Research article Received: 06 June 2013,
Revised: 17 June 2013,
Accepted: 15 August 2013
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2587
A rapid and sensitive method based on magnetic beads for the detection of hepatitis B virus surface antigen in human serum Zhi-Qi Ren,† Tian-Cai Liu,† Jing-Yuan Hou, Mei-Jun Chen, Zhen-Hua Chen, Guan-Feng Lin and Ying-Song Wu* ABSTRACT: Current clinically assays, such as enzyme-linked immunosorbent assay and chemiluminescence immunoassay, for hepatitis B surface antigen (HBsAg) are inferior in terms of either sensitivity and accuracy or rapid and high-throughput analysis. A novel assay based on magnetic beads and time-resolved fluoroimmunoassay was developed for the quantitative determination of HBsAg in human serum. HBsAg was captured using two types of anti-HBsAg monoclonal antibodies (B028, S015) immobilized on to magnetic beads and detected using europium-labeled anti-HBsAg polyclonal detection antibody. Finally, the assay yielded a high sensitivity (0.02 IU/mL) and a wide dynamic range (0.02–700 IU/mL) for HBsAg when performed under optimal conditions. Satisfactory accuracy, recovery and specificity were also demonstrated. The intra- and interassay coefficients of variation were 4.7–8.7% and 3.8–7.5%, respectively. The performance of this assay was further assessed against a well-established commercial chemiluminescence immunoassay kit with 399 clinical serum samples. It was revealed that the test results for the two methods were in good correlation (Y = 1.182X – 0.017, R = 0.989). In the current study, we demonstrated that this novel time-resolved fluoroimmunoassay could be used: as a highly sensitive, automated and high-throughput immunoassay for the diagnosis of acute or chronic hepatitis B virus infection; for the screening of blood or organ donors; and for the surveillance of persons at risk of acquiring or transmitting hepatitis B virus. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: magnetic beads; time-resolved fluoroimmunoassay; hepatitis B surface antibody; hepatitis B virus; rapid and sensitive detection
Introduction Hepatitis B is an infectious liver disease caused by the hepatitis B virus (HBV), and it poses a serious threat to human health on a global scale (1,2). While transmission of HBV has been controlled largely in Western countries, the disease remains epidemic in other highly populated regions, particularly China. About onethird of the 2 billion individuals infected worldwide (3) with HBV reside in China (4). It has been well documented that of all chronic hepatitis B sufferers, 15–25% will develop serious liver problems, including liver damage, cirrhosis and hepatocellular carcinoma. It is estimated that the annual mortality rate from hepatitis B-related liver disease is in excess of 600,000 globally (5,6). HBV surface antigen (HBsAg) is the first serological marker to appear following acute HBV infection and routinely used in the diagnosis of acute or chronic HBV infection (7,8). The detection of HBsAg in serum is pivotal to the discovery of HBV and remains the cornerstone of diagnosis (9,10). Seropositivity for HBsAg indicates that potential infectivity and detection should be conducted for hepatitis B high-risk groups, HBV carriers and pregnant women. In addition, it is also valuable for follow-up treatment in patients with this disease (11). A major concern in hepatitis B today is to continue to improve detection sensitivity of HBV. Traditional methods, such as solid-phase enzyme-linked immunosorbent (ELISAs) (12–14), time-resolved fluoroimmunoassay (TRFIA) (15,16) and chemiluminescence immunoassay (CLIA) (17,18), have been used for the detection
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of HBsAg. Despite considerable advancements in technology, many disadvantages still exist. ELISA and TRFIA are inferior in terms of sensitivity and accuracy, while CLIA is inferior in highthroughput analysis. Thus, they cannot meet the fast, sensitive and high-throughput analysis needed in modern life science and medicine. In this paper, we developed a novel method that integrated the advantages of magnetic beads and TRFIA for the rapid and sensitive detection of HBsAg. Measurement of assay parameters, such as sensitivity, linearity, precision, recovery and specificity, were all addressed in this work. Three hundred and ninety-nine clinical serum samples were analyzed to evaluate the feasibility. The advantages of TRFIA as a basic platform have been described extensively elsewhere (19–23). The biggest innovation came in pushing the limits of magnetic beads (24–26) as the immobilization matrix rather than the 96-well microtiter plates
* Correspondence to: Professor Ying-Song Wu, Institute of Antibody Engineering, School of Biotechnology, Southern Medical University, Guangzhou 510515, Guangdong, People’s Republic of China. E-mail: [email protected] †
These authors contributed equally to this work. Institute of Antibody Engineering, School of Biotechnology, Southern Medical University, Guangzhou 510515, Guangdong, People’s Republic of China
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Z.-Q. Ren et al. of classical TRFIA. This new protocol exhibits superiorities in aspects of rapid testing, specificity, especially in sensitivity and wide detection range due to the advantages of magnetic beads and lanthanide chelates. They offer great promise for use in HBsAg analysis and ultimately for inclusion in clinical diagnosis, prognosis and therapeutic monitoring and blood screening by the immunoassays used in this investigation.
overnight at room temperature. Then the supernatant was removed by the magnetic separator and the beads suspended with blocking buffer. The washing process was repeated three times followed by incubation with 1 mL blocking buffer for 3 h at room temperature. After the final washing step, the antibody–magnetic bead conjugates were resuspended in Tris-buffered saline Tween-20 buffer and stored at 4 °C until required.
Experimental
Preparation of Eu3+-labeled polyclonal antibody
Reagents and apparatus
First, 0.5 mg of polyclonal antibody was diluted in Eu3+ chelatelabeling buffer to a final concentration of 1 mg/mL. Then 0.1 mg of DTTA-Eu3+ was added and the mixture was incubated overnight at room temperature. Free chelates were removed from labeled antibodies using gel filtration on a Sephadex G-50 column (1.5 cm × 40 cm). The concentration of antibody was determined spectrophotometrically at 280 nm. The Eu3+labeled polyclonal antibody (100 μg/mL) was stored in assay buffer at 4 °C until required for use (27,28).
Bovine serum albumin (BSA), 4-morpholineethanesulfonic acid, proclin-300, Tween-20, N-hydroxysulfosuccinimide and 1-ethyl3-(3-dimethylaminopropyl) carbodiimide hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO, USA). DTTA-Eu3+ (N 1 -[p-isothiocyannatobenzyl]-diethylene-triamine-N 1 ,N 2 , N3-tetraacetate-Eu3+) was obtained from PerkinElmer Life and Analytical Sciences (PerkinElmer, Waltham, MA, USA). Sephadex G-50 was purchased from Amersham Pharmacia Biotech (Piscataway, NJ, USA). Magnetic beads were purchased from Merck (Whitehouse Station, NJ, USA). Affinity purified anti-HBsAg monoclonal antibodies (B028, S015) and polyclonal antibodies were all produced in mouse and obtained from Medix (Grankulla, Finland). Monoclonal antibody B028 was specific for HBsAg subtypes adw and adr, and S015 for HBsAg subtype ay. CLIA HBsAg immunoassay kit was obtained from Abbott Laboratories (Chicago, IL, USA). The enhancement solution for Eu3+ dissociation and a 1420 Multilabel Counter (Victor3TM) were purchased from PerkinElmer Wallac (Turku, Finland). All other solvents were of analytical grade. Solutions The coating buffer for monoclonal antibody attachment to magnetic beads was 0.1 mol/L 4-morpholineethanesulfonic acid (pH 5.0). Blocking buffer contained 0.5 mol/L sucrose and 5% BSA (pH 7.0). The washing buffer was 0.05 mol/L Tris–HCl, 0.01% Tween-20 and 0.15 mol/L NaCl (pH 7.8). Tris-buffered saline Tween-20, containing 0.025 mol/L Tris, 0.15 mol/L NaCl and 0.05% Tween-20 (pH 7.4), was used as the preservation and dilution buffer for antibody-coated magnetic beads. The Eu3+ chelate-labeling buffer was 0.05 mol/L Na2CO3 (pH 9.0). TRFIA assay buffer used to dilute the Eu3+ chelate-labeled antibody containing 0.05 mol/L Tris–HCl buffer, 1.5% polyethyleneglycol 6000, 0.3 mmol/L BSA, 0.01% Proclin-300, 0.15 mol/L NaCl, 0.02% (w/v) bovine globulin and 0.01% Tween-20 (pH 7.8). Preparation of monoclonal antibody-coated magnetic beads The conjugates of magnetic beads and antibody were prepared as follows. Briefly, activation of the beads carboxyl groups was achieved by the addition of 25 μL fresh 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (10 mg/mL) and 40 μL N-hydroxysulfosuccinimide (10 mg/mL) to 10 mg of carboxyl-modified magnetic beads in 1 mL coating buffer, and rotated (60 rpm) end-over-end for 30 min at room temperature. Once the carboxyl groups had been activated, the magnetic beads were separated from the supernatant using a magnetic separator. One hundred μg of B028 and 60 μg of S015 monoclonal antibody in 1 mL binding buffer was added to the magnetic beads and mixed by gentle rotation (60 rpm)
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Samples and method The sensitivity of the assay was evaluated by testing serial dilutions of the second WHO HBsAg International Standard with concentrations ranging from 0 to 0.500 IU/mL. A total of 399 human serum samples were kindly provided by the 302 Military Hospital of China (Guangdong, China) and were tested with the present method and CLIA. Data analysis used McNemar’s test by SPSS 13.0 (Chicago, IL, USA), and P < 0.05 was considered statistically significant. Sample means and standard deviations (SD) were prepared using Microsoft Excel. Standard curves were obtained by plotting the logarithm of fluorescence intensity (Y) against the logarithm of analyte concentration (X) and fitting a logistic equation using Origin Pro7.5 (OriginLab Corporation, Northampton, Massachusetts, USA): Log(Y) = A + B × Log(X). Pearson’s linear regression was used to display the linearity and correlations. Assay configuration The prototype assay is a new two-step TRFIA that uses double monoclonal antibody coating on the magnetic beads to capture HBsAg and Eu3+-labeled polyclonal conjugates to detect the captured antigen (Fig. 1). A series of HBsAg standards (0, 0.4, 2, 10, 50, 300 IU/mL) were measured to determine the optimized assay and construct a standard curve. The assay was used to, first establish (a) the optimum temperature (25 °C or 37 °C), and (b) optimum incubation time (10–60 min) in the order as written. The optimum concentration of antibody–magnetic bead conjugates (100–700 μg/mL) and Eu3+labeled polyclonal antibody (0.1–2 μg /mL) was determined at an optimum temperature (25 °C) and incubation time (40 min) established for antigen/antibody complex formation. One hundred μL of HBsAg standard or serum samples and 50 μL antibody–magnetic bead conjugates were dispensed into the wells of 96-well microtiter plates and then incubated for 40 min with gentle shaking at room temperature. The magnetic beads with bound HBsAg were purified from solution using a magnetic separator. After washing three times, 100 μL Eu3+labeled polyclonal antibody conjugates were added. The reaction was allowed to proceed for 40 min with shaking at 25 °C, washed as described previously. The resulting complexes were
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A rapid and sensitive detection of HBsAg
Figure 1. Diagram of the magnetic beads-based time-resolved fluoroimmunoassay method for detection of HBsAg. EDC, N-hydroxysulfosuccinimide; HBsAg, hepatitis B surface antigen; NHS, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride.
resuspended in 200 μL of enhancement solution for 5 min. The concentration of HBsAg was quantified based on the fluorescent signal measured using the Victor3 1420 Multilabel Counter equipped with filters for Eu3+ (613 nm). Results were automatically calculated against the HBsAg index calibration curve. A test value above a signal/negative value ratio of 2.1 was considered positive.
mL and 0.2 μg/mL, respectively. Therefore, the analytical characteristics of the proposed method were obtained under the optimum conditions.
Results Optimization of the incubation time and temperature In the present study, a series of incubation times ranging from 10 to 60 min were tested for the two-step sandwich reaction at 25 °C. The data indicated that the signal-to-noise ratio (Standard B/A) and fluorescence intensity (Standard F) increased with incubation time and did not reach a dynamic balance until 40 min (Fig. 2). Compared with conventional TRFIA (incubation for 120 min) (16), ELISA kit (incubation for 190 min) (13) and CLIA kit (incubation for 90 min) (18), these results demonstrated that the inclusion of magnetic beads played a vital role in increasing the reaction rate and reducing analysis time. As mentioned above, the temperature of 25 °C was chosen as the incubation temperature as no significant difference was observed when the assay was conducted at either 25 °C or 37 °C (not shown). Therefore, performance of the assay was displayed at 25 °C principally due to the convenience of operating. Based on these results, all further optimization steps for the two-step sandwich reaction would be conducted at 25 °C and incubation time of 40 min. Optimization of antibody–magnetic bead conjugates and Eu3+-labeled antibody concentrations The concentration of antibody–magnetic bead conjugates (100, 200, 300, 500 and 700 μg/mL) and Eu3+-labeled polyclonal antibody (0.1, 0.2, 0.4, 0.8 and 2 μg/mL) were optimized with an orthogonal arrangement. The sensitivity was different in each case and the best signal was achieved when the concentration of magnetic beads and Eu3+-labeled antibody were 500 μg/
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Figure 2. Optimization of the incubation time. (A) Influence of incubation time on the signal-to-noise ratio and fluorescence intensity of standard A and B; (B) Influence of incubation time on fluorescence intensity of standard F. CPS, counts/s.
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Z.-Q. Ren et al. Assay sensitivity, linear range and accuracy A series of HBsAg standards (0, 0.4, 2, 10, 50, 300 IU/mL) were measured using the optimized assay and a standard curve constructed (Fig. 3). The linear equation for the standard curve was log(Y) = 0.9215 log(X) + 4.1653 with a correlation coefficient of 0.999 (X is the concentration of HBsAg; Y is the fluorescence intensity). The sensitivity of the assay was evaluated by testing serial dilutions of the second WHO HBsAg International Standard with concentrations ranging from 0 to 0.500 IU/mL (Table 1). The HBsAg cutoff value was 2.1 times that of normal human plasma. The signal/cutoff ratio ≥ 1.00 is considered positive for samples. It was found that the novel TRFIA produced in this study is capable of measuring a wide range of HBsAg concentrations from 0.02 to 700 IU/mL, with a sensitivity of 0.02 IU/mL. The result is equivalent or superior to the ELISA (40–1000 IU/mL) (13), traditional TRFIA (0.4–600 IU/mL) (16) and commercially available CLIA kit (0.02–500 IU/mL) (29). Improvement in HBsAg assay
sensitivity is essential to reduce the window to detect acute HBV infection. Results obtained strongly correlate to the concentration of HBsAg while it is lower than 700 IU/mL. Specimens with an HBsAg value exceeding 700 IU/mL require a 1: 100 or greater dilution to bring them into the range of the calibration curve. In addition, the quantitative determination of the WHO International Reference Panel (calibrated HBsAg concentrations: 33 IU/vial) for HBsAg was essential in assessing the detection accuracy for very low levels of HBsAg in human serum. The detection accuracy was analyzed using recovery (ε = a/A, a is the test value, A is the value provided by WHO reference material) and 90% ≤ ε ≥ 110% considered statistically acceptable (Table 2). Based on these data, the prototype assay displayed an accurate detection for HBsAg in samples. Assay precision and recovery The coefficient of variation (CV) was used to assess the precision of the assay. Three concentrations of serum control (1.6, 84.7, 200 IU/mL) were tested using the optimized method. Each serum sample was tested fivefold and the mean determined for each. The intra-assay CV and interassay CV were calculated by the replicate analysis of these samples. As presented in Table 3, the intra-assay CVs and the interassay CVs did not exceed 10% for each serum concentrations. The three serum controls were also measured to evaluate the recovery of assay (Table 4). As shown in the table, the precisions and recoveries of the proposed assay in our cases complied with international standards well. Assay specificity
Figure 3. Calibration curves for the magnetic beads-based time-resolved fluoroimmunoassay method for detection of HBsAg standards. Fluorescence intensity and standard deviations were calculated from a set of five measurements. CPS, counts/s; HBsAg, hepatitis B surface antigen.
Table 1. Assay sensitivity: test of serially diluted the 2nd International WHO Standard The 2nd WHO International Standard for HBsAg (IU/mL)
S/CO values (magnetic bead-based TRFIA prototype)
0.500 0.250 0.150 0.100 0.050 0.040 0.030 0.020 0.010 0.000 HBsAg concentration (IU/mL) at which the S/CO = 1.00
24.21 12.28 7.43 4.89 2.45 1.98 1.50 1.01 0.82 0.67 0.02
HBsAg, hepatitis B surface antigen; TRFIA, time-resolved fluoroimmunoassay.
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Most false-positive results using current HBsAg detection methods can be observed for pregnant women, for autoimmune diseases, and chronic liver diseases from non-HBV related causes. Thus, a total of 60 specimens with hepatitis B core antigen, hepatitis B e antigen, hepatitis C virus, human immunodeficiency virus, Treponema pallidum and rheumatoid factor were measured to evaluate the assay’s specificity for HBsAg (30). Specimens with an initial test result signal/cutoff ratio ≥ 1.00 were retested in duplicate. As presented in Table 5, the assay specificity was 100%, which indicated that our proposed immunoassay possesses a high specificity for HBsAg, and no cross-reactivity with hepatitis B core antigen, hepatitis B e antigen, hepatitis C virus, human immunodeficiency virus, Treponema pallidum and rheumatoid factor was detected. Compared with chemiluminescence immunoassay To evaluate further the value of the novel TRFIA for clinical applications, 399 blood samples (negative or positive for HBsAg) were screened in parallel using the prototype assay and the commercially available CLIA kit. A single sample for each specimen was analyzed using each assay. Differences in clinical accuracy between the two tests were evaluated with McNemar’s test for correlated proportions (Table 6). The P was obtained by 1 and considered to be statistically insignificant. Among them 231 positive blood samples were selected to demonstrate the correlation between the two methods using linear regression analysis (Fig. 4). The correlation coefficient was R = 0.989 and the equation of the regression curve was Y = 1.182X – 0.017 (X is the
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A rapid and sensitive detection of HBsAg Table 2. Accuracy: quantitative determination of WHO international reference panel HBsAg subtype
Concentration of HBsAg reference panel (IU/mL)
adw adr ay
0.4
1.0
2.0
4.0
– 0.37 (92.5%) –
1.02 (102.0%) 0.99 (99.0%) 1.01 (101.0%)
2.03 (101.5%) 2.06 (103.0%) 1.95 (97.5%)
4.27 (106.7%) – 4.14 (103.5%)
HBsAg, hepatitis B surface antigen.
Table 3. Assay precision: interassay and intra-assay stability of the proposed method
Table 5. Assay specificity: effect of potentially interfering substances on the determination of hepatitis B surface antigen
HBsAg add (IU/mL)
Interfering substance
N
Hepatitis B core antigen Hepatitis B e antigen Hepatitis C virus Human immunodeficiency virus Treponema pallidum Rheumatoid factor Samples tested Reactive (S/CO ≥ 1.00) S/CO range
10 10 10 10 10 10 60
1.6 84.7 200
Interassay precisiona (n = 8)
Intra-assay precisiona (n = 8) Measured (IU/mL)
CV (%)
Measured (IU/mL)
CV (%)
1.48 ± 0.07 85.51 ± 4.69 196.61 ± 17.17
4.7% 5.5% 8.7%
1.54 ± 0.06 81.99 ± 6.15 203.62 ± 10.25
3.8% 7.5% 5.0%
CV, coefficient of variation; HBsAg, hepatitis B surface antigen. a Mean ± standard deviation.
HBsAg concentration estimated with the proposed method; Y is that from CLIA). Linear regression analysis revealed good correlations between the two methods. The novel TRFIA provides sensitivity that is at least equivalent to the clinically approved assay. The sensitive and accurate detection of HBsAg is critical to the identification of infection and the prevention of transfusion-transmitted disease. The analysis and test results proved that the magnetic bead-based TRFIA is accurate, sufficiently effective and will be a potentially powerful tool for diagnostic testing, therapeutic monitoring and blood screening in endemic areas of hepatitis B infection.
Discussion In our study, Eu3+ chelates were used as the fluorescent markers, firstly, due to its superior fluorescence signal strength at wavelengths of 613 nm, and second because, the long decay time of the fluorescent signal (10–6–10–3 s), large Stokes’ shift (> 200 nm), and narrow emission peak allow the label to be read
Reactive S/CO range 0 0 0 0 0 0
0.79–0.83 0.64–0.85 0.71–0.88 0.61–0.82 0.79–0.86 0.67–0.78
0 0.61–0.88
once the nonspecific background has already peaked and decayed (19–23). All these properties result in an enhanced signal-to-noise ratio and a significant improvement in assay sensitivity. Despite these advantages, TRFIA has some significant drawbacks predominantly because of the constraints in using 96-well microtiter plates. Because of the small surface areas within the wells of the microtiter plate, the amount of antigens or antibodies that can be detected is limited. Furthermore, physical adsorption of the coating antigen/antibody to the surface of the plate is inefficient and leads to significant wastage of reagents and materials. It is also time consuming, whereby it generally takes 1 h to attain equilibrium for an immunoreaction because of the small contact area of the solid–liquid interface in the wells of the microtiter plate (27). Over the traditional carriers, the magnetic beads as the immobilization matrix affords a large number of advantages. First, by the very nature of its greater specific surface area, more antibodies can bind to the magnetic beads, leading to the capture of more HBsAg. In contrast to physical adsorption, covalent bond formation between the antibody and magnetic beads
Table 4. Recoveries determined using the proposed method via spiking three different concentration levels of maternal serum controls into HBsAg standards Sample
Original (IU/mL)
Spiked (IU/mL)
Measureda (IU/mL)
Recovery (%)
HBsAg
100 100 100
1.6 84.7 200
97.61 ± 6.07 177.28 ± 4.84 308.19 ± 9.70
96.1% 96.0% 102.7%
HBsAg, hepatitis B surface antigen. a Mean ± standard deviation.
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Z.-Q. Ren et al. Table 6. Comparision between the proposed method and chemiluminescence immunoassay for the test of 399 blood samples Proposed method + – Total
Chemiluminescence immunoassay +
–
231 0 231
0 168 168
Total
231 168 399
+/ , positive/negative.
Funding Sources The National Natural Science Foundation of China (grant no. 81271931), the Natural Science Foundation of Guangdong Province (grant no. S2012010009547) and Scientific Research Foundation of Introducing Talents of Southern Medical University (2009). Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (grant no. 81271931), the Natural Science Foundation of Guangdong Province (grant no. S2012010009547) and Scientific Research Foundation of Introducing Talents of Southern Medical University (2009). We would like to thank all related committees or departments.
Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
References
Figure 4. Plot of the results obtained using the proposed method versus those obtained using CLIA commercial kits for hepatitis B surface antigen detection in 231 serum samples. CLIA, chemiluminescence immunoassay.
leads to a much stronger association. Furthermore, antibodycoated magnetic beads suspended in the reaction solution could increase the reaction rate and consequently reduce reaction times (31–35). All of these advantages result in an appreciable improvement in detection limit, reduction in sampling volume of blood specimens and quicker analysis. In addition, the application of an external magnetic field ensures that the imprinted molecules are separated and concentrated rapidly from unwanted constituents. It is also readily automated as a high-throughput assay for the screening of thousands of samples per day. In conclusion, a novel assay for the enhanced detection of HBsAg in human serum characterized by covalently attaching antibody to the surface of magnetic beads was developed. This method exhibited the superior properties of magnetic beads in combination with TRFIA. The current system using magnetic beads as a solid support provides better flexibility in assay optimization and quantitative determination of human serum. By using a combination of monoclonal and polyclonal antibody conjugates for detection, a high sensitivity and wide linear detection range were obtained under optimal assay conditions. The HBsAg assay offers rapid testing, high sensitivity and precision, good specificity and a wide detection range, while at the same time providing future potential for automation and high throughput.
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27. Hou JY, Liu TC, Lin GF, Li ZX, Zou LP, Li M, Wu YS. Development of an immunomagnetic bead-based time-resolved fluorescence immunoassay for rapid determination of levels of carcinoembryonic antigen in human serum. Anal Chim Acta 2012;734:93–8. 28. Lin GF, Liu TC, Zou LP, Hou JY, Wu YS. Development of a dual-label time-resolved fluoroimmunoassay for the detection of alphafetoprotein and hepatitis B virus surface antigen. Luminescence 2013;28:401–6. 29. Scheiblauer H, Soboll H, Nick S. Evaluation of 17 CE-marked HBsAg assays with respect to clinical sensitivity, analytical sensitivity, and hepatitis B virus mutant detection. J Med Virol 2006;78(Suppl 1):66–70. 30. Lou SC, Pearce SK, Lukaszewska TX, Taylor RE, Williams GT, Leary TP. An improved Abbott ARCHITECT assay for the detection of hepatitis B virus surface antigen (HBsAg). J Clin Virol 2011;51:59–63. 31. Osaki T, Taguchi H, Yamaguchi H, Kamiya S. Detection of Helicobacter pylori in fecal samples of gnotobiotic mice infected with H. pylori by an immunomagnetic-bead separation technique. J Clin Microbiol 1998;36:321–3. 32. DeCory TR, Durst RA, Zimmerman SJ, Garringer LA, Paluca G, DeCory HH, Montagna RA. Development of an immunomagnetic beadimmunoliposome fluorescence assay for rapid detection of Escherichia coli O157:H7 in aqueous samples and comparison of the assay with a standard microbiological method. Appl Environ Microbiol 2005;71:1856–64. 33. Beyor N, Seo TS, Liu P, Mathies RA. Immunomagnetic bead-based cell concentration microdevice for dilute pathogen detection. Biomed Microdevices 2008;10:909–17. 34. Conlan JV, Khounsy S, Blacksell SD, Morrissy CJ, Wilks CR, Gleeson LJ. Development and evaluation of a rapid immunomagnetic bead assay for the detection of classical swine fever virus antigen. Trop Anim Health Prod 2009;41:913–20. 35. McAteer MA, Schneider JE, Ali ZA, Warrick N, Bursill CA, von Zur MC, Greaves DR, Neubauer S, Channon KM, Choudhury RP. Magnetic resonance imaging of endothelial adhesion molecules in mouse atherosclerosis using dual-targeted microparticles of iron oxide. Arterioscler Thromb Vasc Biol 2008;28:77–83.
Copyright © 2013 John Wiley & Sons, Ltd.
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Revised: 17 June 2013,
Accepted: 15 August 2013
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2587
A rapid and sensitive method based on magnetic beads for the detection of hepatitis B virus surface antigen in human serum Zhi-Qi Ren,† Tian-Cai Liu,† Jing-Yuan Hou, Mei-Jun Chen, Zhen-Hua Chen, Guan-Feng Lin and Ying-Song Wu* ABSTRACT: Current clinically assays, such as enzyme-linked immunosorbent assay and chemiluminescence immunoassay, for hepatitis B surface antigen (HBsAg) are inferior in terms of either sensitivity and accuracy or rapid and high-throughput analysis. A novel assay based on magnetic beads and time-resolved fluoroimmunoassay was developed for the quantitative determination of HBsAg in human serum. HBsAg was captured using two types of anti-HBsAg monoclonal antibodies (B028, S015) immobilized on to magnetic beads and detected using europium-labeled anti-HBsAg polyclonal detection antibody. Finally, the assay yielded a high sensitivity (0.02 IU/mL) and a wide dynamic range (0.02–700 IU/mL) for HBsAg when performed under optimal conditions. Satisfactory accuracy, recovery and specificity were also demonstrated. The intra- and interassay coefficients of variation were 4.7–8.7% and 3.8–7.5%, respectively. The performance of this assay was further assessed against a well-established commercial chemiluminescence immunoassay kit with 399 clinical serum samples. It was revealed that the test results for the two methods were in good correlation (Y = 1.182X – 0.017, R = 0.989). In the current study, we demonstrated that this novel time-resolved fluoroimmunoassay could be used: as a highly sensitive, automated and high-throughput immunoassay for the diagnosis of acute or chronic hepatitis B virus infection; for the screening of blood or organ donors; and for the surveillance of persons at risk of acquiring or transmitting hepatitis B virus. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: magnetic beads; time-resolved fluoroimmunoassay; hepatitis B surface antibody; hepatitis B virus; rapid and sensitive detection
Introduction Hepatitis B is an infectious liver disease caused by the hepatitis B virus (HBV), and it poses a serious threat to human health on a global scale (1,2). While transmission of HBV has been controlled largely in Western countries, the disease remains epidemic in other highly populated regions, particularly China. About onethird of the 2 billion individuals infected worldwide (3) with HBV reside in China (4). It has been well documented that of all chronic hepatitis B sufferers, 15–25% will develop serious liver problems, including liver damage, cirrhosis and hepatocellular carcinoma. It is estimated that the annual mortality rate from hepatitis B-related liver disease is in excess of 600,000 globally (5,6). HBV surface antigen (HBsAg) is the first serological marker to appear following acute HBV infection and routinely used in the diagnosis of acute or chronic HBV infection (7,8). The detection of HBsAg in serum is pivotal to the discovery of HBV and remains the cornerstone of diagnosis (9,10). Seropositivity for HBsAg indicates that potential infectivity and detection should be conducted for hepatitis B high-risk groups, HBV carriers and pregnant women. In addition, it is also valuable for follow-up treatment in patients with this disease (11). A major concern in hepatitis B today is to continue to improve detection sensitivity of HBV. Traditional methods, such as solid-phase enzyme-linked immunosorbent (ELISAs) (12–14), time-resolved fluoroimmunoassay (TRFIA) (15,16) and chemiluminescence immunoassay (CLIA) (17,18), have been used for the detection
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of HBsAg. Despite considerable advancements in technology, many disadvantages still exist. ELISA and TRFIA are inferior in terms of sensitivity and accuracy, while CLIA is inferior in highthroughput analysis. Thus, they cannot meet the fast, sensitive and high-throughput analysis needed in modern life science and medicine. In this paper, we developed a novel method that integrated the advantages of magnetic beads and TRFIA for the rapid and sensitive detection of HBsAg. Measurement of assay parameters, such as sensitivity, linearity, precision, recovery and specificity, were all addressed in this work. Three hundred and ninety-nine clinical serum samples were analyzed to evaluate the feasibility. The advantages of TRFIA as a basic platform have been described extensively elsewhere (19–23). The biggest innovation came in pushing the limits of magnetic beads (24–26) as the immobilization matrix rather than the 96-well microtiter plates
* Correspondence to: Professor Ying-Song Wu, Institute of Antibody Engineering, School of Biotechnology, Southern Medical University, Guangzhou 510515, Guangdong, People’s Republic of China. E-mail: [email protected] †
These authors contributed equally to this work. Institute of Antibody Engineering, School of Biotechnology, Southern Medical University, Guangzhou 510515, Guangdong, People’s Republic of China
Copyright © 2013 John Wiley & Sons, Ltd.
Z.-Q. Ren et al. of classical TRFIA. This new protocol exhibits superiorities in aspects of rapid testing, specificity, especially in sensitivity and wide detection range due to the advantages of magnetic beads and lanthanide chelates. They offer great promise for use in HBsAg analysis and ultimately for inclusion in clinical diagnosis, prognosis and therapeutic monitoring and blood screening by the immunoassays used in this investigation.
overnight at room temperature. Then the supernatant was removed by the magnetic separator and the beads suspended with blocking buffer. The washing process was repeated three times followed by incubation with 1 mL blocking buffer for 3 h at room temperature. After the final washing step, the antibody–magnetic bead conjugates were resuspended in Tris-buffered saline Tween-20 buffer and stored at 4 °C until required.
Experimental
Preparation of Eu3+-labeled polyclonal antibody
Reagents and apparatus
First, 0.5 mg of polyclonal antibody was diluted in Eu3+ chelatelabeling buffer to a final concentration of 1 mg/mL. Then 0.1 mg of DTTA-Eu3+ was added and the mixture was incubated overnight at room temperature. Free chelates were removed from labeled antibodies using gel filtration on a Sephadex G-50 column (1.5 cm × 40 cm). The concentration of antibody was determined spectrophotometrically at 280 nm. The Eu3+labeled polyclonal antibody (100 μg/mL) was stored in assay buffer at 4 °C until required for use (27,28).
Bovine serum albumin (BSA), 4-morpholineethanesulfonic acid, proclin-300, Tween-20, N-hydroxysulfosuccinimide and 1-ethyl3-(3-dimethylaminopropyl) carbodiimide hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO, USA). DTTA-Eu3+ (N 1 -[p-isothiocyannatobenzyl]-diethylene-triamine-N 1 ,N 2 , N3-tetraacetate-Eu3+) was obtained from PerkinElmer Life and Analytical Sciences (PerkinElmer, Waltham, MA, USA). Sephadex G-50 was purchased from Amersham Pharmacia Biotech (Piscataway, NJ, USA). Magnetic beads were purchased from Merck (Whitehouse Station, NJ, USA). Affinity purified anti-HBsAg monoclonal antibodies (B028, S015) and polyclonal antibodies were all produced in mouse and obtained from Medix (Grankulla, Finland). Monoclonal antibody B028 was specific for HBsAg subtypes adw and adr, and S015 for HBsAg subtype ay. CLIA HBsAg immunoassay kit was obtained from Abbott Laboratories (Chicago, IL, USA). The enhancement solution for Eu3+ dissociation and a 1420 Multilabel Counter (Victor3TM) were purchased from PerkinElmer Wallac (Turku, Finland). All other solvents were of analytical grade. Solutions The coating buffer for monoclonal antibody attachment to magnetic beads was 0.1 mol/L 4-morpholineethanesulfonic acid (pH 5.0). Blocking buffer contained 0.5 mol/L sucrose and 5% BSA (pH 7.0). The washing buffer was 0.05 mol/L Tris–HCl, 0.01% Tween-20 and 0.15 mol/L NaCl (pH 7.8). Tris-buffered saline Tween-20, containing 0.025 mol/L Tris, 0.15 mol/L NaCl and 0.05% Tween-20 (pH 7.4), was used as the preservation and dilution buffer for antibody-coated magnetic beads. The Eu3+ chelate-labeling buffer was 0.05 mol/L Na2CO3 (pH 9.0). TRFIA assay buffer used to dilute the Eu3+ chelate-labeled antibody containing 0.05 mol/L Tris–HCl buffer, 1.5% polyethyleneglycol 6000, 0.3 mmol/L BSA, 0.01% Proclin-300, 0.15 mol/L NaCl, 0.02% (w/v) bovine globulin and 0.01% Tween-20 (pH 7.8). Preparation of monoclonal antibody-coated magnetic beads The conjugates of magnetic beads and antibody were prepared as follows. Briefly, activation of the beads carboxyl groups was achieved by the addition of 25 μL fresh 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (10 mg/mL) and 40 μL N-hydroxysulfosuccinimide (10 mg/mL) to 10 mg of carboxyl-modified magnetic beads in 1 mL coating buffer, and rotated (60 rpm) end-over-end for 30 min at room temperature. Once the carboxyl groups had been activated, the magnetic beads were separated from the supernatant using a magnetic separator. One hundred μg of B028 and 60 μg of S015 monoclonal antibody in 1 mL binding buffer was added to the magnetic beads and mixed by gentle rotation (60 rpm)
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Samples and method The sensitivity of the assay was evaluated by testing serial dilutions of the second WHO HBsAg International Standard with concentrations ranging from 0 to 0.500 IU/mL. A total of 399 human serum samples were kindly provided by the 302 Military Hospital of China (Guangdong, China) and were tested with the present method and CLIA. Data analysis used McNemar’s test by SPSS 13.0 (Chicago, IL, USA), and P < 0.05 was considered statistically significant. Sample means and standard deviations (SD) were prepared using Microsoft Excel. Standard curves were obtained by plotting the logarithm of fluorescence intensity (Y) against the logarithm of analyte concentration (X) and fitting a logistic equation using Origin Pro7.5 (OriginLab Corporation, Northampton, Massachusetts, USA): Log(Y) = A + B × Log(X). Pearson’s linear regression was used to display the linearity and correlations. Assay configuration The prototype assay is a new two-step TRFIA that uses double monoclonal antibody coating on the magnetic beads to capture HBsAg and Eu3+-labeled polyclonal conjugates to detect the captured antigen (Fig. 1). A series of HBsAg standards (0, 0.4, 2, 10, 50, 300 IU/mL) were measured to determine the optimized assay and construct a standard curve. The assay was used to, first establish (a) the optimum temperature (25 °C or 37 °C), and (b) optimum incubation time (10–60 min) in the order as written. The optimum concentration of antibody–magnetic bead conjugates (100–700 μg/mL) and Eu3+labeled polyclonal antibody (0.1–2 μg /mL) was determined at an optimum temperature (25 °C) and incubation time (40 min) established for antigen/antibody complex formation. One hundred μL of HBsAg standard or serum samples and 50 μL antibody–magnetic bead conjugates were dispensed into the wells of 96-well microtiter plates and then incubated for 40 min with gentle shaking at room temperature. The magnetic beads with bound HBsAg were purified from solution using a magnetic separator. After washing three times, 100 μL Eu3+labeled polyclonal antibody conjugates were added. The reaction was allowed to proceed for 40 min with shaking at 25 °C, washed as described previously. The resulting complexes were
Copyright © 2013 John Wiley & Sons, Ltd.
Luminescence 2013
A rapid and sensitive detection of HBsAg
Figure 1. Diagram of the magnetic beads-based time-resolved fluoroimmunoassay method for detection of HBsAg. EDC, N-hydroxysulfosuccinimide; HBsAg, hepatitis B surface antigen; NHS, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride.
resuspended in 200 μL of enhancement solution for 5 min. The concentration of HBsAg was quantified based on the fluorescent signal measured using the Victor3 1420 Multilabel Counter equipped with filters for Eu3+ (613 nm). Results were automatically calculated against the HBsAg index calibration curve. A test value above a signal/negative value ratio of 2.1 was considered positive.
mL and 0.2 μg/mL, respectively. Therefore, the analytical characteristics of the proposed method were obtained under the optimum conditions.
Results Optimization of the incubation time and temperature In the present study, a series of incubation times ranging from 10 to 60 min were tested for the two-step sandwich reaction at 25 °C. The data indicated that the signal-to-noise ratio (Standard B/A) and fluorescence intensity (Standard F) increased with incubation time and did not reach a dynamic balance until 40 min (Fig. 2). Compared with conventional TRFIA (incubation for 120 min) (16), ELISA kit (incubation for 190 min) (13) and CLIA kit (incubation for 90 min) (18), these results demonstrated that the inclusion of magnetic beads played a vital role in increasing the reaction rate and reducing analysis time. As mentioned above, the temperature of 25 °C was chosen as the incubation temperature as no significant difference was observed when the assay was conducted at either 25 °C or 37 °C (not shown). Therefore, performance of the assay was displayed at 25 °C principally due to the convenience of operating. Based on these results, all further optimization steps for the two-step sandwich reaction would be conducted at 25 °C and incubation time of 40 min. Optimization of antibody–magnetic bead conjugates and Eu3+-labeled antibody concentrations The concentration of antibody–magnetic bead conjugates (100, 200, 300, 500 and 700 μg/mL) and Eu3+-labeled polyclonal antibody (0.1, 0.2, 0.4, 0.8 and 2 μg/mL) were optimized with an orthogonal arrangement. The sensitivity was different in each case and the best signal was achieved when the concentration of magnetic beads and Eu3+-labeled antibody were 500 μg/
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Figure 2. Optimization of the incubation time. (A) Influence of incubation time on the signal-to-noise ratio and fluorescence intensity of standard A and B; (B) Influence of incubation time on fluorescence intensity of standard F. CPS, counts/s.
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Z.-Q. Ren et al. Assay sensitivity, linear range and accuracy A series of HBsAg standards (0, 0.4, 2, 10, 50, 300 IU/mL) were measured using the optimized assay and a standard curve constructed (Fig. 3). The linear equation for the standard curve was log(Y) = 0.9215 log(X) + 4.1653 with a correlation coefficient of 0.999 (X is the concentration of HBsAg; Y is the fluorescence intensity). The sensitivity of the assay was evaluated by testing serial dilutions of the second WHO HBsAg International Standard with concentrations ranging from 0 to 0.500 IU/mL (Table 1). The HBsAg cutoff value was 2.1 times that of normal human plasma. The signal/cutoff ratio ≥ 1.00 is considered positive for samples. It was found that the novel TRFIA produced in this study is capable of measuring a wide range of HBsAg concentrations from 0.02 to 700 IU/mL, with a sensitivity of 0.02 IU/mL. The result is equivalent or superior to the ELISA (40–1000 IU/mL) (13), traditional TRFIA (0.4–600 IU/mL) (16) and commercially available CLIA kit (0.02–500 IU/mL) (29). Improvement in HBsAg assay
sensitivity is essential to reduce the window to detect acute HBV infection. Results obtained strongly correlate to the concentration of HBsAg while it is lower than 700 IU/mL. Specimens with an HBsAg value exceeding 700 IU/mL require a 1: 100 or greater dilution to bring them into the range of the calibration curve. In addition, the quantitative determination of the WHO International Reference Panel (calibrated HBsAg concentrations: 33 IU/vial) for HBsAg was essential in assessing the detection accuracy for very low levels of HBsAg in human serum. The detection accuracy was analyzed using recovery (ε = a/A, a is the test value, A is the value provided by WHO reference material) and 90% ≤ ε ≥ 110% considered statistically acceptable (Table 2). Based on these data, the prototype assay displayed an accurate detection for HBsAg in samples. Assay precision and recovery The coefficient of variation (CV) was used to assess the precision of the assay. Three concentrations of serum control (1.6, 84.7, 200 IU/mL) were tested using the optimized method. Each serum sample was tested fivefold and the mean determined for each. The intra-assay CV and interassay CV were calculated by the replicate analysis of these samples. As presented in Table 3, the intra-assay CVs and the interassay CVs did not exceed 10% for each serum concentrations. The three serum controls were also measured to evaluate the recovery of assay (Table 4). As shown in the table, the precisions and recoveries of the proposed assay in our cases complied with international standards well. Assay specificity
Figure 3. Calibration curves for the magnetic beads-based time-resolved fluoroimmunoassay method for detection of HBsAg standards. Fluorescence intensity and standard deviations were calculated from a set of five measurements. CPS, counts/s; HBsAg, hepatitis B surface antigen.
Table 1. Assay sensitivity: test of serially diluted the 2nd International WHO Standard The 2nd WHO International Standard for HBsAg (IU/mL)
S/CO values (magnetic bead-based TRFIA prototype)
0.500 0.250 0.150 0.100 0.050 0.040 0.030 0.020 0.010 0.000 HBsAg concentration (IU/mL) at which the S/CO = 1.00
24.21 12.28 7.43 4.89 2.45 1.98 1.50 1.01 0.82 0.67 0.02
HBsAg, hepatitis B surface antigen; TRFIA, time-resolved fluoroimmunoassay.
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Most false-positive results using current HBsAg detection methods can be observed for pregnant women, for autoimmune diseases, and chronic liver diseases from non-HBV related causes. Thus, a total of 60 specimens with hepatitis B core antigen, hepatitis B e antigen, hepatitis C virus, human immunodeficiency virus, Treponema pallidum and rheumatoid factor were measured to evaluate the assay’s specificity for HBsAg (30). Specimens with an initial test result signal/cutoff ratio ≥ 1.00 were retested in duplicate. As presented in Table 5, the assay specificity was 100%, which indicated that our proposed immunoassay possesses a high specificity for HBsAg, and no cross-reactivity with hepatitis B core antigen, hepatitis B e antigen, hepatitis C virus, human immunodeficiency virus, Treponema pallidum and rheumatoid factor was detected. Compared with chemiluminescence immunoassay To evaluate further the value of the novel TRFIA for clinical applications, 399 blood samples (negative or positive for HBsAg) were screened in parallel using the prototype assay and the commercially available CLIA kit. A single sample for each specimen was analyzed using each assay. Differences in clinical accuracy between the two tests were evaluated with McNemar’s test for correlated proportions (Table 6). The P was obtained by 1 and considered to be statistically insignificant. Among them 231 positive blood samples were selected to demonstrate the correlation between the two methods using linear regression analysis (Fig. 4). The correlation coefficient was R = 0.989 and the equation of the regression curve was Y = 1.182X – 0.017 (X is the
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A rapid and sensitive detection of HBsAg Table 2. Accuracy: quantitative determination of WHO international reference panel HBsAg subtype
Concentration of HBsAg reference panel (IU/mL)
adw adr ay
0.4
1.0
2.0
4.0
– 0.37 (92.5%) –
1.02 (102.0%) 0.99 (99.0%) 1.01 (101.0%)
2.03 (101.5%) 2.06 (103.0%) 1.95 (97.5%)
4.27 (106.7%) – 4.14 (103.5%)
HBsAg, hepatitis B surface antigen.
Table 3. Assay precision: interassay and intra-assay stability of the proposed method
Table 5. Assay specificity: effect of potentially interfering substances on the determination of hepatitis B surface antigen
HBsAg add (IU/mL)
Interfering substance
N
Hepatitis B core antigen Hepatitis B e antigen Hepatitis C virus Human immunodeficiency virus Treponema pallidum Rheumatoid factor Samples tested Reactive (S/CO ≥ 1.00) S/CO range
10 10 10 10 10 10 60
1.6 84.7 200
Interassay precisiona (n = 8)
Intra-assay precisiona (n = 8) Measured (IU/mL)
CV (%)
Measured (IU/mL)
CV (%)
1.48 ± 0.07 85.51 ± 4.69 196.61 ± 17.17
4.7% 5.5% 8.7%
1.54 ± 0.06 81.99 ± 6.15 203.62 ± 10.25
3.8% 7.5% 5.0%
CV, coefficient of variation; HBsAg, hepatitis B surface antigen. a Mean ± standard deviation.
HBsAg concentration estimated with the proposed method; Y is that from CLIA). Linear regression analysis revealed good correlations between the two methods. The novel TRFIA provides sensitivity that is at least equivalent to the clinically approved assay. The sensitive and accurate detection of HBsAg is critical to the identification of infection and the prevention of transfusion-transmitted disease. The analysis and test results proved that the magnetic bead-based TRFIA is accurate, sufficiently effective and will be a potentially powerful tool for diagnostic testing, therapeutic monitoring and blood screening in endemic areas of hepatitis B infection.
Discussion In our study, Eu3+ chelates were used as the fluorescent markers, firstly, due to its superior fluorescence signal strength at wavelengths of 613 nm, and second because, the long decay time of the fluorescent signal (10–6–10–3 s), large Stokes’ shift (> 200 nm), and narrow emission peak allow the label to be read
Reactive S/CO range 0 0 0 0 0 0
0.79–0.83 0.64–0.85 0.71–0.88 0.61–0.82 0.79–0.86 0.67–0.78
0 0.61–0.88
once the nonspecific background has already peaked and decayed (19–23). All these properties result in an enhanced signal-to-noise ratio and a significant improvement in assay sensitivity. Despite these advantages, TRFIA has some significant drawbacks predominantly because of the constraints in using 96-well microtiter plates. Because of the small surface areas within the wells of the microtiter plate, the amount of antigens or antibodies that can be detected is limited. Furthermore, physical adsorption of the coating antigen/antibody to the surface of the plate is inefficient and leads to significant wastage of reagents and materials. It is also time consuming, whereby it generally takes 1 h to attain equilibrium for an immunoreaction because of the small contact area of the solid–liquid interface in the wells of the microtiter plate (27). Over the traditional carriers, the magnetic beads as the immobilization matrix affords a large number of advantages. First, by the very nature of its greater specific surface area, more antibodies can bind to the magnetic beads, leading to the capture of more HBsAg. In contrast to physical adsorption, covalent bond formation between the antibody and magnetic beads
Table 4. Recoveries determined using the proposed method via spiking three different concentration levels of maternal serum controls into HBsAg standards Sample
Original (IU/mL)
Spiked (IU/mL)
Measureda (IU/mL)
Recovery (%)
HBsAg
100 100 100
1.6 84.7 200
97.61 ± 6.07 177.28 ± 4.84 308.19 ± 9.70
96.1% 96.0% 102.7%
HBsAg, hepatitis B surface antigen. a Mean ± standard deviation.
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Z.-Q. Ren et al. Table 6. Comparision between the proposed method and chemiluminescence immunoassay for the test of 399 blood samples Proposed method + – Total
Chemiluminescence immunoassay +
–
231 0 231
0 168 168
Total
231 168 399
+/ , positive/negative.
Funding Sources The National Natural Science Foundation of China (grant no. 81271931), the Natural Science Foundation of Guangdong Province (grant no. S2012010009547) and Scientific Research Foundation of Introducing Talents of Southern Medical University (2009). Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (grant no. 81271931), the Natural Science Foundation of Guangdong Province (grant no. S2012010009547) and Scientific Research Foundation of Introducing Talents of Southern Medical University (2009). We would like to thank all related committees or departments.
Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
References
Figure 4. Plot of the results obtained using the proposed method versus those obtained using CLIA commercial kits for hepatitis B surface antigen detection in 231 serum samples. CLIA, chemiluminescence immunoassay.
leads to a much stronger association. Furthermore, antibodycoated magnetic beads suspended in the reaction solution could increase the reaction rate and consequently reduce reaction times (31–35). All of these advantages result in an appreciable improvement in detection limit, reduction in sampling volume of blood specimens and quicker analysis. In addition, the application of an external magnetic field ensures that the imprinted molecules are separated and concentrated rapidly from unwanted constituents. It is also readily automated as a high-throughput assay for the screening of thousands of samples per day. In conclusion, a novel assay for the enhanced detection of HBsAg in human serum characterized by covalently attaching antibody to the surface of magnetic beads was developed. This method exhibited the superior properties of magnetic beads in combination with TRFIA. The current system using magnetic beads as a solid support provides better flexibility in assay optimization and quantitative determination of human serum. By using a combination of monoclonal and polyclonal antibody conjugates for detection, a high sensitivity and wide linear detection range were obtained under optimal assay conditions. The HBsAg assay offers rapid testing, high sensitivity and precision, good specificity and a wide detection range, while at the same time providing future potential for automation and high throughput.
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