Characterizing antibody cross-reactivity for immunoaffinity purification of analytes prior to multiplexed liquid chromatography-tandem mass spectrometry

Abstract

Background: Immunoassays for 1α,25-dihydroxyvitamin D [1α,25(OH)(2)D] lack analytical specificity. We characterized the cross-reactivity of an anti-1α,25(OH)(2)D antibody with purified vitamin D metabolites and used these data to map the chemical features of 1α,25(OH)(2)D that are important for antibody binding. Additionally, we hypothesized that when combined with isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS), antibody cross-reactivity could be used to semiselectively enrich for structurally similar metabolites of vitamin D in a multiplexed assay.

Methods: Sample preparation consisted of immunoaffinity enrichment with a solid-phase anti-1α,25(OH)(2)D antibody and derivatization. Analytes were quantified with LC-MS/MS. Supplementation and recovery studies were performed for 11 vitamin D metabolites. We developed a method for simultaneously quantifying 25(OH)D(2), 25(OH)D(3), 24,25(OH)(2)D(3), 1α,25(OH)(2)D(2), and 1α,25(OH)(2)D(3) that included deuterated internal standards for each analyte.

Results: The important chemical features of vitamin D metabolites for binding to the antibody were (a) native orientation of the hydroxyl group on carbon C3 in the A ring, (b) the lack of substitution at carbon C4 in the A ring, and (c) the overall polarity of the vitamin D metabolite. The multiplexed method had lower limits of quantification (20% CV) of 0.2 ng/mL, 1.0 ng/mL, 0.06 ng/mL, 3.4 pg/mL, and 2.8 pg/mL for 25(OH)D(2), 25(OH)D(3), 24,25(OH)(2)D(3), 1α,25(OH)(2)D(2), and 1α,25(OH)(2)D(3), respectively. Method comparisons to 3 other LC-MS/MS methods yielded an r(2) value >0.9, an intercept less than the lower limit of quantification, and a slope statistically indistinguishable from 1.0.

Conclusions: LC-MS/MS can be used to characterize antibody cross-reactivity, a conclusion supported by our multiplexed assay for 5 vitamin D metabolites with immunoenrichment in a targeted metabolomic assay.

Figure 1. Composite chromatogram

Analytes were individually added to an immunoaffinity extract of stripped serum, derivatized, and chromatographically resolved as described for the multiplexed assay. The MRM transition for peaks 1, 2, 3, 4 and 5 is 623.5>298.2; peaks 6, 7, and 8 is 623.5 > 314.2; peak 9 is 635.5 > 314.2; peak 10, 11 and 12 is 607.6 > 298.2; peak 13 and 14 is 619.6 > 298.2. Peak 1: Minor 24,25(OH)₂D₃ PTAD isomer, peak 2: 23(S),25(OH)₂D₃, peak 3: 25,26(OH)₂D₃, peak 4: 24,25(OH)₂D₃, peak 5: 23(R),25(OH)₂D₃, peak 6: 4β,25(OH)₂D₃, peak 7: 1α25(OH)₂D₃, peak 8: 3-epi-1α,25(OH)₂D₃, peak 9: 1α,25(OH)₂D₂, peak 10: Minor 25(OH)D₃ PTAD isomer, peak 11: 3-epi-25(OH)D₃, peak 12: 25(OH)D₃, peak 13: Minor 25(OH)D₂ PTAD isomer, peak 14: 25(OH)D₂. A chromatogram of unsupplemented immunoaffinity extract of a stripped serum sample is shown in Supplemental Figure 1. The derivatization efficiency of 25(OH)D₂ and 25(OH)D₃ is 99.4% and 99.8%, respectively.