2.2.1. Antigens and Couplings

Tetanus toxoid was purchased from Massachusetts Biological Laboratories (Boston, MA, USA) and diphtheria toxoid was purchased from List Biological Laboratories (Campbell, CA, USA). Antigens were coupled to MagPlex microspheres (Luminex Corp, Austin, TX, USA), as described previously [5,23], using antigen concentrations of 12.5 <sup>μ</sup>g/12.5 × <sup>10</sup><sup>6</sup> microspheres for tetanus toxoid and 60 <sup>μ</sup>g/12.5 × <sup>10</sup><sup>6</sup> microspheres for diphtheria toxoid.

### 2.2.2. Sample Preparation and Bead Assay

Samples were diluted to a final serum concentration of 1:400 in Buffer B (PBS pH 7.2 plus 0.5% casein, 0.8% PVP, 0.5% PVA, 0.3% Tween 20, 0.02% sodium azide and 3 μg/mL of Escherichia coli lysate containing GST). All incubation steps were performed at room temperature in 50 μL reaction volumes protected from light while shaking at 600 rpm. Incubation steps were followed by three washes with 200 μL PBS pH 7.2 containing 0.05% Tween 20 (PBST) using a handheld magnet. Diluted samples were incubated with 625 microspheres/antigen/well for 90 min, followed by 45 min incubation with secondary antibodies at a concentration of 50 ng/well for anti-human IgG and 40 ng/well for anti-IgG4 (Southern Biotech, Birmingham, AL, USA). Incubations with 250 ng/well streptavidin-R phycoerythrin (Invitrogen, Waltham, MA, USA) and assay buffer alone were carried out, as previously described [5]. Beads were resuspended in 100 μL PBS pH 7.2 and stored overnight at 4 ◦C prior to reading on MAGPIX (Luminex Corporation, Austin, TX, USA).

#### 2.2.3. Quality Control and Assurance

Data were output as median fluorescence intensity (MFI). To control for background reactivity, each assay included 2 blank wells containing Buffer B only. Samples were run in singlicate. Each plate also contained 1 negative serum control and 2 positive serum controls run in duplicate, as well as a positive pool serum control that was serially diluted to make an 8-point curve to cover the linear range of MFI for most antigens. Samples and controls were analyzed with the average of the plate background subtracted (MFI-BG). As a measure of assay-to-assay variation, the average reactivities of the antigens with the controls was used to create criteria for accepting or rejecting plate data.
