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Sandwich Assay for Tacrolimus
Using 2 Anti-Tacrolimus
Antibodies
T.Q. Wei, Y.F. Zheng, M. Dubowy,
and M. Sharma
April 2014
www.clinchem.org/content/60/4/621.full
© Copyright 2014 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
Introduction: Is a sandwich assay possible?
C32
H3CO
H3CO
H
O
O
OH
O
N
H
C22
14H04 Immunogen:
Tacrolimus linked to KLH via C22
ORH O
O
O
H
OCH3
1E2 Immunogen:
Tacrolimus linked to KLH via C32
OH
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.
© 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. 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
15 - O - Demethyl Tacro
31 - O - Demethyl Tacro
12 - Hydroxyl Tacro
13 - O - Demethyl Tacro
Tacro - 24 - succinate
Tacro
13,15
Tacro
- O-Didesmethyl Tacro
13,31
- O-Didesmethyl Tacro
Tacro
15,31
- O-Didesmethyl Tacro
- 22 - Oxime
- 32 - succinate
M - VIII Tacro
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.
© Copyright 2009 by the American Association for Clinical Chemistry
Methods: Epitope Mapping
32
32
31
31
12
12
13
24
13
24
15
22
15
22
32
31
Binding site for 1E2
Binding aid site for 1E2
12
13
24
15
22
Binding site for 14H04
Binding aid site for 14H04
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.
© 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
Methods: Sandwich Prediction
A
B
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.
© 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. 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 20-30 ng/mL.
© Copyright 2009 by the American Association for Clinical Chemistry
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 nontransplant 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. 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
© Copyright 2009 by the American Association for Clinical Chemistry
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© Copyright 2009 by the American Association for Clinical Chemistry