**4. Development of a multiplex assay for large-scale screening**

At present, many large-scale national and international clinical trials for T1D are in progress, and multiple candidate interventions are being proposed to abrogate or slow progression of T1D among iAb positive subjects. A wider screening of iAbs in the general population, especially in young children, is perspectively in progress or in planning. Currently, four biochemically 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 improved disease specificity will be important and necessary.

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

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.

**4. Development of a multiplex assay for large-scale screening**

At present, many large-scale national and international clinical trials for T1D are in progress, and multiple candidate interventions are being proposed to abrogate or slow progression of T1D among iAb positive subjects. A wider screening of iAbs in the general population, especially in young children, is perspectively in progress or in planning. Currently, four biochemically

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 identify-

recruited for participation in T1D prevention trials.

198 Autoantibodies and Cytokines

ing the environmental triggers to cause the T1D.

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 autoantibody-antigen interactions share the space within one single well in a multiplex assay setting, and each of these interactions might need enough number of their own specific antibodies binding to their corresponding antigen proteins to generate a signal strong enough to be detected, which is particularly important when these autoantibodies are at low levels.

**Figure 6**. The 4-plex assay was based on the same mechanism of our single ECL assay, but linker system was introduced. Four interactions of antibody-antigen are, respectively, restrained on each of four specific linker spots within the same well, and the camera is able to catch the signals from four different sources of spots, respectively. With the limitation of four spots able to accommodate maximum four autoantibody assays in one well, we selected, on purposely, IAA, GADA, IA-2A and TGA. We included TGA instead of ZnT8A because (1) ZnT8A is almost always present with other iAbs and ZnT8A alone is only 1% in subject followed to T1D (8). In two large national clinical trials of TrialNet and TEDDY, ZnT8A is not included in intial screening, and the ZnT8A assay is only performed if any of other three iAbs is positive. (2) We want to screen both T1D and celiac disease as we rationaled earlier. The 4-plex assay retained 100% sensitivity and 100% specificity for all four autoantibodies in terms of positivity identified in patients versus normal controls compared to the corresponding standard RBA and our single ECL assays. In early 2015, MSD company released the new U-Plex™ Development Packs system for creating custom multiplex panels of analytes to replace the QuickPlex 4-Spot system. With a similar principle of multiplex assay mechanism of QuickPlex 4-Spot system, this new system expanded multiplexing up to 10, combining 10 different autoantibody assays in one single well with the same amount of 6 μl serum sample used for a single ECL assay. The UPlex plate assay system is illustrated in **Figure 7** as we currently used it as an UPlex 8-plex assay. With this new system, we have successfully combined eight autoantibody assays within one single well including all four iAbs and four other

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**Figure 7.** Illustration of UPlex 8-plex plate working chart. 8-plex assay is working on the same mechanism of 4-spot

assay. Uplex can combine up to 10 antibody assays in one well.

Very recently, a modified ELISA-based ElisaRSR™ 3-Screen ICA™ is now available for research from the RSR Limited (3-Screen ICA™ ELISA; www.rsrltd.com). It is a combination assay for measuring GADA, IA-2A and ZnT8A. Its single assay platform was validated at multiple IASP workshops for its sensitivity and specificity. The 3 Screen ELISA assay measures three autoantibodies either in three separate wells consuming large volume of serum or in one single well with three assays mixed not able to distinguish which of the three beta cell autoantibodies are present. The biggest disadvantage of the 3 Screen ELISA is its inability to include IAA measurement in the assay. IAA has a very high rate of positivity in young children and is considered as the first iAb on early stages of islet autoimmunity as we discussed earlier. Compared with current single iAb RBA or original single ELISA-based ElisaRSR™ assays, the 3 Screen ELISA assay definitely has its advantage of higher efficiency [52], but screening with its own without including IAA measurement will be a big defect and will not be the best way to persume.

With the platform of our newly established ECL assay technology, we recently published our study of a multiplex assay to accurately measure four autoantibodies in one single well [53] with MesoScale Discovery (MSD) QuickPlex 4-Spot plate as illustrated in

**Figure 6.** Illustration of ECL 4-spot assay. The 4-spot assay is based on the same mechanism of single ECL assay. Each biotinylated antigen will be linked by its corresponding linker which will be captured on the solid phase of the precoated plate. Detection of plate-captured Sulfo-tagged antigen is also accomplished with electrochemiluminescence. 4-spot assay is able to accommodate up to 4 autoantibody assays in one well.

**Figure 6**. The 4-plex assay was based on the same mechanism of our single ECL assay, but linker system was introduced. Four interactions of antibody-antigen are, respectively, restrained on each of four specific linker spots within the same well, and the camera is able to catch the signals from four different sources of spots, respectively. With the limitation of four spots able to accommodate maximum four autoantibody assays in one well, we selected, on purposely, IAA, GADA, IA-2A and TGA. We included TGA instead of ZnT8A because (1) ZnT8A is almost always present with other iAbs and ZnT8A alone is only 1% in subject followed to T1D (8). In two large national clinical trials of TrialNet and TEDDY, ZnT8A is not included in intial screening, and the ZnT8A assay is only performed if any of other three iAbs is positive. (2) We want to screen both T1D and celiac disease as we rationaled earlier. The 4-plex assay retained 100% sensitivity and 100% specificity for all four autoantibodies in terms of positivity identified in patients versus normal controls compared to the corresponding standard RBA and our single ECL assays. In early 2015, MSD company released the new U-Plex™ Development Packs system for creating custom multiplex panels of analytes to replace the QuickPlex 4-Spot system. With a similar principle of multiplex assay mechanism of QuickPlex 4-Spot system, this new system expanded multiplexing up to 10, combining 10 different autoantibody assays in one single well with the same amount of 6 μl serum sample used for a single ECL assay. The UPlex plate assay system is illustrated in **Figure 7** as we currently used it as an UPlex 8-plex assay. With this new system, we have successfully combined eight autoantibody assays within one single well including all four iAbs and four other

autoantibody-antigen interactions share the space within one single well in a multiplex assay setting, and each of these interactions might need enough number of their own specific antibodies binding to their corresponding antigen proteins to generate a signal strong enough to be detected, which is particularly important when these autoantibodies are at low levels.

200 Autoantibodies and Cytokines

Very recently, a modified ELISA-based ElisaRSR™ 3-Screen ICA™ is now available for research from the RSR Limited (3-Screen ICA™ ELISA; www.rsrltd.com). It is a combination assay for measuring GADA, IA-2A and ZnT8A. Its single assay platform was validated at multiple IASP workshops for its sensitivity and specificity. The 3 Screen ELISA assay measures three autoantibodies either in three separate wells consuming large volume of serum or in one single well with three assays mixed not able to distinguish which of the three beta cell autoantibodies are present. The biggest disadvantage of the 3 Screen ELISA is its inability to include IAA measurement in the assay. IAA has a very high rate of positivity in young children and is considered as the first iAb on early stages of islet autoimmunity as we discussed earlier. Compared with current single iAb RBA or original single ELISA-based ElisaRSR™ assays, the 3 Screen ELISA assay definitely has its advantage of higher efficiency [52], but screening with its own without including IAA measurement will be a big defect and will not be the best way to persume.

With the platform of our newly established ECL assay technology, we recently published our study of a multiplex assay to accurately measure four autoantibodies in one single well [53] with MesoScale Discovery (MSD) QuickPlex 4-Spot plate as illustrated in

**Figure 6.** Illustration of ECL 4-spot assay. The 4-spot assay is based on the same mechanism of single ECL assay. Each biotinylated antigen will be linked by its corresponding linker which will be captured on the solid phase of the precoated plate. Detection of plate-captured Sulfo-tagged antigen is also accomplished with electrochemiluminescence.

4-spot assay is able to accommodate up to 4 autoantibody assays in one well.

**Figure 7.** Illustration of UPlex 8-plex plate working chart. 8-plex assay is working on the same mechanism of 4-spot assay. Uplex can combine up to 10 antibody assays in one well.

autoantibody assays, TGA for celiac disease, TPOA and autoantibodies to thyroid globulin (ThGA) for autoimmune thyroiditis, autoantibodies to interferon alpha (IFNaA) for autoimmune polyglandular syndrom-1 (APS-1). With 100th percentile of specificity in 118 healthy normal controls, the 8-plex assay was able to retain 100% sensitivity for all autoantibodies, and the levels of autoantibodies in 8-plex assay were well correlated with their corresponding single RBA or ELISA (for IFNaA) in 168 T1D patients. The further work of assay optimization needs to be done to minimize the interferences of cross-talking between spots, especially an extreme high signal on one spot overspilled to a neighboring spot when it should be negative. The UPlex system made it possible to customize a multiplexed assay according to the needs for screening. It is capable to screen children simultaneously for T1D and other multiple autoimmune diseases often happening in childhood. It is also capable to screen adults simultaneously for T1D and other multiple autoimmune diseases usually seen in adulthood. Such a multiplex ECL assay technology will retain high assay sensitivity and disease specificity as we discussed earlier and provide a great tool for a large scale of screening in the general population with high efficiency and low cost using only a tiny amount of blood sample. We expect these multiplex assays with new technologies, be available in clinic and easily applied for population screening in the near future.

**References**

1213-1218

[1] Atkinson MA, Eisenbarth GS, Michels AW. Type 1 diabetes. Lancet. 2014;**383**(9911):69-82 [2] Secular trends in incidence of childhood IDDM in 10 countries. Diabetes Epidemiology

Development of a Simple Multiplex Electrochemiluminescence (ECL) Assay for Screening Pre…

http://dx.doi.org/10.5772/intechopen.75515

203

[3] Harjutsalo V, Sjoberg L, Tuomilehto J. Time trends in the incidence of type 1 diabetes in

[4] Vehik K et al. Increasing incidence of type 1 diabetes in 0- to 17-year-old Colorado youth.

[5] Ziegler AG et al. Seroconversion to multiple islet autoantibodies and risk of progression

[6] Orban T et al. Pancreatic islet autoantibodies as predictors of type 1 diabetes in the

[7] Bingley PJ. Clinical applications of diabetes antibody testing. Journal of Clinical Endo

[8] Yu L et al. Zinc transporter-8 autoantibodies improve prediction of type 1 diabetes in relatives positive for the standard biochemical autoantibodies. Diabetes Care. 2012;**35**(6):

[9] Steck AK et al. Age of islet autoantibody appearance and mean levels of insulin, but not GAD or IA-2 autoantibodies, predict age of diagnosis of type 1 diabetes: Diabetes auto-

[10] Sosenko JM et al. A longitudinal study of GAD65 and ICA512 autoantibodies during the progression to type 1 diabetes in Diabetes Prevention Trial-Type 1 (DPT-1) participants.

[11] Sosenko JM et al. The prediction of type 1 diabetes by multiple autoantibody levels and their incorporation into an autoantibody risk score in relatives of type 1 diabetic patients.

[12] Vehik K et al. Islet autoantibody seroconversion in the DPT-1 study: Justification for

[13] Pollard DR et al. An immunofluorescence study of anti-pancreatic islet cell antibodies in

[14] Myers MA, Rabin DU, Rowley MJ. Pancreatic islet cell cytoplasmic antibody in diabetes is represented by antibodies to islet cell antigen 512 and glutamic acid decarboxylase.

[15] Petruzelkova L et al. The dynamic changes of zinc transporter 8 autoantibodies in Czech children from the onset of Type 1 diabetes mellitus. Diabetic Medicine. 2014;**31**(2):165-171

repeat screening throughout childhood. Diabetes Care. 2011;**34**(2):358-362

the spontaneously diabetic BB Wistar rat. Diabetologia. 1983;**25**(1):56-59

Research International Group. Diabetes. 1990;**39**(7):858-864

to diabetes in children. JAMA. 2013;**309**(23):2473-2479

crinology and Metabolism. 2010;**95**(1):25-33

Diabetes Care. 2011;**34**(11):2435-2437

Diabetes Care. 2013;**36**(9):2615-2620

Diabetes. 1995;**44**(11):1290-1295

Diabetes Care. 2007;**30**(3):503-509

Finnish children: A cohort study. Lancet. 2008;**371**(9626):1777-1782

Diabetes Prevention Trial-Type 1. Diabetes Care. 2009;**32**(12):2269-2274

immunity study in the young. Diabetes Care. 2011;**34**(6):1397-1399

In conclusion, T1D is now predictable by measuring major iAbs. The ECL assay for iAbs is superior to the current gold standard RBA and other methods in terms of assay sensitivity and specificity for disease risk prediction. With a rapid increasing rate of disease, large scales of population screenings are becoming important for the public health. Large percent of patients with T1D develop other autoimmune diseases, and it has been recommended to screen other relevant automimmune diseases when screening diabetes autoimmunity simultaneously. With the advantages of the ECL assay in its nature of high assay sensitivity and high disease specificity, a simple multiplex assay built on the platform of ECL technology will provide an excellent tool to not only screen multiple iAbs in one single well, but also screen multiple autoimmune diseases simultaneously in large scale of populations efficiently and economically.
