since 1993 , daisy has followed two cohorts of young children at increased risk for type 1 diabetes , including a cohort of relatives of type 1 diabetic patients ( siblings and offspring ) and the general population newborn cohort . susceptible hla - dr / dq genotypes identified through screening of > 31,000 newborns at st . briefly , autoantibodies were tested at 9 , 15 , and 24 months and annually thereafter or at their first visit and annually thereafter if the child was enrolled after birth . children who tested autoantibody positive were given an accelerated testing schedule of every 36 months . a total of 47 daisy children who had development of type 1 diabetes and had their first islet autoantibodies measured by age 18 months were included in this study . subjects were tested for miaa , gad65 , ia2 , and znt8 antibodies at each visit . for ecl - iaa , all first autoantibody - positive samples , all previous samples , and yearly follow - up samples until diabetes were tested . the mean age of diabetes onset was 7.7 years ( range , 0.914.4 years ) . we used radioimmunoassays for autoantibodies to insulin , gad65 , ia-2 , and znt8 using methods previously described ( 1,13 ) . the interassay coefficients of variation were 10 and 5% ( n = 50 ) for gaa and ia-2a , respectively . in the 2009 dasp workshop , the sensitivity and specificity were 72 and 99% , respectively , for gaa , and 62 and 99% , respectively , for ia-2a . for znt8a , the interassay coefficient of variation was 10.4% ( n = 15 ) , and the sensitivity and specificity were 62 and 99% , respectively , in the 2009 dasp workshop . the interassay coefficient of variation was 20% ( n = 100 ) at low positive levels , and the sensitivity and specificity for miaa were 48 and 100% , respectively , in the 2009 dasp workshop . the ecl - iaa using meso scale instrumentation and ruthenium - labeled proinsulin is a nonradioactive iaa assay in which bivalent iaa cross - link two  insulin  moieties in a fluid phase . the iaa in serum will link the sulfo - tagged proinsulin to the biotinylated proinsulin , which will be captured on the solid phase of the streptavidin - coated plate . iaa levels from ecl - iaa assay and our current radioassay ( miaa ) were compared in 150 dasp subjects ; the two assays were correlated ( p < 0.0001 ) , but ecl - iaa assay had higher sensitivity ( 68 vs. 56% ) with the same specificity of 99% for both assays ( 12 ) . the iaa , gaa , ia-2 , and znt8 levels were log - transformed for analyses . multiple linear regression was used to evaluate potential predictors of age of diabetes onset , including age at first positive antibody , initial number of positive antibodies , family history , high - risk hla - dr3/4 , ethnicity , and iaa , gad , ia-2 , and znt8 levels ( both initial and mean levels ) in subjects who had their first autoantibody measurements before age 1.5 years and had progression to diabetes ( n = 47 ) . a two - tailed p value with an  level for significance was set at 0.05 . since 1993 , daisy has followed two cohorts of young children at increased risk for type 1 diabetes , including a cohort of relatives of type 1 diabetic patients ( siblings and offspring ) and the general population newborn cohort . susceptible hla - dr / dq genotypes identified through screening of > 31,000 newborns at st . briefly , autoantibodies were tested at 9 , 15 , and 24 months and annually thereafter or at their first visit and annually thereafter if the child was enrolled after birth . children who tested autoantibody positive were given an accelerated testing schedule of every 36 months . a total of 47 daisy children who had development of type 1 diabetes and had their first islet autoantibodies measured by age 18 months were included in this study . subjects were tested for miaa , gad65 , ia2 , and znt8 antibodies at each visit . for ecl - iaa , all first autoantibody - positive samples , all previous samples , and yearly follow - up samples until diabetes were tested . the mean age of diabetes onset was 7.7 years ( range , 0.914.4 years ) . we used radioimmunoassays for autoantibodies to insulin , gad65 , ia-2 , and znt8 using methods previously described ( 1,13 ) . the interassay coefficients of variation were 10 and 5% ( n = 50 ) for gaa and ia-2a , respectively . in the 2009 dasp workshop , the sensitivity and specificity were 72 and 99% , respectively , for gaa , and 62 and 99% , respectively , for ia-2a . for znt8a , the interassay coefficient of variation was 10.4% ( n = 15 ) , and the sensitivity and specificity were 62 and 99% , respectively , in the 2009 dasp workshop . the interassay coefficient of variation was 20% ( n = 100 ) at low positive levels , and the sensitivity and specificity for miaa were 48 and 100% , respectively , in the 2009 dasp workshop . the ecl - iaa using meso scale instrumentation and ruthenium - labeled proinsulin is a nonradioactive iaa assay in which bivalent iaa cross - link two  insulin  moieties in a fluid phase . the iaa in serum will link the sulfo - tagged proinsulin to the biotinylated proinsulin , which will be captured on the solid phase of the streptavidin - coated plate . iaa levels from ecl - iaa assay and our current radioassay ( miaa ) were compared in 150 dasp subjects ; the two assays were correlated ( p < 0.0001 ) , but ecl - iaa assay had higher sensitivity ( 68 vs. 56% ) with the same specificity of 99% for both assays ( 12 ) . the iaa , gaa , ia-2 , and znt8 levels were log - transformed for analyses . negative or zero values were changed to 0.001 . multiple linear regression was used to evaluate potential predictors of age of diabetes onset , including age at first positive antibody , initial number of positive antibodies , family history , high - risk hla - dr3/4 , ethnicity , and iaa , gad , ia-2 , and znt8 levels ( both initial and mean levels ) in subjects who had their first autoantibody measurements before age 1.5 years and had progression to diabetes ( n = 47 ) . a two - tailed p value with an  level for significance was set at 0.05 . all 47 subjects with progression to diabetes were positive for at least one of the four biochemical antibodies ( iaa , gaa , ia-2 , and znt8 ) . all subjects except one were positive for ecl - iaa ( 46 of 47 , 98% ) during follow - up compared with those positive for miaa assay ( 42 of 47 , 89% ) . the four prediabetic individuals who tested negative for miaa but positive for ecl - iaa all had negative miaa levels for multiple samples over time , although they had several positive ecl - iaa titers over time . on their first antibody - positive visit , 28% subjects had one positive antibody , whereas 43% had two , and 29% subjects had three or more positive antibodies , respectively . ecl - iaa was the most frequent antibody ( 94% ) present at the first positive antibody visit , followed by miaa ( 47% ) , gad ( 36% ) , znt8 ( 26% ) , and ia2 ( 11% ) ( p < 0.0001 ) . table 1 demonstrates initial antibody detection in these 47 prediabetic daisy subjects ; ecl - iaa almost always was the first autoantibody detected : 94% ( 44 of 47 ) total ; 21% alone ( by itself ) ; 23% with only miaa ; 19% with another islet autoantibody ( gad or znt8 ) ; and 30% with two or more other antibodies ( miaa , gad , ia-2 , or znt8 ) . among the 46 subjects who were ecl - iaa positive , the ecl - iaa detected onset of islet autoimmunity earlier than any of the four traditional biochemical antibodies in 10 ( 22% ) children ; i.e. , ecl - iaa was the only positive antibody , whereas all four biochemical antibodies measured by standard radioassays were negative . for those with ecl - iaa as the first antibody by itself , ecl - iaa antedated the onset of other islet autoantibodies by a mean of 2.3 years ( range , 0.37.2 years ) . once positive , ecl - iaa titers tend to be relatively stable overall over time ; i.e. , most subjects found to be positive for ecl - iaa will stay positive for ecl - iaa over time , although there are individual variations . initial antibody detection in daisy prediabetic subjects ( n = 47 ) we additionally tested for ecl - iaa in 1,812 antibody - negative daisy subjects and 14 subjects who had development of diabetes with negative biochemical antibodies ( i.e. , all four biochemical antibodies negative ) . ecl - iaa is very specific , and only 5 of 1,812 ( 0.3% ) antibody - negative subjects tested positive for ecl - iaa . table 2 gives the sensitivity , specificity , and positive and negative predictive values for diabetes outcome for this new ecl - iaa assay in the daisy cohort . when looking at these results , one should take into account that these values were calculated in cohorts of children at increased risk for type 1 diabetes , a cohort of relatives of type 1 diabetes patients , and a general population cohort selected for type 1 diabetes high - risk hla - dr / dq genotypes . among 14 antibody - negative daisy subjects with type 1 diabetes , two were positive for ecl - iaa ; i.e. , ecl - iaa picked up an additional 14% of cases of diabetes . we also tested ecl - iaa among remaining nondiabetic antibody - positive daisy subjects . among 32 daisy subjects positive for miaa alone , only nine ( 28% ) were positive for ecl - iaa . however , ecl - iaa was positive in 18 of 22 ( 82% ) daisy subjects who tested positive for miaa and one or more other antibodies ( gad65 , ia2 , or znt8 ) . sensitivity , specificity , and positive and negative predictive values for ecl - iaa in the daisy cohort of children at increased risk for diabetes as previously published , levels of miaa , but not gad or ia-2 autoantibodies levels , can predict age of diagnosis of type 1 diabetes ( 10 ) . in this study , we confirm that higher mean ecl - iaa levels were correlated with faster progression to diabetes from first appearance of islet autoantibodies ( p = 0.02 ) , whereas levels of znt8 , gad , or ia-2 autoantibodies did not correlate with time to progression to diabetes ( fig . 1 ) . age of onset of diabetes correlates with ecl - iaa levels , but not with znt8 , gad , or ia-2 levels . analysis performed for all subjects who had their first antibody measurement before 1.5 years of age and who had progression to diabetes ( n = 47 ) . antibody levels were log10-transformed for analyses . negative or zero values were changed to 0.001 ( which corresponds to 3 on log scale ) . time to diabetes ( years ) is from first positive antibody visit ( any of the 5 antibodies ) . multiple linear regression was used to evaluate potential predictors of age of diabetes onset , including age at first positive antibody , initial number of positive antibodies , family history , high - risk hla - dr3/4 , ethnicity , and iaa , gad , ia-2 , and znt8 levels ( both initial and mean antibody levels ) . for the first model including initial antibody levels , only initial miaa levels and age at first positive antibody were significant predictors of diabetes onset age ( fig . when analyzing the mean levels of autoantibodies over time for each subject , mean ecl - iaa levels and age of the first positive antibody were significant predictors of age of diabetes diagnosis ( fig . multiple linear regression was used to evaluate potential predictors of age of diabetes onset , including age at first positive antibody ( ab ) , initial number of positive antibodies , family history , high - risk hla - dr3/4 , ethnicity , and iaa , gad , ia-2 , and znt8 levels ( both initial and mean antibody levels ) . for the first model including initial antibody levels , only initial miaa levels ( loginitialmiaa ) and age at first positive antibody ( agefirstab+ ) were significant predictors of diabetes onset age ( a ) . when analyzing mean antibody levels ( b ) , diabetes onset age was best predicted by mean ecl - iaa levels ( logmeanecl - iaa ) and age at first positive antibody ( agefirstab+ ) . in the prospective daisy cohort , ecl - iaa antedated the onset of islet autoimmunity by a mean of 2.3 years in the 22% children initially positive only for ecl - iaa . results from this study indicate that this novel ecl - iaa assay not only is more sensitive but also defines the timing of the initial autoantibody appearance earlier than the previously used miaa radioassay . this earlier detection in timing of onset of islet autoimmunity is of importance in finding potential environmental causes of diabetes and in our understanding of the etiology of type 1 diabetes . however , assays obviating the use of radioactive isotopes also would help to disseminate islet autoantibody testing possibly to point of care . finally , this novel nonradioactive iaa assay seems to be more disease specific ; ecl - iaa is typically positive when two or more autoantibodies are present ( i.e. , subjects at high risk for development of diabetes ) , whereas the majority of miaa - positive - only ( single antibody  positive ) nondiabetic subjects are ecl - iaa negative and these iaa are demonstrated as low - affinity antibodies . these factors are critical in moving from prevention trials in first - degree relatives to the general population , in which 90% of type 1 diabetes cases are found . several studies now have shown that once multiple autoantibodies appear ( two or more autoantibodies ) , almost all these young children have progression to diabetes , although some progression will take > 10 years ( 10,14,15 ) . in children expressing two or more autoantibodies , this progression to diabetes is not influenced by family history , and hla - dr3/4-dqb1 * 0302 genotype is only an additional predictor of progression to diabetes in children expressing one or two autoantibodies but not among patients expressing three autoantibodies ( 10 ) . however , factors influencing rate of progression to diabetes are still largely unknown . in the trialnet study , a diabetes prevention trial  type 1 risk score threshold of 9.00 identifies individuals who are very likely to have progression to diabetes within 2 years ( 16 ) . type 1 risk score includes c - peptide and glucose indexes from oral glucose tolerance testing . in the daisy study , we previously have shown that age of first positive antibody and mean miaa levels ( but not gad65 or ia-2 ) are major determinants of age of onset of diabetes ( 10 ) . in this study , we confirm that ecl - iaa levels ( but not znt8 ) are major determinants of age of onset of diabetes . the unique association of age of diagnosis with levels of iaa perhaps is not unexpected given accumulating evidence that targeting of insulin may be a primary determinant of diabetes of the spontaneous nod mouse model ( 17 ) and the association of polymorphisms of the insulin gene with diabetes risk in humans . for instance , knocking out endogenous insulin genes and mutating a single amino acid of insulin prevents all diabetes of nod mice ( 18,19 ) , whereas knocking out either gad65 or ia-2 has no influence on progression to diabetes of nod mice ( 20,21 ) . the mechanism underlying the specific association of levels of iaa with rate of progression to diabetes is not defined . autoantibodies or the b cells that produce them may play a pathogenic role with evidence from the nod mouse model ( 22 ) and human trials of rituximab ( 23 ) . alternatively , insulin autoantibody levels simply may reflect activity or number of cd4 t cells targeting insulin . this new nonradioactive ecl - iaa assay can define earlier onset of prediabetic autoimmunity and thus may help identify time - dependent events triggering islet autoimmunity . nearly all young children with progression to diabetes are insulin autoantibody positive , and understanding the underlying mechanism of elevated insulin antibodies may contribute to design of future therapies .
objectivewe evaluated a novel electrochemiluminescent assay for insulin / proinsulin autoantibodies ( ecl - iaa ) as a new marker of the onset of islet autoimmunity and as a predictor of type 1 diabetes.research design and methodsthe diabetes autoimmunity study in the young ( daisy ) prospectively follows children at increased genetic risk for development of islet autoimmunity ( defined as presence of autoantibodies to insulin , gad65 , ia-2 , or zinc transporter 8 [ znt8 ] ) and type 1 diabetes ( general population of children and first - degree relatives ) . 
 serial serum samples from subjects who progressed to type 1 diabetes and who had their first islet autoantibodies measured by age 18 months ( n = 47 ) were tested using ecl-iaa.resultsalmost all prediabetic children tested positive for ecl - iaa ( 46 of 47 , 98% ) during follow - up . 
 ecl - iaa was almost always the first autoantibody to appear ( 94% total ; 21% very first [ by itself ] ; 23% with only miaa ; 19% with another islet autoantibody [ gad or znt8 ] ; and 30% with 2 other antibodies [ miaa , gad , ia-2 , or znt8 ] ) . among the 46 subjects who were ecl - iaa positive , ecl - iaa antedated the onset of other islet autoantibodies by a mean of 2.3 years ( range , 0.37.2 years ) 
 . both the age of appearance of autoantibody and iaa levels ( but not gad65 , ia2 , or znt8 levels ) are major determinants of the age of diabetes onset.conclusionsthis new ecl - iaa assay defines more precisely the onset of prediabetic autoimmunity and may help identify events triggering islet autoimmunity , as well as allow earlier intervention for type 1 diabetes . 
 nearly all young children progressing to diabetes are insulin autoantibody positive .