Clin 8iochem, Vol, 24, pp. 55-58, 1991 Printed in Canada. All rights reserved.
0009-9120/91 $3.00 + .00 Copyright © 1991 The Canadian Society of Clinical Chemists.
Cyclosporine Monitoring by Fluorescence Polarization Immunoassay PHILIP P. WANG, ELIZABETH SIMPSON, VICKI MEUCCI, MARJORIE MORRISON, STEVE LUNE'I-I'A, MARIOLA ZAJAC, and ROGER BOECKX Abbott Laboratories, Abbott Park, IL, USA The monitoring of cyclosporine levels in whole blood and serum has become a routine procedure for the post-transplant management of immunosuppression. We have developed fluorescence polarization immunoassays for cyclosporine in whole blood and in serum using a monoclonal antibody. These assays are fast (20 determinations in less than 22 min), sensitive (25 ~.g/L for whole blood samples and 10 ~g/L for serum samples), and precise (CV less than 7% in both assays). Cross-reactivities with AM1 (Metabolite 17) and AM4N (Metabolite 21) are less than 8.5 and 2.5%, respectively, for both assays.
KEY WORDS: cyclosporine; monoclonal antibody; fluorescent polarization immunoassay; renal transplantation. Introduction
C
yclosporine (CsA) therapeutic monitoring has become an essential component of post-transplant immunosuppression. Numerous conferences with specific emphasis on cyclosporine pharmacology, pharmacokinetics, and therapeutic monitoring have resulted in a large number of reviews and communications (1-5). Among the analytical methodologies, high-performance liquid chromatography (HPLC) is considered to be the most accurate, especially for the measurement of parent drug (6,7). A number of nonspecific assays, including polyclonal 3H-radioimmunoassay (RIA) (8), polyclonal 125I-RIA (9-11), polyclonal fluorescence polarization immunoassays (FPIA) (12,13) and nonspecific monoclonal 3H-RIA (14-18) are available. Recently, cyclosporine-specific monoclonal assays using tritiated tracer (14-18) and 125I-labeled tracer (17) have become widely used. The degree of cross-reactivity with cyclosporine metabolites, the technical expertise required, and assay performance characteristics of these assays differ. HPLC is considered to be the most difficult to perform, the most accurate, the least precise, and the most time consuming. The
Correspondence: Philip P. Wang, Department 09TC, Building AP-20, Abbott Laboratories, Abbott Park, IL 60064, USA. Manuscript received May 21, 1990; revised July 21, 1990; accepted August 29, 1990. CLINICAL BIOCHEMISTRY, VOLUME 24, FEBRUARY 1991
polyclonal assays are the least specific, but are simple. We describe here an automated monoclonal antibody FPIA assay for CsA, which is simple and has minimal cross-reactivity with major metabolites. Methods
Monoclonal antibodies, Sandimmun 3H-RIA kits, CsA and its derivatives were provided by Sandoz Ltd., Basel, Switzerland. The TDx Autoanalyzer, its accessories, and other assay components are products of Abbott Laboratories, Abbott Park, IL, USA. The CsA Whole Blood Assay and CsA Serum/Plasma Assay reagents are supplied as fully reconstituted reagent kits. The reagent packs include a pre-treatment solution, a CsA antiserum (mouse monoclonal) and a CsA-fluorescein tracer. In addition, a precipitation reagent and a solubilization reagent are also provided. The FPIA assay procedure requires treatment of the serum, plasma or whole blood specimens prior to analysis by the TDx. This procedure involves the following steps: (1)an aliquot of the sample to be tested (calibrators, controls and patient specimens are treated similarly) is mixed with a precipitation reagent (whole blood samples are, in addition, treated with a solubilization reagent prior to this step); (2) the resulting mixture is thoroughly mixed and centrifuged (9500 × g, 5 rain); and (3) the supernatants are transferred to TDx sample cartridges. All subsequent sample handling steps are performed by the analyzer. A flow chart representation of this procedure is shown in Figure 1. The sample requirements are 150 ~LL for whole blood and 50 ~L for serum or plasma. Calibrators (6 levels) and controls (3 levels) are also supplied in ready-to-use form. The whole blood assay calibrator matrix is reconstituted human blood. The serum/plasma calibrator matrix is reconstituted h u m a n blood. The serum/plasma calibrator matrix is processed human plasma. Known quantities of CsA are mixed with the appropriate matrix to generate calibrators ranging from 0 to 1500 ~g/L and 0 to 400 }zg/L for the whole blood and serum/ plasma assays, respectively. Controls for each 55
W A N G , SIMPSON, MEUCCI, ZT AL.
A Whole Blood Assay 150pL Whole Blood Calibrator, Controlsor patients specimens 50HL ~~ Solubilization Reagent
300HL
B
Serum/Plasma Assay 50pL Serum/Plasma Calibrator, Controlsor patients specimens 150pL
Precipitation Reagent
_1 "-T
I voex i
Vortex
f
?
I
I eo""ooe I
Centrifuge
f
f
I Su0erna'an I
I Supernatant I I,, ,,-- I TDx Analyzer l ~, 11-17 min ,~ I~,~ 14-22 min Results Results
Figure 1--Sample preparation flow chart: A. Whole blood samples; B. Serum/plasma sample. The analysis time on the TDx Analyzer is 11 min for a single whole blood sample and 17 min for 20 whole blood samples; the corresponding analysis times for sera samples are 14 and 22 min. of the assays are prepared in the same manner. The S a n d i m m u n e 3H-RIA assays were performed according to the procedure outlined by the manufacturer.
reactivities of 8.3% for AM1 and 2.5% for AM4N. Similar results were obtained in the serum matrix. Abnormally elevated levels of lipids, serum protein, hemoglobin and bilirubin have no significant effect
Results The assay protocols described will generate 20 cyclosporine levels in less t h a n 22 min after sample preparation. The limit of detection for serum and whole blood specimens is 10 and 25 ~Lg/L, respectively. The serum calibration curve has an upper range of 400 ~Lg/L,whereas the highest whole blood calibrator is 1500 ~Lg/L. The precision of the assay was determined by repeated analytical runs of control samples. The within, between-run and total CV are shown in Table 1. The stored calibration curves have been shown to yield acceptable results within _+ 10% of target concentrations (Table 2) over a two-week period. Major cyclosporine metabolites AM1 and AM4N were tested at concentrations up to 2000 ~g/L in the whole blood matrix in the presence and in the absence of CsA. The results (Table 3) indicate cross-
56
TABLE 1 Reproducibility of Control Samples
Whole Blood Assay Mean ~Lg/L
N
142 369 832
50 50 50
Within-run CV (%) 5.6 4.1 2.3
Between-run CV (%) 2.7 3.9 3.3
Total CV (%) 6.2 5.6 4.0
Serum/Plasma Assay Mean ~g/L
N
45 90 323
25 25 25
Within-run C V (%)
5.3 3.9 3.4
Between-run C V (%)
3.3 1.3 1.8
Total C V (%)
6.3 4.2 3.9
CLINICAL BIOCHEMISTRY, V O L U M E 24, F E B R U A R Y 1991
CYCLOSPORINE MONITORING TABLE2 Stability of Stored Calibration Curve Over a Two-Week Period
TABLE4 Interference Study with Elevated Blood Components Highest Concentration Tested
Calibrator Target ~g/L 100 250 500 1000 1500
Day 7"
Day 14"
101 246 490 998 1500
94 237 481 968 1464
"Concentration of CsA (ng/mL) as determined from a calibration curve generated on Day 0.
on the performance of the FPIA assay (Table 4). Method comparisons of the FPIA whole blood assay with Sandimmune 3H-RIA and with HPLC are shown in Table 5. Renal, heart and liver transplant patient specimens have been included in this study.
Lipid triglyceride cholesterol Serum protein Hemoglobin Bilirubin
16.5 mmol/L 13 rnmol/L 100 g/L 2.67 mmol/L 684 ~mol/L
Interference (%) -
0009-9120/91 $3.00 + .00 Copyright © 1991 The Canadian Society of Clinical Chemists.
Cyclosporine Monitoring by Fluorescence Polarization Immunoassay PHILIP P. WANG, ELIZABETH SIMPSON, VICKI MEUCCI, MARJORIE MORRISON, STEVE LUNE'I-I'A, MARIOLA ZAJAC, and ROGER BOECKX Abbott Laboratories, Abbott Park, IL, USA The monitoring of cyclosporine levels in whole blood and serum has become a routine procedure for the post-transplant management of immunosuppression. We have developed fluorescence polarization immunoassays for cyclosporine in whole blood and in serum using a monoclonal antibody. These assays are fast (20 determinations in less than 22 min), sensitive (25 ~.g/L for whole blood samples and 10 ~g/L for serum samples), and precise (CV less than 7% in both assays). Cross-reactivities with AM1 (Metabolite 17) and AM4N (Metabolite 21) are less than 8.5 and 2.5%, respectively, for both assays.
KEY WORDS: cyclosporine; monoclonal antibody; fluorescent polarization immunoassay; renal transplantation. Introduction
C
yclosporine (CsA) therapeutic monitoring has become an essential component of post-transplant immunosuppression. Numerous conferences with specific emphasis on cyclosporine pharmacology, pharmacokinetics, and therapeutic monitoring have resulted in a large number of reviews and communications (1-5). Among the analytical methodologies, high-performance liquid chromatography (HPLC) is considered to be the most accurate, especially for the measurement of parent drug (6,7). A number of nonspecific assays, including polyclonal 3H-radioimmunoassay (RIA) (8), polyclonal 125I-RIA (9-11), polyclonal fluorescence polarization immunoassays (FPIA) (12,13) and nonspecific monoclonal 3H-RIA (14-18) are available. Recently, cyclosporine-specific monoclonal assays using tritiated tracer (14-18) and 125I-labeled tracer (17) have become widely used. The degree of cross-reactivity with cyclosporine metabolites, the technical expertise required, and assay performance characteristics of these assays differ. HPLC is considered to be the most difficult to perform, the most accurate, the least precise, and the most time consuming. The
Correspondence: Philip P. Wang, Department 09TC, Building AP-20, Abbott Laboratories, Abbott Park, IL 60064, USA. Manuscript received May 21, 1990; revised July 21, 1990; accepted August 29, 1990. CLINICAL BIOCHEMISTRY, VOLUME 24, FEBRUARY 1991
polyclonal assays are the least specific, but are simple. We describe here an automated monoclonal antibody FPIA assay for CsA, which is simple and has minimal cross-reactivity with major metabolites. Methods
Monoclonal antibodies, Sandimmun 3H-RIA kits, CsA and its derivatives were provided by Sandoz Ltd., Basel, Switzerland. The TDx Autoanalyzer, its accessories, and other assay components are products of Abbott Laboratories, Abbott Park, IL, USA. The CsA Whole Blood Assay and CsA Serum/Plasma Assay reagents are supplied as fully reconstituted reagent kits. The reagent packs include a pre-treatment solution, a CsA antiserum (mouse monoclonal) and a CsA-fluorescein tracer. In addition, a precipitation reagent and a solubilization reagent are also provided. The FPIA assay procedure requires treatment of the serum, plasma or whole blood specimens prior to analysis by the TDx. This procedure involves the following steps: (1)an aliquot of the sample to be tested (calibrators, controls and patient specimens are treated similarly) is mixed with a precipitation reagent (whole blood samples are, in addition, treated with a solubilization reagent prior to this step); (2) the resulting mixture is thoroughly mixed and centrifuged (9500 × g, 5 rain); and (3) the supernatants are transferred to TDx sample cartridges. All subsequent sample handling steps are performed by the analyzer. A flow chart representation of this procedure is shown in Figure 1. The sample requirements are 150 ~LL for whole blood and 50 ~L for serum or plasma. Calibrators (6 levels) and controls (3 levels) are also supplied in ready-to-use form. The whole blood assay calibrator matrix is reconstituted human blood. The serum/plasma calibrator matrix is reconstituted h u m a n blood. The serum/plasma calibrator matrix is processed human plasma. Known quantities of CsA are mixed with the appropriate matrix to generate calibrators ranging from 0 to 1500 ~g/L and 0 to 400 }zg/L for the whole blood and serum/ plasma assays, respectively. Controls for each 55
W A N G , SIMPSON, MEUCCI, ZT AL.
A Whole Blood Assay 150pL Whole Blood Calibrator, Controlsor patients specimens 50HL ~~ Solubilization Reagent
300HL
B
Serum/Plasma Assay 50pL Serum/Plasma Calibrator, Controlsor patients specimens 150pL
Precipitation Reagent
_1 "-T
I voex i
Vortex
f
?
I
I eo""ooe I
Centrifuge
f
f
I Su0erna'an I
I Supernatant I I,, ,,-- I TDx Analyzer l ~, 11-17 min ,~ I~,~ 14-22 min Results Results
Figure 1--Sample preparation flow chart: A. Whole blood samples; B. Serum/plasma sample. The analysis time on the TDx Analyzer is 11 min for a single whole blood sample and 17 min for 20 whole blood samples; the corresponding analysis times for sera samples are 14 and 22 min. of the assays are prepared in the same manner. The S a n d i m m u n e 3H-RIA assays were performed according to the procedure outlined by the manufacturer.
reactivities of 8.3% for AM1 and 2.5% for AM4N. Similar results were obtained in the serum matrix. Abnormally elevated levels of lipids, serum protein, hemoglobin and bilirubin have no significant effect
Results The assay protocols described will generate 20 cyclosporine levels in less t h a n 22 min after sample preparation. The limit of detection for serum and whole blood specimens is 10 and 25 ~Lg/L, respectively. The serum calibration curve has an upper range of 400 ~Lg/L,whereas the highest whole blood calibrator is 1500 ~Lg/L. The precision of the assay was determined by repeated analytical runs of control samples. The within, between-run and total CV are shown in Table 1. The stored calibration curves have been shown to yield acceptable results within _+ 10% of target concentrations (Table 2) over a two-week period. Major cyclosporine metabolites AM1 and AM4N were tested at concentrations up to 2000 ~g/L in the whole blood matrix in the presence and in the absence of CsA. The results (Table 3) indicate cross-
56
TABLE 1 Reproducibility of Control Samples
Whole Blood Assay Mean ~Lg/L
N
142 369 832
50 50 50
Within-run CV (%) 5.6 4.1 2.3
Between-run CV (%) 2.7 3.9 3.3
Total CV (%) 6.2 5.6 4.0
Serum/Plasma Assay Mean ~g/L
N
45 90 323
25 25 25
Within-run C V (%)
5.3 3.9 3.4
Between-run C V (%)
3.3 1.3 1.8
Total C V (%)
6.3 4.2 3.9
CLINICAL BIOCHEMISTRY, V O L U M E 24, F E B R U A R Y 1991
CYCLOSPORINE MONITORING TABLE2 Stability of Stored Calibration Curve Over a Two-Week Period
TABLE4 Interference Study with Elevated Blood Components Highest Concentration Tested
Calibrator Target ~g/L 100 250 500 1000 1500
Day 7"
Day 14"
101 246 490 998 1500
94 237 481 968 1464
"Concentration of CsA (ng/mL) as determined from a calibration curve generated on Day 0.
on the performance of the FPIA assay (Table 4). Method comparisons of the FPIA whole blood assay with Sandimmune 3H-RIA and with HPLC are shown in Table 5. Renal, heart and liver transplant patient specimens have been included in this study.
Lipid triglyceride cholesterol Serum protein Hemoglobin Bilirubin
16.5 mmol/L 13 rnmol/L 100 g/L 2.67 mmol/L 684 ~mol/L
Interference (%) -
