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Multiple fluorescent labeling of silica nanoparticles with lanthanide chelates for highly sensitive time-resolved immunofluorometric assays.

Time-resolved fluorometry (TRF) [3] using lanthanide chelates has been widely used in ultrasensitive quantitative immunoassays. (1-5). The unique advantage of TRF is its high sensitivity compared with other routinely used immunoassays, such as ELISA. Currently, the most widely used TRF-based immunoassay is the dissociation-enhanced, lanthanide-induced fluorescence immunoassay (6), in which nonfluorescent Eu(III) chelate is used as label and the amount of Eu(Ill) is measured through a dissociation and fluorescence enhancement step. Another reported system is the enzymatically amplified time-resolved fluorescence immunoassay (7), in which the alkaline phosphatase used as the label is detected through a fluorogenic reaction with Tb(III). These methods both use an indirect labeling strategy that needs a signal development step before fluorescence detection.

The signal development step of lanthanide-based immuno-assays involves extra reaction procedures that are time-consuming and vulnerable to ambient lanthanide contamination. If a fluorescent lanthanide chelate can be used directly as a label, then the signal development step becomes unnecessary and fluorescence can be measured from the solid phase immediately after the immune reactions. Direct labeling has not found wide applications in TRF-based immunoassays, however, mainly because a lanthanide chelate must be highly fluorescent, thermodynamically stable, water soluble, and covalently reactive to serve as a direct label. Such properties are hard to find in any single chelate.

While the development of new chelates is still ongoing, efforts to explore new features of existing fluorescent chelates are generating some intriguing results. An interesting feature found is that the Eu(III) chelate shows fluorescence-enhancing effects in a multiple labeling system (8-10). In such a system, a carrier molecule such as streptavidin (SA) (8) or a polymeric compound (11,12) carrying hundreds of europium chelates is used to label antibody for highly sensitive TRF immunoassays. A breakthrough to further boost the multiple-labeling strategy is the development of nanoparticles containing thousands of fluorescent lanthanide chelates for TRF immunoassays (13-21). These nanoparticles are conjugated to SA or antibody to create a detection reagent, which allows direct measurement of fluorescence from the nanoparticles. Polystyrene nanoparticles containing fluorescent Eu(III) chelates have been used for ultrasensitive detection of prostate-specific antigen (13, 20). Recently, Eu(III), Tb(III), Sm(III), and Dy(III) chelate-dyed latex nanoparticles were also synthesized and used in sandwich-type immunoassays (16). At about the same time, silica nanoparticles containing Eu(III) or Tb(III) chelates were prepared and evaluated for TRF immunoassay (22-25). Silica nanoparticles containing lanthanide chelates are prepared either by physical doping into the preformed silica nanoparticles or prelinking to precursors for on-site silica nanoparticle formation.

In the present study, we covalently linked lanthanide chelates onto the surface of preformed silica nanoparticles. We then conjugated these particles to detection antibodies and used the resulting conjugates in time-resolved immunofluorometric immunoassay (TrIFA) of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg), both individually and simultaneously, with detection limits being 20- to 30-fold lower than those of ELISA. Evaluation of a large number of clinical samples with the current method detected all positive sera identified by ELISA and 5 HbeAg-positive results that were missed by ELISA. These data indicate that our new labeling approach is suitable for highly sensitive TrIFA in clinical settings.

[FIGURE 1 OMITTED]

Materials and Methods

PREPARATION OF LANTHANIDE-COATED NANOPARTICLES

The scheme for preparation of Eu(III)- and Tb(III)-coated nanoparticles is described in Fig. 1A. Preparation details are provided in the Materials and Methods of the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol53/issue8. Briefly, we synthesized bare silica nanoparticles using reverse microemulsion (26, 27). We introduced amino groups to the surfaces of these particles by treatment with (3aminopropyl)trimethoxylsilane (APTMS) (28). We used the aminated nanoparticles for coating Eu(III) ligand 4,4bis(1,1,2,2,3,3-heptafluoro-4,6-hexanedion-6-yl)chlorosulfo-o- terphenyl (BHHCT) or Tb(III) ligand N,N,[N.sup.l], [N.sup.l]-[2,6-bis(3aminomethyl-l-pyrazolyl)-phenylpyridine] tetrakis (BPTA),followed by mixing with Eu[Cl.sub.3] or Tb[C1.sub.3] solutions.

CHARACTERIZATION OF FLUORESCENT NANOPARTICLES

The number of nanoparticles in 1 mL suspension was calculated according to the density of the silica particle (1.96 g/[cm.sup.3]), average particle diameter, and particle concentration, as reported (13,23). The apparent number of chelates per nanoparticle was calculated by comparing the signal of a known number of nanoparticles in washing buffer B (10 mmol/L Tris-HCI, pH 9.1, containing 0.5 g/L Tween 20) with a free chelate calibrator in the same buffer. Spectra analysis and lifetime measurements of the nano-particles are described in the online Data Supplement.

NANOPARTICLE LABELING

We conjugated anti-HBsAg IgG to Eu(III)-coated silica nanoparticles using a site-specific method (29) sketched in Fig. 1B. Conjugation of SA to Tb(lll)-BPTA- coated nanoparticles was bridged with oxidized Dextran 500, sketched in Fig. 1C. Detailed protocols are provided in the online Data Supplement.

NONSPECIFIC BINDING OF ANTIBODY-CONJUGATED LANTHANIDE NANOPARTICLES

We carried out nonspecific binding studies with particle concentrations ranging from 4.1 x [10.sup.-12] mol/L to 1.3 x [10.sup.-10] mol/L, which covers the concentration (6.5 x [10.sup.-11] mol/L) used for calibration curve studies. Detailed protocols are described in the online Data Supplement.

IMMUNOASSAYS

After monoclonal antibody B20 (5 [micro]/mL in 20 mmol/L Tris-HCI buffer, pH 7.4) was physically coated on the microtitration wells (50 /,L/well), we added 50 /[micro]L HBsAg standard solutions or serum samples to each well. We incubated the plate at 37 [degrees]C for 45 min followed by 3 washes with washing buffer A [10 mmol/L PBS (10 mM sodium phosphate, pH 7.4, containing 0.15 M NaCl), pH 7.4, containing 0.5 g/L Tween 20]. Then we added 50 [micro]L Eu(III)BHHCT-coated nanoparticles conjugated with monoclonal anti-HBsAg antibody S04 [diluted 2000-fold with dilution buffer (10 mmol/L Tris-HCI, pH 7.8, containing 2 g/L bovine serum albumin, 1 g/L NaN3, and 9 g/L NaCl)] to each well. The plate was incubated at 37 [degrees]C for 90 min, washed 5 times with washing buffer B, and subjected to TRF measurement. TrIFA of HBeAg was carried out similarly, except that we used biotinylated anti-HBeAg antibody and SA-conjugated, Tb(III)-BPTA-coated silica nanoparticles. Detailed procedures are provided in the online Data Supplement.

We carried out simultaneous assay of HBsAg and HBeAg with Eu(III)-BHHCT-and Tb(lll)-BPTA-coated nanoparticles as follows. Both anti-HBsAg monoclonal antibody B20 and anti-HBeAg monoclonal antibody KE (each diluted to 5 [micro]g/mL with 20 mmol/L Tris-HCI buffer, pH 7.4) were simultaneously coated on the microtitration wells (50 [micro]gL/well) by physical adsorption. Then we added 50 [micro]L HBsAg and HBeAg standard solutions or serum samples to each well. The plate was incubated at 37 [degrees]C for 45 min and washed 3 times with washing buffer A. We added biotinylated anti-HBeAg (50 [micro]L) antibody to each well and incubated the plate at 37 [degrees]C for 45 min. After washing the plate 3 times with washing buffer A, we added 50 / [micro]L of the Eu(III)-BHHCT-conjugated anti-HBsAg and Tb(III)-BPTA-conjugated SA to each well. The plate was incubated at 37 [degrees]C for 90 min, washed 5 times with washing buffer B, and subjected to TRF measurement.

For comparison studies, we comparatively analyzed all serum samples with ELISA reagent sets. For samples showing inconsistent results between TrIFA and ELISA, we performed real-time PCR according to the manufacturer's manual to verify the existence of viral DNA. We carried out analytical recovery of TrIFA by adding standard HBsAg or HBeAg to 3 human serum samples (1 negative sample and 2 positive samples) and detecting the concentrations of HBsAg or HBeAg. We also subjected the reference panels for HBsAg and HBeAg assays to THFA for validation study.

Results

PREPARATION AND CHARACTERIZATION OF LANTHANIDE CHELATE-COATED SILICA NANOPARTICLES

Bare silica nanoparticles had spherical form and uniform size [55 (5) nm in diameter, mean (SD)] (Fig. 2, A and B). After APTMS treatment, we confirmed the existence of amino groups on the surfaces by observing salicylaldehyde-mediated yellow color. When Eu(III)-BHHCT was coated to silica nanoparticles, aggregation occurred immediately after mixing BHHCT with the nanoparticles in ethanol. The aggregated nanoparticles were dispersed by ultrasonic treatment. We used a multiple-round coating strategy to increase overall chelate loading. With Eu(III), maximal loading was achieved after 5 rounds of coating, probably owing to gradual saturation of all BHHCT linkage sites on the nanoparticle (some of these sites might have been blocked in the first 4 rounds of reaction because of aggregation). Coating of Tb(III)/BPTA did not result in any aggregation, and extra rounds of coating did not increase the overall fluorescence intensity.

The Eu(III)-BHHCT- and Th(III)/BPTA-coated nanoparticles showed no change in size and shape compared with the bare silica nanoparticles, but both had clusters of black spots on the particle surface (Fig. 2, C and D). The clustered distribution of Eu(III)-BHHCT on the silica nanoparticles functionalized with APTMS was similar to that of gold nanoparticles when adsorbed on the same functionalized surfaces (30). The fluorescence spectra of coated lanthanide chelates showed no difference from those of free chelates. The lifetimes of free and coated Eu(III)-BHHCT were 0.346 and 0.398 ms, respectively, whereas the lifetimes of the free and coated Tb(III)-BPTA were 2.645 and 2.224 ms. In contrast, the lifetime of Eu(III)-BHHCT encapsulated inside silica nanoparticles prepared by different methods was no longer than 0.300 ms (24), and the lifetime of Tb(III)-BPTA chelates decreased significantly [from 2.68 ms (free chelates) to 1.52 ms (encapsulated) (23)]. As lifetime change is an indicator of fluorescence quenching, the longer lifetime of lanthanide chelates indicated less fluorescence quenching with the surface-coated nanoparticles. Eu(III)-BHHCT even had increased lifetime after coating, suggesting that fluorescence enhancement might have occurred. Such an observation is consistent with multiple labeling of protein carriers (8-10).

[FIGURE 2 OMITTED]

The apparent number of Eu(III)-BHHCT per nanoparticle was calculated at 6.86 x [10.sup.5], which is higher than that observed with commercial polystyrene nanoparticles of even larger size [3.1 x 104 chelates per nanoparticle of 107 nm in diameter (13)]. The apparent number of Tb(III)BPTA per nanoparticle was 4.73 x [10.sup.4], which is also significantly higher than that of physically encapsulated chelates in silica nanoparticles of the similar size [1500 chelates per silica nanoparticle of 42 nm in diameter (23)].

CONJUGATION OF ANTIBODY TO LANTHANIDE-COATED SILICA NANOPARTICLES

The existence of amino groups after lanthanide coating, which was detected by salicylaldehyde-mediated yellow color, allowed us to use a site-specific labeling strategy to conjugate antibody to Eu(III)-BHHCT-coated nanoparticles (Fig. 1B). To achieve the highest sensitivity and specificity, we screened for the best combination of capture and detection antibody pair among 9 anti-HBsAg antibodies including 4 polyclonal antibodies and 5 monoclonal antibodies. The optimal antibody pair with HBsAg was monoclonal antibody S04 as the detection antibody and monoclonal B20 as the capture antibody. The labeling ratio was also optimized with this antibody pair, and we found that 0.8 mg antibody and 4 mg (0.051 nmol) nanoparticles gave the highest signal-to-noise ratio. Unlike Eu(III)-BHHCT-coated nanoparticles, direct conjugation of antibody to Tb(III)-BPTA-coated nanoparticles failed to give any meaningful results. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemistry used for BPTA conjugation might be responsible for this failure, because excess EDC might block part of the amino groups on the silica nanoparticles, making them less accessible to antibody conjugation. By using oxidized Dextran 500 as a neutral and hydrophilic arm, we successfully linked SA to the nanoparticles, which allowed subsequent conjugation of biotinylated anti-HBeAg antibody to nanoparticles (Fig. 1C). The conjugates prepared were fairly stable. Conjugate stock solutions (10 mg/mL; i.e., 1.3 X [10.sup.-7] mol/L) could be kept at 4 [degrees]C for 2 years without obvious loss of detection sensitivity.

[FIGURE 3 OMITTED]

NONSPECIFIC BINDING OF NANOPARTICLE CONJUGATES Overall nonspecific binding of Eu(Ill)-BHHCT-coated nanoparticles was <0.002% (Table 1). The nonspecific binding of antibody-conjugated and nonconjugated EU(III) nanoparticles showed no obvious difference between anti-HBsAg antibody-coated or SA-coated wells; however, the nonspecific binding on SA-coated wells was always lower than that of antibody-coated wells. When additional nanoparticles were added, SA-coated wells showed a slight increase in nonspecific binding, whereas antibody-coated wells showed a negligible increase. The nonspecific binding of Tb(III) nanoparticles onto antibody-coated wells was also <0.002%. The nonspecific binding of antibody conjugates prepared in the present work was comparable with chelate-doped nanospheres (21), though slightly higher than those delicately modified nanospheres with PEG-tethered chains (31).

TrIFA OF HBsAg

The dynamic range of the nanoparticle-based TrIFA of HBsAg was 0.05-111.1 /[micro]g/L (Fig. 3A). The detection limit, defined as the concentration corresponding to a signal that is 3 SD above the zero calibrator, was 0.0092 [micro]g/L. The CV along the entire concentration range was between 1.76% and 8.72% (n = 8). The analytical recovery was in the range of 99.1%-106.5%, CV 2.31%-4.40% (Table 2). In contrast, the linear range of ELISA was 0.2-5.0 /[micro]g/L, with a detection limit of 0.2 /[micro]g/L. When 338 human serum samples were analyzed with both TrIFA and ELISA, the cutoff value was defined as 2.1 times the mean signal of the negative control (Fig. 3B). Based on the cutoff value, the 2 methods gave completely consistent results for all serum samples. Experiments with the reference panel of HBsAg also generated values as expected (data not shown).

TrIFA OF HBeAg

The linear range of the nanoparticle-based TrIFA of HBeAg was 15.0 to 1.11 X [10.sup.4] NCU/L (Fig. 4A). The detection limit was 10.0 NCU/L. The CVs along the whole concentration range were within 3.99% to 12.11% (n = 8). The analytical recovery was in the range of 95.1% to 105.0%, with CVs of 2.39% of 9.09% (Table 2). In contrast, the linear range of ELISA was narrower (3.0 X [10.sup.2] to 9.6 X [10.sup.3] NCU/L), and the detection limit was higher (3.0 x [10.sup.2] NCU/L). When 340 human serum samples were analyzed with both TrIFA and ELISA using 2.1 times the signal of the negative control as the cutoff value, 6 ELISA-negative samples gave positive signals by TrIFA (Fig. 4B). To clarify this difference, these samples were subjected to ELISA tests for HBsAg; HBs, HBe, and HB core antibodies; and real-time PCR assay for hepatitis B virus DNA. Five of 6 samples were confirmed as truly hepatitis B virus-positive samples (see Table S1 in the online Data Supplement). One sample seemed to be false positive, although the result needs further investigation. These results demonstrated that TrIFA had higher sensitivity than ELISA in HBeAg detection. TrIFA of the reference panel of HBeAg also generated expected results (data not shown).

[FIGURE 4 OMITTED]

SIMULTANEOUS TrIFA of HBsAg AND HBeAg

Before simultaneous TrIFA of HBsAg and HBeAg, we first investigated the potential interference between signals of Tb(III) and Eu(III) in simultaneous detection. Under the detection conditions for Th(III), Eu(III) gave negligible interference signal. However, under the detection conditions for Eu(III), Th(III) generated interference signal. We established a correction method (see Correction Methods and Fig. S1 in the online Data Supplement) and used it to build up universal calibration curves for simultaneous TrIFA under different conditions (see Fig. S2 in the online Data Supplement). We then studied the interference between the 2 antibody pairs and noticed that the capture antibody EB3 used in the single assay of HBeAg had strong nonspecific binding to the detection antibody S04 for the HBsAg assay, resulting in high background signals in the HBsAg detection. Thus we changed EB3 to KE in the simultaneous assay after reselection of antibody pairs. Although the substitution of KE for EB3 caused a slight decrease in the assay sensitivity of HBeAg, the interference between the 2 antibody pairs was eliminated. According to the calibration curves of the simultaneous TrIFA of HBsAg and HBeAg (see Fig. S2 in the online Data Supplement), the linear range was 0.11 to 65.41 / [micro]g/L for HBsAg and 33.0 to 8.1 x [10.sup.3] NCU/L for HBeAg; the detection limit was 0.033 [micro]g/L for HBsAg and 27.0 NCU/L for HBeAg. The CVs (n = 8) along the whole concentration range were <10% for both assays.

When 307 human serum samples (including the 6 samples that showed inconsistent results between individual TrIFA and ELISA in the HBeAg assay) were analyzed with simultaneous TrIFA as well as ELISA using the same cutoff value definition as above, concordant results were obtained for all the samples in HBsAg (see Table S2 in the online Data Supplement). The simultaneous assay results for HBeAg showed that, among those 6 differential samples, only 2 (no. 174 and 314) were detected as HBeAg positive in the simultaneous assay. The results of other samples were concordant with ELISA. These results showed that the sensitivity of the simultaneous assay was slightly lower than the individual assay (at least in the case of HBeAg), though still higher than ELISA.

Discussions

Nanoparticle-based TrIFA using lanthanide chelate labels provides an ultrasensitive tool for clinical detection. Taking advantage of both multiple labeling and versatile surface modification of silica nanoparticles, we have directly coated Eu(III) and Tb(III) chelates on the surface of nanoparticles. The particles prepared are successfully used in detection of HBsAg and HBeAg, with a detection limit that is lower than (or equivalent to) other nanoparticle-based TrIFAs and is ~20- to 30-fold lower than ELISA. Moreover, we successfully detected both antigens simultaneously in a single assay. Thus, the present labeling strategy may help lanthanide chelate-based fluorescent TrIFA gain more clinical applications.

Our covalent surface coating approach has many advantages over existing labeling strategies. First, compared with multiple labeling strategy using proteins (8) or polymers (12), our coating process is more straightforward and controllable. Silica nanoparticles are chemically inert, and the coating reaction can be carried out in either organic or water solutions depending on the solubility of the chelate used. Separation of finished nanoparticles from free chelates or unconjugated proteins can be simply conducted by centrifugation. Second, the surface coating approach obviates many drawbacks inherent in the encapsulation strategy currently used for loading lanthanide chelates to latex or silica nanoparticles. For example, covalent coating eliminates the potential leakage problem often encountered in physical doping/dying. So far, physical dying/doping is nearly exclusively used with latex nanoparticles and frequently applied in silica nanoparticle labeling. It has been shown in silica nanoparticles that chelate leakage compromises its performance as label in TrIFA (23-25). Although the leakage problem has not been well explored with latex particles, its potential harm to immunoassay cannot be ignored (32). Third, covalent surface labeling of silica nanoparticles allows flexibly adjusting/maximizing the number of chelates coated on each particle without jeopardizing the uniformity of the nanoparticles. The spongelike, porous nature of silica nanoparticles provides much more surface area than solid nanoparticles do, which allows increased numbers of chelates to be loaded on every silica nanoparticle. In contrast, in either physical doping (23-25) or covalent binding-copolymerization (22,33), the amount of lanthanide chelates has to be delicately controlled to avoid irregular (or even no) nanoparticle formation, limiting the number of chelates that can be loaded on each nanoparticle. That may explain why our surface coating strategy allows more chelates per nanoparticle than the encapsulation methods (16,22). Fourth, fluorescence quenching was not observed with our surface-coated chelates. In contrast, fluorescence quenching is readily observed with encapsulated chelates (23, 24). Lack of quenching further makes the effective chelate number on surface-coated nanoparticles higher than inside-encapsulated nanoparticles. Finally, despite that organic fluorophores encapsulated inside silica nanoparticles are more photostable than free fluorophores (27), the surface-coated lanthanide chelates are also very stable for routine use. Such an observation may be because lanthanide chelates themselves are more photostable than organic fluorophores (23,25) or because in TRF detection, the pulsed excitation light resource causes less photobleaching than constant irradiation in conventional fluorometric detection.

Despite many advantages shown by lanthanide chelate-coated silica nanoparticles, whether they can achieve high sensitivity in immunoassays also depends on many other factors. For example, the binding reagents used, the experimental conditions, and nonspecific binding are all crucial to the sensitivity of the immunoassay, in addition to the delectability of the label (9, 34). On screening antibody pairs, optimizing antibody conjugation format, evaluating nonspecific binding of the conjugated antibodies, and interference correction, ultrahigh sensitivity for both individual and simultaneous detection of HBsAg and HBeAg was realized. Although these factors may mean increased labor intensity for development of a TrIFA assay, it is worthwhile to consider them to achieve the best sensitivity. The low nonspecific binding of the antibody conjugates further confirms the advantages of the hydrophilicity of silica nanoparticles. Through these simple optimizations, our particles give a detection limit for HBsAg of 0.0092 / [micro]/L, which is slightly lower than latex nanoparticles (0.028 [micro]g/L) (17) and silica nanoparticles (0.023 /xg/L) (22). The high sensitivity is especially meaningful for HBeAg, because it is an indicator of hepatitis B virus replication and can remain undetected by classic ELISA in certain samples.

We also established direct, simultaneous detection of 2 analytes using Eu(III) and Tb(III) chelate-coated silica nanoparticles. By using a novel correction algorithm, nearly the same sensitivity was achieved in the simultaneous assays as in the individual assays. Although simultaneous detection using Eu(III) and Tb(III) chelates has been previously achieved by stepwise detections (35, 36), to our knowledge, this is the first time that simultaneous detection was achieved in a single assay. Other simultaneous detection formats have been reported utilizing Eu(III) and Sm(III) chelates (37, 38). However, because Sm(III) chelate has rather low fluorescence yield and relatively short fluorescence lifetime, the Eu(III) and Sm(III) dual-label TrIFA has much lower sensitivity than the individual Eu(III) label assay. Thus, our dual-labeling strategy may allow more dual analysis to be performed simultaneously.

In conclusion, we developed a multiple labeling strategy for coating fluorescent lanthanide chelates on the surface of silica nanoparticles for TrIFA. This strategy was successfully demonstrated in both individual and simultaneous detection of HBsAg and HBeAg, with the detection limit lower than existing labeling approaches. Validation with a large number of clinical samples indicates that our labeling strategy may serve as a solid step toward the clinical use of nanoparticle TRF-based immunoassays.

Grant funding/ support: This work was partially supported by the Xiamen Scientific Development Program (no. 3502Z20055008). Financial disclosures: None declared.

Acknowledgments: We are grateful to Xilin Zhao, Yongyou Zhang, and Jinping Cheng for critical reading of the manuscript and Jieli Zhang for technical assistance. We are also indebted to Dr. H. Matsumoto and Dr. Jingli Yuan for kindly providing BHHCT and BPTA.

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[3] Nonstandard abbreviations: TRF, time-resolved fluorometry; SA, streptavidin; TrIFA, time-resolved immunofluorometric immunoassay; HBsAg, hepatitis B surface antigen; HBeAg, hepatitis B e antigen; APTMS, (3-aminopropyl)trimetho)cylsilane; BHHCT, 4,4-bis(1,1,2,2,3,3-heptafluoro-4,6hexanedion-6-yl)chlorosLdfo-o-terphenyl; BPTA, N,N,[N.sup.], [N.sup.1]-[2,6-bis(3-aminomethyl-l-pyrazolyl)-phenylpyridineI tetrakis; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; NCU, National Centre Unit.

YE Xu [l] and QINGGE Li [1,2] *

[1] Molecular Diagnostics Laboratory, Department of Biomedical Sciences,and the Key Laboratory of the Ministration of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China.

[2] The Key Laboratory of Chemical Biology of Fujian, Xiamen University, Xiamen, Fujian 361005, China.

* Address correspondence to this author at: Department of Biomedical Sciences, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China. Fax 86-592-2187363; e-mail qgli@xmu.edu.cn.

Received August 15, 2006; accepted May 9, 2007. Previously published online at DOI: 10.1373/clinchem.2006.078485
Table 1. Nonspecific binding of Eu(III)-BHHCT-coated
nanoparticles to anti-HBsAg-coated microtitration wells.

 Antibody-labeled Eu(III)-BHHCT-coated
 nanoparticle

Particle concentration, Count (a) CV, % Nonspecific
mol/L binding, %

Anti-HBsAg-coated
microtitration wells

4.1 x [10.sup.-12] 18 5.08 0.0014
8.1 x [10.sup.-12] 64 6.52 0.0025
1.6 x [10.sup.-11] 110 6.70 0.0022
3.3 x [10.sup.-11] 193 9.66 0.0022
6.5 x [10.sup.-11] 346 10.78 0.0017
1.3 x [10.sup.-10] 736 18.39 0.0018

SA-coated
microtitration wells

4.1 x [10.sup.-12] 23 3.99 0.0014
8.1 x [10.sup.-12] 29 5.31 0.0009
1.6 x [10.sup.-11] 54 4.21 0.0009
3.3 x [10.sup.-11] 106 5.45 0.00082
6.5 x [10.sup.-11] 218 5.29 0.00085
1.3 x [10.sup.-10] 439 9.47 0.00085

 Nonlabeled Eu(III)-BHHCT-coated
 nanoparticle

Particle concentration, Count (a) CV, % Nonspecific
mol/L binding, %

Anti-HBsAg-coated
microtitration wells

4.1 x [10.sup.-12] 21 6.54 0.0013
8.1 x [10.sup.-12] 54 5.62 0.0017
1.6 x [10.sup.-11] 66 6.71 0.0011
3.3 x [10.sup.-11] 161 6.47 0.0013

6.5 x [10.sup.-11] 295 10.67 0.0012
1.3 x [10.sup.-10] 861 25.18 0.0017

SA-coated
microtitration wells

4.1 x [10.sup.-12] 1 7.67 0.00006
8.1 x [10.sup.-12] 9 5.78 0.0003
1.6 x [10.sup.-11] 33 8.06 0.00056
3.3 x [10.sup.-11] 64 10.22 0.00053
6.5 x [10.sup.-11] 171 19.04 0.0007
1.3 x [10.sup.-10] 378 23.01 0.00078

n = 12.

(a) Net fluorescence intensity of the nonspecific binding
nanoparticles.

Table 2. Analytical recovery.

Amount added Amount CV, % Amount CV, % Recovery,
 in serum (n = 8) measured (n = 8) %
 sample

HBsAg, ng/mL

5 0 6.19 5.01 4.17 101.7
10 0 10.66 3.64 105.9
5 10 7.40 15.92 4.40 104.3
10 10 21.88 3.39 106.5
5 50 3.97 58.07 3.43 99.9
10 50 62.98 3.31 99.1

HBeAg, NCU/mL

1 0 2.83 1.05 2.39 105.0
2 0 1.95 6.49 97.5
1 2 3.76 2.85 9.09 95.1
2 2 4.02 5.29 100.5
1 5 3.81 5.95 4.90 99.1
2 5 7.27 7.54 103.9
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Title Annotation:Automation and Analytical Techniques
Author:Xu, Ye; Li, Qingge
Publication:Clinical Chemistry
Date:Aug 1, 2007
Words:5497
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