluated by precipitate titration curve in different diabetic groups.  ros modification of gad65 ros directed modification of gad studied previously by our group showed marked structural changes. khan et al., demonstrated that hyperchromicity and tryptophan specific fluorescence for modified gad was found to be significantly higher than native gad and the spectral analysis also showed blue shift of nm in modified gad over native gad far-uv-cd spectropolarimetry of ros-gad exhibited significant changes in secondary structural elements compared to its unmodified form decrease in α-helix and an increase of in β-sheet, random coil and turns was observed. detection of autoantibodies against n-gad and ros-gad65 in a pilot study serum samples from uncomplicated and complicated t1d patients were screened for autoantibodies against n-gad and ros-gad using unmodified and ros-modified gad as antigens. from figure we observed that sera from normal human subjects showed very low level of reactivity towards n-gad or ros-gad. conversely, serum autoantibodies of uncomplicated t1d patients showed significant binding with ros-gad as compared to n-gad. moreover, sera from diabetic nephropathic and diabetic retinopathic patients exhibited statistically higher significant differences in the binding pattern of serum autoantibodies with ros-gad as compared to n-gad. od was considered as a direct measure of the concentrations of autoantibodies in the serum samples. in all the study groups there were higher levels of reactivity of modified antigen as compared to its native form. nh samples showed 14% increased and uncomplicated serum samples exhibited % increased reactivity towards ros-gad as compared to n-gad whereas, nephropathic and retinopathic t1d complicated subjects showed an increase of % and 100% respectively, in the reactivity with modified antigen when compared with unmodified antigen. the binding specificities of serum autoantibodies from uncomplicated and complicated t1d patients were evaluated by competitive elisa using n-gad and ros-gad as inhibitors. significantly higher recognition of modified antigen was observed by the serum autoantibodies from nephropathic and retinopathic patients. n-gad exhibited no marked difference in recognition of serum igg from both complicated and uncomplicated subjects of t1d, table normal humans showed very less or negligible percent inhibition with either of the antigens. n-gad and m-gad represents native gad and modified gad nh represents normal humans as control. the elisa plates were coated with n-gad and m-gad. ros-gad and n-gad were used as inhibitor. * p < vs ros-gad65abs in uncomplicated t1d. estimation of protein bound carbonyl compounds in serum samples in vivo carbonyl content was considered a biomarker of oxidative stress. oxidative stress levels were estimated for every patient of each group of t1d. data showed significant increase in serum protein bound carbonyl contents in complicated subjects as compared to uncomplicated t1d patients. complicated subjects, t1d who had retinopathy exhibited higher amounts of protein bound carbonyl content as compared to nephropathic t1d patients. data are means ± sd or n represents number of smokers from given total respective subjects. for blood glucose estimations, blood was collected in oxalated fluoride containers and the assays were performed immediately. hypertension is defined as sitting systolic blood pressure ≥ mmhg and/or diastolic blood pressure ≥ mmhg or the use of antihypertensive medication. signs * and # represents p values < and < respectively. quantification of apparent association constant the amount of antigen bound to antibody was also evaluated by quantitative precipitin titration curve. igg of uncomplicated and complicated subjects was purified by affinity chromatography on protein a-agarose column. the purified iggs were found to elute in a single symmetrical peak. varying amounts of modified gad were mixed with μg of patient's igg and incubated for h at 37°c and overnight at 4°c. microsurface adsorption-spectral correction technique showed the interaction of igg with modified protein. langmuir equation was used to estimate aac of complicated and uncomplicated t1d samples for ros-gad and was computed to be × 10- m and × 10- m for t1d nephropathic and retinopathic patients respectively. uncomplicated t1d showed × 10- m aac. a maximum of μg and μg of ros-gad was bound to μg and μg of igg from t1d nephropathic and retinopathic subjects respectively. however uncomplicated t1d patients exhibited μg of igg bound with μg of ros-gad no appreciable differences were observed in the aacs calculated for n-gad in same above mentioned serum samples of complicated and uncomplicated t1d patients as shown in figure discussion the levels of ros increase many folds during t1d via formation of sorbitol by polyol pathways, regeneration of cofactors nadph and nad+ by nadh oxidase and glycation reactions. long term association of proteins with high concentrations of hydrogen peroxide and free radical intermediates results in protein modification both at the amino acid and protein levels. this statement is supported by our previous findings based on the structural characterization of native and ros-gad. significant recognition of ros-gad by serum autoantibodies of complicated t1d patients was estimated when compared with autoantibodies from uncomplicated t1d subjects. n-gad did not show any marked differences in recognitions of circulatory autoantibodies from complicated and uncomplicated t1d subjects. the affinity of gad65abs was found to be higher in subjects who had developed t1d with neuropathy or autoimmune polyendocrine disorders. in control normal humans, negligible binding was observed with either of the antigens. from the cohort, twenty serum samples each from complicated and uncomplicated t1d patients were selected and binding affinities of circulating autoantibodies with n-gad and ros-gad were ascertained by competitive elisa. twenty serum samples from normal humans were taken under the same experimental condition as controls. a characteristic difference was observed in the pattern of inhibition elisa assays obtained from complicated and uncomplicated t1d subjects in respect to ros-gad antigen. this indicates that the ros-modified gad is an effective inhibitor showing substantial higher titres of circulating autoantibodies in complicated t1d subjects as compared to uncomplicated t1d. moreover; amongst the complicated patients, retinopathic showed highest recognition for ros gad as compared to nephropathic and uncomplicated patients. further the study also elucidates that with increased duration of disease and poor glycemic control leads to increased oxidative stress and hence the complications. the oxidative stress was further ascertained by the levels of protein bound carbonyl content in patients which is a biomarker of protein oxidation. type diabetes retinopathic patients had highest carbonyl content followed by nephropathic and uncomplicated respectively. possible this heightened state of oxidative stress leads to extensive in vivo gad antigen modifications. thus gad of complicated t1d patients presents more number of epitopes that resemble in vitro ros modified gad and conceivably generates significant number of autoantibodies. gad65ab titers are higher and more prevalent in patients with other associated autoimmune diseases such as thyroiditis. the strong dependence of conformation of protein for autoantibody recognition, blocking experiments and recombinant fab using monoclonal antibodies has been useful for determining conformational gad65ab epitopes. during immune-pathophysiology significant amounts of circulating immune complexes are formed and deposited in kidneys leading to diabetic nephropathy, retinopathy and other tissues causing severe injury. as we discussed in this study continuous long durations of increased levels of ros cause increase in antigenic determinants on gad so, the avidity of gad became more complex and gain increased strength of binding because of interdependency of epitopes. figure and clearly indicate better recognition of ros-gad than n-gad by igg isolated from retinopathic t1d subjects followed by nephropathic and uncomplicated t1d patient. the enhanced recognition of ros-gad by retinopathic t1d patient igg showed the possible participation of oxidative stress and long duration of disease as given in table that might have role in in vivo modification of gad inducing the molecule to express its cryptic epitopes.  in conclusion, significantly high levels of circulating ros-gad65abs were detected in complicated as compared to uncomplicated t1d patients. this risk of the disease may be exemplified due to acceleration in the formation of free radicals with gradual increase in duration of disease. this leads to conformational alterations in n-gad protein which could increase or expose cryptic epitopes. dynamic changes in the gad65abs binding pattern suggest subsequent epitopes spreading with disease progression. this could be one of the etiologies of increased gad65ab immunogenicity that implicated in t1d complications. measurement of these autoantibodies could be shown to be useful in assisting the prediction for the development of t1d progression/or complications. reduction in the levels of ros may lead to decrease in in vivo gad molecules modification thus, leads to delay in the progression of complications. hence antioxidants may play important role in the treatment. 
the apparent association constants indicate greater affinity of igg from retinopathic t1d patients followed by nephropathic and uncomplicated t1d patients for ros-gad compared to n-gad these results were further proven by competitive elisa estimations. however, its association in secondary complications of t1d has not yet been ascertained. hence regulation of ros-gad65abs could offer novel tools for analysing and possibly treating t1d complications. uncomplicated t1d subjects also exhibited reactivity towards ros-gad however, this was found to be less as compared to the binding recorded from complicated subjects. increased oxidative stress and blood glucose levels with extended duration of disease in complicated t1d could be responsible for the gradual formation and/or exposing cryptic epitopes on gad that induce increased production of ros-gad65abs. from the cohort of samples, serum autoantibodies from t1d retinopathic and nephropathic patients showed high recognition of ros-gad as compared to native gad.
 autoantibodies against glutamate decarboxylase- are thought to be a major immunological tool involved in pathogenic autoimmunity development in various diseases. gad65abs are a sensitive and specific marker for type diabetes. these autoantibodies can also be found in 6-10% of patients classified with type diabetes, as well as in 1-2% of the healthy population. the latter individuals are at low risk of developing t1d because the prevalence rate of gad65abs is only about %. it has, therefore, been suggested that the antibody binding to gad in these three different gad65ab-positive phenotypes differ with respect to epitope specificity. the specificity of reactive oxygen species modified gad is already well established in the t1d. however, its association in secondary complications of t1d has not yet been ascertained. hence this study focuses on identification of autoantibodies against ros-gad and quantitative assays in t1d associated complications.  from the cohort of samples, serum autoantibodies from t1d retinopathic and nephropathic patients showed high recognition of ros-gad as compared to native gad. uncomplicated t1d subjects also exhibited reactivity towards ros-gad however, this was found to be less as compared to the binding recorded from complicated subjects. these results were further proven by competitive elisa estimations. the apparent association constants indicate greater affinity of igg from retinopathic t1d patients followed by nephropathic and uncomplicated t1d patients for ros-gad compared to n-gad  increased oxidative stress and blood glucose levels with extended duration of disease in complicated t1d could be responsible for the gradual formation and/or exposing cryptic epitopes on gad that induce increased production of ros-gad65abs. hence regulation of ros-gad65abs could offer novel tools for analysing and possibly treating t1d complications. 