Characterizing antibody cross-reactivity for immunoaffinity purification of analytes prior to multiplexed liquid chromatography--tandem mass spectrometry.
The concentrations of vitamin D metabolites in plasma span >3 orders of magnitude. For example, the concentration of the most active vitamin D metabolite, 1[alpha],25[(OH).sub.2]D, is 1000-fold lower than prohormone 25(OH)D. Previous experiments that demonstrated substantial cross-reactivity of an antibody used in a commercially available competitive RIA for 1[alpha], 25[(OH).sub.2]D [Immunodiagnostic Systems (IDS)] (14) might raise concern regarding the utility of previous clinical and epidemiologic studies that have investigated 1[alpha],25[(OH).sub.2]D as a biomarker in human disease. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to resolve potential interfering analytes and to directly detect the analytes of interest, we previously demonstrated that it is possible to greatly improve the specificity of the antibody-based assay (14). Since the publication of that assay, we have confirmed the results of another study that demonstrated the utility of a different solid-phase antibody (ALPCO Diagnostics) to immunopurify analyte before LC-MS/MS (15). This antibody does not need sample preparation before immunoaffinity enrichment. The simpler work flow and preliminary data demonstrating saturation of the IDS solid-phase reagent with moderate concentrations of spiked 25(OH)D led us to transfer our clinical work flow to the new solid-phase reagent from ALPCO. The chromatographic gradient was similar to that described (14), with the exception that the starting conditions were revised to 56% mobile phase A [Optima water (Fisher), 1 mL/L formic acid (VWR), and 0.5 mmol/L methylamine (Sigma-Aldrich)] and 44% mobile phase B [acetonitrile (Fisher), 1 mL/L formic acid, and 0.5 mmol/L methylamine]. The chromatographic separation of multiple vitamin D analytes is shown in Fig. 1.
To characterize the cross-reactivity of the solidphase antibody, we determined the extraction efficiency(analytical recovery) of the antibodyfor different vitamin D metabolites. Each analyte (Table 1; see Table 1 in the Data Supplement that accompanies the online version of this brief communication at http:// www.clinchem.org/content/vol58/issue/12) was individually added to a vitamin D-depleted human serum matrix (MSG-4000; Golden West Biologicals), extracted with the immunoaffinity reagent from ALPCO, derivatized, and quantified with LC-MS/MS. The chromatographic peak areas obtained from the extraction were compared with the peak area observed after adding each analyte individually to the resulting extract of a nonspiked MSG-4000 sample (Table 1). Three important features of the vitamin D metabolites seemed to determine their affinities for the antibody. First, the extraction efficiencies of 25(OH)[D.sub.2] and 25(OH)[D.sub.3] were substantially lower than for the majority of the dihydroxyvitamin D metabolites, a result indicating that overall polarity was important for binding. Second, vitamin D metabolites with an epimeric orientation of the carbon C3 hydroxyl group of the A ring had a substantially reduced affinity for the antibody, suggesting that this part of the molecule was important for specific binding. Third, 4[beta],25[(OH).sub.2][D.sub.3] had very little affinity, suggesting that this part of the A ring was also important and further highlighting the importance of the A ring and the region near the C3 carbon for antibody affinity. To further support these observations, we determined the apparent dissociation constant ([K.sub.d]) in serum for several of the analytes via standard Scatchard analysis (the [K.sub.d] equals the negative slope of a plot of the ratio of the concentration of bound analyte to that of free analyte vs the concentration of bound analyte). The apparent [K.sub.d] of 1[alpha],25[(OH).sub.2][D.sub.3] was 0.10 [micro]mol/L. That of 1[alpha],25[(OH).sub.2][D.sub.2] was approximately 4-fold lower (0.41 [micro]mol/L), which was similar to that observed for the dihydroxylated 24,25[(OH).sub.2][D.sub.3] metabolite (0.39 [micro]mol/L). The monohydroxylated 25(OH) [D.sub.3] metabolite had a lower affinity (14 [micro]mol/L). The affinity of the carbon C3 epimer of 25(OH)[D.sub.3] could not be measured exactly, but it was noted to be > 140 [micro]mol/L. Taken together, these data suggest that specific A-ring substitutions and overall molecular polarity are important for hapten binding.
[FIGURE 1 OMITTED]
Our chemical characterization of the hapten complementarity of the antibody has 2 important implications. First, the carbon C3 epimer of 25(OH)[D.sub.3] is not well recognized by the antibody. Because the epimer is not easily resolved from native 25(OH)[D.sub.3] in rapid chromatographic methods, the immunoextraction step could lead to shortened LC-MS/MS methods without interference from the epimer (13). Similarly, 4[beta],25[(OH).sub.2][D.sub.3], which is present at concentrations similar to those of 1[alpha],25[(OH).sub.2][D.sub.3], is not well recognized by the antibody. It is difficult to resolve these 2 analytes in short chromatographic methods (16, 17); consequently, methods to quantify 1[alpha],25[(OH).sub.2][D.sub.3] without immunoaffinity extraction need to be carefully evaluated for interference from 4[beta],25[(OH).sub.2][D.sub.3] (18).
Given the favorable affinities of many vitamin D metabolites, we evaluated the possibility of using the immunoextraction of vitamin D metabolites as a step in a multiplexed assay of 25(OH)[D.sub.2], 25(OH)[D.sub.3], 1[alpha],25[(OH).sub.2][D.sub.2],1[alpha],25[(OH).sub.2][D.sub.3], and 24,25[(OH).sub.2][D.sub.3]. Such an assaywould be able to simultaneously evaluate vitamin D stores, production levels of active metabolite, and inactivation levels of metabolites. The multiplexed assay used 400 [micro]L calibrators, controls, or patient sample; 20 [micro]L of a mixture of deuterated internal standards in methanol [500 [micro]g/L each of 25(OH)[D.sub.2]d3, 25(OH)[D.sub.3]-d6, and 24,25[(OH).sub.2][D.sub.3]-d6; 4 [micro]g/L each of 1[alpha],25[(OH).sub.2][D.sub.3]-d6 and 1[alpha],25[(OH).sub.2][D.sub.2]-d6]; and 100 [micro]L immunoaffinity beads. The commercial sources of the deuterated internal standards are listed in Table 1 in the online Data Supplement). The plate was then covered and incubated for 2 h at 45[degrees]C with shaking at 800 rpm in a Thermomixer (Eppendorf). After immunoextraction, the beads were quantitatively transferred to a 2-mL filter plate (Strata Impact; Phenomenex), and washed 10 times with 1 mL Optima-grade water (Fisher). The analytes were eluted from the beads with 0.25 mL acetonitrile into a 1-mL well of a 96-deep-well collection plate (Waters). The eluate was then evaporated in a Turbovap concentrator (Biotage) at 30[degrees]C under nitrogen (20 [ft.sup.3]/h). The residue was reconstituted in 50 [micro]L acetonitrile containing 0.7 g/L 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) (Sigma-Aldrich). After a 30-min incubation at ambient temperature, the excess PTAD was quenched with 70 [micro]L water (Optima), and 40 [micro]L was injected onto a Waters Acquity liquid chromatography system coupled to a Waters Xevo TQ MS tandem mass spectrometer. Values for relevant mass spectrometer parameters are listed in Table 2 in the online Data Supplement. The experiments described spanned 5 lots of affinity beads.
The new multiplexed assay had lower limits of quantification (20% CV) of 1.0 ng/mL, 0.2 ng/mL, 0.06 ng mL, 3.4 pg mL, and 2.8 pg mL for 25(OH)[D.sub.3], 25(OH)[D.sub.2], 24,25[(OH).sub.2][D.sub.3],1[alpha],25[(OH).sub.2][D.sub.3], and 1[alpha],25 [(OH).sub.2][D.sub.2], respectively. Total assay imprecision (Table 1) was [less than or equal to] 17.1% for all analytes at low concentrations (at or below clinical-decision points). We compared the new multiplexed method with liquid-liquid extraction methods optimized for 25(OH)[D.sub.2], 25(OH)[D.sub.3], and 24,25[(OH).sub.2][D.sub.3], which have previously been described (19) or are described in the Supplemental Methods and Table 3 in the online Data Supplement. We also compared the new multiplexed method with the previously described immunoaffinity assay for 1[alpha],25[(OH).sub.2][D.sub.2] and 1[alpha],25[(OH).sub.2][D.sub.3] that uses IDS solidphase antibody (14). Descriptive and plotted data for the method comparisons (Table 1; see Figs. 2-4 in the online Data Supplement) showed that the methods compared acceptably (defined as an [r.sup.2] value >0.9, an intercept less than the lower limit of quantification for each analyte, and a slope statistically indistinguishable from 1.0). In terms of identifying low total 25(OH)D or low total 1[alpha],25[(OH).sub.2]D concentrations in plasma (<20 ng/mL or <17 [micro]g/mL, respectively), the new method reclassified 3.3% and 1.9% of people, respectively, compared with the existing methods. We also compared the new method with a previouslydescribed liquid-liquid extraction method for 1[alpha],25[(OH).sub.2][D.sub.3] (17). The 2 methods were relatively poorly correlated, which may be due to the improved specificity of the immunoaffinity purification approach (see Fig. 5 in the online Data Supplement). Ion suppression for the analytes ranged from -1.2% to 26.3%. We did not perform an extensive mapping experiment with the IDS solid-phase antibody, but the peak areas for a single patient sample were similar with the 2 reagents (see Fig. 6 in the online Data Supplement). Although there is an increased cost of the new assay compared with standard liquid-liquid extraction, the immunoaffinity approach has important advantages. First, interferences from 4[beta],25[(OH).sub.2][D.sub.3] and epi-C3-25(OH)[D.sub.3] are eliminated, which can permit shorter chromatographic runs (16). Second, the lower limit of quantification for 1[alpha],25[(OH).sub.2][D.sub.3] is lower (17), which permits the use of smaller plasma volumes in the assay.
Competitive assays rely on antibody specificity or, in the case of dihydroxyvitamin D analytes, on extensive sample preparation before analysis (e.g., protein precipitation and chromatographic resolution of interfering substances) (8, 20). The thermodynamics of the interference of related compounds with reagent antibodies are most often characterized by equilibrium inhibition studies. In this study, we used equilibrium-binding studies in a human serum matrix to map the chemical features of the vitamin D hapten that most strongly dictate binding to the reagent antibody from a commercially available assay. This work complements competitive-binding studies that have been performed in the past and the many epitope-mapping experiments performed with overlapping synthetic peptides.
The obvious lack of analytical specificity of reagent antibodies in small-molecule immunoassays can be an advantage in multiplexed mass spectrometric assays by simplifying sample preparation and greatly enriching for target analytes. We note that the success of this multiplexed method relies on the large differences in concentration between the analytes being quantified. For example, if the plasma concentrations of 25(OH)[D.sub.3] and 1[alpha],25[(OH).sub.2][D.sub.3] were similar, we might not be able to quantify 25(OH)[D.sub.3] with this approach, owing to the 140-fold lower affinity of the antibody for 25(OH)[D.sub.3] compared with 1[alpha],25[(OH).sub.2][D.sub.3]. In addition, the antibodies for the same analyte used in immunoaffinity enrichment steps before mass spectrometry could differ in important ways between manufacturers. Consequently, competition with similar analytes could differentially affect the recovery of the analytes of interest. Although such differences between ALPCO and IDS did not appear to be a large problem, they could be important in other systems. Projecting forward from our findings in this study, we are interested in seeing if combining antibodies in a single assay will permit the quantification of different classes of analytes simultaneously.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revisingthe article for intellectual content; and (c) final approval of the published article.
Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:
Employment or Leadership: A.N. Hoofnagle, Clinical Chemistry, AACC.
Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: K.E. Thummel, National Institutes of Health (R01 GM63666); A.N. Hoofnagle, National Institutes of Health (P30 DK035816), Clinical Mass Spectrometry Facility of the University of Washington, and Waters.
Expert Testimony: None declared.
Patents: None declared.
Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.
(1.) Holick MF. The use and interpretation of assays for vitamin D and its metabolites. J Nutr 1990; 120(Suppl 11):1464-9.
(2.) Cheng JB, Levine MA, Bell NH, Mangelsdorf DJ, Russell DW. Genetic evidence that the human CYP2R1 enzyme is a key vitamin D 25-hydroxylase. Proc Natl Acad Sci USA 2004;101: 7711-5.
(3.) Zhu J, Deluca HF. Vitamin D 25-hydroxylase Four decades of searching, are we there yet? Arch Biochem Biophys 2012;523:30-6.
(4.) Clements MR, Davies M, Hayes ME, Hickey CD, Lumb GA, Mawer EB, Adams PH. The role of 1,25-dihydroxyvitamin D in the mechanism of acquired vitamin D deficiency. Clin Endocrinol (Oxf) 1992;37:17-27.
(5.) Kanis JA. Vitamin D metabolism and its clinical application. J Bone Joint Surg Br 1982;64:542-60.
(6.) Prosser DE, Jones G. Enzymes involved in the activation and inactivation of vitamin D. Trends Biochem Sci 2004;29:664-73.
(7.) St-Arnaud R. Targeted inactivation of vitamin D hydroxylases in mice. Bone 1999;25:127-9.
(8.) Tanaka Y, DeLuca HF. Measurement of mammalian 25-hydroxyvitamin D3 24R-and 1 alphahydroxylase. Proc Natl Acad Sci USA 1981;78: 196-9.
(9.) DeLuca HF, Suda T, Schnoes HK, Tanaka Y, Holick MF. 25,26-dihydroxycholecalciferol, a metabolite of vitamin D3 with intestinal calcium transport activity. Biochemistry 1970;9:4776-80.
(10.) Wang Z, Lin YS, Zheng XE, Senn T, Hashizume T, Scian M, et al. An inducible cytochrome P450 3A4-dependent vitamin D catabolic pathway. Mol Pharmacol 2011;81:498-509.
(11.) Brown AJ, Ritter C, Slatopolsky E, Muralidharan KR, Okamura WH, Reddy GS. 1[alpha],25-Dihydroxy-3-epi-vitamin D3, a natural metabolite of 1[alpha],25-dihydroxyvitamin D3, is a potent suppressor of parathyroid hormone secretion. J Cell Biochem 1999;73:106-13.
(12.) Fleet JC, Bradley J, Reddy GS, Ray R, Wood RJ 1[alpha],25-[(OH).sub.2]-vitamin D3 analogs with minimal in vivo calcemic activity can stimulate significant transepithelial calcium transport and mRNA expression in vitro. Arch Biochem Biophys 1996; 329:228-34.
(13.) Strathmann FG, Sadilkova K, Laha TJ, LeSourd SE, Bornhorst JA, Hoofnagle AN, Jack R. 3-epi-25 hydroxyvitamin D concentrations are not correlated with age in a cohort of infants and adults. Clin Chim Acta 2012;413:203-6.
(14.) Strathmann FG, Laha TJ, Hoofnagle AN. Quantification of 1[alpha],25-dihydroxy vitamin D by immunoextraction and liquid chromatography-tandem mass spectrometry. Clin Chem 2011;57:127-985.
(15.) Yuan C, Kosewick J, He X, Kozak M, Wang S. Sensitive measurement of serum 1[alpha],25-dihydroxyvitamin D by liquid chromatography/tandem mass spectrometry after removing interference with immunoaffinity extraction. Rapid Commun Mass Spectrom 2011;25:1241-9.
(16.) Aronov PA, Hall LM, Dettmer K, Stephensen CB, Hammock BD. Metabolic profiling of major vitamin D metabolites using Diels-Alder derivatization and ultra-performance liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem 2008;391: 1917-30.
(17.) Wang Z, Senn T, Kalhorn T, Zheng XE, Zheng S, Davis CL, et al. Simultaneous measurement of plasma vitamin D3 metabolites, including 4[beta],25-dihydroxyvitamin D3, using liquid chromatography-tandem mass spectrometry. Anal Biochem 2011; 418:126-33.
(18.) Ding S, Schoenmakers I, Jones K, Koulman A, Prentice A, Volmer DA. Quantitative determination of vitamin D metabolites in plasma using UHPLC-MS/MS. Anal Bioanal Chem 2010;398: 779-89.
(19.) Hoofnagle AN, Laha TJ, Donaldson TF. A rubber transfer gasket to improve the throughput of liquid-liquid extraction in 96-well plates: application to vitamin D testing. J Chromatogr B Analyt Technol Biomed Life Sci 2010;878:1639-42.
(20.) Gray TK, McAdoo T, Pool D, Lester GE, Williams ME, Jones G. A modified radioimmunoassay for 1,25-dihydroxycholecalciferol. Clin Chem 1981; 27:458-63.
Thomas J. Laha,  Frederick G. Strathmann, [2,3] Zhican Wang,  Ian H. de Boer,  Kenneth E. Thummel,  and Andrew N. Hoofnagle [1,5] *
 Department of Laboratory Medicine, University of Washington, Seattle, WA;  Department of Pathology, University of Utah, Salt Lake City, UT;  ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT; Departments of  Pharmaceutics and 5 Medicine, University of Washington, Seattle, WA; * address correspondence to this author at: Department of Laboratory Medicine, University of Washington, Seattle, WA 98195-7110. Fax 206-598-6189; e-mail ahoof@u. washington.edu.
 Nonstandard abbreviations: 25(OH)D, 25-hydroxyvitamin D; 24,25[(OH).sub.2]D, 24,25-dihydroxyvitamin D; LC-MS/MS, liquid chromatography-tandem mass spectrometry; K,, dissociation constant; PTAD, 4-phenyl-1,2,4-triazoline-3,5-dione.
Previously published online at DOI: 10.1373/clinchem.2012.185827
Table 1. Summary data of the new multiplexed vitamin D metabolite method. Recovery, Compound % (a) Structure 25(OH)[D.sub.3] 43.3 (2.1) [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 25(OH)[D.sub.2] 32.2 (3.3) [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 24,25[(OH).sup.2][D.sub.3] 70.8 (9.8) [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 1[alpha],25[(OH).sub.2][D.sub.3] 79.4 (3.5) [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 1[alpha],25[(OH).sub.2][D.sub.2] 78.2 (12.4) [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 23(S),25[(OH).sub.2][D.sub.3] 64.0 (2.1) [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 23(R),25[(OH).sub.2][D.sub.3] 67.0 (3.2) [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 25,26[(OH).sub.2][D.sub.3] 69.2 (4.0) [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 3-epi-25(OH)[D.sub.3] 3.2 (1.0) [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 4/3,25[(OHH).sub.2][D.sub.3] 3.0 (0.01) [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 3-epi-1[alpha],25[(OH).sub.2] 15.0 (0.4) [MATHEMATICAL [D.sub.3] EXPRESSION NOT REPRODUCIBLE IN ASCII] Compound Regression equation (b) [r.sup.2b] 25(OH)[D.sub.3] y = 1.04x + 0.08 ng/mL 0.955 25(OH)[D.sub.2] y = 0.94x - 1.00 ng/mL 0.981 24,25[(OH).sup.2][D.sub.3] y = 0.96x - 0.23 ng/mL 0.922 1[alpha],25[(OH).sub.2][D.sub.3] y = 0.96x - 2.97 pg/mL 0.901 1[alpha],25[(OH).sub.2][D.sub.2] y = 0.89x - 0.54 pg/mL 0.976 23(S),25[(OH).sub.2][D.sub.3] 23(R),25[(OH).sub.2][D.sub.3] 25,26[(OH).sup.2][D.sub.3] 3-epi-25(OH)[D.sub.3] 4/3,25[(OHH).sub.2][D.sub.3] 3-epi-1[alpha],25[(OH).sub.2] [D.sub.3] Concentration Intraassay Compound for CVs CV, % (c) 25(OH)[D.sub.3] 12.3 ng/mL 3.0 25(OH)[D.sub.2] 10.6 ng/mL 4.7 24,25[(OH).sup.2][D.sub.3] 1.6 ng/mL 2.6 1[alpha],25[(OH).sub.2][D.sub.3] 14.6 pg/mL 10.0 1[alpha],25[(OH).sub.2][D.sub.2] 12.8 pg/mL 10.9 23(S),25[(OH).sub.2][D.sub.3] 23(R),25[(OH).sub.2][D.sub.3] 25,26[(OH).sup.2][D.sub.3] 3-epi-25(OH)[D.sub.3] 4/3,25[(OHH).sub.2][D.sub.3] 3-epi-1[alpha],25[(OH).sub.2] [D.sub.3] Concentration rotal LLOQ (d) Compound CV, % (c) 25(OH)[D.sub.3] 3.7 1.0 ng/mL 25(OH)[D.sub.2] 10.2 0.2 ng/mL 24,25[(OH).sup.2][D.sub.3] 6.4 0.06 ng/mL 1[alpha],25[(OH).sub.2][D.sub.3] 15.6 3.4 pg/mL 1[alpha],25[(OH).sub.2][D.sub.2] 17.1 2.8 pg/mL 23(S),25[(OH).sub.2][D.sub.3] 23(R),25[(OH).sub.2][D.sub.3] 25,26[(OH).sup.2][D.sub.3] 3-epi-25(OH)[D.sub.3] 4/3,25[(OHH).sub.2][D.sub.3] 3-epi-1[alpha],25[(OH).sub.2] [D.sub.3] (a) Analytical recovery was calculated as the analyte peak area when spiked before extraction divided by the analyte peak area spiked after extraction. Data are presented as the mean (SD). (b) The equation of the Deming regression (yand x are the concentrations for the new and reference method, respectively) and the Pearson correlation coefficient are presented. (c) Intraassay CV (n = 10) and total assay CV determined at the indicated concentration. Total CV = [[[(Intraassay CV).sup.2] + [(Between-day CV).sup.2].sup.0.5]. (d) For the lower limit of quantification (LLOQ), 5 replicates of linear dilutions were analyzed, and the results for the lowest dilution at which the CV was [less than or equal to] 20% are presented.
|Printer friendly Cite/link Email Feedback|
|Title Annotation:||Brief Communication|
|Author:||Laha, Thomas J.; Strathmann, Frederick G.; Wang, Zhican; de Boer, Ian H.; Thummel, Kenneth E.; Hoofn|
|Date:||Dec 1, 2012|
|Previous Article:||Performance criteria for testosterone measurements based on biological variation in adult males: recommendations from the partnership for the...|
|Next Article:||Introducing tissue microarrays to molecular pathology.|