Detection of Human Islet Autoantibodies Using an Electrochemiluminescence Assay

Published: March 29, 2024

Abstract

Source: Gu, Y. et al., Electrochemiluminescence Assays for Human Islet Autoantibodies. J. Vis. Exp. (2018)

This video showcases the electrochemiluminescence detection of islet autoantibodies in human serum. The antibodies in the serum bridge the ruthenium-conjugated antigen to the biotinylated capture antigen, forming complexes that are detected by electrochemiluminescence measurements.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 1. Buffer Preparation Labeling buffer (2x phosphate-buffered saline, (PBS), pH 7.9): In 400 mL of distilled deionized (DD) water, add 100 mL of 10x PBS, adjust pH to 7.9 with sodium hydroxide (NaOH). 3 mM of Biotin: Dissolve 1 mg of Biotin in 588 µL of labeling buffer. 3 mM of Sulfo-tag: Dissolve 150 nmol of Sulfo-tag in 50 …

Representative Results

Figure 1: The ROC curve for determining the cut-off of assay positivity. The 99th percentile of specificity corresponding to 85% sensitivity was selected and is represented as an index value of 0.023. This upper limit of the assay was taken from 100 healthy controls.

Disclosures

The authors have nothing to disclose.

Materials

Human recombinant proinsulin protein(PINS) AmideBio Human Proinsulin, Rec
Human recombinant GAD65 protein Diamyd rhGAD65
Human recombinant IA-2 protein Creative BioMart IA2
1x Phosphate Buffered Saline (PBS), pH 7.4 Thermo Fisher Scientific 10010-023
10x PBS, pH 7.4 Thermo Fisher Scientific 70011-044
Bovine Serum Albumin (BSA) SIGMA-ALORICH A7906-500G
96-well PCR plate  Fisher 14230232
Streptavidin coated plate MSD L15SA
Zeba sizing spin column Thermo Fisher Scientific 89890
Twen-20 Fisher BP337-500
HCl Fisher A144-500
NaOH Fisher SS255-1
Trizma Base Fisher BP152-5
EZ-Link Biotin Thermo Fisher Scientific PI21329
Sulfo-tag MSD R91AO-1
Blocker A MSD R93AA-1
4x Read Buffer T with Surfactant MSD R92TC-1
EZ-Link NHS-PEG4-Biotin ThermoScience 21329
Sulfo-TAG MSD R91AO-1
96-well polymerase chain reaction (PCR) plate Fisher brand 14230232
96-Well Streptavidin plate MSD GOLD L 15SA-1
Zeba Spin Desalting Column ThermoScience PL208984
96-well Plate Shaker Perkin Elmer 1296-003
Plate Reader MSD QuickPlex SQ120
Benchtop centrifuge with bucket rotary Beckman Allegra X-15R

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Cite This Article
Detection of Human Islet Autoantibodies Using an Electrochemiluminescence Assay. J. Vis. Exp. (Pending Publication), e22098, doi: (2024).

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