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Journal of Immunoassay and Immunochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ljii20
A HIGHLY SENSITIVE POLYCLONAL ANTIBODY-BASED ELISA FOR THERAPEUTIC MONITORING AND PHARMACOKINETIC STUDIES OF LENALIDOMIDE a
a
Nourah Z. Alzoman , Ibrahim A. Darwish & Reem M. Abuhejail
a
a
Click for updates
Department of Pharmaceutical Chemistry, College of Pharmacy , King Saud University , Riyadh , Saudi Arabia Accepted author version posted online: 13 Aug 2013.Published online: 02 Dec 2013.
To cite this article: Nourah Z. Alzoman , Ibrahim A. Darwish & Reem M. Abuhejail (2014) A HIGHLY SENSITIVE POLYCLONAL ANTIBODY-BASED ELISA FOR THERAPEUTIC MONITORING AND PHARMACOKINETIC STUDIES OF LENALIDOMIDE, Journal of Immunoassay and Immunochemistry, 35:2, 130-138, DOI: 10.1080/15321819.2013.824898 To link to this article: http://dx.doi.org/10.1080/15321819.2013.824898
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Downloaded by [Florida Atlantic University] at 10:30 30 January 2015
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Journal of Immunoassay and Immunochemistry, 35:130–138, 2014 Copyright # Taylor & Francis Group, LLC ISSN: 1532-1819 print/1532-4230 online DOI: 10.1080/15321819.2013.824898
Downloaded by [Florida Atlantic University] at 10:30 30 January 2015
A HIGHLY SENSITIVE POLYCLONAL ANTIBODY-BASED ELISA FOR THERAPEUTIC MONITORING AND PHARMACOKINETIC STUDIES OF LENALIDOMIDE
Nourah Z. Alzoman, Ibrahim A. Darwish, and Reem M. Abuhejail Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
& This article describes, for the first time, a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) for therapeutic monitoring and pharmacokinetic studies of lenalidomide (LND), the potent drug for treatment of multiple myeloma (MM). The assay employed a polyclonal antibody that specifically recognizes LND with high affinity, and LND conjugate of bovine serum albumin (LND–BSA) immobilized onto microplate wells as a solid phase. The assay involved a competitive binding reaction between LND, in plasma sample, and the immobilized LND–BSA for the binding sites on a limited amount of the anti–LND antibody. The assay limit of detection was 0.05 ng=mL and the effective working range at relative standard deviations (RSD) of 5% was 0.120 ng=mL. Analytical recovery of LND from spiked plasma was 100.98 3.05. The precisions of the assay were satisfactory; RSD was 2.966.67% and 4.467.14%, for the intra- and inter-assay precision, respectively. The procedure is convenient, and one can analyze 200 samples per working day, facilitating the processing of large-number batch of samples in clinical laboratories. The proposed ELISA has a great value in routine analysis of LND for its therapeutic monitoring and pharmacokinetic studies. Keywords ELISA, lenalidomide, multiple myeloma, pharmacokinetic studies, polyclonal antibody, therapeutic monitoring
INTRODUCTION Lenalidomide (LND) is a potent novel thalidomide analog which demonstrated remarkable clinical activity against multiple myeloma[1] via a multiple-pathways mechanism.[2] The strong evidences-based clinical success of LND in patients has led to its recent approval by US-FDA under the trade name of Revlimid1 capsules by Celgene Corporation.[3] LND has Address correspondence to Ibrahim A. Darwish, Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia. E-mail: [email protected]
Downloaded by [Florida Atlantic University] at 10:30 30 January 2015
Polyclonal Antibody-Based ELISA for Lenalidomide
131
more improved side effects, nevertheless, it causes some dose-dependent side effects such as thrombocytopenia, venous thromboembolism, and myelosuppression.[4] These side effects can be managed by combination therapy and=or careful dose adjustment.[5,6] Because LND is primarily excreted via kidneys, patients with renal insufficiency or failure must be dose adjusted to prevent the exacerbation of its myelosuppressive effects.[2] Furthermore, studies showed large inter-individual pharmacokinetic variability with concentration–toxicity relationship.[7] For these reasons, a risk management, monitoring blood counts, and therapeutic drug monitoring are required to achieve the highest therapeutic benefits of LND and prevent its fatal complications.[8,9] Nevertheless, the therapeutic profile of LND is anticipated to encourage the development of new pharmaceutical preparations for LND. As a consequence, there is an increasing demand for proper analytical technologies for determination of pharmacokinetic parameters in bioequivalence studies for LND, as well as in its therapeutic monitoring. The reported methods (liquid chromatography-coupled with mass spectrometric detectors) for the determination of LND in plasma[10,11] employed the expensive mass detectors that are not available in most laboratories, involved laborious sample extraction procedures that negatively affected the accuracy of the results, and are not suited for screening of large number of specimens. Accordingly, the development of a new alternative analytical technology for the determination of LND in plasma with adequate sensitivity, improved simplicity, lower cost, and higher throughput is urgently needed. Enzyme-linked immunosorbent assay (ELISA) has been proved as a highly selective technique, remarkably quick, easily performed, offers great sensitivity, does not require pretreatment for the samples, and is well suited for the screening of large number of samples.[12] The present article describes, for the first time, the development and validation of a highly sensitive ELISA for determination of LND at concentrations as low as 0.1 ng=mL in plasma samples.
EXPERIMENTAL Instruments Microplate=cuvette reader (Spectramax M5, Molecular Devices, Sunnyvale, CA, USA). Automatic microplate strip washer (MW-12A, Bio-Medical Electronics Co., Ltd., Shenzhen, China). EM-36N microtube shaker (Taitec, Japan). Biofuge Pico centrifuge (Heraeus Instruments, Tuttlingen, Germany). Incubator (KARL KOLB, Scientific Technical Supplies, Dreieich, Germany).
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Downloaded by [Florida Atlantic University] at 10:30 30 January 2015
Materials Lenalidomide (LND) was obtained from LC Laboratories (Woburn, MA, USA). Horseradish peroxidase labeled goat anti-rabbit IgG (HRP-IgG), bovine serum albumin (BSA) and 3,30 ,5,50 -Tetramethylbenzidine (TMB) peroxidase substrate, glutaric anhydride, dialyzing tubes, trinitrobenzene sulphonic acid, Tween-20, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). BCA protein assay kit was purchased from Pierce Chemical Co. (Rockford, IL, USA). ELISA high-binding microwell plates were a product of Corning=Costar, Inc. (Cambridge, MA, USA). Human plasma samples were collected from normal healthy volunteer at King Khalid University Hospital (Riyadh, Saudi Arabia), and they were stored at 20 C until analysis. All other chemicals and solvents used throughout the work were of analytical grade. Procedures Preparation of Anti-LND Antibody and LND-BSA Conjugate Anti-LND polyclonal antibody was induced by immunization of 2–3 month-old female New Zealand white rabbit with LND conjugated with keyhole limpt hemocyanin protein (LND-KLH). The coating conjugate (LND-BSA) was prepared by linking the N-glutaryl-LND (G-LND), via its glutaryl carboxylic group, with BSA protein via its amino groups by EDC reagent. The procedures for preparation of anti-LND antibody and LNDBSA conjugate have been published in our previous article.[13] Determination of the Optimum Concentrations of Antibody and Coating Conjugate The optimum LND-BSA concentration required for coating onto the microwell plates and the best working concentration of the anti-LND antibody were determined by checkerboard titration employing the procedures described by Darwish et al.[14] ELISA Procedures and Data Analysis Samples of LND (50 mL) were diluted 8-fold with PBS. The diluted samples were mixed with equal volumes of anti-LND antibody (1 mg=mL; prepared in phosphate buffer saline [PBS]). Aliquot (50 mL) of the mixture was dispensed into each well of the microwell plate that had been previously coated with LND-BSA solution, and blocked with the blocking solution. The coating of plate wells with LND-BSA and their blocking were described above. After 1 hr incubation at 37 C, the plates were washed with PBS, and
Polyclonal Antibody-Based ELISA for Lenalidomide
133
50 mL of HRP-IgG (1:300 in PBS) was added to each well. After 1 hr incubation, the plates were washed with PBS and the amount of the bound HRP-IgG was quantified using TMB enzyme substrate. The data was acquisitioned by Spectramax1 software (Spectramax M5, Molecular Devices), and transformed to a four-parameter curve fitting using Slide Write software, version 5.011 (Advanced Graphics Software, Inc., Rancho Sanata Fe, CA, USA). Value of the IC50 was that gave the best fit to the following equation:
Downloaded by [Florida Atlantic University] at 10:30 30 January 2015
A ¼ A0 fðA 0 A1 Þ½LND=ðIC50 þ ½LNDÞg; where A is the absorbance at a definite known concentration of LND [LND], A0 is the absorbance in the absence of LND, A1 is the absorbance at the saturating concentration of LND, and IC50 is the LND concentration that produces a 50% inhibition of the absorbance. The concentrations of LND in the samples were then obtained by interpolation on the standard curve.
RESULTS AND DISCUSSION This article describes the development of a highly sensitive ELISA for determination of LND in plasma samples. The assay employed the antibody-capture assay format.[12] Optimization of Assay Variables Choice of Conjugate and Antibody Concentrations In order to determine the optimum concentration of LND-BSA conjugate that is required for immobilization onto the microwells and the concentration of the anti-LND antibody required for effective competitive binding reaction, checkerboard titration[14] of LND-BSA and anti-LND antibody was carried out by the direct ELISA.[13] Various concentrations of LND-BSA conjugate (0.58 mg=mL) were immobilized onto the microwells, and different concentrations of the antibody (0.054 mg=mL) were allowed to bind to each immobilized conjugate concentration. The color signals were generated and the measured absorbances were plotted as a function of the conjugate concentrations at the different antibody concentrations (Figure 1). The conjugate and antibody concentrations that gave 11.5 absorbance unit were considered as reference optimum binding conditions. According to this criterion, the optimum combinations of the immobilized conjugate and anti-LND antibody concentrations (derived from Figure 1) are given in Table 1. Competitive assays were carried out using these combinations and IC50 values were calculated. The lowest
Downloaded by [Florida Atlantic University] at 10:30 30 January 2015
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N. Z. Alzoman et al.
FIGURE 1 Checkerboard titrations for LND-BSA versus anti-LND antibody. Varying concentrations (0.5–8 mg=mL) of LND–BSA conjugate were coated onto the microwell plates. Varying concentrations of anti-LND antibody were allowed to bind to the immobilized LND-BSA. Signals were generated as described in the ‘‘Experimental’’ section. Absorbance values were plotted as a function of LND-BSA concentrations (color figure available online).
IC50 value (highest assay sensitivity) was achieved when the conjugate and antibody concentration was 2 and 1 mg=mL, respectively (Table 1). These concentrations were used in all the subsequent investigations. Immobilization and Lifespan of LND-BSA on the Microplate Wells Optimum immobilization of the LND-BSA conjugate onto the microwells was attained when the incubation time was 2 hr at 37 C, and more than 6 hr at room temperature (25 5 C). The lifespan of the LND-BSA after its immobilization onto the microwell plates was found to be at least 4 and 6 weeks when the coated-blocked plates were stored at 4 and 20 C, respectively. This gives an advantage that the use of pre-coated and blocked plates could reduce the time of coating and blocking the assay plates (3 hr) from the entire assay time. TABLE 1 IC50 Values That Have Been Obtained at Varying Concentrations of the Immobilized LND-BSA Conjugate and Anti-LND Antibody LND-BSA (mg=mL) 1 1 2 2 2 4 4 a
Anti-LND Antibody (mg=mL)
IC50a (ng=mL)
2 4 1 2 4 0.5 1
2 2.1 5 3.2 1 4.1 4 3.5 10 2.6 5 7.8 10 4.1
Values are mean of three determinations RSD (%).
Polyclonal Antibody-Based ELISA for Lenalidomide
135
Downloaded by [Florida Atlantic University] at 10:30 30 January 2015
Validation of the Assay Calibration Curve and Detection Limit The calibration curve for determination of LND by the proposed ELISA is given in (Figure 2). The data that has been obtained from curve showed good correlation coefficients on the 4-parameter curve fit (r ¼ 0.997). The limit of detection (LOD) of the proposed ELISA was defined to be the LND concentration that caused inhibition of 5% of the maximum binding (e.g., at 95% binding). Based on the basis of 3 replicate measurements, the LOD value was found to be 0.05 ng=mL. The sensitivity that has been obtained from the proposed ELISA method described here is enough for determination of LND in plasma as the reported plasma levels of LND is 114 ng= mL after 5 mg single oral dose.[10] Precision Profile The assay precision profile obtained from the result of the calibration standard sample, assayed in triplicate, is shown in (Figure 2). From this profile, the working range of the assay at RSD value less than 5% was derived. This range was found to be 0.1–20 ng=mL for LND. The RSD at the detection limits of the assay was found to be 5.80%. The intra- and inter-assay precisions were tested at three varying concentration levels of drug (low, medium, and high). According to the recommendation of immunoassay validation,[15] the assay gave satisfactory results. The RSD for the intra-assay and inter-assay precisions were 2.966.67% and 4.467.14%, respectively (Table 2).
FIGURE 2 Calibration curve (.) and precision profile (~) of the proposed ELISA for LND. Varying concentrations of standard LND were mixed with anti-LND antibody (1 mg=mL). The reaction mixtures were further manipulated as described in the ‘‘Experimental’’ section. The values plotted are mean SD of three determinations (color figure available online).
136 TABLE 2
N. Z. Alzoman et al. Precision of the ELISA for LND at Three Concentration Levels Intra-Assay
LND (ng=mL) 0.1 1 10
Inter-Assay
Mean SDa (ng=mL)
RSDb (%)
Mean SDa (ng=mL)
RSDb (%)
0.15 0.02 1.04 0.04 10.82 0.32
6.67 3.85 2.96
0.14 0.01 1.12 0.05 9.85 0.44
7.14 4.46 4.47
a
Downloaded by [Florida Atlantic University] at 10:30 30 January 2015
b
Values are mean of eight determinations SD. RSD is the relative standard deviation.
Effect of Plasma Matrix Since the proposed ELISA was designed for quantitation of the LND in plasma samples, it was therefore necessary to study the effect of plasma matrix on the analytical performance of the proposed method. LND-free plasma samples were serially diluted into PBS and each dilution was spiked with 1 ng=mL of LND. The spiked samples were then analyzed by the proposed assay. It was found that the recovery increases with the increase in the plasma dilution, and then leveled off when the plasma dilution was 4-fold. Meanwhile, RSD of the recovery values decreased as the plasma dilution increased, and it leveled off at
Journal of Immunoassay and Immunochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ljii20
A HIGHLY SENSITIVE POLYCLONAL ANTIBODY-BASED ELISA FOR THERAPEUTIC MONITORING AND PHARMACOKINETIC STUDIES OF LENALIDOMIDE a
a
Nourah Z. Alzoman , Ibrahim A. Darwish & Reem M. Abuhejail
a
a
Click for updates
Department of Pharmaceutical Chemistry, College of Pharmacy , King Saud University , Riyadh , Saudi Arabia Accepted author version posted online: 13 Aug 2013.Published online: 02 Dec 2013.
To cite this article: Nourah Z. Alzoman , Ibrahim A. Darwish & Reem M. Abuhejail (2014) A HIGHLY SENSITIVE POLYCLONAL ANTIBODY-BASED ELISA FOR THERAPEUTIC MONITORING AND PHARMACOKINETIC STUDIES OF LENALIDOMIDE, Journal of Immunoassay and Immunochemistry, 35:2, 130-138, DOI: 10.1080/15321819.2013.824898 To link to this article: http://dx.doi.org/10.1080/15321819.2013.824898
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Downloaded by [Florida Atlantic University] at 10:30 30 January 2015
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Journal of Immunoassay and Immunochemistry, 35:130–138, 2014 Copyright # Taylor & Francis Group, LLC ISSN: 1532-1819 print/1532-4230 online DOI: 10.1080/15321819.2013.824898
Downloaded by [Florida Atlantic University] at 10:30 30 January 2015
A HIGHLY SENSITIVE POLYCLONAL ANTIBODY-BASED ELISA FOR THERAPEUTIC MONITORING AND PHARMACOKINETIC STUDIES OF LENALIDOMIDE
Nourah Z. Alzoman, Ibrahim A. Darwish, and Reem M. Abuhejail Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
& This article describes, for the first time, a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) for therapeutic monitoring and pharmacokinetic studies of lenalidomide (LND), the potent drug for treatment of multiple myeloma (MM). The assay employed a polyclonal antibody that specifically recognizes LND with high affinity, and LND conjugate of bovine serum albumin (LND–BSA) immobilized onto microplate wells as a solid phase. The assay involved a competitive binding reaction between LND, in plasma sample, and the immobilized LND–BSA for the binding sites on a limited amount of the anti–LND antibody. The assay limit of detection was 0.05 ng=mL and the effective working range at relative standard deviations (RSD) of 5% was 0.120 ng=mL. Analytical recovery of LND from spiked plasma was 100.98 3.05. The precisions of the assay were satisfactory; RSD was 2.966.67% and 4.467.14%, for the intra- and inter-assay precision, respectively. The procedure is convenient, and one can analyze 200 samples per working day, facilitating the processing of large-number batch of samples in clinical laboratories. The proposed ELISA has a great value in routine analysis of LND for its therapeutic monitoring and pharmacokinetic studies. Keywords ELISA, lenalidomide, multiple myeloma, pharmacokinetic studies, polyclonal antibody, therapeutic monitoring
INTRODUCTION Lenalidomide (LND) is a potent novel thalidomide analog which demonstrated remarkable clinical activity against multiple myeloma[1] via a multiple-pathways mechanism.[2] The strong evidences-based clinical success of LND in patients has led to its recent approval by US-FDA under the trade name of Revlimid1 capsules by Celgene Corporation.[3] LND has Address correspondence to Ibrahim A. Darwish, Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia. E-mail: [email protected]
Downloaded by [Florida Atlantic University] at 10:30 30 January 2015
Polyclonal Antibody-Based ELISA for Lenalidomide
131
more improved side effects, nevertheless, it causes some dose-dependent side effects such as thrombocytopenia, venous thromboembolism, and myelosuppression.[4] These side effects can be managed by combination therapy and=or careful dose adjustment.[5,6] Because LND is primarily excreted via kidneys, patients with renal insufficiency or failure must be dose adjusted to prevent the exacerbation of its myelosuppressive effects.[2] Furthermore, studies showed large inter-individual pharmacokinetic variability with concentration–toxicity relationship.[7] For these reasons, a risk management, monitoring blood counts, and therapeutic drug monitoring are required to achieve the highest therapeutic benefits of LND and prevent its fatal complications.[8,9] Nevertheless, the therapeutic profile of LND is anticipated to encourage the development of new pharmaceutical preparations for LND. As a consequence, there is an increasing demand for proper analytical technologies for determination of pharmacokinetic parameters in bioequivalence studies for LND, as well as in its therapeutic monitoring. The reported methods (liquid chromatography-coupled with mass spectrometric detectors) for the determination of LND in plasma[10,11] employed the expensive mass detectors that are not available in most laboratories, involved laborious sample extraction procedures that negatively affected the accuracy of the results, and are not suited for screening of large number of specimens. Accordingly, the development of a new alternative analytical technology for the determination of LND in plasma with adequate sensitivity, improved simplicity, lower cost, and higher throughput is urgently needed. Enzyme-linked immunosorbent assay (ELISA) has been proved as a highly selective technique, remarkably quick, easily performed, offers great sensitivity, does not require pretreatment for the samples, and is well suited for the screening of large number of samples.[12] The present article describes, for the first time, the development and validation of a highly sensitive ELISA for determination of LND at concentrations as low as 0.1 ng=mL in plasma samples.
EXPERIMENTAL Instruments Microplate=cuvette reader (Spectramax M5, Molecular Devices, Sunnyvale, CA, USA). Automatic microplate strip washer (MW-12A, Bio-Medical Electronics Co., Ltd., Shenzhen, China). EM-36N microtube shaker (Taitec, Japan). Biofuge Pico centrifuge (Heraeus Instruments, Tuttlingen, Germany). Incubator (KARL KOLB, Scientific Technical Supplies, Dreieich, Germany).
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Downloaded by [Florida Atlantic University] at 10:30 30 January 2015
Materials Lenalidomide (LND) was obtained from LC Laboratories (Woburn, MA, USA). Horseradish peroxidase labeled goat anti-rabbit IgG (HRP-IgG), bovine serum albumin (BSA) and 3,30 ,5,50 -Tetramethylbenzidine (TMB) peroxidase substrate, glutaric anhydride, dialyzing tubes, trinitrobenzene sulphonic acid, Tween-20, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). BCA protein assay kit was purchased from Pierce Chemical Co. (Rockford, IL, USA). ELISA high-binding microwell plates were a product of Corning=Costar, Inc. (Cambridge, MA, USA). Human plasma samples were collected from normal healthy volunteer at King Khalid University Hospital (Riyadh, Saudi Arabia), and they were stored at 20 C until analysis. All other chemicals and solvents used throughout the work were of analytical grade. Procedures Preparation of Anti-LND Antibody and LND-BSA Conjugate Anti-LND polyclonal antibody was induced by immunization of 2–3 month-old female New Zealand white rabbit with LND conjugated with keyhole limpt hemocyanin protein (LND-KLH). The coating conjugate (LND-BSA) was prepared by linking the N-glutaryl-LND (G-LND), via its glutaryl carboxylic group, with BSA protein via its amino groups by EDC reagent. The procedures for preparation of anti-LND antibody and LNDBSA conjugate have been published in our previous article.[13] Determination of the Optimum Concentrations of Antibody and Coating Conjugate The optimum LND-BSA concentration required for coating onto the microwell plates and the best working concentration of the anti-LND antibody were determined by checkerboard titration employing the procedures described by Darwish et al.[14] ELISA Procedures and Data Analysis Samples of LND (50 mL) were diluted 8-fold with PBS. The diluted samples were mixed with equal volumes of anti-LND antibody (1 mg=mL; prepared in phosphate buffer saline [PBS]). Aliquot (50 mL) of the mixture was dispensed into each well of the microwell plate that had been previously coated with LND-BSA solution, and blocked with the blocking solution. The coating of plate wells with LND-BSA and their blocking were described above. After 1 hr incubation at 37 C, the plates were washed with PBS, and
Polyclonal Antibody-Based ELISA for Lenalidomide
133
50 mL of HRP-IgG (1:300 in PBS) was added to each well. After 1 hr incubation, the plates were washed with PBS and the amount of the bound HRP-IgG was quantified using TMB enzyme substrate. The data was acquisitioned by Spectramax1 software (Spectramax M5, Molecular Devices), and transformed to a four-parameter curve fitting using Slide Write software, version 5.011 (Advanced Graphics Software, Inc., Rancho Sanata Fe, CA, USA). Value of the IC50 was that gave the best fit to the following equation:
Downloaded by [Florida Atlantic University] at 10:30 30 January 2015
A ¼ A0 fðA 0 A1 Þ½LND=ðIC50 þ ½LNDÞg; where A is the absorbance at a definite known concentration of LND [LND], A0 is the absorbance in the absence of LND, A1 is the absorbance at the saturating concentration of LND, and IC50 is the LND concentration that produces a 50% inhibition of the absorbance. The concentrations of LND in the samples were then obtained by interpolation on the standard curve.
RESULTS AND DISCUSSION This article describes the development of a highly sensitive ELISA for determination of LND in plasma samples. The assay employed the antibody-capture assay format.[12] Optimization of Assay Variables Choice of Conjugate and Antibody Concentrations In order to determine the optimum concentration of LND-BSA conjugate that is required for immobilization onto the microwells and the concentration of the anti-LND antibody required for effective competitive binding reaction, checkerboard titration[14] of LND-BSA and anti-LND antibody was carried out by the direct ELISA.[13] Various concentrations of LND-BSA conjugate (0.58 mg=mL) were immobilized onto the microwells, and different concentrations of the antibody (0.054 mg=mL) were allowed to bind to each immobilized conjugate concentration. The color signals were generated and the measured absorbances were plotted as a function of the conjugate concentrations at the different antibody concentrations (Figure 1). The conjugate and antibody concentrations that gave 11.5 absorbance unit were considered as reference optimum binding conditions. According to this criterion, the optimum combinations of the immobilized conjugate and anti-LND antibody concentrations (derived from Figure 1) are given in Table 1. Competitive assays were carried out using these combinations and IC50 values were calculated. The lowest
Downloaded by [Florida Atlantic University] at 10:30 30 January 2015
134
N. Z. Alzoman et al.
FIGURE 1 Checkerboard titrations for LND-BSA versus anti-LND antibody. Varying concentrations (0.5–8 mg=mL) of LND–BSA conjugate were coated onto the microwell plates. Varying concentrations of anti-LND antibody were allowed to bind to the immobilized LND-BSA. Signals were generated as described in the ‘‘Experimental’’ section. Absorbance values were plotted as a function of LND-BSA concentrations (color figure available online).
IC50 value (highest assay sensitivity) was achieved when the conjugate and antibody concentration was 2 and 1 mg=mL, respectively (Table 1). These concentrations were used in all the subsequent investigations. Immobilization and Lifespan of LND-BSA on the Microplate Wells Optimum immobilization of the LND-BSA conjugate onto the microwells was attained when the incubation time was 2 hr at 37 C, and more than 6 hr at room temperature (25 5 C). The lifespan of the LND-BSA after its immobilization onto the microwell plates was found to be at least 4 and 6 weeks when the coated-blocked plates were stored at 4 and 20 C, respectively. This gives an advantage that the use of pre-coated and blocked plates could reduce the time of coating and blocking the assay plates (3 hr) from the entire assay time. TABLE 1 IC50 Values That Have Been Obtained at Varying Concentrations of the Immobilized LND-BSA Conjugate and Anti-LND Antibody LND-BSA (mg=mL) 1 1 2 2 2 4 4 a
Anti-LND Antibody (mg=mL)
IC50a (ng=mL)
2 4 1 2 4 0.5 1
2 2.1 5 3.2 1 4.1 4 3.5 10 2.6 5 7.8 10 4.1
Values are mean of three determinations RSD (%).
Polyclonal Antibody-Based ELISA for Lenalidomide
135
Downloaded by [Florida Atlantic University] at 10:30 30 January 2015
Validation of the Assay Calibration Curve and Detection Limit The calibration curve for determination of LND by the proposed ELISA is given in (Figure 2). The data that has been obtained from curve showed good correlation coefficients on the 4-parameter curve fit (r ¼ 0.997). The limit of detection (LOD) of the proposed ELISA was defined to be the LND concentration that caused inhibition of 5% of the maximum binding (e.g., at 95% binding). Based on the basis of 3 replicate measurements, the LOD value was found to be 0.05 ng=mL. The sensitivity that has been obtained from the proposed ELISA method described here is enough for determination of LND in plasma as the reported plasma levels of LND is 114 ng= mL after 5 mg single oral dose.[10] Precision Profile The assay precision profile obtained from the result of the calibration standard sample, assayed in triplicate, is shown in (Figure 2). From this profile, the working range of the assay at RSD value less than 5% was derived. This range was found to be 0.1–20 ng=mL for LND. The RSD at the detection limits of the assay was found to be 5.80%. The intra- and inter-assay precisions were tested at three varying concentration levels of drug (low, medium, and high). According to the recommendation of immunoassay validation,[15] the assay gave satisfactory results. The RSD for the intra-assay and inter-assay precisions were 2.966.67% and 4.467.14%, respectively (Table 2).
FIGURE 2 Calibration curve (.) and precision profile (~) of the proposed ELISA for LND. Varying concentrations of standard LND were mixed with anti-LND antibody (1 mg=mL). The reaction mixtures were further manipulated as described in the ‘‘Experimental’’ section. The values plotted are mean SD of three determinations (color figure available online).
136 TABLE 2
N. Z. Alzoman et al. Precision of the ELISA for LND at Three Concentration Levels Intra-Assay
LND (ng=mL) 0.1 1 10
Inter-Assay
Mean SDa (ng=mL)
RSDb (%)
Mean SDa (ng=mL)
RSDb (%)
0.15 0.02 1.04 0.04 10.82 0.32
6.67 3.85 2.96
0.14 0.01 1.12 0.05 9.85 0.44
7.14 4.46 4.47
a
Downloaded by [Florida Atlantic University] at 10:30 30 January 2015
b
Values are mean of eight determinations SD. RSD is the relative standard deviation.
Effect of Plasma Matrix Since the proposed ELISA was designed for quantitation of the LND in plasma samples, it was therefore necessary to study the effect of plasma matrix on the analytical performance of the proposed method. LND-free plasma samples were serially diluted into PBS and each dilution was spiked with 1 ng=mL of LND. The spiked samples were then analyzed by the proposed assay. It was found that the recovery increases with the increase in the plasma dilution, and then leveled off when the plasma dilution was 4-fold. Meanwhile, RSD of the recovery values decreased as the plasma dilution increased, and it leveled off at
