Detection of Human Islet Autoantibodies Using an Electrochemiluminescence Assay

In This Article

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 µL of labeling buffer.
Antigen buffer (5% BSA): In 500 mL of 1x PBS, add 25 g of Bovine Serum Albumin (BSA).
Prepare 0.5 M acetic....

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Results

ROC curve diagram showing sensitivity vs. 1-specificity for model evaluation; dash-dot line.
Figure 1: The ROC curve for determining the cut-off of assay positivity. The 99^th 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 .......