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Rapid quantitation of human epididymis protein 4 in human serum by amplified luminescent proximity homogeneous immunoassay (AlphaLISA)
Journal of Immunological Methods 437 (2016) 64–69
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Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim
Research paper
Rapid quantitation of human epididymis protein 4 in human serum by amplified luminescent proximity homogeneous immunoassay (AlphaLISA) Hui Zhao a,1, Guanfeng Lin b,1, Tiancai Liu c, Junyu Liang c, Zhiqi Ren c, Rongliang Liang c, Baihong Chen b, Wenhua Huang a,⁎, Yingsong Wu c,⁎ a b c
Department of Human Anatomy, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China Teaching and Scientific Research Center, School of Biotechnology, Southern Medical University, Guangzhou, China Institute of Antibody Engineering, School of Biotechnology, Southern Medical University, Guangzhou, China
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Article history: Received 7 July 2016 Received in revised form 23 August 2016 Accepted 23 August 2016 Available online 24 August 2016 Keywords: Amplified luminescent proximity homogeneous immunoassay Ovarian cancer Human epididymis protein 4
a b s t r a c t A sensitive, rapid and homogeneous reaction measurement method for quantitation of human epididymis protein 4 (HE4) in human serum by amplified luminescent proximity homogeneous immunoassay (AlphaLISA) was described. Built on a sandwich-type immunoassay format, analytes in samples were captured by one biotinylated monoclonal antibody combining on the surface of streptavidin coated donor beads, and “sandwiched” by another monoclonal antibody coated on acceptor beads. The coefficient variations of the method were lower than 10%, and the recoveries were in the range of 90–110% for serum samples. A value of 0.88 pmol/l was identified as the minimum detectable dose of the present method for HE4. Compared with the results from electrochemiluminescence immunoassay kit (Roche) in 170 serum samples, there was a satisfied correlation coefficient of 0.984. The present assay demonstrated high sensitivity, wider effective detection range and excellent reproducibility for quantitation of HE4 can be useful for early screening and prognosis evaluation of patients with ovarian cancer. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Ovarian cancer is the second most common gynecologic cancer and the leading cause of death from gynecologic malignancy among women in the world (Granato et al., 2012). Because early symptoms of this disease are common and nonspecific, it is often playing a threat to women health and safety of the silent killer role. It is responsible for 5% of all cancer death in women (Suh et al., 2010). Unfortunately, at present, we are unable to have an effective early diagnosis strategy and hard to distinguish the malignant cases from the benign cases or other gynecological diseases for this malignancy; b20% of ovarian cancer cases are diagnosed in stage I (Bast, 2003), most (N70–80%) of the cases are diagnosed with advanced stages of this disease, which explains its Abbreviations: AlphaLISA, amplified luminescent proximity homogeneous immunoassay; HE4, human epididymis protein 4; BSA, bovine serum albumin; MES, 4morpholineethanesulfonic acid; CMO, carboxy-methoxy lamine; NHS, nhydroxysulfosuccinimide; SA-D beads, streptavidincoated donor beads; HEPES, 2-[4-(2Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid; EDC, 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride; Eu, europium; ECLIA, electrochemiluminescence immunoassay; CV, coefficient of variation; SD, standard deviation; AFP, alpha-fetoprotein; CEA, carcinoembryonic antigen; CA125, cancer antigen 125; CA199, cancer antigen 199. ⁎ Corresponding author. E-mail addresses: [email protected] (W. Huang), [email protected] (Y. Wu). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.jim.2016.08.006 0022-1759/© 2016 Elsevier B.V. All rights reserved.
prevalence and high mortality rate (Havrilesky et al., 2008; Zhou et al., 2015). As mortality is closely related to disease stage at diagnosis (the 5-year survival rate is higher than 70% in stage I or II, but decreases to 40 and 20% in stages III and IV) (Heintz et al., 2006; Nosov et al., 2009; Siegel et al., 2012), an early diagnosis and timely treatment of primary ovarian cancer could improve survival, reduce the burden that patients are bearing and be clinically beneficial for early prediction of responses to treatment and relapses (Jacob et al., 2011; Braicu et al., 2013; Sandri et al., 2013). As we know, serum tumor markers are non-invasive diagnostic tools for identifying malignant tumors, and are commonly used for the early screening of cancer and as an indicator of treatment efficacy. In recent years, significant effort has been made to find new biomarkers for the early diagnosis of ovarian cancer (Lokshin, 2012; Su et al., 2013; Xu et al., 2016). Among new markers, human epididymis protein 4 (HE4) is one of the most promising for ovarian cancer (Anastasi et al., 2010). It is also called whey-acidic-protein four-disulfide core domain protein 2 because of its molecular structure, containing 2 whey acid protein domains and a 4-disulfide-bond core with 8 cysteine residues, and is frequently amplified in ovarian cancer while its expression in normal tissues, including ovary, is low (Schummer et al., 1999; Wang et al., 1999; Ono et al., 2000). HE4 has been recently described as a new marker for early ovarian cancer with higher sensitivity (76.9%) (Abdel-Azeez et al., 2010), and was also recently approved by the Food and Drug
H. Zhao et al. / Journal of Immunological Methods 437 (2016) 64–69
Administration (FDA) to monitor recurrence or progressive disease in ovarian cancer (Fleming et al., 2011). At present, most of the research focused on the application value of HE4, and just a small amount of research reported the methodology for detection of HE4. Zhou LJ et al. reported a sandwich Enzyme-Linked Immunosorbent Assay for detection of HE4 in human serum (Zhou et al., 2015). A Time-Resolved Fluorescence Immunoassay for screening of HE4 was reported by Sun WQ et al. (Sun et al., 2016). Those methods more or less have a number of disadvantages including low sensitivity, instability, nonhomogeneous reaction or the waste of time. We innovatively developed an amplified luminescent proximity homogeneous immunoassay (AlphaLISA), which was designed specifically as a sensitive, precise and rapid measurement method for the quantitation of HE4 in human serum. Thus, the purpose of the present study was the establishment of this novel immunoassay and test its application. This study involved measurement of parameters, such as repeatability, recovery, linearity and feasibility. 2. Materials and methods 2.1. Material and chemicals Bovine serum albumin (BSA), 4-morpholineethanesulfonic acid (MES), N-hydroxysulfosuccinimide (NHS), Sodium cyanoborohydride (NaBH3CN), Carboxy-methoxy lamine (CMO), Sodium azide (NaN3), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), Dimethylsulfoxide (DMSO), Dextran T500, Proclin-300 and Tween-20 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bovine gamma globulin and mouse immunoglobulin were purchased from BioDesign (Memphis, TN, USA). All other chemicals used were of analytical reagent grade and ultra-pure water obtained using a Milli-Q water purification system (Millipore, MA, USA) was used throughout the experiments. Anti-HE4 monoclonal antibodies (H5130715 and 13010,379-100) were obtained from Hotgen Biotech (Beijing, China) and RayBiotech (Norcross, GA, USA), respectively. HE4 antigen (23000,050) was purchased from RayBiotech (Norcross, GA, USA). Unconjugated europium (Eu)-acceptor beads, streptavidincoated donor beads (SA-D beads), EnVision Multilabel Reader and 96-well Optiplates were purchased from PerkinElmer (Waltham, USA). Biotin-NHS (Catalog Number: H1759) was from Sigma-Aldrich (St. Louis, MO). 2.2. Samples and comparison method Samples with the HE4 values measured by an electrochemiluminescence immunoassay (ECLIA) kit which was
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purchased from Roche (Basle, Switzerland) were kindly provided by Nanfang Hospital (Guangzhou, China). Normal female samples were kindly provided by DaAn Clinical Laboratory Center of Sun Yat-sen University (Guangzhou, China). These samples were stored at −20 °C. The collection and storage of the serum samples were carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki). 2.3. Biotinylated antibody One milligram of anti-HE4 antibody (H5130715) was dissolved in 0.05 ml of 0.1 mol/l carbonate buffer (pH 9.5) containing 0.1% NaN3 and Biotin-NHS in DMSO immediately prior to use in the dark at 22 mg/ml. A volume equal to 10% of the total volume of the antibody solution was added into the Biotin-NHS solution with gentle stirring and incubated at room temperature for 4 h. The reaction solution was dialyzed against several changes of PBS buffer (0.01 M sodium phosphate, 0.15 M sodium chloride, pH 7.4) at 4 °C. The dialyzed biotinylated antibody was stored (0.5 mg/ml) at −20 °C until use. 2.4. Coating conjugate preparation Anti-HE4 antibody (4B2-F10-E1), 0.2 mg was dissolved in 0.1 ml sodium phosphate buffer (0.13 mol/l, pH 8.0). The solution was added to 1 mg acceptor beads with 10 μl of 25 mg/ml NaBH3CN and 1.25 μl of 10% Tween-20 and then incubated at 37 °C for 48 h. The total volume of the reaction solution was 200 μl. To block nonconjugated sites, the fresh CMO solution (65 mg/ml) was prepared in 0.8 M NaOH. Ten microliters of CMO solution was added to the reaction and incubated for an hour at 37 °C. The reaction solution was centrifuged (14,000 rpm, 25 min) and the supernatant was removed. Then the bead pellet was resuspended in 200 μl of 0.1 M Tris-HCl, pH 8.0, centrifuged, and washed. After the last centrifugation, the beads were resuspended in storage buffer (200 μl of PBS + 0.05% Proclin-300 as a preservative) at a concentration of 5 mg/ml. 2.5. Preparation of HE4 standards For calibration purposes, a calibrator series was developed by diluting HE4 antigen in a buffer containing 50 mM Tris-HCl, 0.9% NaCl, 0.05% sodium azide, 1.5% BSA, and 0.01% Tween-20, pH 7.8. This step generated the desired standard concentrations of 0, 10, 50, 250, 500, and 1000 pmol/L, designated as A, B C, D, E, and F, correspondingly. The standards were stored at 4 °C.
Fig. 1. Design of a sandwich-type immunoassay based on the amplified luminescent proximity homogeneous assay for the quantitation of HE4.
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H. Zhao et al. / Journal of Immunological Methods 437 (2016) 64–69 Table 1 Analytical recovery test of HE4 added to serum samples. Sample (pmol/L)
Expected value (pmol/L)
Mean (n = 3)
Recovery
41.6
100 200 400 100 200 400 100 200 400
99.5 207.6 391.4 101.2 205.4 395.4 98.9 206.2 416.2
99.5% 103.8% 97.8% 101.2% 102.7% 98.8% 98.9% 103.1% 104.1%
55.6
72.3
3. Results 3.1. Standard curve and signal saturation Fig. 2. Standard curve and intra-assay precision profile of our novel assay for HE4. Each point was based on 10 replicates.
2.6. AlphaLISA assay protocol The proposed immunoassay for the quantitation of HE4 was performed by a sandwich type immunoassay based on the amplified luminescent proximity homogeneous assay, and was shown schematically in Fig. 1. The assay buffer contained 25 mM 2-[4-(2-Hydroxyethyl)-1piperazinyl]ethanesulfonic acid (HEPES), 50 mM NaCl, 5 mM DTPA (tripotassium salt), 2 mg/ml Dextran T500, 0.5% BSA, 0.05 mg/ml bovine gamma globulin, 0.01 mg/ml mouse immunoglobulin, 0.1% Tween-20 (w/v), 0.01% Proclin 300, and 0.01% gentamycin sulfate and was adjusted to pH 7.4. Two-step assay procedures were used. Briefly, the test samples and standards (25 μl) were added to the 96-well plates. A 50 μl mix of antibody-acceptor beads (1:400) and biotinylated antibody (1:400) in the assay buffer was added to the wells. The plates were covered with a lid and incubated at 37 °C for 15 min. Subsequently, 175 μl of SA-D beads (0.012 mg/ml) in assay buffer were added. The plates were covered with a lid and incubated at 37 for 15 min in the dark. The AlphaLISA signal was measured on a 2300 EnVision Multilabel Reader.
2.7. Statistical analysis The data were expressed as mean values from duplicate measurements. All of the data were analyzed with Origin software (version 7.5, OriginLab, MA), and other statistical analyses were performed using Statistical Product and Service Solutions (SPSS) software (version 20.0, SPSS Inc., Chicago, IL). A P value of b0.05 was considered to be statistically significant.
A standard curve for the immunoassay was carried out following our protocol with a series of dilution of standards obtained from 10 separate assays. Standard curve quantitation was carried out using linear regression and log-log regression. For the standard curve depicted in Fig. 2, the best-fit calibration of HE4 was determined to be described by the following equation: LogY = 3.201 + 0.974 × LogX (r2 = 0.996, P b 0.0001). Signal saturation (“hook” effect) were seen when the range exceeded 1000 pmol/L for HE4 (Fig. 3). Within-assay coefficients of variation (n = 10) using standards were b10%. 3.2. Sensitivity The minimum detectable dose was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration. According to this calculation method, a value of 0.88 pmol/L was identified as the minimum detectable dose of the present method for HE4. 3.3. Recovery The analytical recovery was studied by adding purified HE4 antigen to 3 serum samples from different patients. The results were given in Table 1. The recoveries of added analytes were in the range of 90–110%. 3.4. Precision Within-and between-assay imprecision were determined using three serum samples and the same batch of reagents on separate days as shown in Table 2. Total imprecision of the present assay were ranged from 4.1% to 6.9%. As expected, the imprecision of the present assay was remarkably low. 3.5. Linearity Table 3 showed the results of our evaluation of the dilution linearity of this assay when we used samples serially diluted with assay buffer. Expected values were derived from initial concentrations of analytes Table 2 Precision test of our novel assay. Sample
Intra-assay precision (n = 12)
Intra-assay precision (n = 20)
Fig. 3. High-dose signal saturation (hook-effect) of our novel assay for HE4.
1 2 3 1 2 3
CV, coefficient of variation; SD, standard deviation.
HE4 (pmol/L) Mean
SD
CV
27.5 42.6 72.5 28.2 43.3 71.2
1.12 2.64 3.55 1.41 2.98 4.77
4.1% 6.2% 4.9% 5.0% 6.9% 6.7%
H. Zhao et al. / Journal of Immunological Methods 437 (2016) 64–69
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Table 3 Dilution linearity test of our novel assay for HE4. Sample
Dilution
HE4 (pmol/L) Expected
1
2
3
NA 1:2 1:4 1:8 1:16 NA 1:2 1:4 1:8 1:16 NA 1:2 1:4 1:8 1:16
Observed (n = 3) 47.5 23.2 11.6 5.98 2.89 81.6 39.9 20.7 9.9 5.02 135.8 69.4 32.3 17.5 8.8
23.7 11.8 5.93 2.96 40.8 20.4 10.2 5.10 67.9 33.9 16.9 8.5
Recovery 97.8% 98.3% 100.8% 97.6% 97.8% 101.4% 97.0% 98.4% 102.2% 95.3% 103.5% 103.5%
Fig. 4. The concentration distribution of 459 normal serum samples.
NA, not applicable.
in the undiluted samples. Correlating the results obtained from assay with the expected concentrations, we found that expected values were identical with measured values.
assessed. The cross-reactivity was expressed as the percent ratio between measured and expected values. Results in Table 5 showed that no significant cross-reactivity was identified. 3.8. Normal reference range
3.6. Interference The effect of hemolysis, lipemia and bilirubinemia was assessed by adding hemolysate, bilirubin (unconjugated) and triglyceride to the serum samples, respectively. The recoveries were calculated from HE4 values determined before and after addition of interfering substances. The recoveries were between 91.9% and 105.8% (Table 4).
459 samples of normal healthy people were detected by the present method we developed. The result was shown in Fig. 4. When the concentration of HE4 ≤ 90 pmol/l, 96.9% of the samples are successfully classified as normal and when concentration of HE4 ≤ 100 pmol/l, this improves to 97.1%. Furthermore, we made the ROC for our novel assay (Fig. 5). At the concentration of 73.45 pmol/l, the Correct diagnosis index = Sensitivity−(1−Specificity) was the maximum. Comprehensively referencing to the literature for similar results, we set our cutoff value at 90 pmol/l.
3.7. Specificity 3.9. Comparison with ECLIA To investigate the specificity of the developed HE4 assay, the effect of adding other commonly measured proteins alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen 125 (CA125) and cancer antigen 199 (CA199) to normal human serum samples was
Table 4 Interference test of our novel assay for HE4. Interfering substances
Hemoglobin
Bilirubin
Triglyceride
HE4 in 170 clinical samples were analyzed by the present assay. The correlation of the HE4 values obtained by this method and those obtained by ECLIA was excellent; the regression equation was Y = 0.971× X − 0.571 (r2 = 0.984, P b 0.0001). The comparison of HE4 values obtained by the two methods was shown in Fig. 6. 4. Discussion
Concentration (ng/ml)
0 500 1000 0 500 1000 0 500 1000
HE4 (pmol/L) Mean (n = 3)
Recovery
56.3 57.6 59.3 63.5 64.4 58.4 78.3 76.9 82.9
– 102.3% 105.3% – 101.4% 91.9% – 98.2% 105.8%
We established a 96-well plate based homogeneous chemiluminescent sandwich immunoassay for the rapid quantitation of HE4 in human
Table 5 Specificity test of our novel assay for HE4. Interfering substances
Concentration
AFP CEA CA125 CA199
1000 U/ml 560 ng/nl 500 U/ml 600 U/ml
HE4 (pmol/L) Mean (n = 3)
Cross-reactivity rate (%)
0.13 0.11 0.15 0.21
0.01 0.02 0.03 0.03
Fig. 5. The ROC cure for our novel assay.
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H. Zhao et al. / Journal of Immunological Methods 437 (2016) 64–69
Scientific Instrument and Equipment Development Projects of China (Grant No. 61427807). The authors thank the National Natural Science Foundation of China. They also thank Nanfang Hospital, Guangzhou, China, for providing reagents at a reduced cost.
References
Fig. 6. Graphical comparisons of the present assay and ECLIA results for quantitation of HE4.
serum. As shown in Fig.1, one anti-HE4 monoclonal antibody was coated on acceptor beads and another anti-HE4 antibody was biotinylated. It also contains donor beads coated with streptavidin. The resulting complex was quantified in the well by excitation of the donor beads with laser irradiation at 680 nm. Then, specific delayed sharp chemiluminescent emission peak was monitored at about 615 nm. The whole detection time for HE4 only took 30 min in the present assay. AlphaLISA is a simple, small and automatable method (Poulsen and Jensen, 2007; McGiven et al., 2008; Szekeres et al., 2008; Bielefeld-Sevigny, 2009; Zou et al., 2013). The realization of rapid quantitation depends on fast mix-and-measure protocols (Bielefeld-Sevigny, 2009). The assay can be handled manually or as an automatic setup, which facilitates assay development and makes the technology more versatile. Homogenous assay systems are potentially more sensitive to serum interference. The most prominent types of general interference are inner filter effects and singlet oxygen quenchers. By using Eu complex in the acceptor beads emitting light at 615 nm (Bielefeld-Sevigny, 2009), the inner filter effects are minimal when testing serum. Results indicated that the assay was unaffected by interfering substances including hemolysate, bilirubin and triglyceride at concentrations that might be expected in a routine clinical laboratory setting. Notably, the system is free of problems with sample autofluorescence by using the time-resolved mode and because of the large stokes shift between the emission and excitation wavelengths (Lin et al., 2014; Lin et al., 2015a; Lin et al., 2015b). In addition, cross-reactivity data with major interfering tumor markers showed the assay was highly specific for HE4. In summary, we have developed a homogeneous chemiluminescent sandwich immunoassay, which was designed specifically as a rapid, sensitive and homogeneous reaction measurement method for quantitation of the HE4 in human serum. Compared with the results from ECLIA kit (Roche) in 170 serum samples, there was a satisfied correlation coefficient of 0.984. Currently, this method demonstrated high sensitivity, wider effective detection range and excellent reproducibility for the quantitation of HE4, and offered the additional benefit for faster detection, resulting in a substantially faster assay. Based on this investigation, we established a good foundation for further development of other biomarkers using the same platform. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 21575058, 81271931) and National Key
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Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim
Research paper
Rapid quantitation of human epididymis protein 4 in human serum by amplified luminescent proximity homogeneous immunoassay (AlphaLISA) Hui Zhao a,1, Guanfeng Lin b,1, Tiancai Liu c, Junyu Liang c, Zhiqi Ren c, Rongliang Liang c, Baihong Chen b, Wenhua Huang a,⁎, Yingsong Wu c,⁎ a b c
Department of Human Anatomy, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China Teaching and Scientific Research Center, School of Biotechnology, Southern Medical University, Guangzhou, China Institute of Antibody Engineering, School of Biotechnology, Southern Medical University, Guangzhou, China
a r t i c l e
i n f o
Article history: Received 7 July 2016 Received in revised form 23 August 2016 Accepted 23 August 2016 Available online 24 August 2016 Keywords: Amplified luminescent proximity homogeneous immunoassay Ovarian cancer Human epididymis protein 4
a b s t r a c t A sensitive, rapid and homogeneous reaction measurement method for quantitation of human epididymis protein 4 (HE4) in human serum by amplified luminescent proximity homogeneous immunoassay (AlphaLISA) was described. Built on a sandwich-type immunoassay format, analytes in samples were captured by one biotinylated monoclonal antibody combining on the surface of streptavidin coated donor beads, and “sandwiched” by another monoclonal antibody coated on acceptor beads. The coefficient variations of the method were lower than 10%, and the recoveries were in the range of 90–110% for serum samples. A value of 0.88 pmol/l was identified as the minimum detectable dose of the present method for HE4. Compared with the results from electrochemiluminescence immunoassay kit (Roche) in 170 serum samples, there was a satisfied correlation coefficient of 0.984. The present assay demonstrated high sensitivity, wider effective detection range and excellent reproducibility for quantitation of HE4 can be useful for early screening and prognosis evaluation of patients with ovarian cancer. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Ovarian cancer is the second most common gynecologic cancer and the leading cause of death from gynecologic malignancy among women in the world (Granato et al., 2012). Because early symptoms of this disease are common and nonspecific, it is often playing a threat to women health and safety of the silent killer role. It is responsible for 5% of all cancer death in women (Suh et al., 2010). Unfortunately, at present, we are unable to have an effective early diagnosis strategy and hard to distinguish the malignant cases from the benign cases or other gynecological diseases for this malignancy; b20% of ovarian cancer cases are diagnosed in stage I (Bast, 2003), most (N70–80%) of the cases are diagnosed with advanced stages of this disease, which explains its Abbreviations: AlphaLISA, amplified luminescent proximity homogeneous immunoassay; HE4, human epididymis protein 4; BSA, bovine serum albumin; MES, 4morpholineethanesulfonic acid; CMO, carboxy-methoxy lamine; NHS, nhydroxysulfosuccinimide; SA-D beads, streptavidincoated donor beads; HEPES, 2-[4-(2Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid; EDC, 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride; Eu, europium; ECLIA, electrochemiluminescence immunoassay; CV, coefficient of variation; SD, standard deviation; AFP, alpha-fetoprotein; CEA, carcinoembryonic antigen; CA125, cancer antigen 125; CA199, cancer antigen 199. ⁎ Corresponding author. E-mail addresses: [email protected] (W. Huang), [email protected] (Y. Wu). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.jim.2016.08.006 0022-1759/© 2016 Elsevier B.V. All rights reserved.
prevalence and high mortality rate (Havrilesky et al., 2008; Zhou et al., 2015). As mortality is closely related to disease stage at diagnosis (the 5-year survival rate is higher than 70% in stage I or II, but decreases to 40 and 20% in stages III and IV) (Heintz et al., 2006; Nosov et al., 2009; Siegel et al., 2012), an early diagnosis and timely treatment of primary ovarian cancer could improve survival, reduce the burden that patients are bearing and be clinically beneficial for early prediction of responses to treatment and relapses (Jacob et al., 2011; Braicu et al., 2013; Sandri et al., 2013). As we know, serum tumor markers are non-invasive diagnostic tools for identifying malignant tumors, and are commonly used for the early screening of cancer and as an indicator of treatment efficacy. In recent years, significant effort has been made to find new biomarkers for the early diagnosis of ovarian cancer (Lokshin, 2012; Su et al., 2013; Xu et al., 2016). Among new markers, human epididymis protein 4 (HE4) is one of the most promising for ovarian cancer (Anastasi et al., 2010). It is also called whey-acidic-protein four-disulfide core domain protein 2 because of its molecular structure, containing 2 whey acid protein domains and a 4-disulfide-bond core with 8 cysteine residues, and is frequently amplified in ovarian cancer while its expression in normal tissues, including ovary, is low (Schummer et al., 1999; Wang et al., 1999; Ono et al., 2000). HE4 has been recently described as a new marker for early ovarian cancer with higher sensitivity (76.9%) (Abdel-Azeez et al., 2010), and was also recently approved by the Food and Drug
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Administration (FDA) to monitor recurrence or progressive disease in ovarian cancer (Fleming et al., 2011). At present, most of the research focused on the application value of HE4, and just a small amount of research reported the methodology for detection of HE4. Zhou LJ et al. reported a sandwich Enzyme-Linked Immunosorbent Assay for detection of HE4 in human serum (Zhou et al., 2015). A Time-Resolved Fluorescence Immunoassay for screening of HE4 was reported by Sun WQ et al. (Sun et al., 2016). Those methods more or less have a number of disadvantages including low sensitivity, instability, nonhomogeneous reaction or the waste of time. We innovatively developed an amplified luminescent proximity homogeneous immunoassay (AlphaLISA), which was designed specifically as a sensitive, precise and rapid measurement method for the quantitation of HE4 in human serum. Thus, the purpose of the present study was the establishment of this novel immunoassay and test its application. This study involved measurement of parameters, such as repeatability, recovery, linearity and feasibility. 2. Materials and methods 2.1. Material and chemicals Bovine serum albumin (BSA), 4-morpholineethanesulfonic acid (MES), N-hydroxysulfosuccinimide (NHS), Sodium cyanoborohydride (NaBH3CN), Carboxy-methoxy lamine (CMO), Sodium azide (NaN3), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), Dimethylsulfoxide (DMSO), Dextran T500, Proclin-300 and Tween-20 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bovine gamma globulin and mouse immunoglobulin were purchased from BioDesign (Memphis, TN, USA). All other chemicals used were of analytical reagent grade and ultra-pure water obtained using a Milli-Q water purification system (Millipore, MA, USA) was used throughout the experiments. Anti-HE4 monoclonal antibodies (H5130715 and 13010,379-100) were obtained from Hotgen Biotech (Beijing, China) and RayBiotech (Norcross, GA, USA), respectively. HE4 antigen (23000,050) was purchased from RayBiotech (Norcross, GA, USA). Unconjugated europium (Eu)-acceptor beads, streptavidincoated donor beads (SA-D beads), EnVision Multilabel Reader and 96-well Optiplates were purchased from PerkinElmer (Waltham, USA). Biotin-NHS (Catalog Number: H1759) was from Sigma-Aldrich (St. Louis, MO). 2.2. Samples and comparison method Samples with the HE4 values measured by an electrochemiluminescence immunoassay (ECLIA) kit which was
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purchased from Roche (Basle, Switzerland) were kindly provided by Nanfang Hospital (Guangzhou, China). Normal female samples were kindly provided by DaAn Clinical Laboratory Center of Sun Yat-sen University (Guangzhou, China). These samples were stored at −20 °C. The collection and storage of the serum samples were carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki). 2.3. Biotinylated antibody One milligram of anti-HE4 antibody (H5130715) was dissolved in 0.05 ml of 0.1 mol/l carbonate buffer (pH 9.5) containing 0.1% NaN3 and Biotin-NHS in DMSO immediately prior to use in the dark at 22 mg/ml. A volume equal to 10% of the total volume of the antibody solution was added into the Biotin-NHS solution with gentle stirring and incubated at room temperature for 4 h. The reaction solution was dialyzed against several changes of PBS buffer (0.01 M sodium phosphate, 0.15 M sodium chloride, pH 7.4) at 4 °C. The dialyzed biotinylated antibody was stored (0.5 mg/ml) at −20 °C until use. 2.4. Coating conjugate preparation Anti-HE4 antibody (4B2-F10-E1), 0.2 mg was dissolved in 0.1 ml sodium phosphate buffer (0.13 mol/l, pH 8.0). The solution was added to 1 mg acceptor beads with 10 μl of 25 mg/ml NaBH3CN and 1.25 μl of 10% Tween-20 and then incubated at 37 °C for 48 h. The total volume of the reaction solution was 200 μl. To block nonconjugated sites, the fresh CMO solution (65 mg/ml) was prepared in 0.8 M NaOH. Ten microliters of CMO solution was added to the reaction and incubated for an hour at 37 °C. The reaction solution was centrifuged (14,000 rpm, 25 min) and the supernatant was removed. Then the bead pellet was resuspended in 200 μl of 0.1 M Tris-HCl, pH 8.0, centrifuged, and washed. After the last centrifugation, the beads were resuspended in storage buffer (200 μl of PBS + 0.05% Proclin-300 as a preservative) at a concentration of 5 mg/ml. 2.5. Preparation of HE4 standards For calibration purposes, a calibrator series was developed by diluting HE4 antigen in a buffer containing 50 mM Tris-HCl, 0.9% NaCl, 0.05% sodium azide, 1.5% BSA, and 0.01% Tween-20, pH 7.8. This step generated the desired standard concentrations of 0, 10, 50, 250, 500, and 1000 pmol/L, designated as A, B C, D, E, and F, correspondingly. The standards were stored at 4 °C.
Fig. 1. Design of a sandwich-type immunoassay based on the amplified luminescent proximity homogeneous assay for the quantitation of HE4.
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H. Zhao et al. / Journal of Immunological Methods 437 (2016) 64–69 Table 1 Analytical recovery test of HE4 added to serum samples. Sample (pmol/L)
Expected value (pmol/L)
Mean (n = 3)
Recovery
41.6
100 200 400 100 200 400 100 200 400
99.5 207.6 391.4 101.2 205.4 395.4 98.9 206.2 416.2
99.5% 103.8% 97.8% 101.2% 102.7% 98.8% 98.9% 103.1% 104.1%
55.6
72.3
3. Results 3.1. Standard curve and signal saturation Fig. 2. Standard curve and intra-assay precision profile of our novel assay for HE4. Each point was based on 10 replicates.
2.6. AlphaLISA assay protocol The proposed immunoassay for the quantitation of HE4 was performed by a sandwich type immunoassay based on the amplified luminescent proximity homogeneous assay, and was shown schematically in Fig. 1. The assay buffer contained 25 mM 2-[4-(2-Hydroxyethyl)-1piperazinyl]ethanesulfonic acid (HEPES), 50 mM NaCl, 5 mM DTPA (tripotassium salt), 2 mg/ml Dextran T500, 0.5% BSA, 0.05 mg/ml bovine gamma globulin, 0.01 mg/ml mouse immunoglobulin, 0.1% Tween-20 (w/v), 0.01% Proclin 300, and 0.01% gentamycin sulfate and was adjusted to pH 7.4. Two-step assay procedures were used. Briefly, the test samples and standards (25 μl) were added to the 96-well plates. A 50 μl mix of antibody-acceptor beads (1:400) and biotinylated antibody (1:400) in the assay buffer was added to the wells. The plates were covered with a lid and incubated at 37 °C for 15 min. Subsequently, 175 μl of SA-D beads (0.012 mg/ml) in assay buffer were added. The plates were covered with a lid and incubated at 37 for 15 min in the dark. The AlphaLISA signal was measured on a 2300 EnVision Multilabel Reader.
2.7. Statistical analysis The data were expressed as mean values from duplicate measurements. All of the data were analyzed with Origin software (version 7.5, OriginLab, MA), and other statistical analyses were performed using Statistical Product and Service Solutions (SPSS) software (version 20.0, SPSS Inc., Chicago, IL). A P value of b0.05 was considered to be statistically significant.
A standard curve for the immunoassay was carried out following our protocol with a series of dilution of standards obtained from 10 separate assays. Standard curve quantitation was carried out using linear regression and log-log regression. For the standard curve depicted in Fig. 2, the best-fit calibration of HE4 was determined to be described by the following equation: LogY = 3.201 + 0.974 × LogX (r2 = 0.996, P b 0.0001). Signal saturation (“hook” effect) were seen when the range exceeded 1000 pmol/L for HE4 (Fig. 3). Within-assay coefficients of variation (n = 10) using standards were b10%. 3.2. Sensitivity The minimum detectable dose was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration. According to this calculation method, a value of 0.88 pmol/L was identified as the minimum detectable dose of the present method for HE4. 3.3. Recovery The analytical recovery was studied by adding purified HE4 antigen to 3 serum samples from different patients. The results were given in Table 1. The recoveries of added analytes were in the range of 90–110%. 3.4. Precision Within-and between-assay imprecision were determined using three serum samples and the same batch of reagents on separate days as shown in Table 2. Total imprecision of the present assay were ranged from 4.1% to 6.9%. As expected, the imprecision of the present assay was remarkably low. 3.5. Linearity Table 3 showed the results of our evaluation of the dilution linearity of this assay when we used samples serially diluted with assay buffer. Expected values were derived from initial concentrations of analytes Table 2 Precision test of our novel assay. Sample
Intra-assay precision (n = 12)
Intra-assay precision (n = 20)
Fig. 3. High-dose signal saturation (hook-effect) of our novel assay for HE4.
1 2 3 1 2 3
CV, coefficient of variation; SD, standard deviation.
HE4 (pmol/L) Mean
SD
CV
27.5 42.6 72.5 28.2 43.3 71.2
1.12 2.64 3.55 1.41 2.98 4.77
4.1% 6.2% 4.9% 5.0% 6.9% 6.7%
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Table 3 Dilution linearity test of our novel assay for HE4. Sample
Dilution
HE4 (pmol/L) Expected
1
2
3
NA 1:2 1:4 1:8 1:16 NA 1:2 1:4 1:8 1:16 NA 1:2 1:4 1:8 1:16
Observed (n = 3) 47.5 23.2 11.6 5.98 2.89 81.6 39.9 20.7 9.9 5.02 135.8 69.4 32.3 17.5 8.8
23.7 11.8 5.93 2.96 40.8 20.4 10.2 5.10 67.9 33.9 16.9 8.5
Recovery 97.8% 98.3% 100.8% 97.6% 97.8% 101.4% 97.0% 98.4% 102.2% 95.3% 103.5% 103.5%
Fig. 4. The concentration distribution of 459 normal serum samples.
NA, not applicable.
in the undiluted samples. Correlating the results obtained from assay with the expected concentrations, we found that expected values were identical with measured values.
assessed. The cross-reactivity was expressed as the percent ratio between measured and expected values. Results in Table 5 showed that no significant cross-reactivity was identified. 3.8. Normal reference range
3.6. Interference The effect of hemolysis, lipemia and bilirubinemia was assessed by adding hemolysate, bilirubin (unconjugated) and triglyceride to the serum samples, respectively. The recoveries were calculated from HE4 values determined before and after addition of interfering substances. The recoveries were between 91.9% and 105.8% (Table 4).
459 samples of normal healthy people were detected by the present method we developed. The result was shown in Fig. 4. When the concentration of HE4 ≤ 90 pmol/l, 96.9% of the samples are successfully classified as normal and when concentration of HE4 ≤ 100 pmol/l, this improves to 97.1%. Furthermore, we made the ROC for our novel assay (Fig. 5). At the concentration of 73.45 pmol/l, the Correct diagnosis index = Sensitivity−(1−Specificity) was the maximum. Comprehensively referencing to the literature for similar results, we set our cutoff value at 90 pmol/l.
3.7. Specificity 3.9. Comparison with ECLIA To investigate the specificity of the developed HE4 assay, the effect of adding other commonly measured proteins alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen 125 (CA125) and cancer antigen 199 (CA199) to normal human serum samples was
Table 4 Interference test of our novel assay for HE4. Interfering substances
Hemoglobin
Bilirubin
Triglyceride
HE4 in 170 clinical samples were analyzed by the present assay. The correlation of the HE4 values obtained by this method and those obtained by ECLIA was excellent; the regression equation was Y = 0.971× X − 0.571 (r2 = 0.984, P b 0.0001). The comparison of HE4 values obtained by the two methods was shown in Fig. 6. 4. Discussion
Concentration (ng/ml)
0 500 1000 0 500 1000 0 500 1000
HE4 (pmol/L) Mean (n = 3)
Recovery
56.3 57.6 59.3 63.5 64.4 58.4 78.3 76.9 82.9
– 102.3% 105.3% – 101.4% 91.9% – 98.2% 105.8%
We established a 96-well plate based homogeneous chemiluminescent sandwich immunoassay for the rapid quantitation of HE4 in human
Table 5 Specificity test of our novel assay for HE4. Interfering substances
Concentration
AFP CEA CA125 CA199
1000 U/ml 560 ng/nl 500 U/ml 600 U/ml
HE4 (pmol/L) Mean (n = 3)
Cross-reactivity rate (%)
0.13 0.11 0.15 0.21
0.01 0.02 0.03 0.03
Fig. 5. The ROC cure for our novel assay.
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Scientific Instrument and Equipment Development Projects of China (Grant No. 61427807). The authors thank the National Natural Science Foundation of China. They also thank Nanfang Hospital, Guangzhou, China, for providing reagents at a reduced cost.
References
Fig. 6. Graphical comparisons of the present assay and ECLIA results for quantitation of HE4.
serum. As shown in Fig.1, one anti-HE4 monoclonal antibody was coated on acceptor beads and another anti-HE4 antibody was biotinylated. It also contains donor beads coated with streptavidin. The resulting complex was quantified in the well by excitation of the donor beads with laser irradiation at 680 nm. Then, specific delayed sharp chemiluminescent emission peak was monitored at about 615 nm. The whole detection time for HE4 only took 30 min in the present assay. AlphaLISA is a simple, small and automatable method (Poulsen and Jensen, 2007; McGiven et al., 2008; Szekeres et al., 2008; Bielefeld-Sevigny, 2009; Zou et al., 2013). The realization of rapid quantitation depends on fast mix-and-measure protocols (Bielefeld-Sevigny, 2009). The assay can be handled manually or as an automatic setup, which facilitates assay development and makes the technology more versatile. Homogenous assay systems are potentially more sensitive to serum interference. The most prominent types of general interference are inner filter effects and singlet oxygen quenchers. By using Eu complex in the acceptor beads emitting light at 615 nm (Bielefeld-Sevigny, 2009), the inner filter effects are minimal when testing serum. Results indicated that the assay was unaffected by interfering substances including hemolysate, bilirubin and triglyceride at concentrations that might be expected in a routine clinical laboratory setting. Notably, the system is free of problems with sample autofluorescence by using the time-resolved mode and because of the large stokes shift between the emission and excitation wavelengths (Lin et al., 2014; Lin et al., 2015a; Lin et al., 2015b). In addition, cross-reactivity data with major interfering tumor markers showed the assay was highly specific for HE4. In summary, we have developed a homogeneous chemiluminescent sandwich immunoassay, which was designed specifically as a rapid, sensitive and homogeneous reaction measurement method for quantitation of the HE4 in human serum. Compared with the results from ECLIA kit (Roche) in 170 serum samples, there was a satisfied correlation coefficient of 0.984. Currently, this method demonstrated high sensitivity, wider effective detection range and excellent reproducibility for the quantitation of HE4, and offered the additional benefit for faster detection, resulting in a substantially faster assay. Based on this investigation, we established a good foundation for further development of other biomarkers using the same platform. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 21575058, 81271931) and National Key
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