Printer Friendly

Sandwich assay for tacrolimus using 2 antitacrolimus antibodies.

It is a commonly accepted dogma that hapten binding to protein is a one-on-one phenomenon (1, 2). This is because abound hapten is mostly buried in the surface cavity of a binding protein (3-7) and tends to become inaccessible to a second binding event by another protein molecule due to steric hindrance. Therefore, most immunoassays for detection of haptens use a competitive format, in which only 1 antihapten antibody is used and the hapten to be detected competes with a labeled hapten for binding to the antibody. In the few cases in which the sandwich assay format is used for hapten detection, the second binding is achieved by an antimetatype antibody that recognizes the hapten-antibody complex (8-9), by an antiidiotype antibody such as [alpha]-AId (10,11), or by the labeled VH (heavy chain variable domain) chain in the open sandwich assay format (12,13). Until now, the smallest natural molecule for which a true sandwich assay has been reported was angiotensin II, a linear nonhapten (14) octapeptide having a molecular mass of 1048 Da. The 2 antibodies used for this sandwich assay were raised against the molecular fragments of the C and N termini, respectively (15). Recently, a sandwich assay was also reported for a small synthetic molecule composed of 2 haptens connected by a linker using their respective antibodies. The shortest linker between the 2 haptens that allowed sandwich formation was determined to be 5 [Angstrom] (a 5-carbon chain) in length (16).

Tacrolimus (FK506, 804 Da) is a macrolide lactone isolated from the fermentation product of the bacteria Steptomyces tsukubaensis (17). It is commonly used, often along with other immunosuppressant drugs, to reduce graft rejection in allogeneic organ transplants by suppressing the immune system. Because of its narrow therapeutic window, it is critical to monitor blood drug concentrations for optimal efficacy. For drug monitoring, competitive immunoassays employing a single antibody are commercially available. Due to its small size, no sandwich immunoassay has yet been reported for tacrolimus. However, sandwich immunoassays in general offer higher analytical sensitivity and specificity and wider dynamic ranges than the competitive format (18). For example, compared to the competitive format in which only 1 antibody is employed, the sandwich format is more likely to have lower drug metabolite cross-reactivity because of its need for the drug molecule to be simultaneously recognized by 2 antibodies. As a result, a structurally altered metabolite has a lower probability of fitting into all the binding pockets for the parent drug formed by both antibodies. Because of the general lack of targeted biological effects of drug metabolites, lower or no metabolite cross-reactivity is crucial for therapeutic drug monitoring.

Here we report that tacrolimus can be bound simultaneously by 2 antitacrolimus monoclonal antibodies, 14H04 [Kasper et al. (19), US patent US 8,030,458] and 1E2 [Niwa et al. (20), US patent US 5,532,137], in a sandwich immunoassay. To the best of our knowledge, this is the first report ofa true sandwich formation for a natural hapten and for a natural molecule below 1000 Da. Each of the 2 antibodies described here was previously used as the only antibody in its respective competitive assay (21, 22).

Materials and Methods


The 2 mouse monoclonal antibodies employed in simultaneous binding were 14H04 and 1E2. 14H04 was developed using tacrolimus linked to keyhole limpet hemocyanin (KLH) [2] via C22 with an oxime functional group (Fig. 1A). 1E2 was developed using a mixture containing 3 positional isomers of tacrolimus with succinic acid functionality (tacrolimus linked to KLH via C32, C24, or possibly via both C32 and C24 simultaneously). BALB/c mice were immunized with respective tacrolimus-KLH conjugates and standard fusion techniques were employed to generate hybridoma cell lines secreting antibody specific to tacrolimus. On the basis of its lack of binding to the C24 modified succinic compound (Fig. 1A and Table 1), we determined that the immunogen that generated the 1E2 clone was tacrolimus linked to KLH via C32.


Tacrolimus is metabolized by the liver cytochrome P-450 system. All tacrolimus metabolites (Fig. 1) in this study were purchased from Dr. Uwe Christians at the University of Colorado (23, 24). Tacrolimus-C24 and tacrolimus-C32 succinates were prepared (Fig. 1A) by use of 2 previously patented procedures [Niwa et al. (20), US patent US 5,532,137; Steven (25), US patent US 5,164,495].


Chromium dioxide (Cr[O.sub.2]) magnetic particles were coated with 14H04 using 25% glutaraldehyde solution followed by extensive washing using phosphate buffer (10 mmol/L phosphate; 300 mmol/L NaCl, pH 7.0). The detailed coating procedure was described previously [Lau (26), US patent US 4,661,408].


Monoclonal antibody 1E2 was conjugated to [beta]-galactosidase using a standard heterobifunctional succinimidyl trans-4-(N-maleimidylmethyl) cyclohexane-1-carboxylate linker (27). The antibody-[beta]-galactosidase conjugate reagent using 14H04 clone was made in a similar fashion.


The reagent contained 5.5 [micro]mol/L of rapamycin, 20 mmol/L PIPES[TM] (piperazine-N, N'-bis[2-ethanesulfonic acid]), 1.5 mmol/L sodium salt, 1 mmol/L EDTA disodium, and 2.4 mmol/L saponin, pH 6.5.


Two competitive affinity column-mediated immunoassays (ACMIAs), one using 1E2 and the other using 14H04, were conducted as previously described (21, 22). Both assays were performed on the Siemens Dimension[R] RxL Max[R] Integrated Chemistry System using EDTA whole blood (WB) samples. The same pretreatment reagent was dispensed into a reaction vessel. Then, the EDTA WB sample was ultrasonically mixed and added into the same reaction vessel. Following the incubation of the sample with a pretreatment reagent, respective 1E2 or 14H04 antibody-[beta]-galactosidase conjugate reagent was added to the reaction vessel and mixed with the hemolyzed WB sample. This step allows the complex formation of released tacrolimus with the antibody-[beta]-galactosidase conjugate. Next, tacrolimus analog-coated Cr[O.sub.2] particle reagent was added to the reaction mixture. For the 14H04 antibody, the analog used to coat the Cr[O.sub.2] particles was tacrolimus-C22-oxime. For the 1E2 antibody, the analog used was tacrolimus-C32-succinate. After incubation to allow the tacrolimus analog-coated Cr[O.sub.2] particles to scavenge the excess amount of antibody-[beta]-galactosidase conjugates that were not bound to tacrolimus, the Cr[O.sub.2] particles were magnetically separated from the reaction mixture in the vessel and an aliquot of supernatant containing tacrolimus: antibody-[beta]-galactosidase conjugate complexes was transferred from the reaction vessel to a photometric cuvette containing chlorophenol red-[beta]-D-galactopyranoside (CPRG). The rate of the conversion of CPRG to chlorophenol red (CPR) was measured bichromatically at 577 and 700 nm. This rate was proportional to the concentration of tacrolimus in the sample.



Tacrolimus measurement using sandwich ACMIA was performed using EDTA WB samples on the Dimension system. A 50-[micro]L sample was first incubated with the hemolytic pretreatment reagent, then with 1E2-[beta]-galactosidase and with 14H04-coated Cr[O.sub.2] magnetic particles. The magnetic particles were then washed and galactosidase activity on the particles was measured using the detection system as previously described (28). The dose-response curve was generated using tacrolimus calibrators.


ELISA plates were coated with 14H04 or 1E2 followed by tacrolimus and finally with [beta]-galactosidase-conjugated 1E2 or 14H04, respectively. The enzyme reaction was measured using CPRG substrate and read at 577 nm at 1-min intervals for 20 min.


Siemens Clinical Specimen Acquisition Group, Glasgow, DE, collected WB samples from transplant patients. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) results were obtained from the clinical laboratory at the University of Maryland Medical Center, Baltimore, MD, using a previously described methodology (28).


The ANOVA study was performed on the basis of protocol EP15-A2 (section 8.4) published by CLSI. Each sample was run in 5 replicates for 5 days. Samples used in the ANOVA were WB pools from transplant patients taking a tacrolimus drug, except for WBP4 (Table 2), which was a drug-free WB pool from nontransplant patients spiked with tacrolimus (Astellas Pharma).

Limit of blank (LOB), limit of detection (LOD), and limit of quantification (LOQ) studies were conducted based on CLSI protocol EP17-A2. For the LOB, 5 tacrolimus-free WB samples from transplant patients were used. For the LOD and LOQ, 5 tacrolimus-free WB samples from transplant patients to which tacrolimus was added to respective concentrations of 0.8, 0.9, 1.0, 1.1, and 1.3 ng/mL were used.

Recovery was evaluated by adding the drug into a tacrolimus-free WB pool from nontransplant patients to achieve final concentrations of 5.0 and 10.0 ng/mL. The percentage recovery was obtained by dividing the recovered value by the expected value, for which the recovered value is the difference between the measured values of the spiked and control pools.

Physiological interferences were conducted to assess their impacts on the assay. Triglyceride (High Triglyceride Fraction, Lee Biosolutions) was added into WB pools containing approximately 5 or 20 ng/mL tacrolimus to achieve a final concentration of 1000 mg/ dL. Similarly, cholesterol, bilirubin (conjugated and unconjugated), and total protein were added to achieve the final respective concentrations of 400 mg/dL, 60 mg/dL, and 12 g/dL. A cholesterol solution was made of 75% human LDL and 25% human HDL purchased from Lee Biosolutions. Total protein was a mixture of 50% human IgG (immunoglobulin G) and 50% human albumin purchased from Sigma-Aldrich. Conjugated and unconjugated bilirubin were obtained from Frontier Scientific Inc.


Drug analogs used in this report include 8 metabolites and 3 synthetic analogs (Table 1). Their cross-reactivity reflects analog-antibody binding relative to drug-antibody binding. The difference in antibody binding between an analog and its parent drug is a function of the analog's structural deviations from the drug. This is especially true if the deviation occurs at the antibody binding site. A binding site is indicated if a structural deviation at that site results in the loss of binding while the rest of the molecule preserves the drug's basic shape. To assess the ratio of analog-antibody binding to drug-antibody binding, analog cross-reactivity was determined using competitive ACMIA on the Dimension system. It is expressed as a percentage calculated from the measured tacrolimus concentration (nmol/L) divided by the added analog concentration (nmol/L).


The immunogen for 14H04 was tacrolimus linked to KLH via C22, and the immunogen for 1E2 was linked to KLH via C32. There was a separation of 10 carbon atoms between the 2 linkages. Since the antibody-binding region is usually away from the drug-KLH linkage, we surmised that this spatial separation might result in 2 non- or minimally overlapping epitopes. We further hypothesized that the spatially separated epitopes might result in simultaneous binding of the 2 antibodies to the tacrolimus molecule.

On the basis of this hypothesis and available cross-reactivity data, we performed an analysis to locate epitopes for the antibodies to rule out overlaps between their binding sites. To deduce the binding sites for an antibody in the absence of the x-ray cocrystal structure of antibody-drug complexes, we examined the change in binding affinity by altering chemical groups on tacrolimus. If an alteration substantially lowered or eliminated antibody binding, it was reasonable to assume that this chemical group was involved in the binding, provided the alteration did not distort the basic molecular shape of the drug.

These alterations were achieved by hydroxylation on C12; demethylation on C13, C15, and C31; succination of C24 and C32; and addition of an oxime group on C22. The hydroxylation and demethylation products were tacrolimus metabolites purified from an incubation mixture of tacrolimus with liver microsomal enzymes as previously reported (23, 24). The oxime and succinate compounds were obtained by chemical synthesis in our laboratory. The structures of these compounds are shown in Fig. 1A. The binding alteration as expressed by cross-reactivity was measured by competitive ACMIA on the Dimension clinical chemistry system (Table 1).

To rule out distortions of the molecular shape, 3-D structures were generated using CS Chem 3D Model software by CambridgeSoft[R] (Version Chem 3D Pro 12.0.2 1706). The molecular geometric outlines were enhanced by use of a solvent-accessible surface. Fig. 1B shows the enhanced 3-D structures of the molecules from Fig. 1A. Except for metabolite VIII (MVIII), whose 3-D structure deviated significantly from that of tacrolimus due to the formation of a new ring, the rest of the compounds appeared to have conserved the basic 3-D structure observed from various angles (1 angle is shown in Fig. 1B). The minor deviations in 3-D shape from those of tacrolimus were observed in situ where demethylation, hydroxylation, succination, and oxime conjugation occurred. These structural alterations resulted in altered antibody binding as quantified by cross-reactivity.

Because the basic shape of the parent drug is conserved in the drug metabolites and analogs besides MVIII, we assumed that the change of binding affinity occurred in situ of the structural alteration. This provided the basis for using the cross-reactivity data to locate the antibody binding sites. MVIII was not used for epitope analysis due to its drastic deviation. The second-pass metabolites (with 2 or more chemical groups altered) were not used for epitope mapping owing to the possibility of multiple interpretations of the results. As expected, the immunogenic analogs, C22-oxime tacrolimus for 14H04 and C32-succinate tacrolimus for 1E2, demonstrated higher binding than tacrolimus to their respective antibodies (Table 1).

For 14H04, the loss of cross-reactivity with tacrolimus C32 succinate (Table 1 and Fig. 1A) suggested that the hydroxyl group on C32 and possibly adjacent atoms were part of the epitope. By the same token, both C31 methyl (0% cross-reactivity) and C15 methyl (0% cross-reactivity) groups were mapped as part of the epitope. The 28%, 26%, and 25% cross-reactivity demonstrated binding decrease but not binding loss for respective 13-O-desmethyl, 12-O-hydroxyl, and C24 succinate tacrolimus compounds. The decrease suggested that C13 methyl and C12 and C24 hydroxyl groups and atoms adjacent to them might function as aids for the binding sites.

For 1E2, the loss of cross-reactivity for tacrolimus-C22-Oxime, C24-succinate, or 13-O-desmethyl tacrolimus indicated that C22 keto, C24-hydroxyl, and 13-O-methyl groups were part of the epitope. The relative mild cross-reactivity decrease with 31-Odesmethyl (19%) or 15-O-desmethyl tacrolimus (15%) indicated that the C31 and C15 methyl groups might function as aids for the binding sites.

The binding sites for the 2 antibodies were further inspected for overlaps. By spinning the molecule from the angle shown in Fig. 1C clockwise to an angle shown on the right in Fig. 1D, the binding sites for 1E2 were lined up to the upper left and those for 14H04 antibodies were lined up to the lower right. As shown in Fig. 1D, no overlap was found between the binding sites for 1E2 and 14H04, predicting the possibility of simultaneous binding by the 2 antibodies (Fig. 1E). Overlaps between the 1E2 binding-aid sites and 14H04 binding sites (C15 and C31) and overlaps between 14H04 binding-aid sites and 1E2 binding sites (C13 and C24) were also observed. We theorized that these overlaps might still allow the sandwich formation because of the spatial separation between the binding sites; however, they might reduce the binding affinity of each antibody to the hapten during simultaneous binding.


The above hypothesis was tested by the ACMIA and ELISA formats. Both formats showed dose-response to [tacrolimus]. For the ELISA format, better results were obtained when 14H04 was immobilized on the microtiter plate as the capture antibody and 1E2 was conjugated to [beta]-galactosidase as the tag antibody. To confirm true sandwich formation, several controls were employed (Fig. 2). Studies 1 and 2 (Fig. 2A) and 5 and 6 (Fig. 2B) showed that sandwich assay signals were obtained only when 1 antibody of the 2 was used as the capture and the other as the tag. No measurable signal was observed, however, when 14H04 or 1E2 was paired with itself, as shown in studies 3 and 4 (Fig. 2A) and 7 and 8 (Fig. 2B). This experiment ruled out nonspecific binding such as hydrophobic adsorption and confirmed that the simultaneous binding signals were generated only when all 3 components, 14H04, 1E2 antibodies, and tacrolimus, were present. A lack of any 1 of the 3 components yielded no measurable binding signal.

The analytical sensitivity of the sandwich ELISA was explored by testing titration of tacrolimus from 100 ng/mL (124 nmol/L) to 0.4 ng/mL (0.5 nmol/L). Using 14H04 as capture and 1E2 as tag, 0.9 ng/mL (1.1 nmol/L) of tacrolimus was detected in a sample made of PBS spiked at 1:20 with a stock solution containing tacrolimus dissolved in ethanol.

The prediction of sandwich formation was further substantiated by the ACMIA format. By using 14H04-coated Cr[O.sub.2] particles and 1E2-[beta]-galactosidase conjugate, a dose-response curve to [tacrolimus] was obtained (Fig. 3A). This format also revealed expected recovery and desired imprecision for the measurement of tacrolimus drug in transplant patients' WB. Comparison of tacrolimus measurement of the ACMIA sandwich assay was assessed in a split patient correlation study to the LC-MS/MS method using WB samples from transplant patients taking tacrolimus (Fig. 3B). Fifty-five WB samples from kidney and liver transplant patients with LC-MS/MS values were tested with the ACMIA sandwich assay. The results show that the sandwich assay provided tacrolimus drug concentrations similar to the LC-MS/MS values in the WB matrix across an assay range of 0-30 ng/mL (0-37 nmol/L). Biases of the sandwich assay relative to the LC-MS/MS for the ranges of 0-7, 0-20, and 20-30 ng/mL (0-9, 0-25, and 25-37 nmol/L) were assessed and found to be -0.19, 0.15, and -0.54 ng/mL (-0.24, 0.19, and -0.67 nmol/L), respectively (Fig. 3, C and E).


Performance characteristics of the sandwich assay were further evaluated by ANOVA, LOB, LOD, LOQ, recovery, and interference studies (Table 2). The assay demonstrated <6.3% intralaboratory CV in the range of 2.829.1 ng/mL (3.4-36.1 nmol/L, n = 25). LOB, LOD, and LOQ (using an allowed total analytical error of 35%) were determined to be 0.4,0.8, and 1.3 (0.5,1.0, and 1.6 nmol/ L), respectively. The recovery was within [+ or -] 5% of the expected value and < 10% interference was found with high triglyceride (1000 mg/dL), cholesterol (400 mg/dL), bilirubin (60 mg/dL), and total protein (12 g/dL).

To demonstrate that the sandwich format could achieve higher specificity, the percentages of cross-reactivity of metabolites, and analogs measured by the sandwich assay were compared to those measured by the competitive assays (Table 1). As expected, the sandwich assay demonstrated lower cross-reactivity in general because of the need for binding of tacrolimus by both antibodies. Quantitatively, the percentage cross-reactivity for a synthetic analog or metabolite measured by the sandwich assay could be predicted because it should be equal to the product of percentage cross-reactivity for 14H04 times that for 1E2. This was because a sandwich was formed by the tag antibody binding to the hapten molecules already bound to the capture antibody at their respective percentages of cross-reactivity. All metabolites and analogs showed percentages of cross-reactivity that were either identical or very close to the predicted, except for metabolite MIII. Further investigation is needed to elucidate the higher-than-predicted percentages of cross-reactivity for MIII. The consistency between the predicted and measured percentages of cross-reactivity indicated that the sandwich should be a 1E2-hapten-14H04 complex, another proof of true sandwich formation along with the ELISA results shown in Fig. 2.


The limit of molecular mass and shape for a hapten that can be simultaneously bound by 2 hapten-specific receptors has been explored by researchers (1,15,16, 29). To the best of our knowledge to date, tacrolimus is the first natural hapten shown to have such binding. Because of the structural similarity of sirolimus to tacrolimus, we imagine that these types of antibodies for sirolimus might similarly be developed. Our results demonstrate that true sandwich assays for hapten markers such as therapeutic drugs can be developed by using proper techniques. In addition to being analytically sensitive, these assays can achieve higher specificity than their competitive assay counterparts, as exemplified by much lower cross-reactivity with the drug metabolites and synthetic analogs described in this report.

Moreover, several other observations may be of interest. First, on the basis of the epitope analysis, the closest distance found between 2 respective binding sites for 14H04 (at C15) and 1E2 (at C13) is 3 carbon atoms, or roughly 3[Angstrom], if not closer (the angle at C14 was disregarded). This distance is shorter than the previously observed 5 carbon atoms, or 5A, as the shortest linker between 2 small haptens (histamine and homovanillic acid) that allowed a true sandwich formation for the linear synthetic molecule (16). Further studies using more sophisticated techniques such as x-ray cocrystallography may clarify the sandwich binding at the structural and atomic levels. Second, this study demonstrates that antibodies against non-overlapping binding sites on a hapten can be developed by use of an intact hapten linked via different linking positions to a carrier protein as opposed to using fragments of small molecules (15,16). Third, tacrolimus was bound across its macrolide ring by the 2 antibodies, which differs from binding to the 2 opposite ends of a molecule as previously reported in sandwich formation for small linear molecules (15, 16). It is possible that certain conformational changes in the antibody paratope regions and/or adjustment of the 3-D shape of the drug might have been induced to accommodate both antibodies during the sandwich formation. The induced conformational change might explain why the measured percent cross-reactivity for MIII (15desmethyl tacrolimus) in the sandwich assay did not follow the predicted pattern.

The fact that tacrolimus, a ring-structured hydrophobic hapten, was sandwiched by the antitacrolimus antibodies against the odds indicates that such molecular interactions may occur in nature without human intervention. Although it was reported that a hapten-protein complex could bind to another protein in vivo, as in the case of sirolimus-FKBP12-mTOR (mammalian target of rapamycin) (30), it has not been shown that 2 protein receptors that individually bind to a hapten ligand can bind to it simultaneously in biological systems. It will be interesting to explore the existence and functions of such bindings.

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, oranalysis and interpretation of data; (b) drafting or revising the 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: None declared.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: Siemens HDX R&D funding.

Expert Testimony: None declared.

Patents: T.Q. Wei, United States patent US 8,586,322; 2013 Nov 19.

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.

Acknowledgments: We thank C. Schaible and J. Fallon for suggestions and reviewing the manuscript, S. Janas and C. Clark for assistance in experiments, and F. Celano for providing method parameters for the fully automated Dimension RxL Max Integrated Chemistry System.


(1.) Fan M, He J. Recent progress in noncompetitive hapten immunoassays: a review. In: Abuelzein E, ed. Trends in immunolabelled and related techniques. 1st ed. Rijehka: InTech; 2012. p 53-66.

(2.) Jackson M, Ekins, P. Theoretical limitations on immunoassay sensitivity. Current practice and potential advantages of fluorescent Eu3+ chelates as non-radioisotopic tracers. J Immunol Methods 1986;87:13-20.

(3.) Jeffery PD, Schildbach JF, Chang CY, Kussie PH, Margolies MN, Sheriff S. Structure and specificity of the anti-digoxin antibody 40-50. J Mol Biol 1995;248:344-60.

(4.) Tanaka F, Kinoshita K, Tanimura R, Fujii I. Relaxing substrate specificity in antibody-catalyzed reactions: enantioselective hydrolysis of N-Cbz-amino acid esters. J Am Chem Soc 1996;118:2332-9.

(5.) Romesberg F, Spiller B, Schultz P, Stevens R. Immunological origins of binding and catalysis in a Diels-Alderase antibody. Science 1998;279: 1929-33.

(6.) Xu J, Deng QL, Chen JG, Houk KN, Bartek J, Hilvert D, Wilson IA. Evolution of shape complementarity and catalytic efficiency from a primordial antibody template. Science 1999; 286:2345-8.

(7.) Piatesi A, Hilvert D. Immunological optimization of a generic hydrophobic pocket for high affinity hapten binding and Diels-Alder activity. Chembiochem 2004;4:460-6.

(8.) Nagata S, Tsutsumi T, Yoshida F, Ueno Y. A new type sandwich immunoassay for microcystin: production of monoclonal antibodies specific to the immune complex formed by microsystin and an anti-microsystin monoclonal antibody. Nat Toxins 1999;7:49-55.

(9.) Self CH, Dessi JL, Winger LA. High-performance assays of small molecules: enhanced sensitivity, rapidity, and convenience demonstrated with a noncompetitive immunometric anti-immune complex assay system for digoxin. Clin Chem 1994; 40:2035-41.

(10.) Niwa T, Kobayashi T, Sun P, Goto J, Oyama H, Kobayashi N. An enzyme linked immunometric assay for cortical based on idiotype-anti-idiotype reactions. Anal Chim Acta 2009;638:94-100.

(11.) Goletz S, Christensen PA, Kristensen P, Blohm D, Tomlinson I, Winter G, Karsten U. Selection of large diversities of antiidiotypic antibody fragments by phase display. J Mol Biol 2002;315: 1087-97.

(12.) Lim SL, Ichinose H, Shinoda T, Ueda H. Noncompetitive detection of low molecular weight peptides by open sandwich immunoassay. Anal Chem 2007;79:6193-200.

(13.) Ihara M, Suzuki T, Kobayashi N, Goto J, Ueda H. Open-sandwich enzyme immunoassay for one-step noncompetitive detection of corticosteroid 11-deoxycortisol. Anal Chem 2009;81:8298-304.

(14.) Dietrich FM, Frischknecht H. Immunogenicity of synthetic angiotensin II octapeptide and analysis of antibodies by passive haemagglutination. Int Arch Allergy Appl Immunol 1968;34:597-615.

(15.) Grassi J, Creminon C, Frobert Y, Etienne E, Ezan E, Volland H, Pradelles P. Two different approaches for developing immunometric assays of haptens. Clin Chem 1996;42:1532-6.

(16.) Quinton J, Charruault L, Nevers MC, Volland H, Dognon JP, Creminon C, Taran F. Toward the limits of sandwich immunoassay of very low molecular weight molecules. Anal Chem 2010;82: 2536-40.

(17.) Kino T, Hatanaka H, Hashimoto M, Goto T, Okuhara M, Kohsaka M, et al. FK-506, a novel immunosuppressant isolated from a Streptomyces. I. Fermentation, isolation, and physico-chemical and biological characteristics. J Antibiot 1987;40: 1249-55.

(18.) Ekins R. More sensitive immunoassays. Nature 1980;284:14-5.

(19.) Kasper KC, Jeong H, Davalian D, Liu H, Miller PL, Williams DL, inventors; Siemens Healthcare Diagnostics Inc., assignee. Monoclonal antibodies to tacrolimus and immunoassays methods for tacrolimus. United States patent US 8,030,458. 2011 Oct 4.

(20.) Niwa M, Tamura K, Kaizu T, Kobayashi M, inventors; Fujisawa Pharmaceutical Co., Ltd., assignee. Anti-FR-900506 substance antibodies and highly-sensitive enzyme immunoassay method. United States patent US 5,532,137. 1996 July 2.

(21.) Wei TQ, Janas S, Dubowy M, Celano F, Zheng YF, Duffy J, Yang Y. An improved no-manual extraction immunoassay for tacrolimus on the Dimension System [Abstract]. Clin Chem 2011;57:A65.

(22.) Wei TQ, Parker G, Wang CR. Development of a fully automated tacrolimus method for the Dimension[R] clinical chemistry system [Abstract]. Clin Chem 2001;47:A75.

(23.) Christians U, Kruse C, Kownatzki R, Schiebel HM, Schwinzer R, Sattler M, et al. Measurement of FK-506 by HPLC and isolation and characterization of its metabolites. Transplant Proc 1991;23(1 Pt 2):940-1.

(24.) Christians U, Radeke HH, Kownatzki R, Schiebel HM, Schottmann R, Sewing KF. Isolation of an immunosuppressant metabolites of FK-506 generated by human microsome preparations. Clin Biochem 1991;24:271-5.

(25.) Steven LE, inventor; Abbott Laboratories, assignee. Method for preparing a dicarboxylic acid half-acid ester of FK506. United States patent US 5,164,495. 1992 Nov 17.

(26.) Lau HP, Yang EK, Jacobson HW, inventors; E.I. Du Pont de Nemours and Company, assignee. Coated chromium dioxide particles. United States patent US 4,661,408. 1987 Apr 28.

(27.) Bieniarz C, Husain M, Barnes G, King CA, Welch CJ. Extended length heterofunctional coupling agents for protein conjugations. Bioconjugate Chem 1996;7:88-95.

(28.) Griffey MA, Hock KG, Kilgore DC, Wei TQ, Duh SH, Christenson R, Scott MG. Performance of a no-pretreatment tacrolimus assay on the Dade Behring Dimension RxL clinical chemistry analyzer. Clin Chim Acta 2007;384:48-51.

(29.) Vicennati P, Bensel N, Wagner A, Creminon C, Taran F. Sandwich immunoassay as a highthroughput screening method for cross-coupling reactions. Angew Chem Int Ed Engl 2005;44: 6863-6.

(30.) Brown EJ, Albers MW, Shin TB, Ichikawa K, Keith CT, Lane WS, Schreiber SL. A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature 1994;369:756-8.

Tie Q. Wei, [1] * Yi F. Zheng, [1] Michael Dubowy, [1] and Manoj Sharma [1]

[1] Siemens Healthcare Diagnostics Inc., Newark, DE 19714.

* Address correspondence to this author at: Siemens Healthcare Diagnostics Inc., P.O. Box 6101 M/S 707, Newark, DE 19714. Fax 302-631-7478; e-mail

Received August 14, 2013; accepted December 18, 2013.

Previously published online at DOI: 10.1373/clinchem.2013.214023

[2] Nonstandard abbreviations: KLH, keyhole limpet hemocyanin; ACMIA, affinity column mediated immunoassay; WB, whole blood; CPRG, chlorophenol red-[beta]-D-galactopyranoside; CPR, chlorophenol red; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LOB, limit of blank; LOD, limit of detection; LOQ, limit of quantification; MVIII, metabolite VIII.
Table 1. Percentage cross-reactivity of metabolites and
synthetic analogs measured by 2 competitive assays in
comparison to that predicted and measured by the ACMIA
sandwich assay. (a)

Metabolite/analog name (b)               Competitive   Competitive
                                         assay using   assay using
                                          14H04, %       1E2, %

Tacrolimus                                   100           100
13-0-desmethyl tacrolimus (MI)               28             0
31-0-desmethyl tacrolimus (MII)               0            19
15-0-desmethyl tacrolimus (MIII)              0            15
12-0-hydroxyl tacrolimus (MIV)               26            104
15,31-O-didesmethyl tacrolimus (MV)           2             0
13,31-0-didesmethyl tacrolimus (MVI)          2             0
13,15-0-dedesmethyl tacrolimus (MVII)        10            42
MVIII                                         2             0
C22 oxime tacrolimus                         207            0
C24 succinate tacrolimus                     25             0
C32 succinate tacrolimus                      0            197

Metabolite/analog name (b)                 Sandwich      Sandwich
                                            assay          assay
                                         predicted, %   measured, %

Tacrolimus                                   100            100
13-0-desmethyl tacrolimus (MI)                0              0
31-0-desmethyl tacrolimus (MII)               0              0
15-0-desmethyl tacrolimus (MIII)              0             23
12-0-hydroxyl tacrolimus (MIV)                27            20
15,31-O-didesmethyl tacrolimus (MV)           0              0
13,31-0-didesmethyl tacrolimus (MVI)          0              0
13,15-0-dedesmethyl tacrolimus (MVII)         4              4
MVIII                                         0              0
C22 oxime tacrolimus                          0              0
C24 succinate tacrolimus                      0              0
C32 succinate tacrolimus                      0              0

(a) Sandwich cross-reactivity prediction was made by
multiplying the cross-reactivity for 14H04 by that for 1E2.

(b) Each sample was spiked with 40 ng/mL of the metabolite
or 10 ng/mL of the synthetic analog.

Table 2. Performance characteristics of the
tacrolimus sandwich assay.

Precision by ANOVA based on CLSI EP15-A2

Samples                          WBP1 (a)         WBP2 (a)

ng/mL (nmol/L)                  2.8 (3.4)         7.7 (9.5)
Repeatability, CV                  5.8%             1.6%
Intralab, CV                       6.3%             2.3%

LOB, LOD, and LOQ based on CLSI EP17-A2

Metric                        ng/mL (nmol/L)    WB sample (c)

LOB                             0.4 (0.5)         No spike
LOD                             0.8 (1.0)           Spike
LOQ                             1.3 (1.6)           Spike

Spike recovery

WB sample                     Expected ng/mL   Recovered ng/mL
                                 (nmol/L)         (nmol/L)

Sample 1                        5.0 (6.2)         4.9 (6.1)
Sample 2                       10.0 (12.4)       10.3 (12.8)

Physiological interferences

[Tacrolimus]/substance (d)/    Triglyceride      Cholesterol
%interference                  (1000 mg/dL)      (400 mg/dL)

5.0 ng/mL (6.2 nmol/L)            -9.4%             -6.5%

20.0 ng/mL (24.8 nmol/L)          -2.2%             -7.9%

Precision by ANOVA based on CLSI EP15-A2

Samples                             WBP3 (a)           WBP4 (b)

ng/mL (nmol/L)                    11.2 (13.9)         29.1 (36.1)
Repeatability, CV                     2.1%               5.1%
Intralab, CV                          2.2%               5.1%

LOB, LOD, and LOQ based on CLSI EP17-A2



Spike recovery

WB sample                          % Recovery

Sample 1                              98%
Sample 2                              103%

Physiological interferences

[Tacrolimus]/substance (d)/        Bilirubin         Total protein
%interference                      (60 mg/dL)          (12 g/dL)

5.0 ng/mL (6.2 nmol/L)        -4.5% unconjugated;        -7.6%
                                -5.1% conjugated
20.0 ng/mL (24.8 nmol/L)       2.3% unconjugated;        -2.3%
                                -3.1% conjugated
(a) WBP1-WBP3, WB pool from transplant patients.

(b) WPB4, WB pool from nontransplant patients, spiked
with tacrolimus powder.

(c) Five WB 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.

Fig. 2. Confirmation of sandwich formation by ELISA.


Study number          1             2             3             4
Tag Ab           1 E2-b Gal     1E2-b Gal    14HO4-b Gal   14HO4-b Gal
Tacro (ng/mL)       1000            0           1000            0
Capture Ab          14HO4         14HO4         14HO4         14HO4
Milliunits/min      25.3           0.1           0.0           0.0


Study number          5             6             7             8
Tag Ab           14HO4-b Gal   14HO4-b Gal    1E2-b Gal     1E2-b Gal
Tacro (ng/mL)       1000            0           1000            0
Capture Ab           1E2           1E2           1E2           1E2
Milliunits/min       1.6           0.0           0.0           0.0

(A), The sandwich results in milliunits per minute [using 14H04
as capture and 1E2 or 14H04 as tag, with or without tacrolimus
(Tacro)]; (B), the results using 1E2 as capture and 14H04 or
1E2 as tag, with or without tacrolimus. Ab, antibody.
COPYRIGHT 2014 American Association for Clinical Chemistry, Inc.
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2014 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:Drug Monitoring Toxicology: fast track
Author:Wei, Tie Q.; Zheng, Yi F.; Dubowy, Michael; Sharma, Manoj
Publication:Clinical Chemistry
Article Type:Report
Date:Apr 1, 2014
Previous Article:Magnetically promoted rapid immunoreactions using functionalized fluorescent magnetic beads: a proof of principle.
Next Article:Phase I and II cannabinoid disposition in blood and plasma of occasional and frequent smokers following controlled smoked cannabis.

Terms of use | Copyright © 2018 Farlex, Inc. | Feedback | For webmasters