Sandwich Assay for Tacrolimus Using 2 Anti-Tacrolimus Antibodies T.Q. Wei, Y.F. Zheng, M. Dubowy, and M. Sharma April © Copyright 2014 by the American Association for Clinical Chemistry

© Copyright 2009 by the American Association for Clinical Chemistry Introduction – Why a sandwich assay?  Competitive Immunoassays (IAs) have always been format of choice for tacrolimus  No known sandwich IAs for tacrolimus because: −Difficulty in avoiding steric hindrance between two antibody bindings for the hapten drug  Benefits of sandwich over the competitive IAs  Better analytical sensitivity: Antibodies (Abs) in excess −Insensitive to reagent delivery or temperature change  Better specificity: Lower metabolite cross-reactivity −Important for drug assays

© Copyright 2009 by the American Association for Clinical Chemistry Figure 1. Tacrolimus 2D (left) and 3D (right) Structures. Tacrolimus is a hapten drug with molecular mass of 804 daltons. The immunogens for 14H04 and 1E2 antibodies were linked via C22 and C32, respectively. C22 and C32 are separated in both 2D and 3D structures. The 10-carbon separation between the two linkages made it possible for the two antibodies to recognize two distinct epitopes on tacrolimus. Introduction: Is a sandwich assay possible? 1E2 Immunogen: Tacrolimus linked to KLH via C32 14H04 Immunogen: Tacrolimus linked to KLH via C22 C22 C32

© Copyright 2009 by the American Association for Clinical Chemistry Question  Are there better ways to make immunogens for hapten sandwich assays?

© Copyright 2009 by the American Association for Clinical Chemistry Materials & Methods: Epitope Mapping  Modify tacrolimus & measure Ab binding Binding loss as a result of a chemical modification at a position on the carbon backbone indicates the position is a binding site, provided the modification does not alter the basic 3D shape of the drug.  8 metabolites and 3 synthetic analogs Chemical modifications vs. Ab binding change (cross-reactivity) were analyzed  Connected binding sites to draw the epitope for both 1E2 and 14H04 Abs

© Copyright 2009 by the American Association for Clinical Chemistry Methods: Ab Binding of Metabolites/Analogs. Methods: Ab Binding of Metabolites/Analogs. Table 1. Cross-reactivity of Metabolites and Synthetic Analogs Measured by Two Competitive Assays. Each of the two antibodies used in the sandwich format was the assay antibody for its respective competitive assay. Their binding sites (epitope) were deduced from their cross-reactivity data based on the assumption that the loss of binding due to a modification of a chemical group indicates that a binding site is located at or near that group.

© Copyright 2009 by the American Association for Clinical Chemistry Methods: Metabolites & Analogs in 2D Figure 2. 2D Structures of Tacrolimus Metabolites and Synthetic Analogs.

© Copyright 2009 by the American Association for Clinical Chemistry Methods: Metabolites & Analogs in 3D. Methods: Metabolites & Analogs in 3D. Figure 3. 3D Structures of Tacrolimus Metabolites and Synthetic Analogs. Except for metabolite VIII, the rest of the compounds conserved the basic 3-D structure. The minor deviations in 3D shape from that of tacrolimus were observed in situ where demethylation, hydroxylation, succination and oxime conjugation occurred. Carbon atoms are shown as grey balls, oxygen as red balls and the nitrogen as blue balls. Tacro M-VIII Tacro 15,31-O-Didesmethyl Tacro 13,15-O-Didesmethyl Tacro 13,31-O-Didesmethyl Tacro 13-O-Demethyl Tacro Tacro-24-succinate Tacro-32-succinate Tacro-22-Oxime 15-O-Demethyl Tacro 31-O-Demethyl Tacro 12-Hydroxyl Tacro

© Copyright 2009 by the American Association for Clinical Chemistry Figure 4. Deduced Epitopes of tacrolimus for 14H04 and 1E2. The binding and binding-aid sites mapped for 1E2 are depicted as bold blue circles and light blue circles, respectively; and those for 14H04 are depicted as bold black dotted circles and light black dotted circles, respectively. Methods: Epitope Mapping Binding site for 1E2 Binding aid site for 1E2 Binding aid site for 14H04 Binding site for 14H04

© Copyright 2009 by the American Association for Clinical Chemistry Question  What is the weakness of the epitope analysis in the previous slide?

© Copyright 2009 by the American Association for Clinical Chemistry Figure 5. Sandwich Formation of the two Antibodies with Tacrolimus. A: Spinning of the 3-D structure on the left clockwise gives the 3-D structure on the right, which shows that the binding sites for 1E2 (bold blue circles) are spatially separated from the binding sites for 14H04 (bold dotted black circles). B: Speculated schematic of simultaneous binding of FK506 to the two antibodies based on the spatial arrangements of the deduced epitopes. The figure depicts a prediction that the two antibodies approach the drug from two directions. Methods: Sandwich Prediction AB

© Copyright 2009 by the American Association for Clinical Chemistry RESULTS: Sandwich Confirmation Figure 6. Confirmation of Sandwich Formation by ELISA. A: The sandwich results in milliunits/min using 14H04 as capture and 1E2 or 14H04 as tag, with or without tacrolimus; B: The results using 1E2 as capture and 14H04 or 1E2 as tag, with or without tacrolimus.

© Copyright 2009 by the American Association for Clinical Chemistry Results: Curve and Accuracy with ACMIA Results: Curve and Accuracy with ACMIA. Figure 7. Comparison between the ACMIA sandwich assay and HPLC-MS/MS method. A: Dose response curve. B: Method comparison of the assay vs. HPLC-MS/MS. C-E: Bland-Altman plots showing biases vs. HPLC-MS/MS at 0-7, 0 to 20, and ng/mL.

© Copyright 2009 by the American Association for Clinical Chemistry Results: Performance of the Sandwich Assay. Results: Performance of the Sandwich Assay. Table 2. Performance Characteristics of the Tacrolimus Sandwich Assay. a WBP1-3 = whole blood pool from transplant patients. b WPB4= whole blood pool from non- transplant patients spiked with tacrolimus powder. c Five whole blood samples from transplant patients were used for each of the studies. d Substance concentration shown is the final concentration in the sample except for bilirubin which shows the concentration of spike.

© Copyright 2009 by the American Association for Clinical Chemistry Results: Specificity of the Sandwich Assay. Results: Specificity of the Sandwich Assay. Table 3. Cross-reactivity of Metabolites and Synthetic Analogs. The cross-reactivity predicted and measured by the ACMIA sandwich assay is compared to that measured by two competitive assays. Sandwich cross-reactivity prediction was made by multiplying the cross-reactivity for 14H04 and that for 1E2.

© Copyright 2009 by the American Association for Clinical Chemistry Question  What are the possible explanations for 15-O-desmethyl tacrolimus not showing predicted cross-reactivity in the sandwich assay?

© Copyright 2009 by the American Association for Clinical Chemistry Summary  Separation between immunogen linkages made 2 distinct epitopes possible for the two antibodies  Cross-reactivity predicted the sandwich assay  ELISA & ACMIA confirmed sandwich formation  Sandwich assay showed high specificity Low cross-reactivity with drug metabolites & analogs _________________________________________________ Reference: Werena Gounden and Steven J. Soldin. Tacrolimus Measurement: Building a Better Immunoassay. Clin Chem 2014; 60: 575

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