Research Article
Received: 8 April 2008,Revised: 4 May 2008,Accepted: 7 May 2008,Published online 8 September 2008 in Wiley Interscience (https://www.doczj.com/doc/1312247047.html,) DOI 10.1002/bio.1065
Luminescence 2009; 24: 55–61Copyright ? 2008 John Wiley & Sons, Ltd.55
Development of a chemiluminescent imaging assay for the detection of anti-erythropoietin antibody in human sera
Wenjun Wang a, Yanyan Lu a, Sichun Zhang a, Shidong Wang b, Po Cao b, Yaping Tian c and Xinrong Zhang a*
ABSTRACT:Measuring low amounts of anti-erythropoietin antibodies (anti-EPO Abs) is important to evaluate the therapeutic safety of recombinant human erythropoietin (rhEPO). In this work, a simple, sensitive and high-throughput chemiluminescent (CL) imaging assay was developed for the detection of anti-EPO Abs in human sera. The influence of several physicochemical parameters, such as coating conditions, incubation time, detergent concentration and exposure time, were investigated. A calibration curve was established and the range of quantitative detection was 0.12–13.91ng/mL. The limit of detection (LOD, 3s) for the CL-imaging assay was 0.033 ng/mL. Compared to conventional colorimetric enzyme-linked immunosorbent assay (ELISA), the LOD of the CL-imaging assay is 50-fold lower. The recoveries of anti-EPO Abs in the fortified serum were in the range 87.1–116.9% using the present method, which highlighted the validity of the CL-imaging assay system to accurately determine the anti-EPO Abs in serum samples. CL-imaging assay was used to evaluate the presence of anti-EPO Abs in serum samples obtained from chronic renal failure (CRF) patients treated with rhEPO. Contrary to what was expected, the sera from CRF patients did not contain anti-EPO Abs. Copyright ? 2008 John Wiley & Sons, Ltd.
Keywords:erythropoietin; doping; anti-erythropoietin antibodies; chemiluminescent-imaging assay; enzyme-linked immunosorbent assay; serum samples
Introduction
Human erythropoietin (EPO) is a glycoprotein synthesized mainly in the kidney, which stimulates the production of new red blood cell and then enhances muscle oxygenation. The availability of recombinant human erythropoietin (rhEPO) has increased the risk of its illegal use in sports. The misuse of rhEPO has been prohibited by the International Olympic Committee since 1990. Isoelectric focusing (IEF) provides the proof of rhEPO use, based on the charge differences between rhEPO and endogenous EPO (1–3). However, this method is time-consuming and laborious. Therefore, rapid screening methods are necessary to efficiently monitor the abuse of this drug by athletes.
Antibody responses against rhEPO have been reported by several publications (4–8). The antibody was proposed as a candidate of indirect biomarkers for monitoring the misuse of rhEPO and gene doping (9, 10). The challenge to determin-ing the anti-erythropoietin antibodies (anti-EPO Abs) in serum samples was its extremely low concentration. Several immuno-assays have been used for the analysis of anti-EPO Abs, including enzyme-linked immunosorbent assay (ELISA) (4, 5, 11–14), radioimmunoprecipitation assay (RIPA) (14–17), BIAcore immunoassay (18) and bioassay for neutralizing Abs (19–22). However, the methods mentioned are either radioactive or of low sensitivity.
Chemiluminescence (CL) has been exploited in many scien-tific fields, due to its extremely high sensitivity along with wide calibration ranges and simple instrumentation (23, 24). The CL imaging assay provides simple, sensitive and high-throughput means of detection, and therefore would be an ideal method for anti-EPO immunoassay.
The objective of this work was to develop a CL-imaging assay for the detection of anti-EPO Abs in human serum and to evalu-ate whether anti-EPO Abs can be an indirect biomarker of the misuse of rhEPO in sports. To the best of our knowledge, there has been no report so far concerning the detection of anti-EPO Abs based on CL-imaging assay.
Materials and methods
Chemicals and apparatus
rhEPO was obtained from Roche Diagnostics (Penzberg, Germany). Rabbit polyclonal anti-EPO antibody (anti-EPO pAb) and chemi-luminescent substrate were purchased from Millipore (Milford, MA, USA). Bovine serum albumin (BSA), goat anti-rabbit IgG-HRP, and goat anti-human IgG-HRP were purchased from Bo’ao Shen *Correspondence to: X. Zhang, Department of Chemistry, Key Laboratory for the Atomic and Molecular Nanosciences of the Education Ministry, Tsinghua University, 100084 Beijing, People’s Republic of China.
E-mail: xrzhang@https://www.doczj.com/doc/1312247047.html,
a Department of Chemistry, Key Laboratory for Atomic and Molecular Nano-sciences of the Education Ministry, Tsinghua University, 100084 Beijing, People’s Republic of China
b Dongzhimen Hospital, affiliated to Beijing University of Chinese Medicine, 100700 Beijing, People’s Republi
c of China
c Department of Clinical Biochemistry, Chinese PLA General Hospital, 100853 Beijing, People’s Republic of China
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Chemical Reagents Co. (Beijing, China). 3,3′,5,5′-Tetramethylben-zidine (TMB) and Tween 20 were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Unless otherwise stated, all other reagents used in this study were of analytical grade or better.
An ELISA plate reader (Bio-Rad, USA) was used for reading 96-wellsELISA microplates (‘high binding’ grade; Costar, USA).White opaque 96-well microtitre plates (Shenzhen Jincanhua Industry Co. Ltd, China) were used for the CL-imaging assay.Buffers and solution
The following buffers and solutions were used in ELISA and CL-imaging assay: (a) phosphate-buffered saline (PBS), 8.0g NaCl, 2.9g Na 2HPO 4, 0.2g KH 2PO 4 and 0.2g KCl dissolved in 1 L distilled water, pH 7.4; (b) coating buffer, 100mmol/L Na 2CO 3–NaHCO 3, pH 9.6, in distilled water; (c) blocking buffer, 1% (w/v,g/L) gelatin in PBS –this buffer was stored at 4°C and used within a week; (d) washing solution (PBST), 0.05% Tween 20 (v/v)in PBS; (e) antibody dilution buffer, 0.1% (w/v, g/L) gelatin in PBST; (f) ELISA substrate stock solution, 0.1% TMB and H 2O 2 in 0.05 mol/L citrate buffer, pH 4.5; (g) stopping solution, 2N H 2SO 4;(h) CL-imaging HRP substrate stock solution was freshly pre-pared by combining equal volumes of luminol reagent and per-oxide solution. All buffers were prepared using MilliQ H 2O (18M Ω/cm).
Immunoassay procedure
Colorimetric ELISA protocol.High-binding polystyrene 96-well plates were coated with the selected concentration of EPO in
0.1mol/L carbonate buffer at 4°C overnight. On the following day, 150μL blocking solution were added and the mixture was incubated for 30min. The serum samples or anti-erythropoietin antibody standards were added to the wells at 100μL/well. Poly-clonal rabbit anti-EPO antibody (anti-EPO pAbs) was used as a standard for validating the assay because a human anti-EPO antibody standard was not available. Next, goat anti-rabbit IgG-HRP (1:5000 dilutions in PBST, 100μL/well) solution was added and incubated for 1h. After addition of TMB for 15–30min, the enzymatic reaction was stopped by adding 50μL 2M sulphuric acid/well, Absorbance (OD) at 450nm was read and recorded.All incubations were carried out at 37°C. In each step the plates were washed three times with the washing buffer to remove unbound antibody or antigen. The calibration curves were obtained by plotting normalized absorbance values of anti-EPO Abs stand-ards against the logarithm of anti-EPO Abs concentrations.Chemiluminescent imaging assay protocol.This protocol is shown in Figure 1. It was carried out using 96-well white opaque polystyrene microtitre plates instead of transparent plates in ELISA. After immunological steps, 100μL freshly prepared sub-strate solution was added to each well and detected with films.The intensity of the spots was determined using Bio-Rad soft-ware. The wells were individually analysed and the CL intensities were plotted as a function of anti-EPO Abs concentration to yield the calibration curve.Curve fitting and data analysis
Standards were run in triplicate wells, and the mean absorbance
values or CL intensity values were processed. Standard curves
Figure 1.Schematic overview of CL-imaging assay and detection procedure. (a) Microplates were precoated with rhEPO. (b) Anti-EPO Abs (Ab 1) and enzyme-labelled secondary antibody (Ab 2*) were added to microplates. (c) The CL substrate was added, CL emission was seen and then the imaging process was commenced. (d) The image was analysed and intensities quantitated using image analysis software.
CL imaging assay for anti-erythropoietin antibody detection in human sera
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were obtained by plotting absorbance against the logarithm of analyte concentration. Using originPro 7.0 (Microcal Software Inc., Northhampton, MA, USA), sigmoidal curves were fitted to a four-parameter logistic equation:
y={(A–D)/[1+(x/C)]B}+D
where A is the minimum absorbance (background signal), D is the maximum absorbance, C is the concentration producing 50% of the maximal absorbance, and B is the slope at the inflec-tion point of the sigmoid curve.
Analysis of spiked serum samples
The recovery of the CL-imaging assay was evaluated by spiking anti-EPO pAbs to diluted negative human serum in the estab-lished working range, with the same procedure and conditions as described in the section on CL-imaging assay protocol, above. Real sample analysis
Blood samples from randomly chosen rhEPO-treated patients with chronic renal failure (CRF) were provided by the Dongzhimen Hospital (Beijing, China) and the Chinese PLA General Hospital (Beijing, China). Control blood samples from a healthy person were provided by the School Hospital of Tsinghua University (Beijing, China). Serum samples were prepared from whole blood by centrifuging at 10 000×g for 5min in a serum separation vial, and stored as aliquots at –20°C until analysis.
Results and discussion
Optimization
Optimization of coating conditions.In the present study, a solid phase sandwich immunoassay was applied to determine the concentration of anti-EPO antibody. A coating procedure was carried out as the first step of the immunoassay. To achieve opti-mal conditions, coating and blocking buffers as well as coating rhEPO concentrations were optimized. Three types of coating buffers were examined, including carbonate buffer (50mmol/L,
pH 9.6), PBS (10mmol/L, pH 7.4) and citrate buffer (50mmol/L, pH 4.8). The maximum CL intensity was achieved with carbonate buffer compared to PBS buffer and citrate buffer (Figure 2). Car-bonate buffer was therefore chosen as the coating buffer for the subsequent study.
The concentration of coating rhEPO in carbonate buffer was also examined. The result showed that the optimal concentration of rhEPO antigen was in the range 0.5–5.0μg/mL. Outside this range a poor signal:noise (S:N) ratio was observed. Therefore, all subse-quent assays were carried out using 0.5μg/mL rhEPO in carbon-ate buffer, pH 9.6.
To choose the optimal blocking buffer, PBS buffers containing 3% bovine serum albumin (BSA) or 1% gelatin were examined. Both of the blocking buffers were considered acceptable for the blocking buffer (see Fig. 3), and gelatin was used as the blocking agent for the following assay.
Optimization of incubation time.The effect of incubation time on the assay performance was optimized. In the primary experi-ment, two-step and one-step assays under the same conditions were performed. As shown in Figure 4, the one-step assay achieved a slight higher CL intensity than the two-step assay. Therefore, the one-step assay with different incubation times was further examined. 60min incubation provided a higher S:N and was therefore selected for the following CL-imaging assay. Optimization of detergent (Tween-20).Tween 20 is a non-ionic detergent commonly used in immunoassays to reduce non-specific binding and improve sensitivity (25). However, there are quite a few reports of the negative influence of Tween 20 on immunoassay performance (26–28), therefore the influence of Tween-20 con-centration on CL intensities was examined. As shown in Figure 5, the CL intensity and S:N with the buffer to which 0.05% v/v Tween 20 was added were highest. Therefore, 0.05% Tween 20 was selected as the optimal concentration in subsequent experiments. Optimization of exposure time.The effect of exposure time on CL intensity was investigated in the range 30s–5min. The results showed that CL intensity and background were increased simul-taneously. As shown in Figure 6, the best S:N ratio could be obtained when the exposure time was 2min. Thus, an exposure time of 2
min was employed for subsequent research work. The Figure 2.Effect of different coating buffers on CL-imaging assay for anti-EPO Abs. Each value represents the mean of triplicate well replicates.
Figure 3.Effect of different blocking buffers on CL-imaging assay for anti-EPO Abs. Each value represents the mean of triplicate well replicates.
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CL signal from the luminol–HRP reaction rises rapidly and remains stable over a 60min time course, which is enough for a measurement taking only 2min.Performance of the proposed method
Under the optimized conditions, the calibration curve of the CL-imaging assay for anti-EPO Abs was established over the range 0.12–13.91ng/mL (Figure 7). The limit of detection (LOD),calculated as the concentration corresponding to the signal equal to that of the zero standard plus three times its SD, was 0.033ng/mL. The relative standard deviation (RSD) was 8.74%for 0.36ng/mL anti-EPO Abs.
Comparison between colorimetric ELISA and CL-imaging detection
In an effort to determine the relative sensitivity of our strategy,we developed both a CL-imaging assay and a colorimetric ELISA using the same polyclonal antibodies. Figure 7 shows the calibra-
tion curves for anti-EPO Abs obtained from CL-imaging assay and colorimetric ELISA. The LODs of the CL-imaging assay and the colorimetric ELISA were 0.033ng/mL and 1.70ng/mL, respectively,indicating that use of the CL-imaging assay improved the sensi-tivity by 50-fold compared to that of colorimetric ELISA.
A further advantage obtained by using CL-imaging detection was speed of the assay. The one-step procedure selected in the CL-imaging assay increases the simplicity and reduces the assay time, while the colorimetric assay could achieve an high enough absorbance only by using a two-step procedure for low affinity anti-EPO Abs. On the other hand, the CL signal can be measured immediately after substrate addition, while the colorimetric assay requires a 15–30min incubation step as well as an enzyme activity-stopping step prior to signal detection.
Finally, the CL-imaging assay often uses less immunoreagent to achieve better sensitivity than the corresponding colorimetric ELISA. For the developed CL-imaging assay, the optimal concen-tration of coating antigen and developing antibody used was 1/5and 1/250 of that of the colorimetric ELISA, respectively. This may provide a significant cost–benefit in performing such assays.It should be noted that the substrate and microplates used in the CL-imaging assay are relatively more expensive than those used in the colorimetric ELISA.Application
The method of the CL imaging assay was evaluated using forti-fied 50-fold diluted serum samples at different levels of anti-EPO Abs. As shown in Table 1, the average recoveries of anti-EPO Abs from the serum samples were in the range 87.1–116.9% and the average CVs was 6.4–11.9%. Figure 8 shows a good correlation between the fortified concentration and those determined by CL-imaging assay (slope =0.879, R =0.998). No inhibition or interference with the detection of anti-EPO Abs was incurred by rhEPO in the 50-fold diluted serum. These results showed that CL-imaging is suitable for the analysis of anti-EPO Abs in serum samples.
Since ‘doping’ sera were unavailable, sera from rhEPO-treated patients were analysed; 60 serum samples obtained from rhEPO-
treated patients and 40 control serum samples from healthy
Figure 4.Effect of different incubation methods on CL-imaging assay for anti-EPO Abs. Each value represents the mean of triplicate well replicates.
Figure 5.Effect of Tween-20 on CL-imaging assay for anti-EPO Abs. Each value
represents the mean of triplicate well replicates.
Figure 6.Effect of exposure time on CL-imaging assay for anti-EPO Abs. Different exposure times (30s–5min) were evaluated. Each value represents the mean of triplicate well replicates.
CL imaging assay for anti-erythropoietin antibody detection in human sera
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persons were analysed with the developed CL-imaging assay.Since human anti-EPO Abs was not available, rabbit polyclonal anti-EPO Abs added to a 50-fold diluted sample was used as a positive control. The cut-off point for positive samples was cal-culated as the mean CL intensity for the negative controls plus 3 SD. Human samples were analysed in duplicate. As shown in Fig. 9, there was no significant difference in CL intensity between the two groups (mean [SD]: CRF patients, 25.11[0.87], healthy persons, 25.08 [0.96]; p >0.05). The results showed that sera of CRF patients do not contain anti-EPO Abs.
Conclusions
In the present work, a highly sensitive CL-imaging assay for the determination of anti-EPO Abs was established. Anti-EPO Abs could be detected down to 0.033ng/mL using the present method.The method is fast, simple and sensitive and therefore suitable for routine clinical analysis. The ability to measure very low amounts of anti-EPO Abs is very important for evaluating the safety of therapeutic rhEPO. Further study will be carried out to
examine whether there is any anti-EPO Abs in ‘doping’ sera.
Figure 7.Calibration curves obtained in PBST buffer with the immunoassay method for anti-EPO Abs, using (a) colorimetric and (b) CL-imaging detection. The curves are expressed as signal intensity against the log of anti-EPO Abs concentration. The error bars are standard error of mean (SEM), n =3. (c) Image of the plate (exposure time was 2
min; the lanes represented three independent measurements; anti-EPO pAb concentrations, 0.013–200 ng/mL). Each value represents the mean of triplicate well replicates.
Figure 8.Graph showing the correlation between the fortified and the measured concentration values using the optimized CL-imaging assay to analyse anti-EPO pAb in serum diluted 50-fold. Each fortified sample and each spiked sample was tested using three well replicates on every assay.
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Acknowledgements
This work was financially supported by the National Natural Science Council of China (Project No. 20535020; 20635002).
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Table 1.Recovery of anti-EPO Abs from the fortified serum samples Spiked concentration (ng/mL)Determined (ng/mL; n =3)
(mean ±SD)
Inter-assay (CV/%)Average recovery (%)0ND a
––0.6250.70±0.057.1112.31.25 1.46±0.09 6.4116.92.5 2.58±0.228.5103.25 4.80±0.5311.096.07.5 6.53±0.7811.987.110
9.08±0.93
10.1
90.8
a
Not detected.
Figure 9.Distribution of CL intensity of anti-EPO Abs in a healthy person and a CRF patient treated with EPO.
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