**2. Radio-binding assay (RBA) and enzyme-linked immunosorbent assay (ELISA)**

In the USA, 1.4 million people have T1D and as many have multiple islet autoantibodies (iAbs) or pre-T1D with normal glucose homeostasis. Of the latter, 84% will progress to clinical diabetes in 15 years with a remarkable consistency across populations [5]. Although T1D is a T-lymphocyte-mediated autoimmune disease with specific destruction of pancreatic islet β-cells, autoantibodies directed against proteins in insulin-producing beta cells [1] are the best biomarkers for risk prediction and clinical diagnosis. These iAbs usually appear years before overt clinical disease, and the presence of ≥2 iAbs [antibodies-directed against insulin (IAA), glutamic acid decarboxylase (GADA), islet antigen 2 (IA-2A) or zinc transporter 8 (ZnT8A)] predicts the development of clinical T1D in nearly all affected children [5]. Children at risk for T1D need to be identified prior to the onset of symptoms to: (1) prevent life-threatening diabetic ketoacidosis, (2) identify individuals for current and upcoming trials to prevent T1D

Appearance of iAbs is currently the most reliable marker of the autoimmune process leading to T1D, and it determines the disease risk and marks the onset of autoimmune beta cell destruction. The presence of iAbs, their number [6–8] and titer [9–11], has being used extensively to stage diabetes risk and as inclusion criteria into T1D prevention trials [12]. Immunoassays to detect iAbs can be classified into four generations. The first generation is the indirect immunofluorescence on cryostat sections of the pancreas for islet cell antibodies (ICA) [13, 14] present in patients' sera to a variety of autoantigens in islet β-cells. This assay requires the pancreas tissue to conduct the measurement and is very limited in the regular screening. The second generation of iAbs immunoassays is a well-established radio-binding assay (RBA) which is based on various biochemically defined autoantigens and plays a major role as the current gold standard assay for all four major islet autoantibodies IAA, GADA, IA-2A and ZnT8A and ELISAs. Traditional ELISA has been proved not to work well for any iAbs with less sensitivity and less specific in multiple workshops of Islet Autoantibody Standardization Program (IASP, previously DASP). However, a modified bridging ELISA with semifluid-phase interaction between the antibody and antigen has achieved sensitivity and specificity equivalent to the RBA for the measurement of GADA, IA-2A and ZnT8A [15–19]. The third generation is a recently developed and extensively validated nonradioactive iAb assay using electrochemiluminescence (ECL) detection with a higher sensitivity and higher disease specificity compared to the RBAs [20–25]. The ECL assay has been demonstrated to be more sensitive and is able to identify the first iAb of "seroconversion" earlier than RBA by years in young children followed from the birth on pre-T1D who were followed to clinical diabetes. More remarkably, ECL assay is able to discriminate high-affinity, high-risk autoantibodies from those "low risk," low-affinity signals generated by RBA in subjects with single iAb who are less likely to progress to T1D. The fourth generation of autoantibody immunoassays is to develop a simple multiplexed assay to fit for the needs of ongoing and future clinical trials to simultaneously screen multiple iAbs and other autoantibodies in one single well, which will facilitate high-throughput autoantibody screening simultaneously for T1D and other multiple relevant autoimmune diseases in large scale of populations. ECL assay has been illustrated as an excellent platform for a simple multiplex assay with a superior advantage of high sensitivity and disease specificity. In this chapter, we mainly focus on the discussion of: (1) two currently most popular assays for iAbs, a gold standard RBA and a modified bridging ELISA; (2) ECL assay and its comparison with RBA and (3) development of a simple multiplex ECL assay with all advantages considered.

and (3) define the onset of islet autoimmunity and its triggers.

190 Autoantibodies and Cytokines

At present, four major biochemically defined β-cell autoantigens were well characterized including IAA [26], GAD65 [27], IA-2 [28] and ZnT8 [29]. Through laboratory proficiency programs [30, 31] and harmonization efforts [32], RBA, a fluid-phase assay using radiolabeled antigens and precipitating antibody-antigen complex by Protein A/G Sepharose, has been well established as a current "gold" standard assay for autoantibodies to all four major antigens [29, 32, 33].

In RBA, recombinant human insulin is labeled with I-125, while GAD, IA-2 and ZnT8 antigens are expressed from their corresponding recombinant human cDNA and labeled with 35S-methionine by coupled in vitro transcription/translation. To assess general implementation of assay methods and to standardize the iAb assays, the Center for Disease Control and Prevention (CDC) and Immunology and Diabetes Society (IDS) started organizing the Diabetes Autoantibody Standardization Program (DASP) workshop in the year of 2000 [34]. Forty-six laboratories in 13 countries participated in the program. The first proficiency evaluation of DASP showed a high concordance in measurement of GADA (r = 0.96; p < 0.0001) and IA-2A (r = 0.89; p < 0.0001) using the new WHO international reference reagent. In contrast, the workshop demonstrated wide variation among IAA assays, with poor overall performance and low sensitivity. IAA assays were improved after the first proficiency evaluation and the median laboratory assigned sensitivity was 26, 36 and 45% in 2002, 2003 and 2005, respectively (p < 0.0001). However, the IAA assay has proven the most difficult to standardize with relatively wide discrepancies between laboratories in the IASP workshop and has not yet achieved a satisfactory level of sensitivity and specificity. There was still remarkable variation between laboratories and the ranking of IAA levels in patient serum samples was concordant to AUC (p < 0.001), and the AUC ranged from 0.36 to 0.91 [30]. To facilitate comparison of quantitative islet autoantibody results between studies, the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) set up an Islet Autoantibody Harmonization Committee in 2007 to align measurement and reporting of iAbs in all NIDDK-sponsored studies and several different central laboratories are used [32]. With a few years of combined efforts, both GADA and IA-2A assays were successfully harmonized with great concordance as positive or negative between participating laboratories. The IA-2A assay, using a common threshold of 5 DK units/ml, achieved 64% sensitivity with specificity greater than 99% in all laboratories. For GADA, using thresholds equivalent to the 97th percentile of 974 control samples in each laboratory, 1051 (97.9%) of 1074 samples were concordant. ZnT8 is a most recently identified new islet β-cell autoantigen, and ZnT8A assay has been well established through IASP workshop. In combination of current four iAb assays by the standard RBA, the overall sensitivity is able to cover around 95% in newly diagnosed patients with T1D [29]. To lead to more reproducible identification of individuals at risk of type 1 diabetes and improve monitoring in long-term prospective studies, some recommendations by the Islet Autoantibody Harmonization Committee have been applied to current national and international clinical trials for T1D: (1) confirmation of positive results in a second laboratory is likely to be valuable to identify discrepancies; (2) laboratories should use common calibrators against reference standards; (3) the methods used in multiple centers should be compared 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 laboratories.

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

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

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

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**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 plate-

captured Sulfo-tagged antigen is accomplished with electrochemiluminescence.

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

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