Immunoassay For Therapeutic Drug Monitoring Research Articles

B-303 Comparison of Tacrolimus Concentrations Obtained By the Newly Approved Alinity i Tacrolimus Assay With a Reference Liquid-Chromatography Combined With Tandem Mass spectrometry Assay

Abstract Background Tacrolimus belongs to the calcineurin-inhibitor class of medications and used for preventing and maintenance of organ rejection in transplant recipients alone or in combination with other medications. Due to narrow therapeutic window and significant nephrotoxicity, this drug is routinely monitored with a suggested therapeutic range of 5–15 ng/mL. Recently, Abbott Laboratories (Abbott Park, IL) received FDA approval for using tacrolimus immunoassay on the Alinity i platform. We compared tacrolimus values in 101 transplant patients using Alinity i analyzer and a reference method based on liquid chromatography combined with mass tandem spectrometry (LC-MS/MS). Methods Tacrolimus immunoassay on the Alinity i analyzer is a chemiluminescent microparticle immunoassay (CMIA) requiring pretreatment of whole blood specimen prior to analysis. The analytical measurement range of the assay is 2–30 ng/mL. The left-over blood specimens were sent to Eurofins-Viracor reference laboratory in Lenexa, KS for analysis using LC-MS/MS method. After liquid-liquid extraction, specimens were analyzed using ascomycin as the internal standard for the presence of tacrolimus (assay is capable of analyzing other immunosuppressants simultaneously). UPLC C-18 column (waters) was used for separation using two mobile phases (A: 2.0 mM ammonium acetate in 0.1% formic caid in water and B: 2.0 mM ammonium acetate in 0.1% formic acid in methanol). Mass spectrometer was operated in positive ionization electrospray mode (tacrolimus m/z 821.6 > 768.6, ascomycin m/z 809.756.6. Results The total precision of the low control was 3.6% (mean: 4.48 ng/mL; CV 0.16 ng/mL, n = 20) while the total precision of the high control was 5.1% (mean: 25.4 ng/mL, SD: 1.29 ng/mL, n = 20). The linearity of the assay (2.0–30.0 ng/mL) was verified by running various calibrators as unknown. Comparing tacrolimus values in specimens from 101 transplant patients (49 kidney, 19 bone marrow transplant, 16 liver and 15 heart transplant) using the reference method (LC-MS/MS) as the x-axis and the corresponding values obtained by the tacrolimus immunoassay using Alinity I analyzer, we observed the following regression equation: y = 0.97 x + 1.005 (n = 101, r = 0.95). For kidney transplant recipients, the regression equation was y = 0.96 + 1.20 (n = 49, r = 0.94). Discussion Excellent correlation between tacrolimus values obtained by the newly FDA approved tacrolimus immunoassay with the reference LC-MS/MS based assay indicates further improvement in the performance of immunoassays for therapeutic drug monitoring of immunosuppressants. This may be due to reduced cross-reactivity of metabolites as indicated in the package insert. Conclusions The newly approved tacrolimus immunoassay for the application on the Alinity I analyzer can be used for routine therapeutic drug monitoring of tacrolimus in clinical laboratories.

Comparative Evaluation of Four Commercially Available Immunoassays for Therapeutic Drug Monitoring of Infliximab and Adalimumab.

Therapeutic drug monitoring (TDM) of anti-TNF-α is an important tool in clinical practice for inflammatory diseases. In this study, we have evaluated the performance of several assays for drug and antidrug antibodies (ADA) measurement in the serum. 50 sera from patients treated with infliximab (IFX) and 49 sera from patients treated with adalimumab (ADAL) were monitored with four immunoassays. We have compared Promonitor, i-Track10®, and ez-track1 assays to our gold standard Lisa Tracker® ELISA using Cohen's kappa, Passing-Bablok, and Bland-Altman analysis. The qualitative analysis evaluated by Cohen's kappa values found for IFX measurements an "almost perfect" concordance for Promonitor, "moderate" for i-Track10® and "substantial" for ez-Track1. For ADAL, kappa values were "moderate" for all tested methods. For anti-IFX, kappa values were "almost perfect" for Promonitor, "fair" for i-Track10®, and "substantial" for ez-Track1. For anti-ADAL, kappa values were "almost perfect" for all three assays. For quantitative analysis of drug measurements, Pearson's r values were all above 0.9 and Lin's concordance coefficients of all immunoassays were around 0.80. Performances of the four evaluated immunoassays were acceptable for TDM based on our laboratory experience. Nevertheless, concordance between the four methods for IFX measurement was not perfect and we recommend the use of the same assay for the follow-up of a given patient. The performances of the four immunoassays evaluated were similar and are acceptable for TDM based on our laboratory experience.

Comparison of ultra-performance liquid chromatography and ARK immunoassay for therapeutic drug monitoring of voriconazole.

Therapeutic drug monitoring (TDM) of voriconazole is recommended for personalizing doses. The objective of this study was to compare the enzyme immunoassay developed by ARKTM Diagnostics Inc. for the quantification of voriconazole adapted to the Architect C4000 autoanalyzer (Abbott®) with ultra-performance liquid chromatography using ultraviolet detector (UPLC-UV) method. Linearity, precision and accuracy of both methods were validated according to the Food and Drug Administration (FDA) and European Medicines Agency guidelines. The limit of quantification (LOQ) of the UPLC-UV method was determined experimentally. Both methods were applied to the analysis of 62 samples from patients. Correlation was evaluated by Passing-Bablok analysis and the concordance by the Bland-Altman method. Dosage recommendations were generated; the discordances according to the technique were evaluated. All validation parameters determined for UPLC-UV met the criteria set out and LOQ of 0.1μg/mL was established. However, when the enzyme immunoassay was used to determine concentrations ≤1μg/ml, CVs were >20%. A linear correlation between both methods was found. However, an overestimation of immunoassay (systematic error of 0.39μg/mL) was detected. In 11.3% of the samples, the differences in concentrations when they were determined by different techniques would imply a different therapeutic regime. These samples had concentrations close to 1μg/mL. Although both techniques can be used for TDM of voriconazole, when a value close to the lower limit of the therapeutic range is determined by the ARKTM immunoassay, it would be better to verify the result by a non-automated technique to avoid possible underdosing.

Simple and rapid monitoring of doxorubicin using streptavidin-modified microparticle-based time-resolved fluorescence immunoassay.

Developing a simple analytical method suitable for therapeutic drug monitoring in a clinical setting is key to establishing guidelines on accurate dose administration and the advancement of precision medicine. We devised a simple rapid analytical method through the combination of streptavidin-modified microparticles and a time-resolved fluorescence immunoassay for therapeutic drug monitoring. The analytical performance of this method was investigated and validated using clinical samples. By determination of doxorubicin concentration, the proposed assay has shown a satisfactory linear range of detection (3.8–3000 ng mL−1) with a limit of detection of 3.8 ng mL−1 and an IC50 of 903.9 ng mL−1. The intra and inter-assay coefficients of variation were 4.12–5.72% and 5.48–6.91%, respectively, and the recovery was acceptable. The applicability of the proposed assay was assessed by comparing the determined results with those measured by LC-MS/MS, presenting a satisfactory correlation (R2 = 0.9868). The proposed assay, which shows satisfactory analytical performance, has great potential for application in the field of TDM in the future.

You Should Have the Same Area Counts.

Mass spectrometry is a sensitive technique used to detect, identify, and quantify molecules based on their m/z . High-performance liquid chromatography (HPLC)4 is a common separation technique used before analysis of biological samples by LC-MS/MS. LC-MS/MS is preferred over immunoassay for therapeutic drug monitoring of immunosuppressive drugs due to its specificity and sensitivity in detecting analytes in liquid and nonvolatile samples. However, time-consuming sample preparation and interferences resulting from ion suppression are among the disadvantages of LC-MS/MS because they can compromise or invalidate results. Also, although the instruments used are more robust, computer-interfaced, user-friendly, and easier to maintain compared with decades ago (1), their technical complexity leads to difficulties in troubleshooting and can be intimidating to laboratorians. Creating exhaustive system checklists …

Design of a surface plasmon resonance immunoassay for therapeutic drug monitoring of amikacin

The therapeutic drug monitoring (TDM) of pharmaceutical drugs with narrow therapeutic ranges is of great importance in the clinical setting. It provides useful information towards the enhancement of drug therapies, aiding in dosage control and toxicity risk management. Amikacin is an aminoglycoside antibiotic commonly used in neonatal therapies that is indicated for TDM due to the toxicity risks inherent in its use. Current techniques for TDM such as high performance liquid chromatography (HPLC) and gas chromatography–mass spectrometry (GC–MS) are costly, time consuming, and cannot be performed at the site of action. Over the last decades, surface plasmon resonance (SPR) biosensors have become increasingly popular in clinical diagnostics due to their ability to detect biomolecular interactions in real-time.We present an SPR-based competitive immunoassay for the detection of the antibiotic amikacin, suitable for TDM in both adults and neonates. We have obtained high specificity and sensitivity levels with an IC50 value of 1.4ng/mL and a limit of detection of 0.13ng/mL, which comfortably comply with the drug's therapeutic range. Simple dilution of serum can therefore be sufficient to analyze low-volume real samples from neonates, increasing the potential of the methodology for TDM. Compared to current TDM conventional methods, this SPR-based immunoassay can provide advantages such as simplicity, potential portability, and label-free measurements with the possibility of high throughput. This work is the foundation towards the development of an integrated, simple use, highly sensitive, fast, and point-of-care sensing platform for the opportune TDM of antibiotics and other drugs in a clinical setting.

Comparability and Imprecision of 8 Frequently Used Commercially Available Immunoassays for Therapeutic Drug Monitoring

The development of new immunoassays for therapeutic drug monitoring and the adaptation of current assays on new analytical platforms has led to a plethora of different tests. This variability may influence comparability between the methods and affect interpretation of test results used for guiding drug treatment. Comparability and imprecision of 8 immunoassays for therapeutic drug monitoring were evaluated using 6 different analyzers from 3 manufacturers. Current and previous generation analytical systems used were Architect and AxSym (Abbott Laboratories), cobas c 501 module and COBAS INTEGRA 800 analyzer (Roche Diagnostics GmbH), and Dimension Vista and Dimension Xpand (Siemens Healthcare Diagnostics Inc). Carbamazepine, digoxin, phenobarbital, phenytoin, theophylline, tobramycin, vancomycin, and valproic acid were measured using leftover routine samples. Good performance and comparability of the drug assays was seen on all systems for carbamazepine, phenytoin, and valproic acid except for phenytoin on the Dimension Vista. Deviations from the acceptance criteria were found with digoxin, phenobarbital, theophylline, tobramycin, and vancomycin. Despite a change of the analytical principle on the Roche systems for most drugs, the results demonstrated a very good between-system comparability of the concentration measured. Systematic deviations were found between AxSYM and ARCHITECT for digoxin, phenobarbital, and tobramycin, and between Dimension Xpand and Dimension Vista for digoxin and vancomycin. The study revealed differences between assays, platforms, and assay principles. Care should be taken if methods or instruments are replaced in a laboratory. Laboratories are advised to perform their own method comparison studies and to inform their customers about the effect on the therapeutic ranges in case of observed clinically significant deviations.

Analytical and clinical evaluation of a new immunoassay for therapeutic drug monitoring of infliximab and adalimumab

In the last decade, biological agents have revolutionised rheumatology. Tumour necrosis factorα (TNF α ) neutralising antibody constructs as infl iximab and adalimumab are widely used as treatment for rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis and psoriasis. Infl iximab is a chimeric (mouse-human) monoclonal antibody against TNF α . It has been shown to induce the formation of human antichimeric antibodies. Adalimumab is a fully human antibody against TNF α and therefore thought to be less immunogenic than chimeric antibodies. However, even fully human antibodies may lead to the production of human antihuman antibodies. Unfortunately, despite the overall effectiveness of these biological agents, a signifi cant proportion of patients do not respond or lose response over time. An explanation may be that antibodies are formed against these therapeutic agents. Another reason could be the necessity of achieving trough anti-TNF α drug concentrations in a target range (1, 2) . In addition, the incidence of anti-drug antibodies could be associated with adverse events. Higher concentrations of anti-infl iximab antibodies were associated with a higher risk of infusion reactions (3) , and severe arterial and/or venous thromboembolic events have been related with the presence of anti-adalimumab antibodies (4) . Until now, in the absence of direct measurement of drug levels and anti-drug antibodies, decision-making in the case of failure to anti-TNF α treatment was based on clinical outcome alone. Recently, a new enzyme linked immunosorbent assay (ELISA) has been developed to detect soluble drug levels and antibody formation among patients receiving infl iximab or adalimumab (Promonitor ® , Proteomika, Derio, Vizcaya, Spain, distributed by Menarini Diagn o stics S.A. ® , Badalona, Barcelona, Spain). The aim of this work was to evaluate the analytical performance and the clinical validation of these four assays with the purpose of testing the hypothesis that they are suitable for therapeutic drug monitoring of infl iximab and adalimumab in treated patients. Following the recommendations for the optimisation of immunoassays (5) , we performed our evaluation of these assays during a period of 6 months and two different reagent lots were used. Intraand inter-assay variability were evaluated on three different days with two replicates of the same sample in each assay. The linearity was performed by diluting different proportions of a standard from the upper and lower end of the dynamic range for each analyte to be evaluated. For the clinical validation study, serum was collected from 69 patients with rheumatic diseases treated with infl iximab (Remicade ® , Schering-Plough) or adalimumab (Humira ® , Abbott Laboratories). Informed consent of the patients was required. Before each infusion of infl iximab or subcutaneous injection of adalimumab, 10 mL of serum was collected and stored at – 20 ° C. All sera were tested under standardised conditions specifi ed by the fabricant. Six dilutions of serum samples and 10 for each standard curve were made. All the analytical development was carried out without knowledge of clinical data. Briefl y, in the anti-TNF α drug assay, recombinant human TNF α was coated overnight at room temperature on the solid phase and recognised by infl iximab or adalimumab in each case. The therapeutic monoclonal antibody was detected by an anti-human inmunoglobulin specifi c antibody conjugated to biotine. Serum concentrations of antibodies against these drugs were measured using a double-antigen ELISA based on their capture by drug-coated microplates and their detection by biotine-coupled anti-TNF α drug. This ELISA has a detection limit of 2 ng/mL for infl i ximab and 0.4 ng/mL for adalimumab. And the cut-off value for the drug is 0.053 mg/L for infl iximab and 0.002 mg/L for adalimumab. And for antibodies, the cut-off value is 37 UA/mL for infl iximab and 8 UA/mL for adalimumab. Descriptive statistics were provided using median (range) or percentages. Statistical analysis was performed using the Sigma Plot ® program. Differences between independent groups were traced with the use of Student t-test for normally distributed values. p-Values < 0.05 were considered statistically signifi cant. *Corresponding author: Francisca Llinares-Tello c/o Col o n 120 2 ° A, 03570, Villajoyosa, Alicante, Spain E-mail: paquillinares@eresmas.com Received January 25, 2012; accepted March 9, 2012 ; previously published online March 26, 2012

Establishment of norvancomycin fluorescence polarization immunoassay for therapeutic drug monitoring

To establish a rapid and simple fluorescence polarization immunoassay method for determination of norvancomycin serum concentration, we collected 300 serum samples from the patients receiving norvancomycin in the hospitals localized in Shanghai, China. The drug concentrations were measured by the established HPLC method and FPIA with vancomycin kit. A FPIA algorithm for the determination of norvancomycin concentration was established according to the correlation between the FPIA and HPLC results. The methods and algorithm were validated in another 70 clinical samples. HPLC determination showed a good linear correlation within the range of 0.5-100 mg l(-1) of norvancomycin concentrations. The method was validated via extraction recovery, intra- and inter-day methodological recovery and stability of norvancomycin in serum. Correlation analysis between the measurements of HPLC and FPIA in 300 serum samples gave the linear regression equation: (concentration by HPLC)=0.760 × (concentration by FPIA)-0.577 (P<0.001, R(2)=0.982). An algorithm was derived from this correlation for measuring the serum norvancomycin concentrations with FPIA. When it was validated in additional 70 serum samples from patients, 'FPIA algorithm' showed good accuracy versus HPLC: 'FPIA algorithm'=0.93 (HPLC)+0.63, R(2)=0.962, and 94.3% of the results from FPIA algorithm fell within the range of -20%/+20% of HPLC. This algorithm developed in this study can be easily used for determination of norvancomycin using TDx analyzer with vancomycin kit indirectly. It may also be useful for norvancomycin therapeutic drug monitoring.

145 Novel Immunoassay for Therapeutic Drug Monitoring of mTOR-Inhibitors after Heart and Lung Transplantation

Therapeutic drug monitoring (TDM) of immunosuppressive drugs after transplantation like the mTOR-inhibitors sirolimus (SRL) or everolimus (ERL) is based on measuring blood levels alone. But this often results in under- or over-immunosuppression leading to rejection or infection, respectively. Earlier studies have shown the potential value of measuring pharmacodynamic drug effects for TDM. Therefore we developed an assay to measure drug effects on the mTOR-pathway.

Analytical and Pharmacological Aspects of Therapeutic Drug Monitoring of mTOR Inhibitors

Mammalian Target Of Rapamycin (mTOR) inhibitors represent a new class of immunosuppressant drugs extensively used for the prevention and the treatment of graft rejection in organ transplant recipients. Their current use is due to referred low nephrotoxic effects, particularly important in kidney transplanted and/or patients with renal failure. The most representative drugs of such class are Sirolimus (Siro) and Everolimus (Rad). Both drugs show a narrow therapeutic window, therefore, monitoring of whole-blood drug levels is recommended in order to optimize the therapy. Among the available assays, Liquid Chromatography coupled with UltraViolet or Electrospray Tandem Mass Spectrometry methods (LC/UV or LC/ESI-MSMS) are the most accurate and specific ones. A reliable alternative is represented by immunoassays, which offer the opportunity to minimize sample pre-treatment, thus reducing the time between drawing blood sample and measuring the drug concentration, an important aspect in high-throughput analyses. Despite this, a limitation in the use of immunoassays for therapeutic drug monitoring is the lower specifity compared with the chromatographic methods when analysing structurally-related drugs. New insights to optimize mTOR inhibitors regimens seem to be offered by the evaluation of CYP450 3A activity by using the probe drug approach. To such purpose, there are a number of major probe drugs used for in vivo studies including: midazolam, cortisol, lidocaine, nifedipine, dextromethorphan, erythromycin, dapsone and alfentanil. The aim of the present paper is to report the most recent knowledge concerning this issue, supplying a critical and comprehensive review for whom are involved both in the clinical and analytical areas.

Evaluation of a New Immunoassay for Therapeutic Drug Monitoring of Tacrolimus in Adult Liver Transplant Recipients

Therapeutic drug monitoring is necessary when using tacrolimus (FK) due to the associated side effects. The aim of this study was to compare the chemiluminescent assay (CMIA) system with the previously established Abbott IMx Tacrolimus II microparticle enzyme immunoassay (MEIA) in liver transplant recipients and evaluate its accuracy. Between March and June 2008, all blood samples from the liver transplant recipients at the hospital were tested for FK trough level using 2 different methods, CMIA and MEIA. The posttransplant time, hematocrit, and other clinical parameters during the study period were recorded. FK trough level was analyzed in 398 samples from 57 liver transplant recipients by CMIA and MEIA. The correlation in FK level between the 2 methods was excellent (r(2) = 0.941). However, the FK level was underestimated in MEIA by more than 23% in samples with an FK level of less than 3.5 ng/mL and by 6.8% in those with an FK level between 3.5 and 5 ng/mL. CMIA is superior to MEIA in measuring low FK level, allowing the FK level to be maintained at less than 5 ng/mL in selected liver transplant recipients. The effects of maintaining low levels of FK should be evaluated in liver transplant recipients.

Design, synthesis and studies of triphosphate analogues for the production of anti AZT-TP antibodies

Triphosphates anabolites are the active chemical species of nucleosidic reverse transcriptase inhibitors in HIV-therapy. Herein, we describe (i) the design of stable triphosphate analogues of AZT using molecular modelling, (ii) their synthesis and (iii) their use for producing anti AZT-TP antibodies in the aim of developing an immunoassay for therapeutic drug monitoring.

Comparison of gas—liquid chromatography and fluorescence polarization immunoassay for therapeutic drug monitoring of flecainide acetate

A gas chromatographic (GC) method for quantitation of flecainide acetate in human plasma is described and compared with a fluorescence polarization immunoassay (FPIA) for therapeutic drug monitoring. The GC method includes a solid-phase extraction procedure and electron capture detection (ECD) without the need of derivatization. Within-day and between-day coefficients of variation were <7% for GC and FPIA. Recovery was between 89–101% for the GC method. Plasma from 36 patients were analysed by both GC and FPIA and the results showed a good correlation (slope = 0.96; intercept = 0.009 μg ml −1; r = 0.987).

Four fluorescence polarization immunoassays for therapeutic drug monitoring evaluated.

We evaluated four fluorescence polarization immunoassays--those for phenytoin, procainamide, N-acetylprocainamide (NAPA), and quinidine--from Roche Diagnostic Systems, done in the Cobas Bio FP centrifugal analyzer. The assays for phenytoin, NAPA, and quinidine demonstrated a linear response over the expected ranges of concentrations, and analytical recovery of test drug added to drug-free sera was greater than 90%. However, recovery in the procainamide assay was poor (69-82%) for samples containing greater than 8 mg/L, owing to nonlinearity. Results of method-comparison studies of the four assays paralleled the recovery studies, although the quinidine assay demonstrated a bias (1.0 mg/L higher) when compared with EMIT (Syva). The precision of the phenytoin assay was acceptable at all concentrations tested (total CV less than 7.0%). Imprecision of the other assays was significant at certain concentrations: total CV greater than 10.0% at subtherapeutic values for NAPA and quinidine, and greater than 9.0% for low (2.4 mg/L) and moderate concentrations (9.6 mg/L) of procainamide. Interferences were not significant for hemolyzed, icteric, or lipemic specimens or for specimens with added drug metabolites. The calibration curves for all four assays had good stability (greater than 60 days).

Reliability of antidepressant assays: a reference laboratory perspective on antidepressant monitoring.

Chromatography (gas and liquid) and immunoassays are used for monitoring the commonly prescribed tricyclic antidepressants. Many commercially available immunoassays are known to cross react with structurally similar compounds. Chromatographic methods make it possible simultaneously to resolve and quantify amitriptyline, nortriptyline, imipramine, desipramine, trimipramine, doxepin, desmethyldoxepin, protriptyline, and maprotiline-and potentially crossreactive compounds can be separated from the tricyclics. Immunoassays may have a valuable role in initial toxicological screening for the presence of a tricyclic-like compound, and they also may be helpful in a laboratory dedicated to a well-controlled patient group. However, 10% of our specimens contain more or different antidepressants than we are requested to analyze for. With our analysis, we are able to report which antidepressants are present, and in what concentrations. Further, in the case of a potential overdose of tricyclic, the primary purpose for early toxicological analysis to anticipate subsequent clinical complications. Therefore, even in the case of toxicological analysis, it is important to know exactly what tricyclic antidepressant is present rather than just the semiquantitative presence of one or more structurally related compounds, because these various compounds differ markedly in their potential for adverse effects. There are too many potential, and possibly yet unknown, interactions for a reference laboratory routinely to rely on immunoassays for therapeutic drug monitoring or toxicological identification of antidepressants.