Detection of Human Islet Autoantibodies Using an Electrochemiluminescence Assay

Overview

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

1. 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).
2. 3 mM of Biotin: Dissolve 1 mg of Biotin in 588 µL of labeling buffer.
3. 3 mM of Sulfo-tag: Dissolve 150 nmol of Sulfo-tag in 50 µL of labeling buffer.
4. Antigen buffer (5% BSA): In 500 mL of 1x PBS, add 25 g of Bovine Serum Albumin (BSA).
5. Prepare 0.5 M acetic acid solution.
6. 1.0 M of Tris-HCl buffer pH 9.0: Prepare 1 M Tris-HCl buffer, and adjust pH to 9.0 with HCl.
7. Coating buffer (3% Blocker A): In 500 mL of 1x PBS, add 15 g of Blocker A.
8. Washing buffer (0.05 % Tween 20, PBST): In 5000 mL of 1x PBS, add 2.5 mL of Tween 20.
9. Reading buffer (2x Read Buffer T with surfactant): In 500 mL of DD water, add 500 mL of 4x Read Buffer T with Surfactant (Table of Materials).
10. Store Biotin and Sulfo-tag in a -20 °C freezer.  
    NOTE: Both Biotin and Sulfo-tag solutions should always be freshly prepared just before the labeling procedure.

2. Label the Human Islet Autoantigen with Biotin and Sulfo-tag

NOTE: A high concentration of antigen, ≥0.5 mg/mL, is recommended for a more efficient labeling reaction.

1. Determine the molar ratio of human islet autoantigen for Biotin and Sulfo-tag.
   1. Obtain the antigen molar number by dividing the antigen weight by the molecular weight.
   2. Use the molar ratio of 1:5 for the antigen with smaller molecular weight (≤10 kd), and the molar ratio of 1:20 for the antigen with larger molecular weight (>50 kd).
   3. Calculate the volume for Biotin or Sulfo-tag by dividing the molar number by its concentration.
2. Mix the human islet autoantigen with Biotin or Sulfo-tag with the molar ratio of 1:5.
   NOTE: The protein in either tris or glycine buffer systems should be exchanged to 2x PBS buffer with pH 7.9 using the sizing spin column.
3. Since Biotin and Sulfo-tag are light sensitive, cover the reaction tubes with aluminum foil. Incubate the reaction at room temperature (RT) for 1 h.
4. During the incubation, prime the spin column by dispensing 2x PBS buffer into the column three times. Centrifuge the solution at 1000 x g for 2 min each time.
   NOTE: Currently the labeling reaction, through bridging, is still occurring and needs to be stopped.
5. Stop the labeling reaction by purifying the labeled human islet autoantigen. To purify, pass the autoantigen through the spin column and centrifuge the column at 1000 x g for 2 min.
6. Determine the labeled antigen concentration (µg/µL) using the amount of antigen protein and the final volume. 
   NOTE: Roughly, there will be 90 - 95% retention of labeled antigen after every spin column pass.
7. Aliquot the labeled antigen and store labeled antigen at -80 °C.

3. Define the Best Concentrations and Ratios for the Two Labeled Antigens for the Assay (Checker Board Assay)

1. Prepare two serum samples, one high positive and one negative, with a total volume of 200 µL for each.
2. Aliquot 4 µL of serum and add 1x PBS until the final volume is 20 µL per well for a 96-well polymerase chain reaction (PCR) plate. Use half of a plate for the high positive sample and the other half for the negative sample, as shown in Figure 1A.
3. Add 10 µL of Biotin and 10 µL of Sulfo-tag labeled antigen and conduct serial dilutions for the two differently labeled antigens, as shown in Figure 1A. Run a horizontal serial dilution for one labeled antigen and a vertical serial dilution for the other labeled antigen.
4. Continue the rest of the assay steps described in 5.3 to 9.1.
5. Calculate the ratio of signals from the high positive sample against each of the corresponding signals from the negative sample shown in Figure 1B.
6. Identify the best concentrations for Biotin and Sulfo-tag labeled antigens by selecting a point with the highest or near highest ratio of positive to negative. Consider the low background signal from the negative sample to identify the ratio.
   NOTE: Assay Day 1 includes Step 4 to Step 6.

4. Prepare the Antigen Buffer Using the Correct Concentration of Biotin/Sulfo-tag Labeled Antigen

1. Select the rational concentration of each antigen based on the checker board assay.
2. Prepare 3 mL of antigen solution per plate, using the rational concentration of Biotin/Sulfo-tag labeled antigen for the antigen buffer.

5. Incubate Serum Samples with Labeled Antigen

NOTE: There are two protocols in this section, one without serum acid treatment and one with serum acid treatment. All islet autoantibody assays except insulin autoantibody (IAA) assay use regular protocol without serum acid treatment from steps 5.1 to 5.5, whereas IAA assay skips these steps and uses protocol with serum acid treatment from steps 5.6 to 5.9.

1. Aliquot 4 µL of serum and add 1x PBS until the final volume is 20 µL per well for a 96-well PCR plate.
2. Add 20 µL of labeled antigen solution per well.
3. Cover the PCR plate with sealing foil to avoid light.
4. Put the plate on a shaker (low speed) at RT for 2 h.
5. Put the plate in the 4 °C refrigerator and incubate overnight (18 - 24 h).
   NOTE: The following protocol of steps from 5.6 to 5.9 is only designed for IAA assay and other autoantibody assays skip these steps.
6. Mix 15 µL of serum with 18 µL of 0.5 M acetic acid for each serum sample. Incubate this mixture at RT for 45 min.
7. Prepare the antigen solution with the rational concentration of Biotin and Sulfo-tag labeled antigen, based on the checker board assay, using antigen buffer. In each well, aliquot 35 µL of antigen buffer, containing Biotin and Sulfo-tag labeled antigen, into a new PCR plate.
8. Before the 45 min incubation (step 5.6) step is finished, add 8.3 µL of 1 M Tris pH 9.0 buffer to the side of each well on the antigen plate (step 5.6). It is important to limit the mixing between the Tris buffer and the antigen. Immediately transfer 25 µL of the serum, which is treated with acid, in step 5.6, into each well and agitate the solution. Cover the PCR plate with sealing foil to avoid light.
9. Put the plate on a shaker (low speed) at RT for 2 h. Put the plate in the 4 °C refrigerator and allow the plate to incubate overnight (18 - 24 h).

6. Prepare the Streptavidin Plate

1. Take a streptavidin plate from the 4 °C refrigerator and allow the plate to come to RT.
2. Once the streptavidin plate is at RT, add 150 µL of 3% Blocker A to each well.
3. Cover the PCR plate with sealing foil.
4. Incubate the plate in the 4 °C refrigerator overnight.   
   NOTE: Assay Day 2 includes Step 7 to Step 9.

7. Transfer Serum/Antigen Incubates to the Streptavidin Plate

1. The next day, take out the streptavidin plate from the refrigerator and discard the buffer from the plate. Set some dry paper towels out on the table and tap the plate upside down until there is no more buffer inside of the wells.
2. Fill the empty streptavidin plate with 150 µL of PBST per well and discard the PBST from the plate for a total of three washes.
3. Transfer 30 µL of serum/antigen incubates per well into the streptavidin plate.
4. Cover the plate with foil to avoid light. Shake the plate, at a low setting, at RT for 1 h.

8. Wash the Plate and Add Read Buffer

1. Discard incubates from the plate. Add 150 µL of PBST per well and discard the PBST from the plate for a total of three washes.
2. After washing, add 150 µL per well of Reading buffer.
   NOTE: It is important to prevent air bubbles in the solution because this will affect how the plate is read on the plate reader machine.

Representative Results

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 healthy controls.

Materials

Name	Company	Catalog Number	Comments
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