Trimellitic anhydride

Trimellitic anhydride (TMA) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and was dissolved in a mixture of acetone (Junsei, Tokyo, Japan) and olive oil (Filippo Berio, Lucca, Italy) (A/O; vehicle 4 : 1) immediately before application. Formamide solution and Evans blue were purchased from Junsei (Tokyo, Japan) and Sigma-Aldrich (St. Louis, MO, USA), respectively.

In this investigation, we followed the methods of chemical binding to proteins from our previous publication, in which we ascertained elevated levels of antibodies against xenobiotics in a subgroup of healthy subjects [19 (link)]. In that study, we identified antibodies to the same chemical haptens in about 20% of tested individuals. For this study, we used the same binding methods.
We purchased the HSA, BSA, hemoglobin, formaldehyde, tolylene-2.4-diisocyanate, trimellitic anhydride, p-amino benzoic acid, bisphenol-A (BPA), tetrabromobisphenol-A (TBBPA), isopropyl benzoic acid, cyanoethyl benzoic acid, propyl 4-hydroxybenzoic acid, permethrin, mercury chloride, nickel sulfate, cobalt acetate, cadmium chloride, lead acetate, and arsenic oxide from Sigma Aldrich (St. Louis, MO, USA).

Three chemical skin sensitizers, oxazolone (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one; OXA, purity ≥90%, E0753), formaldehyde (FA, purity 36.5–38%, F8775), and 2,4-dinitrochlorobenzene (DNCB, purity 97%, 138630), and three respiratory sensitizers, ortho-phthaldialdehyde (OPA, purity ≥97%, P1378), hexamethylene-1,6-diisocyanate (HDI, purity ≥98%, 52650), and trimellitic anhydride (TMA, purity 97%, B4600), were purchased from Sigma-Aldrich (Table S1 in Supplementary Material). A magnetic bead conjugated with monoclonal antibody (mAb) against CD14 was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). mAbs for human CD14 (HCD14), CD11c (cllne 3.9), and CD11c (EP1347Y) were obtained from BioLegend (San Diego, CA, USA), eBioscience (La Jolla, CA, USA), and Abcam (Cambridge, UK), respectively. Alex Fluor 647 anti-rabbit IgG was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Human recombinant granulocyte macrophage colony-stimulating factor (GM-CSF) was purchased from Miltenyi Biotec. Human recombinant IL-4 was kindly provided from Schering-Plough (currently, Merck, Kenilworth, NJ, USA).