Sequential Determination of Two Proteins by Temperature-Triggered

Moreover, highly sensitive CL detection of HRP was applied, and the detection limits of IgG and IgA were as low as 2.0 and 1.5 ng/mL, respectively. Wi...
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Anal. Chem. 2006, 78, 5920-5924

Sequential Determination of Two Proteins by Temperature-Triggered Homogeneous Chemiluminescent Immunoassay Yang Zhou,† Yuhao Zhang,‡ Choiwan Lau,† and Jianzhong Lu*,†

School of Pharmacy and Zhongshan Hospital, Fudan University, 138 Yixueyuan Road, Shanghai 200032, China

A novel protocol for performing a sequential dual-protein immunoassay, based on a temperature-triggered separation/mixing process and HRP-catalyzed chemiluminescence (CL) detection, is described. In contrast to current multilabel-based detection techniques, a single HRP label is employed in this proposed method. Herein we introduce poly(N-isopropylacrylamide) (PNIP) and magnetic beads as bimolecular immobilizing carriers to separate different targets by taking advantage of thermal response, as demonstrated by sequential detection of human IgG and IgA. PNIP is known to aggregate and precipitate out of water when the temperature is raised above the lower critical solution temperature (LCST) of 31 °C; thus, it can be separated from supernatant by centrifugation. Besides, magnetic beads can be separated from PNIP by magnetic force as the temperature is lower than LCST. A homogeneous noncompetitive ELISA was employed, formed by primary antibodies immobilized onto the surface of magnetic beads and PNIP, antigen as IgG and IgA in the sample, and HRP-labeled second antibodies. Moreover, highly sensitive CL detection of HRP was applied, and the detection limits of IgG and IgA were as low as 2.0 and 1.5 ng/mL, respectively. Within the calibrated amount, the protocol had excellent precision within 11% for each target and was comparable in performance to commercial single-analyte ELISAs. Furthermore, the proposed method has been sucessfully applied to the determination of dual analyte in real samples without cross-reaction, and a good correlation was achieved after comparison with the conventional assay for IgG and IgA in 40 human serum samples. Simultaneous detection of multiple proteins has gained considerable interest in clinical, environmental, and biodefense applications. Numerous multianalyte immunoassays have been developed due to their unique advantages, such as less sample, reduced analysis time, minimized repetitions of tedious procedures, and lower cost per test, as compared to parallel singleanalyte assays.1 In recent years, considerable effort is mainly * To whom correspondence should be addressed. E-mail:[email protected]. † School of Pharmacy. ‡ Zhongshan Hospital. (1) Brecht, A.; Abuknesha, R. TrAC, Trends Anal. Chem. 1995, 14, 361371.

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focused on developing multilabel-based multianalyte assay methods. Typically, these multianalyte immunoassay methods employ more than one label, such as several fluorescent, enzyme, or metal ion labels.2-5 However, there are some restrictions in number of labels and, therefore, of analytes that could be determined simultaneously.6 For instance, multicolor fluorescence-linked immunoassays are often complicated by the requirement of an elaborate excitation and detection scheme and by the broad emission bands.7 In addition, single-label-based multianalyte analysis was generally achieved by using an array of immunosensing electrodes, with each electrode containing a different immobilized capture antibody. The spatial separation of the electrodes enabled individual immunoassays to be performed at each electrode without interference due to amperometric cross talk. For example, Kojima et al.8 reported the qualitative detection of two tumor markers using an electrode array. Recently, Wilson9 reported the quantitative simultaneous electrochemical multianalyte immunoassay of two tumor markers by spotting two antibodies on different iridium oxide electrodes. Still, it is a great challenge for the chemists to develop novel single-label-based multianalyte assays based on other detection strategies, though it would also be important to extend the applications of current multilabel-based detection techniques. Chemiluminescence (CL) has been exploited within a wide range of applications in various fields, due to their extremely high sensitivity along with their extra advantages such as simple instrumentation, wide calibration ranges, and suitability for miniaturization in analytical chemistry.10-13 In addition, it is well (2) Eriksson, S.; Vehnianen, M.; Jansen, T.; Meretoja, V.; Saviranta, P.; Timo, L. Clin. Chem. 2000, 46, 658-666. (3) Swartzman, E. E.; Miraglia, S. J.; Mellentin-Michelotti, J.; Evangelista, L.; Yuan, P. M. Anal. Biochem. 1999, 271, 143-151. (4) Guzman-Va´zquez de Prada, A.; Pen ˜a, N.; Parrado, C.; Reviejo, A. J.; Pingarro´n, J. M. Talanta. 2004, 62, 896-903. (5) Hayes, F. J.; Halsall, H. B.; Heineman, W. R. Anal. Chem. 1994, 66, 18601865. (6) Kricka, L. J. Clin. Chem. 1992, 38, 327-328. (7) Goldman, E. R.; Clapp, A. R..; Anderson, G. P.; Uyeda, H. T.; Medintz, I. L.; Mattoussi, H.; Mauro, J. M. Anal. Chem. 2004, 76, 684-688. (8) Kojima, K.; Hiratsuka, A.; Suzuki, H.; Yano, K.; Ikebukuro, K.; Karube, I. Anal. Chem. 2003, 75, 1116-1122. (9) Wilson, M. S. Anal. Chem. 2005, 77, 1496-1502. (10) Garcia-Campana, A. M.; Baeyens, W. R. G. Chemiluminescence in Analytical Chemistry; Marcel Dekker: New York, 2001. (11) Agbaria, R. A.; Oldham, P. B.; McCarroll, M.; McGown, L. B.; Warner, I. M. Anal. Chem. 2002, 74, 3952-3962. (12) Roda, A.; Guardigli, M.; Michelini, E.; Mirasoli, M.; Pasini, P. Anal. Chem. 2003, 75, 463A-470A. 10.1021/ac060783s CCC: $33.50

© 2006 American Chemical Society Published on Web 07/20/2006

known that the special thermosensitive poly(N-isopropylacrylamide) (PNIP) has been widely utilized as the separation carrier since it was exploited in immunoassays by Hoffman, while magnetic beads as special biomolecular immobilizing carrier offer a promising alternative to conventional methodology.14-21 In contrast to current multilabel-based detection techniques, we took good advantage of these two different kinds of carriers to separate targets by thermotriggered precipitation, and one label rather than multiple was employed. A fast homogeneous immunoreaction as well as a simple heterogeneous separation process was carried out in the light of some certain characteristics of water-soluble PNIP and magnetic beads, and thus, lower nonspecific affinity and higher sensitivity were accomplished. To demonstrate our original idea, we illustrate an attempt to investigate a highly sensitive CL detection for sequentially determining the amount of IgG and IgA in the present work. The results indicated that the detection limits for IgG and IgA were estimated as low as 2.0 and 1.5 ng/mL, respectively, which is equal to or better than other reported assays.18-21 Moreover, no cross-reaction was found in this proposed method, while good correlation has been achieved after comparison with conventional assay for IgG and IgA in 40 serum samples. EXPERIMENT SECTION Chemicals. All chemicals were of analytical reagent grade and were used as received. The water was prepared using Milli-XQ equipment. Human IgG, human IgA, anti-IgA, 4-iodophenol, and anti-human polyvalent immunoglobulins (G, A, M)-peroxidase antibody (A8400) were purchased from Sigma. The anti-IgG-coated magnetic beads (1.6 µm, 5 mg/mL) were obtained from Qiagen GmbH, and BSA was bought from Sino-American Biotechnology Co. N-Isopropylacrylamide (NIP) was purchased from TCI, and N,N,N′,N′-tetramethylethylenediamine (TEMED) was obtained from Acros. Other chemical reagents were bought from Sinopharm Chemical Reagent Co. Ltd. Apparatus. CL measurements were performed with a BPCL chemiluminescence analyzer (Beijing, China). Preparation of the PNIP-Anti-IgA Conjugates. A total of 250 µg of anti-IgA in 1.7 mL of PBS was first coupled with 1% N-acryloxysuccinimde in 300 µL of DMSO according to previous work26 and then immobilized to 104 mg of NIP at 25 °C by using (13) Powe, A. M.; Fletcher, K. A.; St Luce, N. N.; Lowry, M.; Neal, S.; McCarroll, M. E.; Oldham, P. B.; McGown, L. B.; Warner, I. M. Anal. Chem. 2004, 76, 4614-4634. (14) Bromberg, L. E.; Ron E. S. Adv. Drug. Delivery Rev. 1998, 31, 197-221. (15) Hoffman, A. S. Clin. Chem.. 2000, 46, 1478-1486. (16) Chen, J. P.; Yang, H. J.; Hoffman, A. S. Biomaterials 1990, 11, 625630. (17) Monji, N.; Hoffman, A. S. Appl. Biochem. Biotechnol. 1987, 14, 107120. (18) Hafeli, U.; Schutt, W.; Teller, J.; Zborowski, M. Scientific and Clinical Applications of Magnetic Carriers; Plenum: New York, 1997. (19) Wang, J.; Liu, G.; Munge, B.; Lin, L.; Zhu, Q. Angew. Chem., Int. Ed. 2004, 43, 2158-2161. (20) Lu, J.; Lau, C.; Kai, M. Chem. Commun. 2003, 2888-2889. (21) Malmstadt, N.; Yager, P.; Hoffman, A. S.; Stayton, P. S. Anal. Chem. 2003, 75, 2943-2949. (22) Ni, J.; Lipert, R. J.; Dawson, G. B.; Porter, M. D. Anal. Chem. 1999, 71, 4903-4908. (23) Fan, A. P.; Lau, C. W.; Lu, J. Z. Anal. Chem. 2005. 77, 3238-3242. (24) Dequaire, M.; Degrand, C.; Limoges, B. Anal. Chem. 2000, 72, 55215528. (25) Sato, K.; Tokeshi, M.; Odake, T.; Kimura, H.; Ooi, T.; Nakao, M.; Kitamori, T. Anal. Chem. 2000, 72, 1144-1147.

10 mg of ammonium persulfate as the initiator and 10 µL of TEMED as the accelerator in 5 mL of PBS. After polymerization for 2 h by continuous shaking, PNIP was precipitated at 35 °C and separated from unreacted monomers and anti-IgA by centrifugation for 5 min at 35 °C (4000 rpm). The procedure above was repeated for 3 times, and 100 mg of PNIP-anti-IgA conjugates were resuspended in 5 mL of PBS and stored at 4 °C for use. Immunoassay Procedure. In a typical experiment, anti-IgGcoated magnetic beads were washed 3 times with PBS-0.05% Tween 20 buffer before use. Ten microliters of PNIP (20 mg/ mL) and 1.25 µL of magnetic beads were pipetted into each tube, and 100 µL of calibrator or serum diluted with 2% BSA was then added. The mixture was incubated with continuous shaking at 25 °C for 1 h and then washed 3 times by centrifugation at 35 °C (4000 rpm). The precipitate was redissolved prior to the addition of HRP-labeled antibodies, incubated for 1 h at 25 °C, and then followed by temperature-triggered precipitation at 35 °C for 3 times to wipe off the supernatant. Subsequently, the aggregated complex was redissolved in 100 µL of PBS. CL Detection Procedure. Following the magnetic isolation from PNIP, the magnetic beads were rinsed with PBS-0.05% Tween 20 buffer twice and then resuspended in 10 µL of PBS. The supernatant were preconcentrated by centrifugation at 35 °C (4000 rpm) and then resuspended in 10 µL of PBS. A 10-µL aliquot of the resultant magnetic beads or PNIP solution was introduced into 14 × 40 mm glass tubes in which 90 µL of 0.5 mM luminol and 1 mM 4-iodophenol in 0.05 M Tris-HCl solution were contained, and then 100 µL of hydrogen peroxide was injected to the tube. The CL signal was then integrated for 50 s. RESULTS AND DISCUSSION The principle of CL detection with PNIP and magnetic beads as bimolecular immobilizing carriers to separate different targets is depicted in Scheme 1, which was applied to sequential determination of IgG and IgA. Scheme 1 included four steps: (i) adding serum into the mixture of anti-IgG-coated magnetic beads and PINP-anti-IgA conjugates to induce the antibody-antigen immunoreaction and then removing free segments by thermotriggered centrifugation; (ii) employing the HRP-labeled second antibody to form the sandwich complex and then removing free HRP label by thermotriggered centrifugation; (iii) separating magnetic beads from PNIP by magnetic force under the temperature of lower critical solution temperature (LCST); (iiii) detecting the CL signal of PNIP and magnetic beads, respectively (Scheme 1). Optimization of Assay Conditions. Several parameters were investigated systematically in order to establish optimal conditions for sequential CL detection of IgG and IgA, including the amounts of anti-IgG-coated magnetic beads, PNIP-anti-IgA conjugates, and incubation time. Figure 1 illustrated the CL ratio versus different dilution coefficients of HRP-labeled antibodies. As the amount of antibodies increased, the CL ratio increased at the beginning and leveled off between the dilution coefficient of 1:1000 and 1:500. Hence, subsequent work employed the dilution of 1:1000 HRP-labeled antibodies. (26) Chen, J.P.; Hoffman, A. S. Biomaterials 1990, 11, 631-634.

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Scheme 1. Schematic Representation of CL Detection of Dual-Analyte Assay by Using PNIP and Magnetic Beads

The amounts of the PNIP and magnetic beads versus the CL intensity were optimized as shown in Figures 2 and 3. A higher CL ratio was achieved with 10 µL of PNIP and 1.25 µL of magnetic beads, which were then selected for further studies. Most importantly, the CL ratio was unchanged for IgG determination with the increase of PNIP-anti-IgA conjugates, while the CL ratio was almost constant for IgA determination with increasing amount of anti-IgG-coated magnetic beads. This indicated that no significant cross-reaction was found between IgA and IgG, leading to sequential determination of IgG and IgA. 5922

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The effect of incubation time on the CL ratio was examined and optimized. It was found that the CL ratio for both IgG and IgA reached a maximum after incubation for 1 h at 25 °C. After that, the CL ratio was slightly decreased (Figure 4). A calibration graph in the concentration range of 0.5-25.0 ng showed a good linear correlation (R2 ) 0.998) between the amount of IgG and the CL intensity, represented by I ) 106905C - 230.25, while the dynamic range for IgA extended between 0 and 1.0 ng and 1-20.0 ng, represented by I ) 230171C - 4960.2 (R2 ) 0.9945) and I ) 112202C + 111690 (R2 ) 0.9993), respectively.

Figure 1. CL ratio versus the dilution coefficient of HRP-labeled antibodies. Experimental conditions: 50 µL of PNIP-anti-IgA conjugates and 2.5 µL of anti-IgG-coated magnetic beads incubated with 100-µL samples containing 10 ng of IgG (O) and 5 ng of IgA (4). After centrifugation, different dilutions of HRP-labeled antibodies were added for 1 h and followed by thermotriggered precipitation. Sequential detection was carried out as described in the Experimental Section.

Figure 3. CL ratio versus the amount of magnetic beads. Experimental conditions: 10 µL of PNIP and different amounts of magnetic beads were incubated with 100-µL samples containing 10 ng of IgG (O) and 5 ng of IgA (4). After centrifugation, 1:1000 HRP-labeled antibodies was added for 1 h and followed by thermotriggered precipitation. Sequential detection was carried out as described in the Experimental Section.

Figure 2. CL ratio versus the amount of PNIP. Experimental conditions: different amounts of PNIP-anti-IgA conjugates and 2.5 µL of anti-IgG-coated magnetic beads were incubated with 100-µL samples containing 10 ng of IgG (O) and 5 ng of IgA (4). After centrifugation, a 1:1000 dilution of HRP-labeled antibodies was added for 1 h and followed by thermotriggered precipitation. Sequential detection was carried out as described in the Experimental Section.

Figure 4. CL ratio versus the reaction time. Experimental conditions: 10 µL of PNIP and 1.25 µL of magnetic beads were incubated with 100-µL samples containing 10 ng of IgG (O) and 5 ng of IgA (4). After centrifugation, 1:1000 HRP-labeled antibodies was added and incubated for different times, followed by thermotriggered precipitation. Sequential detection was carried out as described in the Experimental Section.

The average integrated background response for IgG was 28 785, and the detection limits were thus estimated to be 2.0 ng/mL IgG (3σ, n ) 5), i.e., 1.2 × 10-11 M human IgG (1.2 fmol in the 100-µL sample), while the mean integrated background response for IgA was 31 527 and the detection limits were thus estimated to be 1.2 ng/mL IgA (3σ, n ) 5), i.e., 7.5 × 10-12 M human IgA (0.75 fmol in the 100-µL sample), which are competitive with or even better

than other immunoassay formats as shown in Table 1. The intraassay precisions (n ) 4) were 4-9% for IgA and 5-10% for IgG, while the interassay precision for analytes over 4 days was 4.2-10.4% for IgA and 6.3-10.3% for IgG. The recoveries of IgA (0.5, 5, and 20 ng) in the presence of 20 ng of IgG were 95-104%, while the recoveries of IgG (0.5, 5, and 20 ng) in the presence of 20 ng of IgA were 89-97%. In addition, comparison of the CL intensities for IgA in the presence and absence of IgG yielded a

Table 1. Comparison of Immunoassay Methods Developed for IgG and IgA analyte

label

immunoassay format

analytical technique

detection limit (ng/mL)

IgG IgG IgG IgG IgG IgG IgG IgG IgA IgA

Au Au Au AP Au CdS AP HRP Au HRP

single analyte single analyte single analyte single analyte single analyte multiple analyte multiple analyte dual-analyte single analyte dual-analyte

Raman scattering CL ASV at a SPE time-resolved fluorescence surface plasmon resonance electrochemical coding electrochemical CL (this work) TLM CL (this work)

3022 0.523 0.524 0.0327 128 1029 330 2.0 100025 1.2

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Figure 5. Relationship between the proposed method and the immunoturbidimetric assay for IgA (4) and IgG (O).

correlation coefficient of 0.9993, while that for IgG with and without IgA provided a correlation coefficient of 0.9998. These results further showed no significant cross-reaction in the proposed dual-analyte immunoassay technique. Comparison with Immunoturbidimetric Assay. Comparisons with the immunoturbidimetric test results offered by Zhongshan Hospital for IgA and IgG in 40 human serum samples are shown in Figure 5. The distributions of IgA and IgG levels in 40 serum samples were approximated by a normal distribution. The correlation was calculated with a linear least-squares method, and the 95% confidence intervals of the slope and intercept were 0.9402-1.012 and -0.2899 to 0.7289 for IgG, respectively, as well as 0.9194-1.0051 and -0.0403 to 0.1916 for IgA, respectively. The confidence intervals for the slope and intercept values include the unit and zero values, respectively; thus, the results indeed showed no significant difference between the conventional assay and the proposed one, as confirmed by the Student t-test (P >0.05). CONCLUSION Herein we have demonstrated a novel protocol to integrating two homogeneous immunoassay procedures for performing (27) Evangelista, R. A.; Pollak, A.; Gudgin, T. E. F. Anal. Biochem. 1991, 197, 213-224. (28) Lyon, L. A.; Musick, M. D.; Natan, M. J. Anal. Chem. 1998, 70, 51775183. (29) Liu, G.; Wang, J.; Kim, J.; Jan, M. R.; Collins, G. E. Anal. Chem. 2004, 76, 7126-7130. (30) Wilson, M. S.; Nie, W. Anal. Chem. 2006, 78, 2507-2513. (31) Jia, Z.; Chen, H.; Zhu, X.; Yan, D. J. Am. Chem. Soc. In press (ja062314d).

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sequential dual-protein determination, based on a temperaturetriggered separation/mixing process and HRP-catalyzed CL detection. First, PNIP and magnetic beads as special bimolecular immobilizing carriers have the advantages of enabling assays to be performed homogeneously and also provide means to concentrate the signal-generating samples, in contrast to single-labelbased multianalyte analysis by using an array of immunosensing electrodes; second, the majority of the current methods for the multilabel-based detection techniques are coupled with sophisticated and expensive fluorescent detection instrumentation, while the single HRP label-based CL technique was employed in this research due to its high sensitivity for the determination of enzyme, plus its extra advantages such as simple instrumentation, wide calibration ranges, and suitability for miniaturization in analytical chemistry. According to these unique advantages and the data presented above, we come to the conclusion that IgG and IgA can be sequentially and sensitively detected by using the proposed CL technique. In addition, it may be possible for multiplexing, in terms of the different thermal properties of thermosensitive aggregates, and employing other labels such as colloidal gold to further increase the sensitivity. For example, by employing magnetic beads and three thermosensitive polymers with 22, 30, and 37 °C LCST,31 it should be possible for the determination of four proteins together. However, in comparison with current single-label-based multianalyte analysis, this homogeneous system would likely be cumbersome, requiring multiple carefully controlled temperature changes plus intermittent centrifuging to separate different analytes before CL quantification. Moreover, the proposed CL technique may also be extended to the multilabel-based detection techniques, for example, by employing two labels such as HRP and ALP plus two carriers, four proteins could be simply determined together. Overall, the work presented here validates the design and concept of our dualanalyte immunoassay and provides the foundation for the development of highly sensitive techniques with increased multianalyte capability. In view of the analytical point, this protocol can be extended to fields of clinical bioaffinity assays of analytes and detection of DNA hybridization, especially for clinical diagnosis. Currently, the application of this protocol in the sequential determination of three DNA sequences is still in process in our laboratory and the results will further prove the feasibility of this conception. ACKNOWLEDGMENT We gratefully acknowledge financial support from National Natural Science Foundation of China (20575014), the Program for New Century Excellent Talents in University, Shanghai Key Basic Research Program (05JC14010), and “Shu Guang” project supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation.

Received for review April 27, 2006. Accepted June 19, 2006. AC060783S