High-throughput multiplexed autoantibody detection to screen type 1 diabetes and multiple autoimmune diseases simultaneously

Gu, Yong; Zhao, Zhiyuan; Waugh, Kathleen; Miao, Dongmei; Jia, Xiaofan; Cheng, Jeremy; Michels, Aaron; Rewers, Marian; Yang, Tao; Yu, Liping

doi:10.1016/j.ebiom.2019.08.036

Cited by 27 publications

(12 citation statements)

References 30 publications

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“…Furthermore, a multiplexed approach is optimal when limited amounts of antigen, such as tissue lysates from patients, are available. Thus, multiplex assays are preferred if assay sensitivity and specificity are sufficiently preserved, and indeed a number of multiplex immunoassays have been developed to detect target molecules such as autoantibodies and cytokines (50)(51)(52). We used various fluorescent proteins to identify cell lines, and combinations of two fluorochromes clearly distinguish each cell line.…”

Section: Discussionmentioning

confidence: 99%

Multiplex T Cell Stimulation Assay Utilizing a T Cell Activation Reporter-Based Detection System

et al. 2020

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Recent advancements in single cell sequencing technologies allow for identification of numerous immune-receptors expressed by T cells such as tumor-specific and autoimmune T cells. Determining antigen specificity of those cells holds immense therapeutic promise. Therefore, the purpose of this study was to develop a method that can efficiently test antigen reactivity of multiple T cell receptors (TCRs) with limited cost, time, and labor. Nuclear factor of activated T cells (NFAT) is a transcription factor involved in producing cytokines and is often utilized as a reporter system for T cell activation. Using a NFAT-based fluorescent reporter system, we generated T-hybridoma cell lines that express intensely fluorescent proteins in response to antigen stimulation and constitutively express additional fluorescent proteins, which serve as identifiers of each T-hybridoma expressing a unique TCR. This allows for the combination of multiple T-hybridoma lines within a single reaction. Sensitivity to stimulation is not decreased by adding fluorescent proteins or multiplexing T cells. In multiplexed reactions, response by one cell line does not induce response in others, thus preserving specificity. This multiplex assay system will be a useful tool for antigen discovery research in a variety of contexts, including using combinatorial peptide libraries to determine T cell epitopes.

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Section: Discussionmentioning

confidence: 99%

Multiplex T Cell Stimulation Assay Utilizing a T Cell Activation Reporter-Based Detection System

et al. 2020

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“…TGA screening was performed in a multiplex ECL assay format combined with other three islet autoantibodies, and all positive results were retested for confirmation using corresponding single ECL assays. The method of multiplex ECL assay has been published previously [ 13 ]. The mechanisms of both single and multiplex ECL assays are shown in Figure 1 .…”

Section: Methodsmentioning

confidence: 99%

“…Unfortunately, there is no easy and inexpensive tool to screen for these conditions. With the platform of our established ECL assay, we successfully developed a multiplex ECL assay [ 13 ] to determine multiautoantibodies simultaneously in one single well for multiple autoimmune diseases. In the present study, we applied our multiplex ECL assay in an ongoing clinical trial, the Autoimmunity Screening for Kids (ASK) [ 14 ], to screen for both CD and T1D in children from general population in Denver metropolitan area.…”

Section: Introductionmentioning

confidence: 99%

Large-Scale Screening in General Population Children for Celiac Disease with a Multiplex Electrochemiluminescence (ECL) Assay

Jia

et al. 2020

Journal of Immunology Research

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Background. Autoimmunity Screening for Kids (ASK) study was launched to screen general population children for type 1 diabetes (T1D) and celiac disease (CD). Methods. A total of 23,319 children from general population were screened. A high throughput multiplex electrochemiluminescence (ECL) assay to screen multiautoantibodies in a single well was applied, parallel with a standard radiobinding assay (RBA). All children with any positive autoantibodies in screening were revisited within one month for confirmation and followed every 6 months. Results. Among 23,319 children, 2.6% (606/23,319) of children were tested positive for TGA. Multiplex ECL assay detected more TGA (584/23,319) in the initial screening than RBA (490/23,319, p = 0.004 ) and was able to detect TGA earlier than RBA in a subset of children by 0.8 to 34.8 months. Prevalence of TGA by either ECL or RBA in children with islet autoantibodies was found significantly higher than overall prevalence in general population screened. Conclusions. A multiplex ECL assay was more sensitive than standard RBA by identifying more TGA positivity and detecting TGA earlier in general population screening. It also provides a high efficient tool with its unique advantage of multiplexing measurements to screen for multiple autoimmune diseases simultaneously in general population.

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“…Another example is the multiplex electrochemiluminescence assay (ECL). The ECL employs an acid dissociation step to process the serum sample, which is then incubated overnight (>16 hr) with probes before subsequent signal analysis for GAD, IA-2 and insulin autoantibodies [9][10][11][12][13]. Recently, this method was expanded to measure 7 autoantibodies in a single assay [13].…”

Section: Plos Onementioning

confidence: 99%

“…Alternative methods such as ELISAs have been developed to address some of the limitations of radioassay; however, they do not detect insulin autoantibodies, one of the earliest biomarkers for type 1 diabetes, and still consume 20-50 μL of serum per autoantibody [6][7][8]. Other multiplex approaches such as nanoparticle enhanced immunoassays and electrochemical luminescence (ECL) assays require specialized instrumentation for assay readout, sample pretreatment or longer time-to-result (>16 hr) [5,[9][10][11][12][13]. Thus, detection of islet autoantibodies for disease diagnosis and risk screening can benefit substantially from a sample-sparing, nonradioactive, multiplexed, highly sensitive/specific assay that is easily deployable in routine clinical laboratories.…”

Section: Introductionmentioning

confidence: 99%

Sensitive detection of multiple islet autoantibodies in type 1 diabetes using small sample volumes by agglutination-PCR

Cortez¹,

Gebhart²,

Robinson³

et al. 2020

PLoS ONE

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Islet autoantibodies are predominantly measured by radioassay to facilitate risk assessment and diagnosis of type 1 diabetes. However, the reliance on radioactive components, large sample volumes and limited throughput renders radioassay testing costly and challenging. We developed a multiplex analysis platform based on antibody detection by agglutination-PCR (ADAP) for the sample-sparing measurement of GAD, IA-2 and insulin autoantibodies/antibodies in 1 μL serum. The assay was developed and validated in 7 distinct cohorts (n = 858) with the majority of the cohorts blinded prior to analysis. Measurements from the ADAP assay were compared to radioassay to determine correlation, concordance, agreement, clinical sensitivity and specificity. The average overall agreement between ADAP and radioassay was above 91%. The average clinical sensitivity and specificity were 96% and 97%. In the IASP 2018 workshop, ADAP achieved the highest sensitivity of all assays tested at 95% specificity (AS95) rating for GAD and IA-2 autoantibodies and top-tier performance for insulin autoantibodies. Furthermore, ADAP correctly identified 95% high-risk individuals with two or more autoantibodies by radioassay amongst 39 relatives of T1D patients tested. In conclusion, the new ADAP assay can reliably detect the three cardinal islet autoantibodies/antibodies in 1μL serum with high sensitivity. This novel assay may improve pediatric testing compliance and facilitate easier community-wide screening for islet autoantibodies.

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High-throughput multiplexed autoantibody detection to screen type 1 diabetes and multiple autoimmune diseases simultaneously

Cited by 27 publications

References 30 publications

Multiplex T Cell Stimulation Assay Utilizing a T Cell Activation Reporter-Based Detection System

Multiplex T Cell Stimulation Assay Utilizing a T Cell Activation Reporter-Based Detection System

Large-Scale Screening in General Population Children for Celiac Disease with a Multiplex Electrochemiluminescence (ECL) Assay

Sensitive detection of multiple islet autoantibodies in type 1 diabetes using small sample volumes by agglutination-PCR

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