**3. Electrochemiluminescence (ECL) assays**

While the current standard RBA for iAbs has been greatly improved through the laboratory proficiency programs and harmonization efforts, there are still works in progress. The IAA assay, especially, has not yet achieved a satisfactory level of sensitivity and specificity. Importantly, IAA has a high prevalence among young children [36] and is usually the first iAb to appear in young children [37]. The ability of prospective clinical studies, for example, The Environmental Determinants of Diabetes in the Young (TEDDY), which aims to identify triggers of islet autoimmunity, depends on accurate detection of the timing of appearance of the first iAb to mark the very beginning of islet autoimmunity. We have recently developed and extensively validated a new generation of nonradioactive iAb assay using ECL detection, as we described in the previous section, with an excellent sensitivity and specificity [20–25] for both IAA and GADA. The assay is based on the principle that interaction of autoantibodies with antigen molecules is in liquid phase, and the detection signals are directly from the labeled antigen molecules bound to specific autoantibodies without applying the labeled second antibody (labeled anti-human IgG antibody usually used in conventional ELISA) for detection, which usually causes a very high background in autoantibody assays with human samples. As illustrated for ECL assay protocol in **Figure 1**, autoantibodies in serum help bridge the Sulfo-tagged antigen molecule to the biotinylated antigen molecule, which will be captured on the solid phase of the streptavidin-coated plate. Detection of plate-captured Sulfo-tagged antigen is accomplished with electrochemiluminescence. The fact that the ECL assay is able to capture all immunoglobulin subgroups no matter IgG, IgM, IgA or IgE allows this assay a higher sensitivity than current standard RBA based on IgG detection. With the new ECL assays, we have analyzed thousands of participants in Diabetes Autoimmunity Study in the Young (DAISY) [21], TrialNet Pathway to Prevention subjects [23] and very recently TEDDY subjects (unpublished data). Compared with gold standard RBA, the ECL assays, especially ECL-IAA assays, were demonstrated more sensitive. In DAISY longitudinally follow-up study, we analyzed 427 sequential samples from 63 pre-T1D who were closely followed to clinical T1D. Nearly all of these children (62/63) were detected ECL-IAA years before disease onset, including 10 children who were completely IAA negative by RBA-IAA during the follow-up. Remarkably, 25% of these early longitudinally followed samples during

and harmonized to eliminate potential factors that will contribute to discrepancies; (4) a common set of samples which can provide knowledge of concordance, sensitivity and specificity including a large number of samples from healthy controls, and patients should be identified to establish working thresholds of positivity that are similar between the participating

T1D is now able to be predicted by measuring iAbs present in the peripheral blood, and these iAbs have been used to determine disease risk and onset of autoimmune beta cell destruction. Almost all children with two or more of these iAbs will progress to clinical diabetes with a remarkable consistency across populations, while progression to diabetes in children persistently positive for a single iAb is only 15% in 15 years [30], as most of these children have a

In summary, the current method most commonly used for screening iAbs is the "gold" standard RBA, a fluid-phase assay using radiolabeled antigens and immunoprecipitation, to quantify four major iAbs named IAA, GADA, IA-2A and ZNT8A. These four RBAs are currently able to cover 95% of sensitivity in newly diagnosed patients with T1D. Through the efforts of iAb proficiency programs and harmonization consortia, current standard RBA for iAbs have been greatly improved, but there are still work to be done, especially (1) IAA is not achieved its sensitivity and specificity and (2) there is a low risk prediction in subjects with single iAb positivity.

A direct enzyme-linked immunosorbent assay (ELISA) format (binding of antigen to plate and detection of bound autoantibody with labeled anti-antibodies) has proven difficult to develop. To date, only one ELISA-based ElisaRSR™ for GADA, IA-2A and ZnT8A, distributed by Kronus that utilizes capture of solution-phase antigen by one chain of immunoglobulin (Ig) while being bound by its other chain to plate-bound antigen has demonstrated sensitivity and specificity similar to the fluid-phase RBA (www.rsrltd.com). But there is no any ELISAbased assay that works well for IAA measurement according to IASP workshop [30]. ELISA assays were found only to detect insulin antibodies induced by exogenous insulin injection [38], but not natural insulin autoantibodies. Our group reported a high sensitive ELISA-based competition Europium IAA assay in 2009 [39], and it worked very well for mouse IAA assay, but unfortunately it did not work at all for human samples, although RBA-IAA assay works

While the current standard RBA for iAbs has been greatly improved through the laboratory proficiency programs and harmonization efforts, there are still works in progress. The IAA assay, especially, has not yet achieved a satisfactory level of sensitivity and specificity. Importantly, IAA has a high prevalence among young children [36] and is usually the first iAb to appear in young children [37]. The ability of prospective clinical studies, for example, The Environmental Determinants of Diabetes in the Young (TEDDY), which aims to identify triggers of islet autoimmunity, depends on accurate detection of the timing of appearance of the first iAb to mark the very beginning of islet autoimmunity. We have recently developed

low-affinity iAb not associated with disease [35–37].

equally well for both human and mouse samples [33].

**3. Electrochemiluminescence (ECL) assays**

laboratories.

192 Autoantibodies and Cytokines

**Figure 1.** Illustration of the bivalent plate capture ECL assay. The antibody in serum will link the Sulfo-tagged antigen to the biotinylated antigen, which will be captured on the solid phase of the streptavidin-coated plate. Detection of platecaptured Sulfo-tagged antigen is accomplished with electrochemiluminescence.

pre-diabetes stage which were positive for ECL-IAA were negative for RBA-IAA (**Figure 2**). ECL-IAA were found to antedate the onset of islet autoimmunity in these children by a mean of 2.3 years (range: 0.3–7.2 years) [21]. The earlier identification of IAA among young children was validated from a later TEDDY study cohort (unpublished data). Results from these studies indicate that this novel ECL-IAA assay is not only more sensitive but also defines the timing of the initial autoantibody appearance earlier than the previously used RBA-IAA. This earlier detection and accuracy in timing of onset of islet autoimmunity in young children followed from the birth is important to find potential environmental causes of diabetes and our understanding of the etiology of T1D as the ability to identify triggers of islet autoimmunity, for example, TEDDY study depends on the sensitivity and validity of iAb assays used to pinpoint the "seroconversion" to islet autoimmunity.

Remarkably, both ECL-IAA and ECL-GADA assays were selectively detecting the positivity from the high-risk subjects who were followed to progression of clinical T1D or the subjects who were multiple iAbs positive. In the study of DAISY children [20, 22], we analyzed all pre-T1D children who were followed to clinical diabetes and all non-diabetic children who were persistently iAbs positive, either multiple iAbs or single iAb, and compared them with RBA. Almost all samples positive by RBA (IAA or GADA) were ECL assay positive in children on pre-T1D or children with presence of multiple iAbs. In contrast, only around 25% of nondiabetic children with either single IAA or single GADA positive by RBA were ECL positive. **Figure 3** shows positivity of IAA among some children in the DAISY study as an example, persistently expressing IAA with other iAbs (multiple iAbs) or as a single iAb. Panel A: ECL-IAA

was well correlated with RBA-IAA for all five subjects (multiple follow-up positive for both assays), who were multiple iAbs positive. Panel B: 7/8 subjects with single iAb (IAA only) were consistently ECL-IAA negative. The antibody affinity study was performed to compare these IAA or GADA detected by RBA, but differentiated by ECL assay as positive or negative. The results of affinity analysis discovered that IAA or GADA detected by RBA, but negative with ECL assay, in children who were absent of other iAbs had low affinity, while IAA or GADA positive by both RBA and ECL assays had high affinity. **Figure 4** illustrates the IAA (panel A) or GADA (panel B) competition assays with unlabeled proinsulin or GAD65 protein. The IAA or GADA not detected by the ECL assay required a 10- to 100-fold higher concentration of unlabeled insulin/proinsulin or GAD65 protein for 50% inhibition of binding of IAA or GADA to labeled insulin or GAD65 protein. These results demonstrated that both ECL-IAA and ECL-GADA were able to discriminate high-affinity, high-risk iAbs from those "low risk," low-affinity signals generated by RBA in subjects who are less likely to progress to T1D. In our later validation study with a large TrialNet cohort of Pathway to Prevention, identical results were obtained [23] by characteristics of disease specificity with both ECL-IAA and ECL-GADA. The study analyzed 3500 subjects in blind with 571 multiple iAbs, 1727 single iAb and 384 pre-T1D who were followed to clinical T1D later in the study. The ECL assay and RBA were found congruent in pre-diabetics and in the subjects with multiple autoantibodies, but only 24% of single RBA-IAA (p < 0.0001) and 46% of single RBA-GADA (p < 0.0001) were confirmed by the ECL-IAA and ECL-GADA assays, respectively. With the prospective following up for the subjects with single IAA or GADA, 51% of RBA-IAA and 63% of RBA-GADA subjects not confirmed by ECL were found lost their iAbs and became iAb negative after a mean follow-up time of 2.4 years, behaving as "transient" iAb positivity. Only a few subjects converted to multiple iAbs or progressed to clinical diabetes. In contrast, only small percentages of RBA-IAA and RBA-GADA confirmed by ECL assays became negative (p < 0.0001 for both IAA and GADA) during the follow-up as significant number of subjects progressed to clinical diabetes or multiple iAbs. In the study, 2944 subjects were studied with their very first initial screening samples

were consistently ECL-IAA negative.

**Figure 3.** Sera from children in the DAISY study persistently expressing IAA as a single or with multiple iAbs were analyzed with the ECL-IAA assay. Panel A: ECL-IAA was well correlated with RBA-IAA for all 5 subjects (multiple follow-up positive for both assays) who were multiple iAbs positive. Panel B: 7/8 subjects with single iAb (IAA only)

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**Figure 2.** IAA identification in pre-diabetic children by ECL-IAA. The x-axis represents results of ECL-IAA assay and the y-axis represents results of mIAA RIA assay. About 25% of pre-diabetic samples were positive for ECL-IAA but negative for mIAA RIA assay.

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pre-diabetes stage which were positive for ECL-IAA were negative for RBA-IAA (**Figure 2**). ECL-IAA were found to antedate the onset of islet autoimmunity in these children by a mean of 2.3 years (range: 0.3–7.2 years) [21]. The earlier identification of IAA among young children was validated from a later TEDDY study cohort (unpublished data). Results from these studies indicate that this novel ECL-IAA assay is not only more sensitive but also defines the timing of the initial autoantibody appearance earlier than the previously used RBA-IAA. This earlier detection and accuracy in timing of onset of islet autoimmunity in young children followed from the birth is important to find potential environmental causes of diabetes and our understanding of the etiology of T1D as the ability to identify triggers of islet autoimmunity, for example, TEDDY study depends on the sensitivity and validity of iAb assays used to pin-

Remarkably, both ECL-IAA and ECL-GADA assays were selectively detecting the positivity from the high-risk subjects who were followed to progression of clinical T1D or the subjects who were multiple iAbs positive. In the study of DAISY children [20, 22], we analyzed all pre-T1D children who were followed to clinical diabetes and all non-diabetic children who were persistently iAbs positive, either multiple iAbs or single iAb, and compared them with RBA. Almost all samples positive by RBA (IAA or GADA) were ECL assay positive in children on pre-T1D or children with presence of multiple iAbs. In contrast, only around 25% of nondiabetic children with either single IAA or single GADA positive by RBA were ECL positive. **Figure 3** shows positivity of IAA among some children in the DAISY study as an example, persistently expressing IAA with other iAbs (multiple iAbs) or as a single iAb. Panel A: ECL-IAA

**Figure 2.** IAA identification in pre-diabetic children by ECL-IAA. The x-axis represents results of ECL-IAA assay and the y-axis represents results of mIAA RIA assay. About 25% of pre-diabetic samples were positive for ECL-IAA but

point the "seroconversion" to islet autoimmunity.

194 Autoantibodies and Cytokines

negative for mIAA RIA assay.

**Figure 3.** Sera from children in the DAISY study persistently expressing IAA as a single or with multiple iAbs were analyzed with the ECL-IAA assay. Panel A: ECL-IAA was well correlated with RBA-IAA for all 5 subjects (multiple follow-up positive for both assays) who were multiple iAbs positive. Panel B: 7/8 subjects with single iAb (IAA only) were consistently ECL-IAA negative.

was well correlated with RBA-IAA for all five subjects (multiple follow-up positive for both assays), who were multiple iAbs positive. Panel B: 7/8 subjects with single iAb (IAA only) were consistently ECL-IAA negative. The antibody affinity study was performed to compare these IAA or GADA detected by RBA, but differentiated by ECL assay as positive or negative. The results of affinity analysis discovered that IAA or GADA detected by RBA, but negative with ECL assay, in children who were absent of other iAbs had low affinity, while IAA or GADA positive by both RBA and ECL assays had high affinity. **Figure 4** illustrates the IAA (panel A) or GADA (panel B) competition assays with unlabeled proinsulin or GAD65 protein. The IAA or GADA not detected by the ECL assay required a 10- to 100-fold higher concentration of unlabeled insulin/proinsulin or GAD65 protein for 50% inhibition of binding of IAA or GADA to labeled insulin or GAD65 protein. These results demonstrated that both ECL-IAA and ECL-GADA were able to discriminate high-affinity, high-risk iAbs from those "low risk," low-affinity signals generated by RBA in subjects who are less likely to progress to T1D. In our later validation study with a large TrialNet cohort of Pathway to Prevention, identical results were obtained [23] by characteristics of disease specificity with both ECL-IAA and ECL-GADA. The study analyzed 3500 subjects in blind with 571 multiple iAbs, 1727 single iAb and 384 pre-T1D who were followed to clinical T1D later in the study. The ECL assay and RBA were found congruent in pre-diabetics and in the subjects with multiple autoantibodies, but only 24% of single RBA-IAA (p < 0.0001) and 46% of single RBA-GADA (p < 0.0001) were confirmed by the ECL-IAA and ECL-GADA assays, respectively. With the prospective following up for the subjects with single IAA or GADA, 51% of RBA-IAA and 63% of RBA-GADA subjects not confirmed by ECL were found lost their iAbs and became iAb negative after a mean follow-up time of 2.4 years, behaving as "transient" iAb positivity. Only a few subjects converted to multiple iAbs or progressed to clinical diabetes. In contrast, only small percentages of RBA-IAA and RBA-GADA confirmed by ECL assays became negative (p < 0.0001 for both IAA and GADA) during the follow-up as significant number of subjects progressed to clinical diabetes or multiple iAbs. In the study, 2944 subjects were studied with their very first initial screening samples

**Figure 4.** Illustration of the IAA (Panel A) or GADA (Panel B) competition assays with unlabeled proinsulin or GAD65 protein. IAA /GADA negative by ECL-IAA / ECL-GADA assay (blue line), compared with IAA /GADA positive by ECL-GADA/ECL-IAA assay (red line), and required higher concentrations of unlabeled insulin/proinsulin or GAD65 protein for 50% maximal inhibition, which is consistent with low affinity.

to low affinity were seen over time. There were 14 subjects in the group who progressed to clinical T1D during the study period. All except one were ECL-positive and had a high affinity for IAA or GADA. These results implicate that a more disease-specific iAb measurement like ECL assay can identify high affinity iAbs with high disease risk on the early stage of initial screening. It is generally assumed that the very low risk of those with only a single iAb, either single IAA or single GADA, spreading of autoimmunity to other autoantigens is needed to increase risk or marks a stage closer to overt diabetes, and multiple studies of both relatives of T1D patients and general populations have documented that expression of multiple iAbs are associated with extreme risk of progression to T1D. The pattern of expression of iAbs detected by ECL assays predicts both epitope spreading and diabetes risk. Differences in iAbs reactivity can be assessed prior to epitope spreading consistent with early determination of both

**RBA positive, GADA only RBA positive, IAA only**

PS6M (mg/dl) 22 ± 90 −19 ± 71 0.001 36 ± 75 −9 ± 70 0.009 Progressed to T1D 14.0% (n = 15) 1.3% (n = 1) 0.002 16.7% (n = 4) 0% (n = 0) 0.005

PS6M is a 6-month progression scale based on worsening of the 30–120-min OGTT glucose.

**Table 1.** ECL iAb identify progressors to T1D among children and adults with single iAb by RBA.

**ECL+ (n = 107) ECL− (n = 78) p-value ECL+ (n = 24) ECL− (n = 63) p-value**

**Figure 5.** Predictive values of IAA and GADA assays in the TrialNet Initial Screening study. A: Positive predictive values of RIA-IAA and ECL-IAA assays, RIA-GADA and ECL-GADA assays. B: Negative predictive values of RIA-IAA and ECL-IAA assays, RIA-GADA and ECL-GADA assays. Positive and negative predictive values of ECL-IAA and ECL-

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GADA were significantly higher than those of RIA-IAA and RIA-GADA assays, respectively.

and prospectively followed to clinical T1D during the study period. The positive and negative predictive values of RBA and ECL assays, in terms of progression to T1D, were analyzed and compared, and the data are illustrated in **Figure 5**. The positive predictive values for ECL-IAA and ECL-GADA were 32 and 24%, significantly higher than those RBA-IAA and RBA-GADA (21 and 16%, respectively; both p < 0.0001) (panel A). Similarly, the negative predictive values for ECL-IAA and ECL-GADA were 94 and 96%, significantly higher than those for RBA-IAA and RBA-GADA (92%; p < 0.05 and 94%; p = 0.007, respectively) (panel B). Compared with HLA analysis, the highest risk genotype for T1D, HLA-DR3/4, DQB1\*0302 was significantly higher in subjects with IAA or GADA confirmed by ECL than those not confirmed by ECL assays (p < 0.0017 and p < 0.0001, respectively). The frequency of this high-risk HLA genotype in subjects with either single IAA or GADA not confirmed by ECL assays was found identical to subjects with negative results for all iAbs, a very low-risk population who are very unlikely to progress to T1D.

Autoantibody affinity in sequential follow-up samples from their initial positive screening to their later follow-up with a mean follow-up of 5.3 years were investigated [25] in a subset of subjects in TrialNet Pathway to Prevention study who were persistent single RBA-IAA or single RBA-GADA positive confirmed or not confirmed by ECL assays. Among either single IAA or single GADA subjects, all subjects who were confirmed by ECL assays were found to have high affinity autoantibodies at their very first initial positive visit. Affinity results stayed consistent over time. Similarly, those who were negative for ECL assays showed lower affinity at initial visit and affinity stayed low over time. No converting events from low to high or high Development of a Simple Multiplex Electrochemiluminescence (ECL) Assay for Screening Pre… http://dx.doi.org/10.5772/intechopen.75515 197

**Figure 5.** Predictive values of IAA and GADA assays in the TrialNet Initial Screening study. A: Positive predictive values of RIA-IAA and ECL-IAA assays, RIA-GADA and ECL-GADA assays. B: Negative predictive values of RIA-IAA and ECL-IAA assays, RIA-GADA and ECL-GADA assays. Positive and negative predictive values of ECL-IAA and ECL-GADA were significantly higher than those of RIA-IAA and RIA-GADA assays, respectively.

and prospectively followed to clinical T1D during the study period. The positive and negative predictive values of RBA and ECL assays, in terms of progression to T1D, were analyzed and compared, and the data are illustrated in **Figure 5**. The positive predictive values for ECL-IAA and ECL-GADA were 32 and 24%, significantly higher than those RBA-IAA and RBA-GADA (21 and 16%, respectively; both p < 0.0001) (panel A). Similarly, the negative predictive values for ECL-IAA and ECL-GADA were 94 and 96%, significantly higher than those for RBA-IAA and RBA-GADA (92%; p < 0.05 and 94%; p = 0.007, respectively) (panel B). Compared with HLA analysis, the highest risk genotype for T1D, HLA-DR3/4, DQB1\*0302 was significantly higher in subjects with IAA or GADA confirmed by ECL than those not confirmed by ECL assays (p < 0.0017 and p < 0.0001, respectively). The frequency of this high-risk HLA genotype in subjects with either single IAA or GADA not confirmed by ECL assays was found identical to subjects with negative results for all iAbs, a very low-risk population who are very unlikely

for 50% maximal inhibition, which is consistent with low affinity.

**Figure 4.** Illustration of the IAA (Panel A) or GADA (Panel B) competition assays with unlabeled proinsulin or GAD65 protein. IAA /GADA negative by ECL-IAA / ECL-GADA assay (blue line), compared with IAA /GADA positive by ECL-GADA/ECL-IAA assay (red line), and required higher concentrations of unlabeled insulin/proinsulin or GAD65 protein

Autoantibody affinity in sequential follow-up samples from their initial positive screening to their later follow-up with a mean follow-up of 5.3 years were investigated [25] in a subset of subjects in TrialNet Pathway to Prevention study who were persistent single RBA-IAA or single RBA-GADA positive confirmed or not confirmed by ECL assays. Among either single IAA or single GADA subjects, all subjects who were confirmed by ECL assays were found to have high affinity autoantibodies at their very first initial positive visit. Affinity results stayed consistent over time. Similarly, those who were negative for ECL assays showed lower affinity at initial visit and affinity stayed low over time. No converting events from low to high or high

to progress to T1D.

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to low affinity were seen over time. There were 14 subjects in the group who progressed to clinical T1D during the study period. All except one were ECL-positive and had a high affinity for IAA or GADA. These results implicate that a more disease-specific iAb measurement like ECL assay can identify high affinity iAbs with high disease risk on the early stage of initial screening. It is generally assumed that the very low risk of those with only a single iAb, either single IAA or single GADA, spreading of autoimmunity to other autoantigens is needed to increase risk or marks a stage closer to overt diabetes, and multiple studies of both relatives of T1D patients and general populations have documented that expression of multiple iAbs are associated with extreme risk of progression to T1D. The pattern of expression of iAbs detected by ECL assays predicts both epitope spreading and diabetes risk. Differences in iAbs reactivity can be assessed prior to epitope spreading consistent with early determination of both


**Table 1.** ECL iAb identify progressors to T1D among children and adults with single iAb by RBA.

epitope spreading and diabetes risk. This is of particular value in children and young adults who are positive for a single iAb, as demonstrated by TrialNet Pathway to Prevention (**Table 1**, J Sosenko, manuscript submitted). Among TrialNet participants who were positive for a single iAb by RBA, 52% (141/272) were not confirmed by ECL. These ECL-negative subjects showed no worsening of glycemia and little progression to T1D during a median follow-up of 4.7 years. In contrast, OGTT glycemia worsened significantly in the ECL-positive single iAb participants, comparably with the worsening in 90 multiple iAb + subjects (PS6M 23 ± 96 mg/dl); the latter group had a high risk for progression to T1D (30%). The ECL assay can substantially refine the selection of single iAb positive individuals at high risk, who possibly could be recruited for participation in T1D prevention trials.

defined iAbs including IAA, GADA, IA-2A and ZnT8A are equally important in prediction and evaluation of risk of progression to T1D in both relatives of patients with T1D and general population. The screening methods using current standard RBA with single iAb measurement are laborious and inefficient for such a large scale of screening. While significant progress has been made in standardization of iAb assays and high-throughput technologies, the cost and logistic complexity of currently used methods preclude their widespread use in population-based screening. The determination of initial iAb positivity is very important, which may represent the initiation of islet autoimmunity. However, the results of iAb measurement at this early stage in subjects with a single iAb are not reliable with current standard RBA since the majority of these single iAb subjects identified by RBA have low affinity antibodies, most of them transient, and therefore biologically appear to be "false positives" with respect to T1D development as we discussed earlier. Poor assay specificity is likely to be more problematic for screening process in general population with lower frequency of risk for T1D than high risk relative cohort as in TrialNet Pathway to Prevention we studied. A high-throughput assay technology with

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One in four children at risk for T1D develops islet, celiac, thyroid or rheumatoid autoimmunity in the DAISY study. Interestingly, there is little overlap of these phenotypes in an individual child, but this overlap increases with age. The incidence of autoantibodies to thyroid peroxidase (TPOA) surges after the age of 12. Importantly, up to 40% of patients with T1D have an additional autoimmune condition [42–44]. It is important and urgent to screen biomarkers of other autoimmune diseases when screening diabetes, simultaneously. Unfortunately, there is no easy and inexpensive tool to screen for these conditions. With a big effort, all DAISY and TEDDY study participants are screened for autoantibodies to tissue transglutaminase (TGA) for celiac disease autoimmunity. Persistent TGA positivity and celiac disease are secondary endpoints in both studies [45, 46]. DAISY data suggest that, by age 18, at least 7% of the general population persistently express one or more of the nine autoantibodies: IAA, GADA, IA-2A, ZnT8A, TPOA, TGA and three other autoantibodies for rheumatoid arthritis, Addison's disease and autoimmune gastritis. If confirmed, this would argue for a universal screening. We have carried out a pilot of such screening for iAbs and TGA, in children of 2–6 years old attending general pediatric care offices in Denver [47]. Participating parents and providers ranked the

combined screening for iAbs and TGA as more valuable than screening for iAbs alone.

To fit the purpose of large-scale screening in national clinical trials and the general population, people are starting to seek a possible method of a multiplexed assay combining multiple autoantibody assays together in one single well. Recently, a few studies of multiplex antibody assays were reported with different technologies [48–51], but none of these assay platforms has neither compared with currently used "gold" standard RBA for its sensitivity and specificity in T1D study, especially in subjects with risk to T1D, nor validated in an international Islet Autoantibody Standardization Program (IASP) workshop or in a large cohort of clinical trial. From previous multiple studies, none of the conventional ELISA methods worked well for any iAb measurements, especially for IAA according to multiple IASP workshop [30]. Interaction of iAbs with their corresponding antigen proteins in liquid phase will still be necessary in a multiplex assay setting to achieve a proper sensitivity and specificity and it is a particularly essential condition for IAA assay. The capacity of specific autoantibodyantigen binding might be a new consideration with our recent experiences in a multiplex assay setting, which has never been an issue in any single antibody assay format. Multiple

improved disease specificity will be important and necessary.

Presently, clinical prevention trials in T1D TrialNet study are only selecting relatives of patients with T1D who have multiple iAbs. It is generally agreed that some clinical intervening on early stages of islet autoimmune processes could result in a better outcome as some evidence in animal model for prevention [40], but the clinical trial studies using subjects with single iAb, either single IAA or GADA, are not available since the risk is too low. However, the subjects with single iAb (IAA or GADA) detected by more disease-specific assays like ECL assay with the nature of ability to detect high affinity antibodies may qualify for enrollment into prevention trials as their risk for diabetes is much higher and those with low affinity, low-risk signals generated by RBA will be removed [20, 22, 23, 25]. On the other hand, subjects found to be negative for ECL assays may benefit from less intensive monitoring in these longitudinal prospective studies [41] to save the efforts and costs of these studies.

In summary, islet autoimmunity of T1D can be identified at the very beginning of the disease process by measurements of iAbs. Two major iAbs, IAA and GADA, usually appear earlier than other iAbs and are often detected in isolation as single iAb in the screening of relatives and general population. Most of these single iAbs detected by current gold standard RBA are at low risk, low affinity and non-disease relevant as "biologically" false positives while part of these single iAbs does represent the early stage of islet autoimmunity in T1D progression. With more disease-specific assays like an ECL assay method, high-risk and disease-relevant iAbs are able to be identified at the very beginning of the disease process in subjects with single iAb before the development of multiple iAbs closer to overt clinical diabetes, which will be greatly appreciated for clearing the current confusions of single iAb positivity and aid the T1D clinical trials for both identifying environmental triggers of islet autoimmunity and intervening with islet autoimmune process on very beginning stage to prevent the disease. The ECL assay was demonstrated its superiority to RBA in sensitivity and especially ECL-IAA was able to antedated the onset of islet autoimmunity by years than RBA, which will be very important to accurately pinpoint the very beginning of islet autoimmunity for identifying the environmental triggers to cause the T1D.
