title: 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.
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle, WA 98195, USA.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Laha
authors.forename: Frederick G
authors.initials: FG
authors.lastname: Strathmann
authors.forename: Zhican
authors.initials: Z
authors.lastname: Wang
authors.forename: Ian H
authors.initials: IH
authors.lastname: de Boer
authors.forename: Kenneth E
authors.initials: KE
authors.lastname: Thummel
authors.forename: Andrew N
authors.initials: AN
authors.lastname: Hoofnagle
mesh_terms: D000906:Antibodies / Q000276:immunology*; D002853:Chromatography, Liquid; D003429:Cross Reactions; D003903:Deuterium; D006801:Humans; D006887:Hydroxycholecalciferols / Q000737:chemistry / Q000276:immunology*; D007201:Indicator Dilution Techniques; D057230:Limit of Detection; D015394:Molecular Structure; D013329:Structure-Activity Relationship; D053719:Tandem Mass Spectrometry
chemical_list: D000906:Antibodies; D006887:Hydroxycholecalciferols; D003903:Deuterium