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).