A novel, high-performance, low-volume, rapid luciferase immunoprecipitation system (LIPS) assay to detect autoantibodies to zinc transporter 8

Claire L Williams; Ilaria Marzinotto; Cristina Brigatti; Kathleen M Gillespie; Group The Box Study; Vito Lampasona; Alistair J K Williams; Anna E Long

doi:10.1093/cei/uxad139

A novel, high-performance, low-volume, rapid luciferase immunoprecipitation system (LIPS) assay to detect autoantibodies to zinc transporter 8

Claire L Williams^*, Ilaria Marzinotto, Cristina Brigatti, Kathleen M Gillespie, Group The Box Study, Vito Lampasona, Alistair J K Williams, Anna E Long

^*Corresponding author for this work

Research output: Contribution to journal › Article (Academic Journal) › peer-review

1 Citation (Scopus)

Abstract

Background:
Zinc transporter 8 autoantibodies (ZnT8A) are thought to appear close to type 1 diabetes (T1D) onset and can identify high-risk multiple (≥2) autoantibody positive individuals. Radiobinding assays (RBA) are widely used for ZnT8A measurement but have limited sustainability. We sought to develop a novel, high-performance, non-radioactive luciferase immunoprecipitation system (LIPS) assay to replace RBA.

Methods:
A custom dual C-terminal ZnT8 (aa268-369; R325/W325) heterodimeric antigen, tagged with a Nanoluciferase (TM) (Nluc-ZnT8) reporter, and LIPS assay was developed. Assay performance was evaluated by testing sera from new onset T1D (n=573), healthy schoolchildren (n=521), and selected first-degree relatives (FDRs) from the Bart’s Oxford family study (n=617; 164 progressed to diabetes).

Results:
In new-onset T1D, ZnT8A levels by LIPS strongly correlated with RBA [Spearman’s r=0.89; p<0.0001), and positivity was highly concordant (94.3%). At a high specificity (95%), LIPS and RBA had comparable assay performance [LIPS pROC-AUC(95) 0.032 (95% CI: 0.029-0.036); RBA pROC-AUC(95) 0.031 (95% CI: 0.028-0.034); p=0.376]. Overall, FDRs found positive by LIPS or RBA had a comparable 20-year diabetes risk (52.6% and 59.7%, respectively), but LIPS positivity further stratified T1D risk in FDRs positive for at least one other islet autoantibody detected by RBA (p=0.0346).

Conclusion:
This novel, high-performance, cheaper, quicker, higher throughput, low blood volume Nluc-ZnT8 LIPS assay is a safe, non-radioactive alternative to RBA with enhanced sensitivity and ability to discriminate T1D progressors. This method offers an advanced approach to current strategies to screen the general population for T1D risk for immunotherapy trials and to reduce rates of diabetic ketoacidosis at diagnosis.

Original language	English
Pages (from-to)	215-224
Number of pages	10
Journal	Clinical and Experimental Immunology
Volume	215
Issue number	3
Early online date	27 Dec 2023
DOIs	https://doi.org/10.1093/cei/uxad139
Publication status	Published - 1 Mar 2024

Bibliographical note

Funding Information:
We are grateful to Diabetes UK for providing financial support to The BOX study (14/0004472), for awarding a research grant to KMG (14/0004869), jointly funding AEL with JDRF via an RD Lawrence Fellowship (18/0005778 and 3-APF-2018-591-A-N), and funding CLW via a PhD studentship (16/0005556 and 18/0005778) and a post-doctoral research grant awarded to AEL and KGM (21/0006332).

Publisher Copyright:
© The Author(s) 2023.

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The natural history of the autoimmune response to zinc transporter 8 in type 1 diabetes (PhD; Diabetes UK funded)
Williams, C. L. (Principal Investigator)
2/10/17 → 10/10/21
Project: Research

Diabetes UK Basic Science Digital Poster Award 2021
Williams, C. L. (Recipient), 30 Apr 2021
Prize: Prizes, Medals, Awards and Grants

File

Diabetes UK Professional Conference 2021
Williams, C. L. (Participant)
16 Apr 2021 → 30 Apr 2021
Activity: Participating in or organising an event types › Participation in conference

Cite this

Williams, C. L., Marzinotto, I., Brigatti, C., Gillespie, K. M., The Box Study, G., Lampasona, V., Williams, A. J. K., & Long, A. E. (2024). A novel, high-performance, low-volume, rapid luciferase immunoprecipitation system (LIPS) assay to detect autoantibodies to zinc transporter 8. Clinical and Experimental Immunology, 215(3), 215-224. https://doi.org/10.1093/cei/uxad139

@article{281f35c6b084480584a892b198f4bb14,

title = "A novel, high-performance, low-volume, rapid luciferase immunoprecipitation system (LIPS) assay to detect autoantibodies to zinc transporter 8",

abstract = "Background: Zinc transporter 8 autoantibodies (ZnT8A) are thought to appear close to type 1 diabetes (T1D) onset and can identify high-risk multiple (≥2) autoantibody positive individuals. Radiobinding assays (RBA) are widely used for ZnT8A measurement but have limited sustainability. We sought to develop a novel, high-performance, non-radioactive luciferase immunoprecipitation system (LIPS) assay to replace RBA.Methods:A custom dual C-terminal ZnT8 (aa268-369; R325/W325) heterodimeric antigen, tagged with a Nanoluciferase (TM) (Nluc-ZnT8) reporter, and LIPS assay was developed. Assay performance was evaluated by testing sera from new onset T1D (n=573), healthy schoolchildren (n=521), and selected first-degree relatives (FDRs) from the Bart{\textquoteright}s Oxford family study (n=617; 164 progressed to diabetes). Results:In new-onset T1D, ZnT8A levels by LIPS strongly correlated with RBA [Spearman{\textquoteright}s r=0.89; p<0.0001), and positivity was highly concordant (94.3%). At a high specificity (95%), LIPS and RBA had comparable assay performance [LIPS pROC-AUC(95) 0.032 (95% CI: 0.029-0.036); RBA pROC-AUC(95) 0.031 (95% CI: 0.028-0.034); p=0.376]. Overall, FDRs found positive by LIPS or RBA had a comparable 20-year diabetes risk (52.6% and 59.7%, respectively), but LIPS positivity further stratified T1D risk in FDRs positive for at least one other islet autoantibody detected by RBA (p=0.0346).Conclusion:This novel, high-performance, cheaper, quicker, higher throughput, low blood volume Nluc-ZnT8 LIPS assay is a safe, non-radioactive alternative to RBA with enhanced sensitivity and ability to discriminate T1D progressors. This method offers an advanced approach to current strategies to screen the general population for T1D risk for immunotherapy trials and to reduce rates of diabetic ketoacidosis at diagnosis.",

author = "Williams, {Claire L} and Ilaria Marzinotto and Cristina Brigatti and Gillespie, {Kathleen M} and {The Box Study}, Group and Vito Lampasona and Williams, {Alistair J K} and Long, {Anna E}",

note = "Funding Information: We are grateful to Diabetes UK for providing financial support to The BOX study (14/0004472), for awarding a research grant to KMG (14/0004869), jointly funding AEL with JDRF via an RD Lawrence Fellowship (18/0005778 and 3-APF-2018-591-A-N), and funding CLW via a PhD studentship (16/0005556 and 18/0005778) and a post-doctoral research grant awarded to AEL and KGM (21/0006332). Publisher Copyright: {\textcopyright} The Author(s) 2023.",

year = "2024",

month = mar,

day = "1",

doi = "10.1093/cei/uxad139",

language = "English",

volume = "215",

pages = "215--224",

journal = "Clinical and Experimental Immunology",

issn = "0009-9104",

publisher = "Wiley-Blackwell",

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A novel, high-performance, low-volume, rapid luciferase immunoprecipitation system (LIPS) assay to detect autoantibodies to zinc transporter 8. / Williams, Claire L; Marzinotto, Ilaria; Brigatti, Cristina et al.
In: Clinical and Experimental Immunology, Vol. 215, No. 3, 01.03.2024, p. 215-224.

Research output: Contribution to journal › Article (Academic Journal) › peer-review

TY - JOUR

T1 - A novel, high-performance, low-volume, rapid luciferase immunoprecipitation system (LIPS) assay to detect autoantibodies to zinc transporter 8

AU - Williams, Claire L

AU - Marzinotto, Ilaria

AU - Brigatti, Cristina

AU - Gillespie, Kathleen M

AU - The Box Study, Group

AU - Lampasona, Vito

AU - Williams, Alistair J K

AU - Long, Anna E

N1 - Funding Information: We are grateful to Diabetes UK for providing financial support to The BOX study (14/0004472), for awarding a research grant to KMG (14/0004869), jointly funding AEL with JDRF via an RD Lawrence Fellowship (18/0005778 and 3-APF-2018-591-A-N), and funding CLW via a PhD studentship (16/0005556 and 18/0005778) and a post-doctoral research grant awarded to AEL and KGM (21/0006332). Publisher Copyright: © The Author(s) 2023.

PY - 2024/3/1

Y1 - 2024/3/1

N2 - Background: Zinc transporter 8 autoantibodies (ZnT8A) are thought to appear close to type 1 diabetes (T1D) onset and can identify high-risk multiple (≥2) autoantibody positive individuals. Radiobinding assays (RBA) are widely used for ZnT8A measurement but have limited sustainability. We sought to develop a novel, high-performance, non-radioactive luciferase immunoprecipitation system (LIPS) assay to replace RBA.Methods:A custom dual C-terminal ZnT8 (aa268-369; R325/W325) heterodimeric antigen, tagged with a Nanoluciferase (TM) (Nluc-ZnT8) reporter, and LIPS assay was developed. Assay performance was evaluated by testing sera from new onset T1D (n=573), healthy schoolchildren (n=521), and selected first-degree relatives (FDRs) from the Bart’s Oxford family study (n=617; 164 progressed to diabetes). Results:In new-onset T1D, ZnT8A levels by LIPS strongly correlated with RBA [Spearman’s r=0.89; p<0.0001), and positivity was highly concordant (94.3%). At a high specificity (95%), LIPS and RBA had comparable assay performance [LIPS pROC-AUC(95) 0.032 (95% CI: 0.029-0.036); RBA pROC-AUC(95) 0.031 (95% CI: 0.028-0.034); p=0.376]. Overall, FDRs found positive by LIPS or RBA had a comparable 20-year diabetes risk (52.6% and 59.7%, respectively), but LIPS positivity further stratified T1D risk in FDRs positive for at least one other islet autoantibody detected by RBA (p=0.0346).Conclusion:This novel, high-performance, cheaper, quicker, higher throughput, low blood volume Nluc-ZnT8 LIPS assay is a safe, non-radioactive alternative to RBA with enhanced sensitivity and ability to discriminate T1D progressors. This method offers an advanced approach to current strategies to screen the general population for T1D risk for immunotherapy trials and to reduce rates of diabetic ketoacidosis at diagnosis.

AB - Background: Zinc transporter 8 autoantibodies (ZnT8A) are thought to appear close to type 1 diabetes (T1D) onset and can identify high-risk multiple (≥2) autoantibody positive individuals. Radiobinding assays (RBA) are widely used for ZnT8A measurement but have limited sustainability. We sought to develop a novel, high-performance, non-radioactive luciferase immunoprecipitation system (LIPS) assay to replace RBA.Methods:A custom dual C-terminal ZnT8 (aa268-369; R325/W325) heterodimeric antigen, tagged with a Nanoluciferase (TM) (Nluc-ZnT8) reporter, and LIPS assay was developed. Assay performance was evaluated by testing sera from new onset T1D (n=573), healthy schoolchildren (n=521), and selected first-degree relatives (FDRs) from the Bart’s Oxford family study (n=617; 164 progressed to diabetes). Results:In new-onset T1D, ZnT8A levels by LIPS strongly correlated with RBA [Spearman’s r=0.89; p<0.0001), and positivity was highly concordant (94.3%). At a high specificity (95%), LIPS and RBA had comparable assay performance [LIPS pROC-AUC(95) 0.032 (95% CI: 0.029-0.036); RBA pROC-AUC(95) 0.031 (95% CI: 0.028-0.034); p=0.376]. Overall, FDRs found positive by LIPS or RBA had a comparable 20-year diabetes risk (52.6% and 59.7%, respectively), but LIPS positivity further stratified T1D risk in FDRs positive for at least one other islet autoantibody detected by RBA (p=0.0346).Conclusion:This novel, high-performance, cheaper, quicker, higher throughput, low blood volume Nluc-ZnT8 LIPS assay is a safe, non-radioactive alternative to RBA with enhanced sensitivity and ability to discriminate T1D progressors. This method offers an advanced approach to current strategies to screen the general population for T1D risk for immunotherapy trials and to reduce rates of diabetic ketoacidosis at diagnosis.

U2 - 10.1093/cei/uxad139

DO - 10.1093/cei/uxad139

M3 - Article (Academic Journal)

C2 - 38150393

SN - 0009-9104

VL - 215

SP - 215

EP - 224

JO - Clinical and Experimental Immunology

JF - Clinical and Experimental Immunology

IS - 3

ER -

A novel, high-performance, low-volume, rapid luciferase immunoprecipitation system (LIPS) assay to detect autoantibodies to zinc transporter 8

Abstract

Bibliographical note

UN SDGs

Access to Document

Handle.net

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Projects

The natural history of the autoimmune response to zinc transporter 8 in type 1 diabetes (PhD; Diabetes UK funded)

Prizes

Diabetes UK Basic Science Digital Poster Award 2021

Activities

Diabetes UK Professional Conference 2021

Cite this