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Journal of Immunoassay and Immunochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ljii20
Development of an ELISA for the Pharmacokinetic Evaluation of a Murine Anti CD66 Monoclonal Antibody in Human Serum a
a
b
J. Langford , F. Chowdhury & K. Orchard a
Cancer Sciences Unit, Faculty of Medicine, Southampton University, United Kingdom b
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Department of Haematology, Southampton University Hospitals Trust, Southampton, United Kingdom Accepted author version posted online: 25 Feb 2015.
To cite this article: J. Langford, F. Chowdhury & K. Orchard (2015) Development of an ELISA for the Pharmacokinetic Evaluation of a Murine Anti CD66 Monoclonal Antibody in Human Serum, Journal of Immunoassay and Immunochemistry, 36:6, 579-596, DOI: 10.1080/15321819.2015.1017107 To link to this article: http://dx.doi.org/10.1080/15321819.2015.1017107
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Journal of Immunoassay and Immunochemistry, 36:579–596, 2015 Copyright © Taylor & Francis Group, LLC ISSN: 1532-1819 print/1532-4230 online DOI: 10.1080/15321819.2015.1017107
DEVELOPMENT OF AN ELISA FOR THE PHARMACOKINETIC EVALUATION OF A MURINE ANTI CD66 MONOCLONAL ANTIBODY IN HUMAN SERUM
J. Langford,1 F. Chowdhury,1 and K. Orchard2 1
Cancer Sciences Unit, Faculty of Medicine, Southampton University, United Kingdom Department of Haematology, Southampton University Hospitals Trust, Southampton, United Kingdom
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2
An enzyme-linked immunosorbent assay (ELISA) was needed to assist in the pharmacokinetic evaluation of the murine antibody conjugate CHX A” DTPA Besilesomab in serum samples in a clinical trial . A search failed to locate a validated assay that quantified murine antibodies in human serum so the purpose of this article was to develop a robust assay, validated against current guidelines. A detailed method for an ELISA to measure a murine antibody in human serum is described. The assay was validated as fit for purpose against the target values of coefficient of variation < 20% and accuracy ± 20%. Keywords ELISA, pharmacokinetic
murine
antibody,
human
serum,
validation,
immunoassay,
INTRODUCTION Targeted radioimmunotherapy using monoclonal antibodies as vectors to transport beta-emitting cytotoxic radionuclides to tumor-associated antigen targets is now a recognized therapy; for example, 90 Yttrium ibritumomab tiuxetan “Zevalin”® (Schering Health Care) targets the CD20 antigen expressed on plasma B cells and may be used in the treatment of non-Hodgkin lymphoma. Further research into optimal vectors and radionuclides, including use of alpha particle-emitting radionuclides, is ongoing.[1-4] At the University of Southampton we are currently conducting a phase 1 clinical trial which involves administration of the murine monoclonal antibody Besilesomab (Therapharm, Germany), conjugated to a bifunctional Address correspondence to J. Langford, Cancer Sciences Unit, Faculty of Medicine, Southampton University, SO16 6YD, UK. E-mail: [email protected]
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chelator ITC DTPA CHX A” (Macrocyclics,Texas), which facilitates radiolabeling with the cytotoxic, beta-emitting, radionuclide 90 Yttrium (90 Y). This trial is assessing the use of 90 Y anti-CD66 monoclonal antibody as an adjunct therapy to patients about to undergo standard conditioning and allogeneic peripheral stem cell transplantation (PSCT) for hematological malignancies such as multiple myeloma. The CD 66 antigens are expressed from the promyelocyte stage to the mature granulocyte which makes them suitable targets for bone marrow irradiation.[5,6] Prior to the treatment stage of the trial, dosimetry and bio distribution are assessed by injection of the same antibody conjugate but radiolabeled with a different radionuclide, the gamma-emitting 111 Indium. This use of 111 Indium as a dosimetry surrogate for 90 Yttrium has been commonly utilized in the past.[7−10] Gamma camera images are visually assessed and imaged-based dosimetry based on the Committee of Medical Internal Radiation Dose (MIRD) principles are performed to ensure compliance with trial protocol defined organ limits.[11,12] In the dosimetry phase each patient in the trial received a radioactive infusion of approximately 185 MBq 111 Indium labeled CHX A” DTPA Besilesomab. Less than 1.5 mg of the conjugate was used in each patient and serial duplicate blood samples were taken over the course of one week. For each time point one whole blood sample was lysed to evenly disperse the radioactivity throughout the sample and counted in a gamma counter (Perkin Elmer Wizard II) to determine radioactive clearance, and the other centrifuged and reserved as serum for this assay. Following satisfactory dosimetry, 90 Yttrium-labeled antibody conjugate was given, with an activity based on lean body weight. Serial blood samples taken over the week following administration are also measured using a gamma counter against a standard of the same radionuclide, to track the clearance of radiolabeled conjugate from the blood and thus calculate the cumulative radioactivity in the blood. As this measurement involves the radioactive component, which may or may not still be totally bound to the antibody conjugate in vivo, it was considered that a second assay to quantify the antibody element of the sample would be important to better define the pharmacokinetics of the immunological component. As a well-established, and relatively cheap, technique with documented validation guidelines[13-19] an enzyme-linked immunosorbent assay (ELISA) study based on the murine origin of the antibody was chosen. A search failed to locate a commercially available, validated ELISA that quantified mouse antibody in human serum so it was decided to design and validate a method able to reliably quantify mouse antibody levels in human serum.
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This article details the optimized, simple method for a direct sandwich ELISA to quantify murine antibodies in human serum samples using readily available, low-cost reagents, validated against stated criteria. With resurgent interest in the use of monoclonal antibody conjugates, it is hoped that this method may prove useful to other researchers. EXPERIMENTAL
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Reagents/Equipment Capture Antibody: Goat anti-mouse IgG, affinity isolated, adsorbed with human serum proteins. Sigma-Aldrich, nominal 1.20 mg/mL. SigmaAldrich, Gillingham, UK. Secondary Antibody Conjugate: Peroxidase-conjugated sheep anti-mouse IgG, affinity isolated, adsorbed against human IgG, 1.0 mg/mL Sigma-Aldrich. Murine Antibody/Conjugate: (Analyte) for clinical use, standard and positive control. ITC CHX A” DTPA Besilesomab. ITC CHX A” bifunctional chelate supplied by Macrocyclics, Texas. Besilesomab supplied by Therapharm, Germany, conjugated under GMP conditions by Clinical Biotechnology Centre, Bristol. Coating Buffer : Carbonate-bicarbonate buffer capsules, Sigma-Aldrich. Peroxidase Substrate: (o-phenylenediamine dihydrochloride) OPD peroxidase substrate tablet set; Sigma-Aldrich Microtitre plates. Nunc Maxisorb Immuno F96 microplates Thermo Scientific (Hemel Hempstead, UK). Wash Buffer : PBS with 0.05% Tween 20 Sigma-Aldrich Blocking buffer ; PBS with 5% bovine serum albumin PBS Sigma-Aldrich and BSA SigmaAldrich. Stop Reagent: 2.5 M H2 SO4 diluted from Sigma -Aldrich 95%. Positive Control: 350 ng/mL antibody in human serum. Patient-Specific Negative Control. Baseline serum sample taken from each patient after consent but prior to any involvement in the clinical trial. Microplate Washer : Thermo Scientific Wellwash 3. Benchtop Incubator : Thermo Scientific, model B15ELISA. Plate Reader : Thermo Scientific Multiskan Ex, filter set at 492 nm. Sample Dilution Plate: BD Biosciences, San Jose, CA, USA. Assay Development and Optimization Overview Batches of six, 96 well flat bottomed microtitre plates were coated with goat anti-mouse antibody and potential non specific binding sites
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blocked using phosphate buffered saline (PBS) with 5% Bovine Serum Albumin (BSA). The plates were washed and standards, controls, and samples containing mouse antibody added and incubated for one hour at room temperature. The secondary antibody conjugate, sheep anti-mouse conjugated with horseradish peroxidase was added and incubated for 1 hr at room temperature. The plates were washed and the OPD substrate added and incubated for 30 min in the dark. The H2 SO4 stop reagent was added and the plates read at 492 nM. Results calculated from interpolation using a four-parameter logistic fit of the individual assay standard curve. The variables that required optimization were the coating antibody concentration, the secondary antibody conjugate dilution, and the blocking step,
Establishing Assay Range Each vial of antibody conjugate contained 0.5 mL at a concentration of 2.87 mg/mL, i.e., 1.435 mg per vial. Each vial was radiolabelled under GMP conditions by the radiopharmacy department at University Hospitals Southampton Trust and the final volume adjusted to 8 mL. Following successful QC testing(radiochemical purity greater than 90% bound) the product was signed off by a Qualified Person. As the specific radioactive concentration was known, and the residual activity in the infusion apparatus was subtracted, the amount of antibody conjugate administered to each patient was known. Assuming a nominal blood volume of 5000 mL this would give an initial maximum concentration in the blood after mixing of approximately 287 ng/mL. Nine serial blood samples were taken over the course of 5 days with the final concentration expected to be in single figures. An upper limit of 700 ng/mL was chosen to ensure that the highest sample would be well within the assay range allowing for variation in blood volumes, and that there was capacity for higher levels in case of future dose escalations. The lower limit was determined by assay performance. 700, 350, 175, 87.5, 43.75, 21.9,10.9, 5.47 ng/mL range is conveniently 8 doubling dilutions and fits a column on a standard 96 well plate.
Choice of Plates and Reagents The microtitre plates, blocking buffer, wash buffer, and peroxidase substrate listed in the reagents section were chosen as they were readily available products and were shown to be satisfactory in this ELISA. The percentage of bovine serum albumin in the blocking buffer was raised to 5% to combat non specific binding. Incubation time for the peroxidase substrate was determined empirically at 30 min in the dark, which was within the reaction plateau and gave a maximum OD for the highest standard of just less than 3.0 at 492 nm.
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As it took more than 10 min to load the samples and dilute the standards, an uncoated, non adsorbent “dilution plate” was used in order to avoid unequal incubation times with the capture antibody. All samples and diluted standards were added to this plate, which had been blocked under the same conditions as the assay plate as an additional precaution. When all controls standards and samples had been added, the contents of all wells were promptly transferred to the coated assay plate using a multichannel pipette. Optimization of Capture Antibody and Secondary Antibody Dilutions Plates were coated with 1, 2, 5, 20, 40 µg/mL of coating antibody and run with secondary antibody dilutions of 1: 1.25 K, 2.5 K, 5 K, 10 K, 20 K, and 40 K in a “checkerboard” fashion to determine the combination that gave the best signal to noise ratio for the chosen assay range. This was found to be 20 µg for the coating antibody and 1:2.5 K dilution for the secondary antibody conjugate, which gave a sigmoidal curve typical of a biological assay (Figure 1). Optimization of Blocking Buffer The concentration of the blocking buffer was increased in increments until non specific binding was minimized as apparent from blanks,reagent and negative controls. A concentration of 5% bovine serum albumin (BSA)
FIGURE 1 Standard curves for varying dilutions of secondary antibody with the coating antibody at 20 µg per mL. Besilesomab concentration range 0.177–2900 ng/mL.
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in phosphate buffered saline (PBS) was found to be effective, while maintaining good assay sensitivity.
Final Optimized Protocol for Validation Coating Plates
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Day 1: 0.135M saline was made by diluting 8.8 mL normal saline to 10 mL with distilled water. The goat anti mouse capture antibody was diluted with 1 mL of 0.135 M saline which resulted in a concentration of 1.21 mg/mL. The carbonate/bicarbonate coating buffer was made by dissolving the contents of 1 capsule in 100 mL distilled water. 529 µL antibody solution was diluted with 31.47 mL coating buffer which gave 32 mL at a concentration of 20 µg/mL. This was sufficient to coat 6 plates. The remaining ∼0.5 mL capture antibody was labeled and stored frozen. Six microplates were coated by adding 50 µL to each well (96 × 50 = 4.8 mL for 1 plate), incubated at 37◦ for 1 hr and then overnight at 4◦ . Day 2: 1000 mL wash buffer was made up (2 tabs in distilled water). The plates were washed five times using the automatic plate washer and wash buffer. Prior to the final wash the plates were allowed to soak for 5 min. Coated plates were stored at −20◦ C sealed with tape and wrapped in cling film until required.
Blocking 5% blocking buffer was made up by dissolving 1 tab PBS in 200 mL of distilled water, 20 mL was removed and reserved. 10 g of BSA was dissolved using a magnetic stirrer and the final volume adjusted to 200 mL with the reserved PBS then stored at 4◦ and used within 2 days. A coated microplate was allowed to reach room temp and then blocked by adding 200 µL blocking buffer to each well and incubating for 1 hr at 37◦ C. The dilution plate was blocked at the same time. After blocking, the plates were used on the same day. All wells were washed ×5 with wash buffer as before.
Standards Standards were prepared from ITC CHX A” DTPA Besilesomab batch AB012, 2879 µL/1000 µL, diluted to 700 ng/mL with normal human serum.
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Positive Control A batch of positive controls at 350 ng/mL was made from the antibody conjugate as above and 150 µL aliquots frozen. A single aliquot was allowed to reach room temperature before use in each assay. Assay Procedure All reagents were allowed to reach room temperature. 50 µL blocking buffer was added to dilution plate wells A1, B1, C1. 50 µL normal undiluted serum was added to dilution plate wells D1, E1, B2, and 3, to H2 and 3. 50 µL positive control was added to dilution plate wells F1 and G1. 100 µL of the 700 ng/mL standard was added to dilution plate wells A2, A3. Doubling dilutions were performed as per plan, using normal undiluted serum as diluent. 50 µL was removed and discarded from H2,3. 50 µL patient undiluted samples were added to dilution plate wells as per plan. The contents of all dilution plate wells were transferred to the assay plate, shaked to mix and incubated at room temperature for 1 hr. A typical microplate layout is shown in Figure 3. Secondary Antibody Conjugate 10 µL secondary antibody was thawed and diluted to 25 mL with blocking buffer (1:2500 dilution). 50 µL was added to all wells except the blank (A1), mixed and incubated at room temperature for 1 hr. All wells were washed with wash buffer as before. Substrate 2 OPD tablets and 2 urea hydrogen peroxide/buffer tablets were dissolved in 40 mL distilled water, 200 µL added to all wells, and mixed well. The microplate was incubated in the dark at room temp for 30 min. Stop Reagent 50 µL of stop reagent (2.5 M Sulphuric acid) was added to all wells forcibly using a multichannel pipette to halt the enzyme reaction and the microplate was read on the platereader with the 492 nm filter selected. Calculation of Results Results were calculated using the inbuilt “Ascent” software of the Thermo Multiskan platereader interpolating sample concentrations from a four parameter logistical fit of the standard curve.
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FIGURE 2 Finalized protocol summary flowchart.
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ELISA for Measurement of Murine Antibody Levels in Human Serum 1 A
2
3
4
5
6
7
8
9
10
11
12
STD
STD
Pat A
Pat A
Pat A
Pat A
Pat A
Pat A
Pat A
Pat A
Pat A
700
700
PSNC
1
2
3
4
5
6
7
8
STD
STD
Pat A
Pat A
Pat A
Pat A
Pat A
Pat A
Pat A
Pat A
Pat A
350
350
PSNC
1
2
3
4
5
6
7
8
STD
STD
Pat B
Pat B
Pat B
Pat B
Pat B
Pat B
Pat B
Pat B
Pat B
175
175
PSNC
2
2
3
4
5
6
7
8
STD
STD
Pat B
Pat B
Pat B
Pat B
Pat B
Pat B
Pat B
Pat B
Pat B
87.5
87.5
PSNC
2
2
3
4
5
6
7
8
STD
STD
Pat C
Pat C
Pat C
Pat C
Pat C
Pat C
Pat C
Pat C
Pat C
43.75
43.75
PSNC
1
2
3
4
5
6
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8
STD
STD
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Pat C
Pat C
Pat C
Pat C
Pat C
Pat C
Pat C
Pat C
21.9
21.9
PSNC
1
2
3
4
5
6
7
8
STD
STD
Pat D
Pat D
Pat D
Pat D
Pat D
Pat D
Pat D
Pat D
Pat D
10.9
10.9
PSNC
1
2
3
4
5
6
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STD
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Pat D
Pat D
Pat D
Pat D
Pat D
Pat D
Pat D
Pat D
5.45
5.45
PSNC
1
2
3
4
5
6
7
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BL B RC C RC D NC E NC F
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PC G PC H
FIGURE 3 Typical microplate layout, Capture antibody 20µg/ml. Secondary antibody dilution 1:2500. BL(Blank) is blocking buffer + substrate; RC(Reagent Control) is blocking buffer + conjugate + substrate; NC(Negative Control) is normal serum + conjugate + substrate; PSNC(Patient Specific Negative Control) is patient serum + conjugate + substrate; PC positive control 350 ng/mL (In normal serum) + conjugate + substrate PAT (patient sample); STD (standard).
RESULTS AND DISCUSSION Assay Validation Parameters that were assessed for assay validation were selectivity and specificity, accuracy, precision, robustness, linearity of standards, and stability. Selectivity and Specificity Selectivity is the ability of an analytical method to discriminate between the required analyte and interfering substances in the sample and specificity is the ability of the assay to differentiate the analyte from structurally similar substances in the matrix.[20] To explore this all assays were performed with blanks, negative controls, and reagent controls, the latter two in pooled donor supplied normal human serum, the same matrix as the samples.
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TABLE 1 Selectivity Data from 5 assays showing low absorbencies for blanks, reagent controls, and patient specific controls demonstrating no non specific binding problems. Mean correlation coefficient of the standard curves is greater than 0.99
Selectivity data OD at 492 nm
Plate Blank
Assay #1
0.094
Assay #2
0.071
Assay #3
0.080
Assay #4
0.080
Assay #5
0.080
Means sd %cv
0.081 0.007 8.312
Reagent Control RC
Negative Control NC
0.090 0.098 0.078 0.081 0.092 0.101 0.082 0.088 0.102 0.094 0.091 0.008 8.409
0.096 0.087 0.090 0.097 0.107 0.106 0.092 0.099 0.104 0.104 0.098 0.006 6.467
Patient Specific Negative Control(PSNC)
Mean OD at Correlation at LLOQ R2 std 5.47 ng/mL curve
nd
0.328
0.998
nd
0.276
0.998
nd
0.290
1.000
0.073
0.297
0.999
0.089 0.081 0.007 8.064
0.289 0.296 0.016 5.361
1.000 0.999
(nd = not determined)
Patients enrolled in the trial did not receive any concomitant mouse based antibodies that could interfere with the assay, however as a check “Patient Specific Negative Controls” (sera taken after patient consent to be enrolled in the trial) were included. These samples were taken prior to any participation in the clinical trial and were surplus to human anti mouse antibody screening (HAMA).This fulfilled the criterion of checking for possible interference in both healthy and disease state matrices.[21] Blank wells contained blocking buffer and substrate, negative controls contained normal serum + conjugate + substrate, Patient Specific Negative Controls contained patient serum + conjugate + substrate. Results show low absorbencies for all blanks and negative controls (Table 1). Accuracy and Precision Accuracy is the degree to which the result of a measurement conforms to a true result and precision is the degree to which repeated measurements of the same sample show the same result. Target values were set at: coefficient of variation (%cv) < 20%; Accuracy ± 20% at LLOQ. Five sets of validation samples were prepared by spiking normal human serum with CHX A” DTPA Besilesomab at 700, 350, 87.5, and 5.47 ng/mL. Accuracy and precision data were obtained from 5 separate assays with the
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TABLE 2 Inter-assay (between studies) accuracy and precision results from 5 validation samples showing good accuracy and percentage coefficient of variation of less than 20% Validation samples n = 5
Inter-assay accuracy spiked ng/mL 700.00 350.00 87.50 5.47
mean ng/ml
residual
sd
cv%
accuracy %
710.81 342.14 86.34 5.45
−10.81 7.86 1.16 0.02
76.04 32.50 3.65 0.39
10.70 9.50 4.23 7.21
98.46 97.75 98.68 99.62
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results tabulated in Tables 2 and 3. Both inter-assay accuracy and intra assay precision had a percentage coefficient of variation (%cv) of less than 20%.
Robustness Robustness is the ability of the assay to remain reliable in the face of variable parameters. Shankar et al.[20] recommend assessment of conditions that are likely to change in the laboratory environment and testing the parameters that are considered critical. Incubation temperatures of 37◦ C were controlled using a thermostatically controlled incubator and those of 4◦ by a monitored refrigerator both maintained under GLP conditions, and incubation times were easily controlled accurately, so the effects of variation in these parameters was not examined. As there was only a single operator who would perform these assays in this lab it was deemed unecessary to assess interoperator variation at this juncture. The effect of variation in laboratory ambient temperature and different reagent batches was explored with the results presented in table 4. All assays were run with a positive control of 350 ng/mL that had been made in a batch and frozen as aliquots. When this batch is depleted a new positive control batch will be made and validated in an assay containing both old and new batches. In practice, inclusion of a positive control, negative control, reagent control, and blank in every assay run ensures intra-assay quality and therefore any failure of robustness would be immediately apparent. Dilutional Linearity This is of greater importance when different matrices are used for standard and samples. Patient samples did not need dilution to combat non-specific binding which enabled high levels of sensitivity to be maintained. The high correlation coefficient of the standard curves demonstrate no discordance in dilutional variation.
590
sd
mean ng/mL
760.854 313.713 87.942 6.029
std ng/mL
700.00 350.00 87.50 5.47
96.252 12.041 2.844 0.335
1
Assay #
Intra- Assay precision
12.650 3.838 3.233 5.554
cv%
n=3
691.720 344.225 81.250 5.103
mean ng/mL
Assay #
32.980 33.625 1.026 0.023
sd
2
4.768 9.768 1.262 0.451
cv%
n=2
694.996 353.745 88.515 5.296
mean ng/mL
Assay #
20.619 14.705 4.045 0.514
sd
3
2.967 4.157 4.570 9.705
cv%
n=2
700.330 352.329 87.500 5.469
mean ng/mL
Assay #
81.810 37.513 0.570 0.062
sd
4
11.682 10.647 0.651 1.134
cv%
n=2
681.129 360.915 85.705 5.058
mean ng/mL
Assay #
37.990 22.385 0.745 0.192
sd
5
5.577 6.202 0.869 3.786
cv%
n=2
TABLE 3 Intra-assay (between replicates) precision. Results from 5 assays with standards in triplicate (assay 1) and in duplicate for remaining 4 assays. %cv less than 20% in all cases
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Positive
Control
350 ng/mL
Assay Robustness
Assay 1
Assay 2
Assay 3
Assay 4
Assay 5
Reagent batch # Lab Temp ◦ C Rep 1 ng/mL Rep 2 ng/mL Mean ng/mL Sd CV% Mean value ng/mL (n = 5) Mean sd (n = 5)
1 20.0 410.648 324.598 367.62 43.02 11.70 320.87
1 23.0 352.634 272.97 312.80 39.83 12.73
1 19.0 328.147 328.043 328.10 0.05 0.02
1 22.0 298.23 325.69 311.96 13.73 4.40
2 24.0 276.57 291.21 283.89 7.32 2.58
20.79
Mean CV% (n = 5)
6.29
Mean Accuracy % (n = 5)
91.11
Standard Curves The desired working range had been determined as 5.47–700 ng/mL and the standard curves were produced using spiked samples of human serum at 700 ng/mL. Doubling dilutions were performed using a dilution plate and pooled normal human serum as diluent to give 700, 350, 175, 87.5, 43.8, 21.9, 10.9, and 5.5 ng/mL. It was found to be unnecessary to change pipette tips when performing doubling dilutions for standards. Using eight points for the standard curve conforms with the recommendation to use at least six points when defining a nonlinear standard curve A four parameter logistical fit was used as provided in the microplate reader “Ascent” software (Figure 4A). Standard curves from five assays showed good reproducibility (Figure 4B). Standard Curves 700–5.47 ng/ml
Typical Calibration Curve 2.6
2.2 1.8 1.4
Optical Density at 492nm
2.6
od at 492
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TABLE 4 Robustness. Positive control of 350 ng/mL measured in 5 assays showed a mean value of 318.87 ng/mL with a %cv of < 20% and an accuracy of 91.11%
4 parameter logistic fit Linear correlation coefficient2 (r2) = 0.9987
1
2.2 1.8 1.4 1 0.6 0.2
0.6
10
0.2 10
100 ng/ml
a
4 parameter logistic fit Assay 1 Assay 2 Assay 3 Assay 4 Assay 5
ng/ml
b
FIGURE 4 Standard curves. A typical standard curve (a). Standard curves from 5 assays including different reagent batches showed good replication (b).
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Limits of Detection Lower limit of Detection The mean plate blank optical density of 5 assays was 0.081 ± 0.007sd. The mean optical density of the 5.47 ng standard was 0.296, which is greater than the blank value plus 3 standard deviations (0.102). This suggests that that the 5.47 value is statistically valid assuming normal distributions about the means[22,23]
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Upper Level of Detection The initial standard curves in Figure 1 show that the assay has capability for detection of higher concentrations of antibody, but the maximum level required by this study was 700 ng/mL. Stability Analyte (Besilesomab, Anti CD66 antibody Conjugate) Radiochemical purity testing of the 111 Indium labeled Anti CD66 antibody conjugate by High Performance Liquid Chromatography/Size Exclusion Chromatography (HPLC) has shown it to maintain radiochemical purity in excess of 90%, thus proving its structural integrity for at least 3 years when stored at −70◦ C (Figure 5A). Analyte Short-Term Stability Our routine quality control procedures prior to patient infusion and 1 day post infusion on dose remnants show that it is stable at room temperature and 4◦ C for at least 24 hr (Figure 5B). Assay Reagent Stability Analysis of 3 standards (700, 87.5, and 5.47 ng/mL) show minimal reduction in calculated concentration over a four month period using the same batches of coated plates and all reagents with the exception of the blocking buffer which was made fresh each run (Figure 5C). Patient Sample Stability To test patient sample degradation with time, three patient samples were re-assayed on three occasions at varying times after storage. There were only small variations in assay measurements with all % cvs being less than 20% when samples were re-analysed up to one year after initial storage at −20◦ C (Figure 5D).
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ELISA for Measurement of Murine Antibody Levels in Human Serum CHX A̕̕ ANTI CD66 CONJUGATE AB012111 INDIUM STABILITY HPLC
CHX A̕̕ ANTI CD66 CONJUGATE AB012 111 INDIUM STABILITY POST RADIOLABELLING HPLC
105
105 INDIUM STABILITY HPLC
95
Linear (PASS LINE)
90
85
pat 2 Room Temp
90
pat 2 4deg C
85
pat 3 Room Temp Pat 3 4 deg c
0 0
01
01
Pat 1 Room Temp
95
80
80 /2 /01
PASSLINE
100 % RCP 111 INDIUM
% RCP 111 INDIUM
100
0
01
/2 /07
20
05
1
1
01
01
/2 /02
/2 /08
24
11
2
2
01
01
/2 /03
/2 /09
27
3
01
/2 /04
01
15
3
01
/2 /11
5
10
4
01
/2 /05
15
20
25
HOURS AFTER PREP
pat 1 4 deg C
20
b
DATE
a
Concentration ng/ml 450.00
Effect of Reagent Age on Standard results
Patient Sample Stability in Storage
400.00 350.00
800.000
MEASURED NG/ML
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300.00 700.000
250.00
600.000
200.00 150.00
500.000
100.00 std 700 ng/ml
400.000
50.00 0.00 10.0
std 87.5 ng/ml
300.000
15.0
20.0
25.0
std 5.47 ng/ml
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100.000 0.000
4
3/
/0
19
4
1 20
4/
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4
4
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1 20
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/0
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7/
/0
19
1 20
30.0
35.0
40.0
45.0
50.0
55.0
Weeks after storage
200.000 Pat 1
Pat 2
Pat 3
mean
%cv
mean
%cv
mean
1
361.62
5.02
296.90
2.38
362.13
4
155.40
5.89
77.95
6.56
142.05
6.68
7
24.15
16.69
7.03
17.00
6.84
13.32
c
%cv 4.44
d Freeze/Thaw Stability at –20 and –70 °C in Human Serum. OD at 492 nm 2.500
2.000 700ng/ml-20C 700ng/ml-70c 87.5ng/ml-20c 87.5ng/ml-70c 5.47ng/ml-20c 5.47ng/ml-70c
1.500
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e FIGURE 5 Stability. (a) Conjugate stability. Results of radiolabelling Besilesomab conjugate with 111 Indium showing no obvious deterioration over three year period. (b) Short-term stability. Results of 5 patient dose remnant samples show short term stability of greater than 24 hr after labelling with 111 Indium, both at room temperature and 4◦ C. (c) Reagent stability. Calculated concentration shows minimal reduction over the course of 4 months using the same assay reagents. (d) Patient sample stability in storage. Data from 3 samples from 3 patients which had been re-assayed three times were reviewed. Sample storage ranged from 10 weeks to 52 weeks after initial storage. There is only a slight reduction trend in assay response with %CV values less than 20% in all cases. (e)Freeze/thaw cycle stability. Two groups of three validation standards(700, 87.5, and 5.47 ng/mL were assessed at −20◦ C and −70◦ C over 5 freeze/thaw cycles with no loss of optical density observed. Error bars are ± 2 sd.
Freeze Thaw Cycle Stability As there is a possibility for antibody damage resulting from freeze/thaw cycles[24] this was assessed by the following method. Two groups (A and B)
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each containing five sets of three standard concentrations (700, 87.5, and 5.47 ng/mL) were made. Group A was stored at −20◦ C and group B at −70◦ C. Both groups were allowed to freeze for at least 24 hr. On Day 1, one set of three standards from each group was defrosted at room temperature and then refrozen. On Day 2, two groups were thawed and refrozen. This was repeated adding a group on each day until Day 5 when all samples were analyzed together in a test assay (Figure 5E).
Reagent Storage Coated plates were stored at −20◦ C, blocking and wash buffers were made fresh each day. Capture and secondary antibody conjugates, positive controls patient samples, and 700 ng/mL standards were stored in aliquots at −20◦ C. Other reagents were stored at manufacturers’ recommendations. When fresh batches of assay reagents were introduced they were run in parallel with the expiring batch and when new plates were coated the negative and positive control results were examined for consistency. Comparison of Results with Previously Published Studies The results of this study showed that the assay described was fit for purpose with a mean precision of 9.39 (%cv) inter study variation, 5.17 (%cv) intra study variation, and a mean accuracy of 98.5%.These figures are well within the limits of precision 25%cv and accuracy ± 25% recommended by Findlay et al.[19]. Others[15,18] suggest lower limits of 20%cv for precision and ± 20% accuracy. Alciato et al.[22] reported figures of < 15% cv for both intra and inter study precision and an accuracy of 94 ± 5% in their assay for quantifying GAS6 protein. Acevedo et al.[25] developing an ELISA for measuring PSA concentrations achieved an accuracy range of 96–103% with an intra study precision of 2.8–6.3(%cv) and an inter study precision of 2.1–3.2 %cv. CONCLUSION The aim of this article, namely the design and validation of an ELISA to quantify Besilesomab levels within the range of 5.47–700 ng/mL in human serum, has been achieved. The assay is relatively cheap and simple to perform and 36 patient samples can be run in duplicate on each plate along with controls and blanks with the suggested layout. In excess of 30 assays have been run to date and factors optimized along the way to arrive at this protocol.
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This is a robust, reliable assay which could be easily adapted and translated to other mouse antibody human studies, for which a commercial validated assay is unavailable. FUNDING Funding for this work was provided by The Experimental Cancer Medicine Centre (ECMC). The Experimental Cancer Medicine Centre initiative is jointly funded by Cancer Research UK, the National Institute for Health Research in England and the Departments of Health for Scotland, Wales and Northern Ireland.
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REFERENCES 1. Milenic, D.E.; Brady, E.D.; Brechbiel, M.W. Antibody-Targeted Radiation Cancer Therapy. Nat. Rev. Drug Discov. 2004, 3(6), 488–499. 2. Zalutsky, M.R.; Pozzi, O.R. Radioimmunotherapy with Alpha-Particle Emitting Radionuclides. Quart. J. Nucl. Med. Mol. Imaging . 2004, 48(4), 289–296. 3. Abbas, N.; Heyerdahl, H.; Bruland, O.S.; Borrebæk, J.; Nesland, J.; Dahle, J. Experimental α-particle Radioimmunotherapy of Breast Cancer using 227 Th-labeled p-benzyl-DOTA-trastuzumab. EJNMMI Res. 2011, 1, 18. doi:10.1186/2191-219X-1-18. 4. Shankey, R.; Goldenberg, D. Use of Antibodies and Immunoconjugates for the Therapy of More Accessible Cancers. Adv. Drug Deliv. Rev. 2008, 60(12), 1407–1420. 5. Orchard, K.; Cooper, M.; Lewington, V.; Tristam, M.; Zivanovic, M.; Thom, J.; Quadri, S.; Richardson, D.; Causer, L.; Johnson, P. Targeted Radiotherapy in the Conditioning prior to Haematopoetic Stem Cell Transplantation: Results of a Phase I Trial using an Yttrium 90 Labelled anti CD66 Murine Monoclonal Antibody Demonstrating Consistently High BM Uptake. Biol. Blood Marrow Transplant. 2006, 12(2,Supplement 1),10–11. 6. Kletting, P.; Reske, S.; Glatting, G. Dependence of the Anti CD66 Antibody Biodistribution on the Dissociation Constant: A Simulation Study. Z. Med. Phys. 2011, 21, 301–304. 7. Chakravarty, R.; Chakraborty S.; Dash A. Comparative Evaluation of Bifunctional Chelators for Preparation of 90Y based Radiopharmaceuticals. J. Nucl. Med. 2014, 55 (Supplement 1), 218. 8. Buchmann, I.; Meyer, R.G.; Mier, W.; Haberkorn, U. Myeloablative Radioimmunotherapy in Conditioning prior to Haematological Stem Cell Transplantation: Closing the Gap between Benefit and Toxicity? Eur. J. Nucl. Med. Mol. Imag . 2009, 36(3), 484–498. doi:10.1007/s00259-008-0996-6. Epub January 8, 2009. 9. Guinn, B-A.; Bosslet, K.; Lee, C.; Richardson, D.; Orchard, K. Expression of CD66 in Non-Hodgkin Lymphomas and Multiple Myeloma. Eur. J. Haematol. 2011, 87 (6), 554–555. 10. Carrasquillo, J.A.; White, J.D.; Paik, C.H.; Raubitschek, A.; Le, N.; Rotman, M.; Brechbiel, M.W.; Gansow, O.A.; Top, L.E.; Perentesis, P.; Reynolds, J.C.; Nelson, D.L.; Waldmann, T.A. Similarities and Differences in 111In- and 90Y-labeled 1B4M-DTPA AntiTac Monoclonal Antibody Distribution. J. Nucl. Med. 1999, 40(2), 268–276. 11. Fisher, D.R.; Shen, S.; Meredith, R.F. MIRD Dose Estimate Report No. 20: Radiation AbsorbedDose Estimates for 111In- and 90Y-ibritumomab Tiuxetan. J. Nucl. Med. 2009, 50(4), 644–652. doi:10.2967/jnumed.108.057331. Epub March 16, 2009. 12. Dewaraja, Y.K.; Frey, E.C.; Sgouros, G.; Brill, A.B.; Roberson, P.; Zanzonico, P.B.; Ljungberg, M. MIRD Pamphlet No. 23: Quantitative SPECT for Patient-specific 3-dimensional Dosimetry in Internal Radionuclide Therapy. J. Nucl. Med. 2012, 53(8), 1310–1325. doi:10.2967/jnumed.111.100123. Epub June 28, 2012. 13. Chau, C.H.; Rixe, O.; McLeod, H.; Figg, W.D. Validation of Analytic Methods for Biomarkers Used in Drug Development. Clin. Cancer Res. 2008, 14(19), 5967–5976.
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14. Chowdhury, F.; Tutt, A.L.; Chan, C.; Glennie, M.; Johnson, P.W. Development, Validation and Application of ELISAs for Pharmacokinetic and HACA Assessment of a Chimeric anti-CD40 Monoclonal Antibody in Human Serum. J. Immunol. Meth. 2010, 363(1), 1–8. doi:10.1016/j.jim.2010.09.023. Epub September 24, 2010. 15. FDA. Food and Drug Administration U.S. Department of Health and Human Services Guidance For Industry Bioanalytical Method Validation, May 2001; http://www.fda.gov/downloads/drugs/ guidancecomplianceregulatoryinformation/guidances/ucm070107.pdf. Accessed November 14, 2014. 16. Mander, A.; Chowdhury, F.; Low, L.; Ottensmeier, C.H. Fit for purpose? A Case Study: Validation of Immunological Endpoint Assays for the Detection of Cellular and Humoral Responses to Anti-tumour DNA Fusion Vaccines. Cancer Immunol. Immunother . 2009, 58(5), 789–800. doi:10.1007/s00262-008-0633-z. Epub December 10, 2008. 17. Lee, J.W.; Devanarayan, V.; Barrett, Y.C.; Weiner, R.; Allinson, J.; Fountain, S.; Keller, S.; Weinryb, I.; Green, M.; Duan, L.; Rogers, J.A.; Millham, R.; O’Brien, P.J.; Sailstad, J.; Khan, M.; Ray, C.; Wagner, J.A. Fit-for-Purpose Method Development and Validation for Successful Biomarker Measurement. Pharm. Res. 2006, 23(2), 312–328. Epub January 12, 2006. 18. Cummings, J.; Ward, T,H.; Greystoke, A.; Ranson, M.; Dive, C. Biomarker Method Validation in Anticancer Drug Development. Br. J. Pharmacol. 2008, 153(4), 646–656. Published online September 17, 2007. doi:10.1038/sj.bjp.0707441PMCID:PMC2259203 19. Findlay, J.W.; Smith, W.C.; Lee, J.W.; Nordblom. G.D.; Das, I.; DeSilva, B.S.; Khan, M.N.; Bowsher, R.R. Validation of Immunoassays for Bioanalysis: A Pharmaceutical Industry Perspective. J. Pharm. Biomed. Anal. 2000, 21(6), 1249–1273. 20. Shankar, G.; Devanarayan, V.; Amaravadi, L.; Barrett, Y.C.; Bowsher, R.; Finco-Kent, D.; Fiscella, M.; Gorovits, B.; Kirschner, S.; Moxness, M.; Parish, T.; Quarmby, V.; Smith, H.; Smith, W.; Zuckerman, L.A.; Koren, E. Recommendations for the Validation of Immunoassays Used for Detection of Host Antibodies against Biotechnology Products. J. Pharmaceut. Biomed. Anal. 2008, 48(5), 1267–1281. 21. Mire-Sluis, A.R.; Barrett, Y.C.; Devanarayan, V.; Koren, E.; Liu, H.; Maia, M.; Parish, T.; Scott, G.; Shankar, G.; Shores, E.; Swanson, S.J.; Taniguchi, G.; Wierda, D.; Zuckerman, L.A. Recommendations for the Design and Optimization of Immunoassays Used in the Detection of Host Antibodies against Biotechnology Products. J. Immunol. Meth. 2004, 289(1–2), 1–16. 22. Alciato, F.; Sainaghi, P.P.; Castello, L.; Bergamasco, L.; Carnieletto, S.; Avanzi. G.C. Development and Validation of an ELISA Method for Detection of Growth Arrest Specific 6 (GAS6) Protein in Human Plasma. J. Immunoass. Immunochem. 2008, 29(2), 167–180. 23. Butler, J.E.; Enzyme Linked Immunosorbent Assay. J. Immunoass. 2008, 21(2–3),165–209. 24. Pinsky, N.A.; Huddleston, J.M.; Jacobson, R.M.; Wollan, P.C.; Poland, G.A. Effect of Multiple FreezeThaw Cycles on Detection of Measles, Mumps, and Rubella Virus Antibodies. Clin. Diagn. Lab. Immunol. 2003, 10(1), 19–21. 25. Acevedo, B.; Yasser, P.; Ruiz, M.; Rojas, G.; Benitez, J.; Ayala, M.; Gavilondo, J. Development and Validation of a Quantitative ELISA for the Measurement of PSA Concentration. Clinica Chimica Acta 2002, 317 , 55–63.
Journal of Immunoassay and Immunochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ljii20
Development of an ELISA for the Pharmacokinetic Evaluation of a Murine Anti CD66 Monoclonal Antibody in Human Serum a
a
b
J. Langford , F. Chowdhury & K. Orchard a
Cancer Sciences Unit, Faculty of Medicine, Southampton University, United Kingdom b
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Department of Haematology, Southampton University Hospitals Trust, Southampton, United Kingdom Accepted author version posted online: 25 Feb 2015.
To cite this article: J. Langford, F. Chowdhury & K. Orchard (2015) Development of an ELISA for the Pharmacokinetic Evaluation of a Murine Anti CD66 Monoclonal Antibody in Human Serum, Journal of Immunoassay and Immunochemistry, 36:6, 579-596, DOI: 10.1080/15321819.2015.1017107 To link to this article: http://dx.doi.org/10.1080/15321819.2015.1017107
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Journal of Immunoassay and Immunochemistry, 36:579–596, 2015 Copyright © Taylor & Francis Group, LLC ISSN: 1532-1819 print/1532-4230 online DOI: 10.1080/15321819.2015.1017107
DEVELOPMENT OF AN ELISA FOR THE PHARMACOKINETIC EVALUATION OF A MURINE ANTI CD66 MONOCLONAL ANTIBODY IN HUMAN SERUM
J. Langford,1 F. Chowdhury,1 and K. Orchard2 1
Cancer Sciences Unit, Faculty of Medicine, Southampton University, United Kingdom Department of Haematology, Southampton University Hospitals Trust, Southampton, United Kingdom
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2
An enzyme-linked immunosorbent assay (ELISA) was needed to assist in the pharmacokinetic evaluation of the murine antibody conjugate CHX A” DTPA Besilesomab in serum samples in a clinical trial . A search failed to locate a validated assay that quantified murine antibodies in human serum so the purpose of this article was to develop a robust assay, validated against current guidelines. A detailed method for an ELISA to measure a murine antibody in human serum is described. The assay was validated as fit for purpose against the target values of coefficient of variation < 20% and accuracy ± 20%. Keywords ELISA, pharmacokinetic
murine
antibody,
human
serum,
validation,
immunoassay,
INTRODUCTION Targeted radioimmunotherapy using monoclonal antibodies as vectors to transport beta-emitting cytotoxic radionuclides to tumor-associated antigen targets is now a recognized therapy; for example, 90 Yttrium ibritumomab tiuxetan “Zevalin”® (Schering Health Care) targets the CD20 antigen expressed on plasma B cells and may be used in the treatment of non-Hodgkin lymphoma. Further research into optimal vectors and radionuclides, including use of alpha particle-emitting radionuclides, is ongoing.[1-4] At the University of Southampton we are currently conducting a phase 1 clinical trial which involves administration of the murine monoclonal antibody Besilesomab (Therapharm, Germany), conjugated to a bifunctional Address correspondence to J. Langford, Cancer Sciences Unit, Faculty of Medicine, Southampton University, SO16 6YD, UK. E-mail: [email protected]
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chelator ITC DTPA CHX A” (Macrocyclics,Texas), which facilitates radiolabeling with the cytotoxic, beta-emitting, radionuclide 90 Yttrium (90 Y). This trial is assessing the use of 90 Y anti-CD66 monoclonal antibody as an adjunct therapy to patients about to undergo standard conditioning and allogeneic peripheral stem cell transplantation (PSCT) for hematological malignancies such as multiple myeloma. The CD 66 antigens are expressed from the promyelocyte stage to the mature granulocyte which makes them suitable targets for bone marrow irradiation.[5,6] Prior to the treatment stage of the trial, dosimetry and bio distribution are assessed by injection of the same antibody conjugate but radiolabeled with a different radionuclide, the gamma-emitting 111 Indium. This use of 111 Indium as a dosimetry surrogate for 90 Yttrium has been commonly utilized in the past.[7−10] Gamma camera images are visually assessed and imaged-based dosimetry based on the Committee of Medical Internal Radiation Dose (MIRD) principles are performed to ensure compliance with trial protocol defined organ limits.[11,12] In the dosimetry phase each patient in the trial received a radioactive infusion of approximately 185 MBq 111 Indium labeled CHX A” DTPA Besilesomab. Less than 1.5 mg of the conjugate was used in each patient and serial duplicate blood samples were taken over the course of one week. For each time point one whole blood sample was lysed to evenly disperse the radioactivity throughout the sample and counted in a gamma counter (Perkin Elmer Wizard II) to determine radioactive clearance, and the other centrifuged and reserved as serum for this assay. Following satisfactory dosimetry, 90 Yttrium-labeled antibody conjugate was given, with an activity based on lean body weight. Serial blood samples taken over the week following administration are also measured using a gamma counter against a standard of the same radionuclide, to track the clearance of radiolabeled conjugate from the blood and thus calculate the cumulative radioactivity in the blood. As this measurement involves the radioactive component, which may or may not still be totally bound to the antibody conjugate in vivo, it was considered that a second assay to quantify the antibody element of the sample would be important to better define the pharmacokinetics of the immunological component. As a well-established, and relatively cheap, technique with documented validation guidelines[13-19] an enzyme-linked immunosorbent assay (ELISA) study based on the murine origin of the antibody was chosen. A search failed to locate a commercially available, validated ELISA that quantified mouse antibody in human serum so it was decided to design and validate a method able to reliably quantify mouse antibody levels in human serum.
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This article details the optimized, simple method for a direct sandwich ELISA to quantify murine antibodies in human serum samples using readily available, low-cost reagents, validated against stated criteria. With resurgent interest in the use of monoclonal antibody conjugates, it is hoped that this method may prove useful to other researchers. EXPERIMENTAL
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Reagents/Equipment Capture Antibody: Goat anti-mouse IgG, affinity isolated, adsorbed with human serum proteins. Sigma-Aldrich, nominal 1.20 mg/mL. SigmaAldrich, Gillingham, UK. Secondary Antibody Conjugate: Peroxidase-conjugated sheep anti-mouse IgG, affinity isolated, adsorbed against human IgG, 1.0 mg/mL Sigma-Aldrich. Murine Antibody/Conjugate: (Analyte) for clinical use, standard and positive control. ITC CHX A” DTPA Besilesomab. ITC CHX A” bifunctional chelate supplied by Macrocyclics, Texas. Besilesomab supplied by Therapharm, Germany, conjugated under GMP conditions by Clinical Biotechnology Centre, Bristol. Coating Buffer : Carbonate-bicarbonate buffer capsules, Sigma-Aldrich. Peroxidase Substrate: (o-phenylenediamine dihydrochloride) OPD peroxidase substrate tablet set; Sigma-Aldrich Microtitre plates. Nunc Maxisorb Immuno F96 microplates Thermo Scientific (Hemel Hempstead, UK). Wash Buffer : PBS with 0.05% Tween 20 Sigma-Aldrich Blocking buffer ; PBS with 5% bovine serum albumin PBS Sigma-Aldrich and BSA SigmaAldrich. Stop Reagent: 2.5 M H2 SO4 diluted from Sigma -Aldrich 95%. Positive Control: 350 ng/mL antibody in human serum. Patient-Specific Negative Control. Baseline serum sample taken from each patient after consent but prior to any involvement in the clinical trial. Microplate Washer : Thermo Scientific Wellwash 3. Benchtop Incubator : Thermo Scientific, model B15ELISA. Plate Reader : Thermo Scientific Multiskan Ex, filter set at 492 nm. Sample Dilution Plate: BD Biosciences, San Jose, CA, USA. Assay Development and Optimization Overview Batches of six, 96 well flat bottomed microtitre plates were coated with goat anti-mouse antibody and potential non specific binding sites
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blocked using phosphate buffered saline (PBS) with 5% Bovine Serum Albumin (BSA). The plates were washed and standards, controls, and samples containing mouse antibody added and incubated for one hour at room temperature. The secondary antibody conjugate, sheep anti-mouse conjugated with horseradish peroxidase was added and incubated for 1 hr at room temperature. The plates were washed and the OPD substrate added and incubated for 30 min in the dark. The H2 SO4 stop reagent was added and the plates read at 492 nM. Results calculated from interpolation using a four-parameter logistic fit of the individual assay standard curve. The variables that required optimization were the coating antibody concentration, the secondary antibody conjugate dilution, and the blocking step,
Establishing Assay Range Each vial of antibody conjugate contained 0.5 mL at a concentration of 2.87 mg/mL, i.e., 1.435 mg per vial. Each vial was radiolabelled under GMP conditions by the radiopharmacy department at University Hospitals Southampton Trust and the final volume adjusted to 8 mL. Following successful QC testing(radiochemical purity greater than 90% bound) the product was signed off by a Qualified Person. As the specific radioactive concentration was known, and the residual activity in the infusion apparatus was subtracted, the amount of antibody conjugate administered to each patient was known. Assuming a nominal blood volume of 5000 mL this would give an initial maximum concentration in the blood after mixing of approximately 287 ng/mL. Nine serial blood samples were taken over the course of 5 days with the final concentration expected to be in single figures. An upper limit of 700 ng/mL was chosen to ensure that the highest sample would be well within the assay range allowing for variation in blood volumes, and that there was capacity for higher levels in case of future dose escalations. The lower limit was determined by assay performance. 700, 350, 175, 87.5, 43.75, 21.9,10.9, 5.47 ng/mL range is conveniently 8 doubling dilutions and fits a column on a standard 96 well plate.
Choice of Plates and Reagents The microtitre plates, blocking buffer, wash buffer, and peroxidase substrate listed in the reagents section were chosen as they were readily available products and were shown to be satisfactory in this ELISA. The percentage of bovine serum albumin in the blocking buffer was raised to 5% to combat non specific binding. Incubation time for the peroxidase substrate was determined empirically at 30 min in the dark, which was within the reaction plateau and gave a maximum OD for the highest standard of just less than 3.0 at 492 nm.
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As it took more than 10 min to load the samples and dilute the standards, an uncoated, non adsorbent “dilution plate” was used in order to avoid unequal incubation times with the capture antibody. All samples and diluted standards were added to this plate, which had been blocked under the same conditions as the assay plate as an additional precaution. When all controls standards and samples had been added, the contents of all wells were promptly transferred to the coated assay plate using a multichannel pipette. Optimization of Capture Antibody and Secondary Antibody Dilutions Plates were coated with 1, 2, 5, 20, 40 µg/mL of coating antibody and run with secondary antibody dilutions of 1: 1.25 K, 2.5 K, 5 K, 10 K, 20 K, and 40 K in a “checkerboard” fashion to determine the combination that gave the best signal to noise ratio for the chosen assay range. This was found to be 20 µg for the coating antibody and 1:2.5 K dilution for the secondary antibody conjugate, which gave a sigmoidal curve typical of a biological assay (Figure 1). Optimization of Blocking Buffer The concentration of the blocking buffer was increased in increments until non specific binding was minimized as apparent from blanks,reagent and negative controls. A concentration of 5% bovine serum albumin (BSA)
FIGURE 1 Standard curves for varying dilutions of secondary antibody with the coating antibody at 20 µg per mL. Besilesomab concentration range 0.177–2900 ng/mL.
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in phosphate buffered saline (PBS) was found to be effective, while maintaining good assay sensitivity.
Final Optimized Protocol for Validation Coating Plates
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Day 1: 0.135M saline was made by diluting 8.8 mL normal saline to 10 mL with distilled water. The goat anti mouse capture antibody was diluted with 1 mL of 0.135 M saline which resulted in a concentration of 1.21 mg/mL. The carbonate/bicarbonate coating buffer was made by dissolving the contents of 1 capsule in 100 mL distilled water. 529 µL antibody solution was diluted with 31.47 mL coating buffer which gave 32 mL at a concentration of 20 µg/mL. This was sufficient to coat 6 plates. The remaining ∼0.5 mL capture antibody was labeled and stored frozen. Six microplates were coated by adding 50 µL to each well (96 × 50 = 4.8 mL for 1 plate), incubated at 37◦ for 1 hr and then overnight at 4◦ . Day 2: 1000 mL wash buffer was made up (2 tabs in distilled water). The plates were washed five times using the automatic plate washer and wash buffer. Prior to the final wash the plates were allowed to soak for 5 min. Coated plates were stored at −20◦ C sealed with tape and wrapped in cling film until required.
Blocking 5% blocking buffer was made up by dissolving 1 tab PBS in 200 mL of distilled water, 20 mL was removed and reserved. 10 g of BSA was dissolved using a magnetic stirrer and the final volume adjusted to 200 mL with the reserved PBS then stored at 4◦ and used within 2 days. A coated microplate was allowed to reach room temp and then blocked by adding 200 µL blocking buffer to each well and incubating for 1 hr at 37◦ C. The dilution plate was blocked at the same time. After blocking, the plates were used on the same day. All wells were washed ×5 with wash buffer as before.
Standards Standards were prepared from ITC CHX A” DTPA Besilesomab batch AB012, 2879 µL/1000 µL, diluted to 700 ng/mL with normal human serum.
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Positive Control A batch of positive controls at 350 ng/mL was made from the antibody conjugate as above and 150 µL aliquots frozen. A single aliquot was allowed to reach room temperature before use in each assay. Assay Procedure All reagents were allowed to reach room temperature. 50 µL blocking buffer was added to dilution plate wells A1, B1, C1. 50 µL normal undiluted serum was added to dilution plate wells D1, E1, B2, and 3, to H2 and 3. 50 µL positive control was added to dilution plate wells F1 and G1. 100 µL of the 700 ng/mL standard was added to dilution plate wells A2, A3. Doubling dilutions were performed as per plan, using normal undiluted serum as diluent. 50 µL was removed and discarded from H2,3. 50 µL patient undiluted samples were added to dilution plate wells as per plan. The contents of all dilution plate wells were transferred to the assay plate, shaked to mix and incubated at room temperature for 1 hr. A typical microplate layout is shown in Figure 3. Secondary Antibody Conjugate 10 µL secondary antibody was thawed and diluted to 25 mL with blocking buffer (1:2500 dilution). 50 µL was added to all wells except the blank (A1), mixed and incubated at room temperature for 1 hr. All wells were washed with wash buffer as before. Substrate 2 OPD tablets and 2 urea hydrogen peroxide/buffer tablets were dissolved in 40 mL distilled water, 200 µL added to all wells, and mixed well. The microplate was incubated in the dark at room temp for 30 min. Stop Reagent 50 µL of stop reagent (2.5 M Sulphuric acid) was added to all wells forcibly using a multichannel pipette to halt the enzyme reaction and the microplate was read on the platereader with the 492 nm filter selected. Calculation of Results Results were calculated using the inbuilt “Ascent” software of the Thermo Multiskan platereader interpolating sample concentrations from a four parameter logistical fit of the standard curve.
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FIGURE 2 Finalized protocol summary flowchart.
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ELISA for Measurement of Murine Antibody Levels in Human Serum 1 A
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Pat C
Pat C
Pat C
Pat C
Pat C
Pat C
Pat C
43.75
43.75
PSNC
1
2
3
4
5
6
7
8
STD
STD
Pat C
Pat C
Pat C
Pat C
Pat C
Pat C
Pat C
Pat C
Pat C
21.9
21.9
PSNC
1
2
3
4
5
6
7
8
STD
STD
Pat D
Pat D
Pat D
Pat D
Pat D
Pat D
Pat D
Pat D
Pat D
10.9
10.9
PSNC
1
2
3
4
5
6
7
8
STD
STD
Pat D
Pat D
Pat D
Pat D
Pat D
Pat D
Pat D
Pat D
Pat D
5.45
5.45
PSNC
1
2
3
4
5
6
7
8
BL B RC C RC D NC E NC F
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PC G PC H
FIGURE 3 Typical microplate layout, Capture antibody 20µg/ml. Secondary antibody dilution 1:2500. BL(Blank) is blocking buffer + substrate; RC(Reagent Control) is blocking buffer + conjugate + substrate; NC(Negative Control) is normal serum + conjugate + substrate; PSNC(Patient Specific Negative Control) is patient serum + conjugate + substrate; PC positive control 350 ng/mL (In normal serum) + conjugate + substrate PAT (patient sample); STD (standard).
RESULTS AND DISCUSSION Assay Validation Parameters that were assessed for assay validation were selectivity and specificity, accuracy, precision, robustness, linearity of standards, and stability. Selectivity and Specificity Selectivity is the ability of an analytical method to discriminate between the required analyte and interfering substances in the sample and specificity is the ability of the assay to differentiate the analyte from structurally similar substances in the matrix.[20] To explore this all assays were performed with blanks, negative controls, and reagent controls, the latter two in pooled donor supplied normal human serum, the same matrix as the samples.
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TABLE 1 Selectivity Data from 5 assays showing low absorbencies for blanks, reagent controls, and patient specific controls demonstrating no non specific binding problems. Mean correlation coefficient of the standard curves is greater than 0.99
Selectivity data OD at 492 nm
Plate Blank
Assay #1
0.094
Assay #2
0.071
Assay #3
0.080
Assay #4
0.080
Assay #5
0.080
Means sd %cv
0.081 0.007 8.312
Reagent Control RC
Negative Control NC
0.090 0.098 0.078 0.081 0.092 0.101 0.082 0.088 0.102 0.094 0.091 0.008 8.409
0.096 0.087 0.090 0.097 0.107 0.106 0.092 0.099 0.104 0.104 0.098 0.006 6.467
Patient Specific Negative Control(PSNC)
Mean OD at Correlation at LLOQ R2 std 5.47 ng/mL curve
nd
0.328
0.998
nd
0.276
0.998
nd
0.290
1.000
0.073
0.297
0.999
0.089 0.081 0.007 8.064
0.289 0.296 0.016 5.361
1.000 0.999
(nd = not determined)
Patients enrolled in the trial did not receive any concomitant mouse based antibodies that could interfere with the assay, however as a check “Patient Specific Negative Controls” (sera taken after patient consent to be enrolled in the trial) were included. These samples were taken prior to any participation in the clinical trial and were surplus to human anti mouse antibody screening (HAMA).This fulfilled the criterion of checking for possible interference in both healthy and disease state matrices.[21] Blank wells contained blocking buffer and substrate, negative controls contained normal serum + conjugate + substrate, Patient Specific Negative Controls contained patient serum + conjugate + substrate. Results show low absorbencies for all blanks and negative controls (Table 1). Accuracy and Precision Accuracy is the degree to which the result of a measurement conforms to a true result and precision is the degree to which repeated measurements of the same sample show the same result. Target values were set at: coefficient of variation (%cv) < 20%; Accuracy ± 20% at LLOQ. Five sets of validation samples were prepared by spiking normal human serum with CHX A” DTPA Besilesomab at 700, 350, 87.5, and 5.47 ng/mL. Accuracy and precision data were obtained from 5 separate assays with the
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TABLE 2 Inter-assay (between studies) accuracy and precision results from 5 validation samples showing good accuracy and percentage coefficient of variation of less than 20% Validation samples n = 5
Inter-assay accuracy spiked ng/mL 700.00 350.00 87.50 5.47
mean ng/ml
residual
sd
cv%
accuracy %
710.81 342.14 86.34 5.45
−10.81 7.86 1.16 0.02
76.04 32.50 3.65 0.39
10.70 9.50 4.23 7.21
98.46 97.75 98.68 99.62
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results tabulated in Tables 2 and 3. Both inter-assay accuracy and intra assay precision had a percentage coefficient of variation (%cv) of less than 20%.
Robustness Robustness is the ability of the assay to remain reliable in the face of variable parameters. Shankar et al.[20] recommend assessment of conditions that are likely to change in the laboratory environment and testing the parameters that are considered critical. Incubation temperatures of 37◦ C were controlled using a thermostatically controlled incubator and those of 4◦ by a monitored refrigerator both maintained under GLP conditions, and incubation times were easily controlled accurately, so the effects of variation in these parameters was not examined. As there was only a single operator who would perform these assays in this lab it was deemed unecessary to assess interoperator variation at this juncture. The effect of variation in laboratory ambient temperature and different reagent batches was explored with the results presented in table 4. All assays were run with a positive control of 350 ng/mL that had been made in a batch and frozen as aliquots. When this batch is depleted a new positive control batch will be made and validated in an assay containing both old and new batches. In practice, inclusion of a positive control, negative control, reagent control, and blank in every assay run ensures intra-assay quality and therefore any failure of robustness would be immediately apparent. Dilutional Linearity This is of greater importance when different matrices are used for standard and samples. Patient samples did not need dilution to combat non-specific binding which enabled high levels of sensitivity to be maintained. The high correlation coefficient of the standard curves demonstrate no discordance in dilutional variation.
590
sd
mean ng/mL
760.854 313.713 87.942 6.029
std ng/mL
700.00 350.00 87.50 5.47
96.252 12.041 2.844 0.335
1
Assay #
Intra- Assay precision
12.650 3.838 3.233 5.554
cv%
n=3
691.720 344.225 81.250 5.103
mean ng/mL
Assay #
32.980 33.625 1.026 0.023
sd
2
4.768 9.768 1.262 0.451
cv%
n=2
694.996 353.745 88.515 5.296
mean ng/mL
Assay #
20.619 14.705 4.045 0.514
sd
3
2.967 4.157 4.570 9.705
cv%
n=2
700.330 352.329 87.500 5.469
mean ng/mL
Assay #
81.810 37.513 0.570 0.062
sd
4
11.682 10.647 0.651 1.134
cv%
n=2
681.129 360.915 85.705 5.058
mean ng/mL
Assay #
37.990 22.385 0.745 0.192
sd
5
5.577 6.202 0.869 3.786
cv%
n=2
TABLE 3 Intra-assay (between replicates) precision. Results from 5 assays with standards in triplicate (assay 1) and in duplicate for remaining 4 assays. %cv less than 20% in all cases
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Positive
Control
350 ng/mL
Assay Robustness
Assay 1
Assay 2
Assay 3
Assay 4
Assay 5
Reagent batch # Lab Temp ◦ C Rep 1 ng/mL Rep 2 ng/mL Mean ng/mL Sd CV% Mean value ng/mL (n = 5) Mean sd (n = 5)
1 20.0 410.648 324.598 367.62 43.02 11.70 320.87
1 23.0 352.634 272.97 312.80 39.83 12.73
1 19.0 328.147 328.043 328.10 0.05 0.02
1 22.0 298.23 325.69 311.96 13.73 4.40
2 24.0 276.57 291.21 283.89 7.32 2.58
20.79
Mean CV% (n = 5)
6.29
Mean Accuracy % (n = 5)
91.11
Standard Curves The desired working range had been determined as 5.47–700 ng/mL and the standard curves were produced using spiked samples of human serum at 700 ng/mL. Doubling dilutions were performed using a dilution plate and pooled normal human serum as diluent to give 700, 350, 175, 87.5, 43.8, 21.9, 10.9, and 5.5 ng/mL. It was found to be unnecessary to change pipette tips when performing doubling dilutions for standards. Using eight points for the standard curve conforms with the recommendation to use at least six points when defining a nonlinear standard curve A four parameter logistical fit was used as provided in the microplate reader “Ascent” software (Figure 4A). Standard curves from five assays showed good reproducibility (Figure 4B). Standard Curves 700–5.47 ng/ml
Typical Calibration Curve 2.6
2.2 1.8 1.4
Optical Density at 492nm
2.6
od at 492
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TABLE 4 Robustness. Positive control of 350 ng/mL measured in 5 assays showed a mean value of 318.87 ng/mL with a %cv of < 20% and an accuracy of 91.11%
4 parameter logistic fit Linear correlation coefficient2 (r2) = 0.9987
1
2.2 1.8 1.4 1 0.6 0.2
0.6
10
0.2 10
100 ng/ml
a
4 parameter logistic fit Assay 1 Assay 2 Assay 3 Assay 4 Assay 5
ng/ml
b
FIGURE 4 Standard curves. A typical standard curve (a). Standard curves from 5 assays including different reagent batches showed good replication (b).
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Limits of Detection Lower limit of Detection The mean plate blank optical density of 5 assays was 0.081 ± 0.007sd. The mean optical density of the 5.47 ng standard was 0.296, which is greater than the blank value plus 3 standard deviations (0.102). This suggests that that the 5.47 value is statistically valid assuming normal distributions about the means[22,23]
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Upper Level of Detection The initial standard curves in Figure 1 show that the assay has capability for detection of higher concentrations of antibody, but the maximum level required by this study was 700 ng/mL. Stability Analyte (Besilesomab, Anti CD66 antibody Conjugate) Radiochemical purity testing of the 111 Indium labeled Anti CD66 antibody conjugate by High Performance Liquid Chromatography/Size Exclusion Chromatography (HPLC) has shown it to maintain radiochemical purity in excess of 90%, thus proving its structural integrity for at least 3 years when stored at −70◦ C (Figure 5A). Analyte Short-Term Stability Our routine quality control procedures prior to patient infusion and 1 day post infusion on dose remnants show that it is stable at room temperature and 4◦ C for at least 24 hr (Figure 5B). Assay Reagent Stability Analysis of 3 standards (700, 87.5, and 5.47 ng/mL) show minimal reduction in calculated concentration over a four month period using the same batches of coated plates and all reagents with the exception of the blocking buffer which was made fresh each run (Figure 5C). Patient Sample Stability To test patient sample degradation with time, three patient samples were re-assayed on three occasions at varying times after storage. There were only small variations in assay measurements with all % cvs being less than 20% when samples were re-analysed up to one year after initial storage at −20◦ C (Figure 5D).
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ELISA for Measurement of Murine Antibody Levels in Human Serum CHX A̕̕ ANTI CD66 CONJUGATE AB012111 INDIUM STABILITY HPLC
CHX A̕̕ ANTI CD66 CONJUGATE AB012 111 INDIUM STABILITY POST RADIOLABELLING HPLC
105
105 INDIUM STABILITY HPLC
95
Linear (PASS LINE)
90
85
pat 2 Room Temp
90
pat 2 4deg C
85
pat 3 Room Temp Pat 3 4 deg c
0 0
01
01
Pat 1 Room Temp
95
80
80 /2 /01
PASSLINE
100 % RCP 111 INDIUM
% RCP 111 INDIUM
100
0
01
/2 /07
20
05
1
1
01
01
/2 /02
/2 /08
24
11
2
2
01
01
/2 /03
/2 /09
27
3
01
/2 /04
01
15
3
01
/2 /11
5
10
4
01
/2 /05
15
20
25
HOURS AFTER PREP
pat 1 4 deg C
20
b
DATE
a
Concentration ng/ml 450.00
Effect of Reagent Age on Standard results
Patient Sample Stability in Storage
400.00 350.00
800.000
MEASURED NG/ML
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300.00 700.000
250.00
600.000
200.00 150.00
500.000
100.00 std 700 ng/ml
400.000
50.00 0.00 10.0
std 87.5 ng/ml
300.000
15.0
20.0
25.0
std 5.47 ng/ml
Sample
100.000 0.000
4
3/
/0
19
4
1 20
4/
/0
19
1 20
5/
/0
19
4
4
4
1 20
6/
/0
19
1 20
7/
/0
19
1 20
30.0
35.0
40.0
45.0
50.0
55.0
Weeks after storage
200.000 Pat 1
Pat 2
Pat 3
mean
%cv
mean
%cv
mean
1
361.62
5.02
296.90
2.38
362.13
4
155.40
5.89
77.95
6.56
142.05
6.68
7
24.15
16.69
7.03
17.00
6.84
13.32
c
%cv 4.44
d Freeze/Thaw Stability at –20 and –70 °C in Human Serum. OD at 492 nm 2.500
2.000 700ng/ml-20C 700ng/ml-70c 87.5ng/ml-20c 87.5ng/ml-70c 5.47ng/ml-20c 5.47ng/ml-70c
1.500
1.000
0.500
0.000 1
2
3
4
5
Freeze Thaw Cycles
e FIGURE 5 Stability. (a) Conjugate stability. Results of radiolabelling Besilesomab conjugate with 111 Indium showing no obvious deterioration over three year period. (b) Short-term stability. Results of 5 patient dose remnant samples show short term stability of greater than 24 hr after labelling with 111 Indium, both at room temperature and 4◦ C. (c) Reagent stability. Calculated concentration shows minimal reduction over the course of 4 months using the same assay reagents. (d) Patient sample stability in storage. Data from 3 samples from 3 patients which had been re-assayed three times were reviewed. Sample storage ranged from 10 weeks to 52 weeks after initial storage. There is only a slight reduction trend in assay response with %CV values less than 20% in all cases. (e)Freeze/thaw cycle stability. Two groups of three validation standards(700, 87.5, and 5.47 ng/mL were assessed at −20◦ C and −70◦ C over 5 freeze/thaw cycles with no loss of optical density observed. Error bars are ± 2 sd.
Freeze Thaw Cycle Stability As there is a possibility for antibody damage resulting from freeze/thaw cycles[24] this was assessed by the following method. Two groups (A and B)
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each containing five sets of three standard concentrations (700, 87.5, and 5.47 ng/mL) were made. Group A was stored at −20◦ C and group B at −70◦ C. Both groups were allowed to freeze for at least 24 hr. On Day 1, one set of three standards from each group was defrosted at room temperature and then refrozen. On Day 2, two groups were thawed and refrozen. This was repeated adding a group on each day until Day 5 when all samples were analyzed together in a test assay (Figure 5E).
Reagent Storage Coated plates were stored at −20◦ C, blocking and wash buffers were made fresh each day. Capture and secondary antibody conjugates, positive controls patient samples, and 700 ng/mL standards were stored in aliquots at −20◦ C. Other reagents were stored at manufacturers’ recommendations. When fresh batches of assay reagents were introduced they were run in parallel with the expiring batch and when new plates were coated the negative and positive control results were examined for consistency. Comparison of Results with Previously Published Studies The results of this study showed that the assay described was fit for purpose with a mean precision of 9.39 (%cv) inter study variation, 5.17 (%cv) intra study variation, and a mean accuracy of 98.5%.These figures are well within the limits of precision 25%cv and accuracy ± 25% recommended by Findlay et al.[19]. Others[15,18] suggest lower limits of 20%cv for precision and ± 20% accuracy. Alciato et al.[22] reported figures of < 15% cv for both intra and inter study precision and an accuracy of 94 ± 5% in their assay for quantifying GAS6 protein. Acevedo et al.[25] developing an ELISA for measuring PSA concentrations achieved an accuracy range of 96–103% with an intra study precision of 2.8–6.3(%cv) and an inter study precision of 2.1–3.2 %cv. CONCLUSION The aim of this article, namely the design and validation of an ELISA to quantify Besilesomab levels within the range of 5.47–700 ng/mL in human serum, has been achieved. The assay is relatively cheap and simple to perform and 36 patient samples can be run in duplicate on each plate along with controls and blanks with the suggested layout. In excess of 30 assays have been run to date and factors optimized along the way to arrive at this protocol.
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This is a robust, reliable assay which could be easily adapted and translated to other mouse antibody human studies, for which a commercial validated assay is unavailable. FUNDING Funding for this work was provided by The Experimental Cancer Medicine Centre (ECMC). The Experimental Cancer Medicine Centre initiative is jointly funded by Cancer Research UK, the National Institute for Health Research in England and the Departments of Health for Scotland, Wales and Northern Ireland.
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