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Fluorescence Immunoassay System Based on the Use of a pH-Sensitive Phase-Separating Polymer
Analytical Biochemistry 296, 167–173 (2001) doi:10.1006/abio.2001.5184, available online at http://www.idealibrary.com on
Fluorescence Immunoassay System Based on the Use of a pH-Sensitive Phase-Separating Polymer Huang-Hao Yang,* Qing-Zhi Zhu,* Shi Chen,* Dong-Hui Li,† Xiao-Lan Chen,* Ma-tai Ding,* and Jin-Gou Xu* ,1 *Key Laboratory of Analytical Science of MOE and Department of Chemistry, and †Cancer Research Center, Xiamen University, Xiamen 361005, People’s Republic of China
Received December 1, 2000; published online August 15, 2001
Poly(N-isopropylacrylamide-co-methacrylic acid) [P(NIPAAm-co-MAA)], a linear water-soluble pH-sensitive phase-separating polymer, was synthesized and used as a novel separation carrier for the reactants in immunoassay. This polymer precipitates out of water below a critical pH 5.8 at 37°C and redissolves when the pH of solution is above 6.2. The characteristic of this polymer makes it possible to carry out the immunochemical steps of an immunoassay in a true solution and then to quickly separate the resulting product from the reaction mixture. The above approach was applied to determination of ␣-fetoprotein with the competitive immunoassay format. Compared with traditional ELISA using the same reactants, the proposed method was much faster (the assay time decreased from 100 –120 to 30 min) and showed similar sensitivity, i.e., 0.04 ng/mL. In addition, a sandwich immunoassay method for the determination of hepatitis B surface antigen was also studied, and the results showed that the pH phase-separating immunoassay could be carried out through a sandwich or a competitive method. This general technique may also be used for a wide variety of separation processes in addition to immunoassay, in which a specific component is to be isolated for analysis, recovery, or disposal. © 2001 Academic Press
Key Words: immunoassay; pH-sensitive phase-separating polymer; ␣-fetoprotein; hepatitis B surface antigen.
etc., not only in serum but also in water, foods and other consumer products (1–5). Generally, immunoassays can be divided into two categories, homogeneous and heterogeneous. Homogeneous immunoassays, such as the enzyme-mediated immunoassay test (EMIT) 2 (6), have the advantages of being rapid, easy to perform, and readily amenable to automation. However, the drawbacks of these methods, such as poor sensitivity and being prone to interference, have limited their application. On the other hand, heterogeneous immunoassays such as enzyme-linked immunosorbent assay (ELISA), are generally more sensitive than homogeneous immunoassays, less prone to interference, and can be used for both low and high molecular weight analytes. However, heterogeneous immunoassays tend to be time consuming in order to reach chemical equilibrium (7). Different approaches have been proposed to solve the problems noted above for both homogeneous and heterogeneous immunoassays. Among them is using certain temperature-sensitive polymers to carry out a fast homogeneous immune reaction and a simple heterogeneous separation process. The polymers are known to precipitate when the temperature is raised above the lower critical solution temperature (LCST). Among the polymers, poly-N-isopropylacrylamide (PNIPAAm) is known to have a LCST of about 31°C and has been 2
Immunoassay techniques are now widely applied in measurements of toxins, drugs, hormones, pesticides, 1
To whom correspondence should be addressed at Department of Chemistry, Xiamen University, Xiamen 361005, People’s Republic of China. Fax: ⫹86-592-2188054. E-mail: [email protected]. 0003-2697/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Abbreviations used: P(NIPAAm-co-MAA), poly(N-isopropylacrylamide-co-methacrylic acid); HRP, horseradish peroxidase; AFP, ␣-fetoprotein; HBsAg, hepatitis B surface antigen; EMIT, enzyme-mediated immunoassay test; ELISA, enzyme-linked immunosorbent assay; LCST, lower critical solution temperature; LCSP, lower critical solution pH; NIPAAm, N-isopropylacrylamide; TEMED, N,N,N⬘,N⬘-tetramethylethylenediamine; MAA, methacrylic acid; APS, ammonium persulfate; FITC, fluorescein isothiocyanate; NAS, N-hydroxysuccinimidyl acrylate; p-HPA, p-hydroxyphenylacetic acid; H 2O 2, hydrogen peroxide; BSA, bovine serum albumin; PBS, phosphate-buffered saline; RSD, relative standard deviation. 167
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FIG. 1.
Protocol for the pH-sensitive phase-separating immunoassay method.
widely utilized as the separation carrier in immunoassays (8 –10). However, such assays can only be conducted under the LCST of the polymers, and therefore, this would inevitably affect the immune reaction efficiency (11). pH-sensitive polymers are another kind of environmentally sensitive polymer, which contain ionizable groups and can lead to pH-dependent phase transition (12). However, the polymers previously developed are not fit for use as the carrier in immunoassays due to their pH values for precipitation being in the low pH range. In this work, a series of pH-sensitive phase-separating polymers containing both temperature- and pHsensitive components were synthesized by copolymerizing N-isopropylacrylamide and methacrylic acid. Poly(methacrylic acid) (PMAA) is one of the pH-sensitive polymers which contains ionizable groups as the anionic polyelectrolyte. The pH of the environment determines the ionization degree of carboxylic acid groups of the PMAA. The influence of the concentration of MAA on the lower critical solution pH (LCSP) of P(NIPAAM-co-MAA) polymers was investigated in the
pH range of 3– 8 at 37°C. The LCSPs were determined by cloud point measurement. As a result, the polymer containing 2.5 mol% MAA, with a LCSP of 5.8 at 37°C, was chosen to use as the separation carrier for the immunoassay of AFP. With the use of P(NIPAAm-coMAA) as a support, the whole immunoassay process, including the homogeneous immune reaction of the P(NIPAAm-co-MAA)- conjugated monoclonal antibody and ␥-fetoprotein (AFP), the heterogeneous separation of the P(NIPAAm-co-MAA)-bound immune complex, and the homogeneous fluorescence detection of the final product, can be carried out by control of the pH. The scheme for the pH-based polymer– enzymatic immunoassay is shown in Fig. 1. In a competitive immunoassay, the standard AFP and the horseradish peroxidase labeled AFP (AFP–HRP) first reacted with the antibody immobilized on P(NIPAAm-co-MAA), and then the pH of solution was adjusted below the polymer’s LCSP to precipitate the polymer–immune complex, the polymer–immune complex precipitate was separated and redissolved by the adjustment of pH, and finally the HRP-labeled AFP in the immune complex was quantified by fluorescence measurement. A
FLUORESCENCE IMMUNOASSAY SYSTEM USING pH-SENSITIVE POLYMER
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sandwich immunoassay method for the determination of HBsAg was also developed showing that the pHsensitive phase-separating immunoassay could be performed in the sandwich method as well as the competitive method. The proposed method offers several advantages over the thermal phase-separating immunoassay. First, the immune process is conducted at 37°C; thus shorter incubation time is needed. Second, this method can be applied to the processes in which temperature change is not allowed or hardly conducted. Furthermore, this polymer can also be used as a pH-switching control of selective removal and delivery in human bodies since the pH-dependent phase transition is conducted at 37°C.
Synthesis of P(NIPAAm-co-MAA)
MATERIALS AND METHODS
Cloud-Point Determination
Reagents
The absorbance at 450 nm was measured against the pH of solution, which was raised from 3 to 8 in a 0.2 pH increment per minute, at 37°C using a Beckman DU7400 UV/VIS diode array spectrophotometer. The cloud point was defined as the pH at the inflection point in the normalized absorbance vs pH curve and the cloud point of solution which does not exhibit an infection point was determined at 10% absorbance in the curve (14).
␣-Fetoprotein, anti-AFP monoclonal antibody (MAb), horseradish peroxidase-labeled AFP (AFP– HRP, 3 mg/mL), and bovine serum albumin (BSA) were purchased from Sino-American Biotechnology Company (Shanghai, China). Hepatitis B surface antigen (HBsAg), anti-HBsAg monoclonal antibody, and horseradish peroxidase-labeled goat anti-HBsAg antibody (7 mg/mL) were purchased from Xinchuang Biotechnology Company (Xiamen, China). N-Isopropylacrylamide (NIPAAm) and N,N,N’,N’-tetramethylethylenediamine (TEMED) were purchased from TCI-EP (Tokyo Kasei, Japan) and used without further purification. Methacrylic acid, ammonium persulfate (APS), and fluorescein isothiocyanate (FITC) were obtained from Shanghai Chemical Reagent Co. (Shanghai, China). N-Hydroxysuccinimidyl acrylate (NAS) was synthesized as described (13). Hydrogen peroxide (H 2O 2, 30%) was obtained from Shanghai Taopu Chemical Factory (Shanghai, China), and the stock solution (3% H 2O 2) was standardized by titration with a standard solution of KMnO 4. p-Hydroxyphenylacetic acid (p-HPA) was obtained from Sigma and used without further purification. All the reagents are of analytical grade. The distilled, deionized water was used throughout.
Redox polymerization was used to prepare linear polymer samples of NIPAAm and MAA. Prior to the reaction, MAA was separated from the reaction inhibitor, p-methoxyphenol, by distillation under vacuum. Polymers were prepared using molar MAA fractions of 0.2, 0.1, 0.05, 0.025, 0.02, and 0 with the balance being NIPAAm. In addition, 10 mol% (of total monomers) APS was used as the initiator and 1 mol% TEMED as the accelerator. After polymerization at 37°C for 2 h, the polymers were precipitated by addition of a constant amount of 0.1 M HCl and separated from unreacted monomers by centrifugation at 37°C for 5 min (10,000 rpm).
Preparation of Monomer-Conjugated and Monomer, FITC-co-Labeled Anti-AFP Monoclonal Antibody A 0.2 mL of NAS (a 10% solution in DMSO, v/v) was added to 2.0 mL of phosphate-buffered saline solution (PBS, pH 7.0) containing 4 mg anti-AFP MAb. The resultant mixture was vibrated for 1 h at 37°C and then passed through a column of Sephadex G-25 that had been equilibrated with PBS, pH 7.0. The fractions of the elute that contained antibody were collected and stored at 4°C. An aliquot of this monomer-conjugated MAb (MAb M) was further reacted with a 25-fold molar excess of FITC. Excess, unconjugated FITC was removed by filtration on a Sephadex G-25 column. The resultant, double-conjugated anti-AFP MAb (MAb M,F) with both NAS and FITC, was stored at 4°C.
Apparatus A Hitachi 650-10S spectrofluorimeter (Tokyo, Japan) equipped with a plotter unit and a 1-cm quartz cell was used for recording fluorescence spectra and making fluorescence measurements. The absorption spectrum was obtained on a Beckman DU-7400 UV/VIS diode array spectrophotometer (Fullerton, CA). All the pH measurements were made with a Model PHS-301 pH meter (Xiamen, China). A TGL-16G supercentrifuge (Shanghai, China) and a SHZ-88 water-bath vibrator (Jiangshu, China) were used.
Immobilization of Anti-AFP MAb M and Anti-AFP MAb M,F to the P(NIPAAm-co-MAA) The product of MAb M or MAb M,F (2 mg) was diluted to 4 mL with PBS, and then 975 L of 1 mol/L NIPAAm, 250 L of 0.1 mol/L MAA, 100 L of 0.1 mol/L APS, and 10 L of 0.1 mol/L TEMED were added and the mixture was allowed to stand for 2 h in a 37°C water bath to carry out the copolymerization reaction. The P(NIPAAm-co-MAA)–MAb and P(NIPAAm-co-MAA)– MAb F conjugates were precipitated by the addition of
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200 L 0.2 mol/L HAc-NaAc buffer (pH 5.2) and the precipitate was collected by centrifugation at 37°C for 5 min (10,000 rpm). After the supernatant was removed, the precipitate was dissolved in PBS (pH 7.0). The precipitation-centrifugation-dissolution procedure noted above was repeated three times. Finally, the conjugates were dissolved in 10 mL of PBS (pH 7.4) and stored at 4°C. Immobilization of Anti-HBsAg Monoclonal Antibody to the P(NIPAAm-co-MAA) The immobilization of anti-HBsAg monoclonal antibody to the P(NIPAAm-co-MAA) polymers used the same procedure described as above for the anti-AFP antibodies. The immobilized antibodies were also dissolved in 10 mL of PBS (pH 7.4) and stored at 4°C. Competitive Enzyme Immunoassay for AFP AFP standards with different concentrations were prepared in 0.01 M PBS (pH 7.0, containing 1% BSA). A horseradish peroxidase-labeled AFP (AFP–HRP) solution (4 mg/mL) was diluted by 1:2000 with the PBS/ BSA. Fifty microliters of the standard AFP solution or the serum sample of a patient (or 500 L of normal human serum sample) and 100 L diluted AFP–HRP were added to the mixed solution of 50 L of P(NIPAAmMAA)–MAb plus 200 L of PBS (pH 7.0, containing 5% BSA). The mixture solution was diluted to 1.0 mL with 0.01 mol/L PBS solution (pH 7.0) and stirred by vibration for 5 min at 37°C to carry out the specific binding, and then 50 L 0.2 mol/L HAc-NaAc buffer (pH 5.2) was added to precipitate the polymer with antigen– antibody complex. The resultant precipitate was separated by centrifugation at 10,000 rpm for 5 min at 37°C and redissolved in 1.0 mL of 0.01 mol/L PBS (pH 7.0), and then 50 L HAc-NaAc buffer was added to precipitate the polymer again. This procedure was repeated three times, and the final precipitate obtained was mixed with 500 L 0.05 mol/L NaH 2PO 4-NaOH buffer (pH 5.8), 200 L of 0.01 mol/L p-HPA solution, and 200 L of 1 ⫻ 10 ⫺4 mol/L H 2O 2. The mixture was incubated for 15 min at room temperature followed by the addition of 100 l of 0.01 mol/L NaOH. The final fluorescence intensities of the solutions were measured at 410 nm with the excitation at 325 nm.
To a 1.5-mL plastic centrifuge tube 50 L of standard HBsAg solution or patient serum samples (or normal human blood serum) and 20 L of P(NIPAAmco-MAA)-(anti-HBsAg MAb) and 50 L of BSA (5%) were added and diluted to 1.0 mL with 0.01 mol/L PBS (pH 7.4). The mixture was vibrated at 37°C for 5 min for the specific binding to occur, and then 50 L 0.2 mol/L HAc-NaAc buffer (pH 5.2) was added to precipitate the polymer with antigen–antibody complex. The resultant precipitate was separated by centrifugation at 10,000 rpm for 5 min at 37°C and redissolved in 1.0 mL of 0.01 mol/L PBS (pH 7.0), and then 50 L HAcNaAc buffer was added to precipitate the polymer again. This procedure was repeated three times. The resultant precipitate was mixed with 100 L of diluted antibody–HRP and diluted with PBS to 1.0 mL. The solution was vibrated at 37°C for 10 min, and then the final immune complex, P(NIPAAm-co-MAA)–MAb– HBsAg–antibody–HRP, was separated by using the same procedure as that for the P(NIPAAm-co-MAA)– MAb–HBsAg complex described above. Finally, precipitate obtained was mixed with 500 L 0.05 mol/L NaH 2PO 4-NaOH buffer (pH 5.8), 200 L of 0.01 mol/L p-HPA solution and 200 L of 1 ⫻ 10 ⫺4 mol/L H 2O 2. The mixture was incubated for 15 min at room temperature followed by the addition of 100 L of 0.01 mol/L NaOH and measurement of the final fluorescence intensity of the solution. A calibration graph of fluorescence intensity versus HBsAg concentration was plotted. RESULTS AND DISCUSSION
Lower Critical Solution pH of the Polymers Polymers were formed with compositions ranging from 0 to 20% MAA, and the effect of polymer composition on pH sensitivity was determined by the cloudpoint method. In Fig. 2, the polymers with 2.5% or higher MAA content demonstrated sharp phase transitions as a function of pH. Polymers with higher LCSP were obtained using smaller amounts of MAA. It is thought that the LCSP of polymers is due to inter- or intramolecular hydrogen bonding between the amide group of NIPAAm and the carboxylic acid group of MAA. The polymer containing 2.5% MAA had the highest LCSP of 5.8, a pH that should not affect greatly the antibody–antigen binding. This polymer was therefore conjugated with antibody and used as a carrier of the reactants for immunoassay.
Sandwich Enzyme Immunoassay for HBsAg HBsAg standards with different concentrations were prepared in 0.01 M PBS (pH 7.0, containing 1% BSA). A horseradish peroxidase-labeled goat anti-HBsAg antibody (antibody-HRP) solution (7 mg/mL) was diluted by 1:1000 in PBS:BSA.
Preparation of P(NIPAAm-co-MAA)–MAb Conjugate In order to prepare a conjugate of MAb and P(NIPAAm-co-MAA), the MAb was first conjugated with an acrylamide monomer using NASI, as shown in Fig. 1. The monomer-conjugated antibody was then
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FLUORESCENCE IMMUNOASSAY SYSTEM USING pH-SENSITIVE POLYMER TABLE 1
Determination of AFP in Colon Carcinoma Patient Serum Patient serum
AFP levels a (ng/mL)
RSD (n ⫽ 12, %)
Added (ng)
Found (ng)
1 2 3 4 5
426 507 493 512 478
3.2 6.5 4.7 5.6 6.1
20 20 20 20 20
20.8 21.4 19.2 21.2 19.4
a
FIG. 2. pH dependence of absorbance at 450 nm for aqueous solutions of a series of P(NIPAAm-co-MAA)s with different initial molar ratios of methacrylic acid: (■) 0.2, (F) 0.1, (Œ) 0.05, () 0.025, (})0.01, and (⫻)0.
polymerized with NIPAAm and MAA in a free radical polymerization using the redox initiator system of APS and TEMED. The P(NIPAAm-co-MAA)–MAb conjugate was purified by repeated precipitation, washing, and redissolution. The reliability of the synthesis of the conjugate was tested. The results indicated that the synthesis of these substances showed a good reproducibility. In order to prove that MAb was covalently bound to P(NIPAAm-co-MAA) in P(NIPAAm-co-MAA)–MAb conjugate, we prepared two kinds of solution. One was a solution of P(NIPAAm-co-MAA)–MAb F conjugate prepared according to the procedure described above,
FIG. 3. Calibration curve for the determination of AFP. The plotted values are the means of three replicate determinations for each point. The standard The standard deviations for all the plotted values were less than 2.5%. The standard AFP and the AFP–HRP first reacted with the antibody immobilized on P(NIPAAm-co-MAA), then the pH of solution was adjusted below the polymer’s LCSP to precipitate the polymer–immune complex, and the polymer–immune complex precipitate was separated and redissolved by the adjustment of pH, finally the HRP-labeled AFP in the immune complex was quantified by measuring the fluorescence produced in a solution containing p-HPA and H 2O 2.
Mean of 12 determinations.
the other was prepared in a similar way but MAb F was not activated by NAS. Then, both of the solutions were precipitated and centrifuged as described above and the precipitates redissolved in PBS (pH 7.0, 0.01 mol/ L). It was found that about 90% of MAb F remained in the P(NIPAAm-co-MAA)–MAb F conjugate, whereas only 3% of MAb F remained in P(NIPAAm-co-MAA) obtained from the mixed solution of P(NIPAAm-co-MAA) and MAb F without added NAS. These experiments demonstrated that MAb was chemically conjugated to P(NIPAAm-co-MAA) in the P(NIPAAm-co-MAA)–MAb conjugate but not absorbed. Optimization of Competitive Immunoassay Conditions In the competitive immunoassay for AFP using P(NIPAAm-co-MAA), the results showed that the optimal concentration for the HRP–AFP was in the range of 1:2500 to 1:1000 times dilution of the original solution. A concentration of 1:2000 dilution of the original solution was recommended. The influence of the vol-
FIG. 4. Correlation between pH phase-separating immunoassay method and the ELISA method of AFP in human sera (n ⫽ 25). The ELISA was performed in a 96-well plate. Nonlabeled and HRPlabeled AFP competes for binding to the plate-bound antibody. After the immunoreaction, the immunochemically adsorbed HRP–AFP conjugate moiety was determined by measurement of the fluorescence produced in a solution containing p-HPA and H 2O 2.
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the results are shown in Fig. 3. A good linear relationship was observed between the fluorescence intensity and the AFP concentration in the range of 0.1–100 ng/mL. The detection limit (3/S) for AFP was calculated from the standard deviation of blank (n ⫽ 12) to be 0.04 ng/mL. The correlation coefficient was 0.996 (n ⫽ 7). Immunoassay of AFP in Human Blood Sera
FIG. 5. Calibration curve for the determination of HBsAg. In a sandwich immunoassay, the standard HBsAg first reacted with the antibody immobilized on P(NIPAAm-co-MAA), then the pH of solution was adjusted below the polymer’s LCSP to precipitate the polymer–immune complex, and the polymer–immune complex precipitate was separated and redissolved by the adjustment of pH. The resultant precipitate was further reacted with antibody–HRP, the final immune complex, P(NIPAAm-co-MAA)–MAb–HBsAg–antibody–HRP, was separated by controlling the pH, and finally the HRP-labeled AFP in the immune complex was quantified by measuring the fluorescence produced in a solution containing p-HPA and H 2O 2.
ume of P(NIPAAm-co-MAA)–MAb was studied, and 50 L of 500 ng/mL P(NIPAAm-co-MAA)–MAb in the system was shown to be optimal. The results also showed that the immune reaction of P(NIPAAm-co-MAA)–MAb and antigen would reach equilibrium within 4 min, which is much shorter than that of solid immunoreaction, and thermal-sensitive polymers modulated immuoreactions (8 –11). Calibration Graph for AFP The calibration curve for the determination of AFP was constructed under the optimal conditions, and
The AFP levels in colon carcinoma patient sera were determined by the proposed assay, the results are summarized in Table 1. The relative standard deviation within a batch was below 7% (n ⫽ 12). To evaluate the accuracy of the assay, a standard solution of AFP (20 ng) was added to 50 L of each serum sample solution containing different AFP concentrations. After measurement of the concentrations of AFP in sera samples and in sera samples with standard AFP, the recoveries of the AFP added were calculated. The results in Table 1 show that the recoveries are satisfactory, and the reproducibility of the determination is good. The AFP concentrations in 25 human sera were determined with both the pH phase-separating immunoassay method and the ELISA method. Correlation of the two methods is showed in Fig. 4. The correlation coefficient of AFP determination was 0.990. Calibration Graph for HBsAg The calibration graph for HBsAg was constructed under optimum conditions and the result are shown in Fig. 5. A good linear relationship was observed between the fluorescence intensity and the HBsAg con-
TABLE 2
Determination of HBsAg in Human Serum HBsAg levels Sample No. Healthy serum c 1 2 patient serum d 3 4 5 6 7 8 9 a
This method (ng/mL) a
ELISA b
RSD (%)
Added (ng)
Recovery (%)
0 0
⫺ ⫺
— —
— —
— —
3224 1785 2173 3574 2563 2736 1475
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
8.8 7.4 9.2 6.9 7.2 8.3 4.2
100 100 100 100 100 100 100
107 104 107 96 106 94 109
Mean of six determinations. Results supplied by the Hospital of Xiamen University. c The sample was not diluted. d The original sera were diluted by 10-fold. b
FLUORESCENCE IMMUNOASSAY SYSTEM USING pH-SENSITIVE POLYMER
centration in the range of 0.5–250 ng/mL. The detection limit (3/S) calculated from the standard deviation of the blank determination (n ⫽ 9) was found to be 0.2 ng/mL HBsAg. The correlation coefficient was 0.991. To evaluate the accuracy of the assay, a standard solution of HBsAg (100 ng) was added to 50 L of each serum sample solution containing different HBsAg concentrations. After measurement of the concentrations of HBsAg in sera samples and in sera samples with standard HBsAg, the recoveries of the HBsAg added were calculated. The results in Table 2 show that the recoveries are satisfactory, and the reproducibility of the determination is good. Immunoassay of HBsAg in Blood Sample The HBsAg levels in healthy human serum and hepatitis B patient sera were determined by the proposed assay. These samples were also analyzed in the hospital of Xiamen University by the conventional ELISA method. Results obtained by these two methods are summarized in Table 2. The relative standard deviation within a batch was below 10% (n ⫽ 6) for the determination of HBsAg in human serum. The possibility of using the proposed method for the analysis of samples was further confirmed by determining the recovery of known amounts of HBsAg added to the sample. The results in Table 2 show that the proposed method is feasible in practice.
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ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of Fujian Province (No. 9810004) and the Research Fund for the Doctoral Program of Higher Education.
REFERENCES 1. Ngo, T. T., and Lenhoff, H. M. (1985) Enzyme Mediated Immunoassay, Plenum, New York. 2. Wild, D. (Ed) (1994) The Immunoassay Handbook; Stockton Press, New York. 3. Sugawara, Y., Gee, S. J., Sanborn, J. R., Gilman, S. D., and Hammock, B. D. (1998) Anal. Chem. 70, 1092–1099. 4. Knopp, D., Schedl M., Achatz. S, Kettrup, A., and Niessner, R. (1999) Anal. Chim. Acta 399, 115–126. 5. Dzgoev, A. B., Gazaryan, I. G., Lagrimini., L. M., Ramanathan, K., and Danielsson, B. (1999) Anal. Chem. 71, 5258 –5261. 6. Li, T. M., Benovic, J. L., Buckier, R. T., and Burd, J. F. (1981) Clin. Chem. 27, 22–26. 7. Sciutto, E., Garat, B., Ortega, A., and Larralde, C. (1987) Mol. Immunol. 24, 577–585. 8. Monji, N., and Hoffman, A. S. (1987) Appl. Biochem. Biotechnol. 14, 107–120. 9. Zhu, Q. Z., Liu, F. H., Xu, J. G., Su, W. J., and Huang, J. W. (1998) Analyst 123, 1131–1134. 10. Zhu, Q. Z. Liu, F. H., Li, D. H., Xu, J. G., Su, W. J., and Huang, J. W. (1998) Anal. Chim. Acta 375, 177–185. 11. Zhu, Q. Z., Yang, H. H., Li, D. H., Xu, J. G., Zhang, Y., and Zhang, C. G. (1999) Chem. J. Chin. Univ. 20, 544 –548. 12. Yoo, M. K., Sung, Y. K., Cho, C. S., and Lee, Y. M. (1997) Polymer 38, 2759 –2765. 13. Heskins, M., and Guillet, J. E. (1968) J. Macronol. Sci. Chem. A2 8, 1441–1442. 14. Chen, G., and Hoffman, A. S. (1995) Nature 373, 49 –52.
Fluorescence Immunoassay System Based on the Use of a pH-Sensitive Phase-Separating Polymer Huang-Hao Yang,* Qing-Zhi Zhu,* Shi Chen,* Dong-Hui Li,† Xiao-Lan Chen,* Ma-tai Ding,* and Jin-Gou Xu* ,1 *Key Laboratory of Analytical Science of MOE and Department of Chemistry, and †Cancer Research Center, Xiamen University, Xiamen 361005, People’s Republic of China
Received December 1, 2000; published online August 15, 2001
Poly(N-isopropylacrylamide-co-methacrylic acid) [P(NIPAAm-co-MAA)], a linear water-soluble pH-sensitive phase-separating polymer, was synthesized and used as a novel separation carrier for the reactants in immunoassay. This polymer precipitates out of water below a critical pH 5.8 at 37°C and redissolves when the pH of solution is above 6.2. The characteristic of this polymer makes it possible to carry out the immunochemical steps of an immunoassay in a true solution and then to quickly separate the resulting product from the reaction mixture. The above approach was applied to determination of ␣-fetoprotein with the competitive immunoassay format. Compared with traditional ELISA using the same reactants, the proposed method was much faster (the assay time decreased from 100 –120 to 30 min) and showed similar sensitivity, i.e., 0.04 ng/mL. In addition, a sandwich immunoassay method for the determination of hepatitis B surface antigen was also studied, and the results showed that the pH phase-separating immunoassay could be carried out through a sandwich or a competitive method. This general technique may also be used for a wide variety of separation processes in addition to immunoassay, in which a specific component is to be isolated for analysis, recovery, or disposal. © 2001 Academic Press
Key Words: immunoassay; pH-sensitive phase-separating polymer; ␣-fetoprotein; hepatitis B surface antigen.
etc., not only in serum but also in water, foods and other consumer products (1–5). Generally, immunoassays can be divided into two categories, homogeneous and heterogeneous. Homogeneous immunoassays, such as the enzyme-mediated immunoassay test (EMIT) 2 (6), have the advantages of being rapid, easy to perform, and readily amenable to automation. However, the drawbacks of these methods, such as poor sensitivity and being prone to interference, have limited their application. On the other hand, heterogeneous immunoassays such as enzyme-linked immunosorbent assay (ELISA), are generally more sensitive than homogeneous immunoassays, less prone to interference, and can be used for both low and high molecular weight analytes. However, heterogeneous immunoassays tend to be time consuming in order to reach chemical equilibrium (7). Different approaches have been proposed to solve the problems noted above for both homogeneous and heterogeneous immunoassays. Among them is using certain temperature-sensitive polymers to carry out a fast homogeneous immune reaction and a simple heterogeneous separation process. The polymers are known to precipitate when the temperature is raised above the lower critical solution temperature (LCST). Among the polymers, poly-N-isopropylacrylamide (PNIPAAm) is known to have a LCST of about 31°C and has been 2
Immunoassay techniques are now widely applied in measurements of toxins, drugs, hormones, pesticides, 1
To whom correspondence should be addressed at Department of Chemistry, Xiamen University, Xiamen 361005, People’s Republic of China. Fax: ⫹86-592-2188054. E-mail: [email protected]. 0003-2697/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Abbreviations used: P(NIPAAm-co-MAA), poly(N-isopropylacrylamide-co-methacrylic acid); HRP, horseradish peroxidase; AFP, ␣-fetoprotein; HBsAg, hepatitis B surface antigen; EMIT, enzyme-mediated immunoassay test; ELISA, enzyme-linked immunosorbent assay; LCST, lower critical solution temperature; LCSP, lower critical solution pH; NIPAAm, N-isopropylacrylamide; TEMED, N,N,N⬘,N⬘-tetramethylethylenediamine; MAA, methacrylic acid; APS, ammonium persulfate; FITC, fluorescein isothiocyanate; NAS, N-hydroxysuccinimidyl acrylate; p-HPA, p-hydroxyphenylacetic acid; H 2O 2, hydrogen peroxide; BSA, bovine serum albumin; PBS, phosphate-buffered saline; RSD, relative standard deviation. 167
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FIG. 1.
Protocol for the pH-sensitive phase-separating immunoassay method.
widely utilized as the separation carrier in immunoassays (8 –10). However, such assays can only be conducted under the LCST of the polymers, and therefore, this would inevitably affect the immune reaction efficiency (11). pH-sensitive polymers are another kind of environmentally sensitive polymer, which contain ionizable groups and can lead to pH-dependent phase transition (12). However, the polymers previously developed are not fit for use as the carrier in immunoassays due to their pH values for precipitation being in the low pH range. In this work, a series of pH-sensitive phase-separating polymers containing both temperature- and pHsensitive components were synthesized by copolymerizing N-isopropylacrylamide and methacrylic acid. Poly(methacrylic acid) (PMAA) is one of the pH-sensitive polymers which contains ionizable groups as the anionic polyelectrolyte. The pH of the environment determines the ionization degree of carboxylic acid groups of the PMAA. The influence of the concentration of MAA on the lower critical solution pH (LCSP) of P(NIPAAM-co-MAA) polymers was investigated in the
pH range of 3– 8 at 37°C. The LCSPs were determined by cloud point measurement. As a result, the polymer containing 2.5 mol% MAA, with a LCSP of 5.8 at 37°C, was chosen to use as the separation carrier for the immunoassay of AFP. With the use of P(NIPAAm-coMAA) as a support, the whole immunoassay process, including the homogeneous immune reaction of the P(NIPAAm-co-MAA)- conjugated monoclonal antibody and ␥-fetoprotein (AFP), the heterogeneous separation of the P(NIPAAm-co-MAA)-bound immune complex, and the homogeneous fluorescence detection of the final product, can be carried out by control of the pH. The scheme for the pH-based polymer– enzymatic immunoassay is shown in Fig. 1. In a competitive immunoassay, the standard AFP and the horseradish peroxidase labeled AFP (AFP–HRP) first reacted with the antibody immobilized on P(NIPAAm-co-MAA), and then the pH of solution was adjusted below the polymer’s LCSP to precipitate the polymer–immune complex, the polymer–immune complex precipitate was separated and redissolved by the adjustment of pH, and finally the HRP-labeled AFP in the immune complex was quantified by fluorescence measurement. A
FLUORESCENCE IMMUNOASSAY SYSTEM USING pH-SENSITIVE POLYMER
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sandwich immunoassay method for the determination of HBsAg was also developed showing that the pHsensitive phase-separating immunoassay could be performed in the sandwich method as well as the competitive method. The proposed method offers several advantages over the thermal phase-separating immunoassay. First, the immune process is conducted at 37°C; thus shorter incubation time is needed. Second, this method can be applied to the processes in which temperature change is not allowed or hardly conducted. Furthermore, this polymer can also be used as a pH-switching control of selective removal and delivery in human bodies since the pH-dependent phase transition is conducted at 37°C.
Synthesis of P(NIPAAm-co-MAA)
MATERIALS AND METHODS
Cloud-Point Determination
Reagents
The absorbance at 450 nm was measured against the pH of solution, which was raised from 3 to 8 in a 0.2 pH increment per minute, at 37°C using a Beckman DU7400 UV/VIS diode array spectrophotometer. The cloud point was defined as the pH at the inflection point in the normalized absorbance vs pH curve and the cloud point of solution which does not exhibit an infection point was determined at 10% absorbance in the curve (14).
␣-Fetoprotein, anti-AFP monoclonal antibody (MAb), horseradish peroxidase-labeled AFP (AFP– HRP, 3 mg/mL), and bovine serum albumin (BSA) were purchased from Sino-American Biotechnology Company (Shanghai, China). Hepatitis B surface antigen (HBsAg), anti-HBsAg monoclonal antibody, and horseradish peroxidase-labeled goat anti-HBsAg antibody (7 mg/mL) were purchased from Xinchuang Biotechnology Company (Xiamen, China). N-Isopropylacrylamide (NIPAAm) and N,N,N’,N’-tetramethylethylenediamine (TEMED) were purchased from TCI-EP (Tokyo Kasei, Japan) and used without further purification. Methacrylic acid, ammonium persulfate (APS), and fluorescein isothiocyanate (FITC) were obtained from Shanghai Chemical Reagent Co. (Shanghai, China). N-Hydroxysuccinimidyl acrylate (NAS) was synthesized as described (13). Hydrogen peroxide (H 2O 2, 30%) was obtained from Shanghai Taopu Chemical Factory (Shanghai, China), and the stock solution (3% H 2O 2) was standardized by titration with a standard solution of KMnO 4. p-Hydroxyphenylacetic acid (p-HPA) was obtained from Sigma and used without further purification. All the reagents are of analytical grade. The distilled, deionized water was used throughout.
Redox polymerization was used to prepare linear polymer samples of NIPAAm and MAA. Prior to the reaction, MAA was separated from the reaction inhibitor, p-methoxyphenol, by distillation under vacuum. Polymers were prepared using molar MAA fractions of 0.2, 0.1, 0.05, 0.025, 0.02, and 0 with the balance being NIPAAm. In addition, 10 mol% (of total monomers) APS was used as the initiator and 1 mol% TEMED as the accelerator. After polymerization at 37°C for 2 h, the polymers were precipitated by addition of a constant amount of 0.1 M HCl and separated from unreacted monomers by centrifugation at 37°C for 5 min (10,000 rpm).
Preparation of Monomer-Conjugated and Monomer, FITC-co-Labeled Anti-AFP Monoclonal Antibody A 0.2 mL of NAS (a 10% solution in DMSO, v/v) was added to 2.0 mL of phosphate-buffered saline solution (PBS, pH 7.0) containing 4 mg anti-AFP MAb. The resultant mixture was vibrated for 1 h at 37°C and then passed through a column of Sephadex G-25 that had been equilibrated with PBS, pH 7.0. The fractions of the elute that contained antibody were collected and stored at 4°C. An aliquot of this monomer-conjugated MAb (MAb M) was further reacted with a 25-fold molar excess of FITC. Excess, unconjugated FITC was removed by filtration on a Sephadex G-25 column. The resultant, double-conjugated anti-AFP MAb (MAb M,F) with both NAS and FITC, was stored at 4°C.
Apparatus A Hitachi 650-10S spectrofluorimeter (Tokyo, Japan) equipped with a plotter unit and a 1-cm quartz cell was used for recording fluorescence spectra and making fluorescence measurements. The absorption spectrum was obtained on a Beckman DU-7400 UV/VIS diode array spectrophotometer (Fullerton, CA). All the pH measurements were made with a Model PHS-301 pH meter (Xiamen, China). A TGL-16G supercentrifuge (Shanghai, China) and a SHZ-88 water-bath vibrator (Jiangshu, China) were used.
Immobilization of Anti-AFP MAb M and Anti-AFP MAb M,F to the P(NIPAAm-co-MAA) The product of MAb M or MAb M,F (2 mg) was diluted to 4 mL with PBS, and then 975 L of 1 mol/L NIPAAm, 250 L of 0.1 mol/L MAA, 100 L of 0.1 mol/L APS, and 10 L of 0.1 mol/L TEMED were added and the mixture was allowed to stand for 2 h in a 37°C water bath to carry out the copolymerization reaction. The P(NIPAAm-co-MAA)–MAb and P(NIPAAm-co-MAA)– MAb F conjugates were precipitated by the addition of
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200 L 0.2 mol/L HAc-NaAc buffer (pH 5.2) and the precipitate was collected by centrifugation at 37°C for 5 min (10,000 rpm). After the supernatant was removed, the precipitate was dissolved in PBS (pH 7.0). The precipitation-centrifugation-dissolution procedure noted above was repeated three times. Finally, the conjugates were dissolved in 10 mL of PBS (pH 7.4) and stored at 4°C. Immobilization of Anti-HBsAg Monoclonal Antibody to the P(NIPAAm-co-MAA) The immobilization of anti-HBsAg monoclonal antibody to the P(NIPAAm-co-MAA) polymers used the same procedure described as above for the anti-AFP antibodies. The immobilized antibodies were also dissolved in 10 mL of PBS (pH 7.4) and stored at 4°C. Competitive Enzyme Immunoassay for AFP AFP standards with different concentrations were prepared in 0.01 M PBS (pH 7.0, containing 1% BSA). A horseradish peroxidase-labeled AFP (AFP–HRP) solution (4 mg/mL) was diluted by 1:2000 with the PBS/ BSA. Fifty microliters of the standard AFP solution or the serum sample of a patient (or 500 L of normal human serum sample) and 100 L diluted AFP–HRP were added to the mixed solution of 50 L of P(NIPAAmMAA)–MAb plus 200 L of PBS (pH 7.0, containing 5% BSA). The mixture solution was diluted to 1.0 mL with 0.01 mol/L PBS solution (pH 7.0) and stirred by vibration for 5 min at 37°C to carry out the specific binding, and then 50 L 0.2 mol/L HAc-NaAc buffer (pH 5.2) was added to precipitate the polymer with antigen– antibody complex. The resultant precipitate was separated by centrifugation at 10,000 rpm for 5 min at 37°C and redissolved in 1.0 mL of 0.01 mol/L PBS (pH 7.0), and then 50 L HAc-NaAc buffer was added to precipitate the polymer again. This procedure was repeated three times, and the final precipitate obtained was mixed with 500 L 0.05 mol/L NaH 2PO 4-NaOH buffer (pH 5.8), 200 L of 0.01 mol/L p-HPA solution, and 200 L of 1 ⫻ 10 ⫺4 mol/L H 2O 2. The mixture was incubated for 15 min at room temperature followed by the addition of 100 l of 0.01 mol/L NaOH. The final fluorescence intensities of the solutions were measured at 410 nm with the excitation at 325 nm.
To a 1.5-mL plastic centrifuge tube 50 L of standard HBsAg solution or patient serum samples (or normal human blood serum) and 20 L of P(NIPAAmco-MAA)-(anti-HBsAg MAb) and 50 L of BSA (5%) were added and diluted to 1.0 mL with 0.01 mol/L PBS (pH 7.4). The mixture was vibrated at 37°C for 5 min for the specific binding to occur, and then 50 L 0.2 mol/L HAc-NaAc buffer (pH 5.2) was added to precipitate the polymer with antigen–antibody complex. The resultant precipitate was separated by centrifugation at 10,000 rpm for 5 min at 37°C and redissolved in 1.0 mL of 0.01 mol/L PBS (pH 7.0), and then 50 L HAcNaAc buffer was added to precipitate the polymer again. This procedure was repeated three times. The resultant precipitate was mixed with 100 L of diluted antibody–HRP and diluted with PBS to 1.0 mL. The solution was vibrated at 37°C for 10 min, and then the final immune complex, P(NIPAAm-co-MAA)–MAb– HBsAg–antibody–HRP, was separated by using the same procedure as that for the P(NIPAAm-co-MAA)– MAb–HBsAg complex described above. Finally, precipitate obtained was mixed with 500 L 0.05 mol/L NaH 2PO 4-NaOH buffer (pH 5.8), 200 L of 0.01 mol/L p-HPA solution and 200 L of 1 ⫻ 10 ⫺4 mol/L H 2O 2. The mixture was incubated for 15 min at room temperature followed by the addition of 100 L of 0.01 mol/L NaOH and measurement of the final fluorescence intensity of the solution. A calibration graph of fluorescence intensity versus HBsAg concentration was plotted. RESULTS AND DISCUSSION
Lower Critical Solution pH of the Polymers Polymers were formed with compositions ranging from 0 to 20% MAA, and the effect of polymer composition on pH sensitivity was determined by the cloudpoint method. In Fig. 2, the polymers with 2.5% or higher MAA content demonstrated sharp phase transitions as a function of pH. Polymers with higher LCSP were obtained using smaller amounts of MAA. It is thought that the LCSP of polymers is due to inter- or intramolecular hydrogen bonding between the amide group of NIPAAm and the carboxylic acid group of MAA. The polymer containing 2.5% MAA had the highest LCSP of 5.8, a pH that should not affect greatly the antibody–antigen binding. This polymer was therefore conjugated with antibody and used as a carrier of the reactants for immunoassay.
Sandwich Enzyme Immunoassay for HBsAg HBsAg standards with different concentrations were prepared in 0.01 M PBS (pH 7.0, containing 1% BSA). A horseradish peroxidase-labeled goat anti-HBsAg antibody (antibody-HRP) solution (7 mg/mL) was diluted by 1:1000 in PBS:BSA.
Preparation of P(NIPAAm-co-MAA)–MAb Conjugate In order to prepare a conjugate of MAb and P(NIPAAm-co-MAA), the MAb was first conjugated with an acrylamide monomer using NASI, as shown in Fig. 1. The monomer-conjugated antibody was then
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FLUORESCENCE IMMUNOASSAY SYSTEM USING pH-SENSITIVE POLYMER TABLE 1
Determination of AFP in Colon Carcinoma Patient Serum Patient serum
AFP levels a (ng/mL)
RSD (n ⫽ 12, %)
Added (ng)
Found (ng)
1 2 3 4 5
426 507 493 512 478
3.2 6.5 4.7 5.6 6.1
20 20 20 20 20
20.8 21.4 19.2 21.2 19.4
a
FIG. 2. pH dependence of absorbance at 450 nm for aqueous solutions of a series of P(NIPAAm-co-MAA)s with different initial molar ratios of methacrylic acid: (■) 0.2, (F) 0.1, (Œ) 0.05, () 0.025, (})0.01, and (⫻)0.
polymerized with NIPAAm and MAA in a free radical polymerization using the redox initiator system of APS and TEMED. The P(NIPAAm-co-MAA)–MAb conjugate was purified by repeated precipitation, washing, and redissolution. The reliability of the synthesis of the conjugate was tested. The results indicated that the synthesis of these substances showed a good reproducibility. In order to prove that MAb was covalently bound to P(NIPAAm-co-MAA) in P(NIPAAm-co-MAA)–MAb conjugate, we prepared two kinds of solution. One was a solution of P(NIPAAm-co-MAA)–MAb F conjugate prepared according to the procedure described above,
FIG. 3. Calibration curve for the determination of AFP. The plotted values are the means of three replicate determinations for each point. The standard The standard deviations for all the plotted values were less than 2.5%. The standard AFP and the AFP–HRP first reacted with the antibody immobilized on P(NIPAAm-co-MAA), then the pH of solution was adjusted below the polymer’s LCSP to precipitate the polymer–immune complex, and the polymer–immune complex precipitate was separated and redissolved by the adjustment of pH, finally the HRP-labeled AFP in the immune complex was quantified by measuring the fluorescence produced in a solution containing p-HPA and H 2O 2.
Mean of 12 determinations.
the other was prepared in a similar way but MAb F was not activated by NAS. Then, both of the solutions were precipitated and centrifuged as described above and the precipitates redissolved in PBS (pH 7.0, 0.01 mol/ L). It was found that about 90% of MAb F remained in the P(NIPAAm-co-MAA)–MAb F conjugate, whereas only 3% of MAb F remained in P(NIPAAm-co-MAA) obtained from the mixed solution of P(NIPAAm-co-MAA) and MAb F without added NAS. These experiments demonstrated that MAb was chemically conjugated to P(NIPAAm-co-MAA) in the P(NIPAAm-co-MAA)–MAb conjugate but not absorbed. Optimization of Competitive Immunoassay Conditions In the competitive immunoassay for AFP using P(NIPAAm-co-MAA), the results showed that the optimal concentration for the HRP–AFP was in the range of 1:2500 to 1:1000 times dilution of the original solution. A concentration of 1:2000 dilution of the original solution was recommended. The influence of the vol-
FIG. 4. Correlation between pH phase-separating immunoassay method and the ELISA method of AFP in human sera (n ⫽ 25). The ELISA was performed in a 96-well plate. Nonlabeled and HRPlabeled AFP competes for binding to the plate-bound antibody. After the immunoreaction, the immunochemically adsorbed HRP–AFP conjugate moiety was determined by measurement of the fluorescence produced in a solution containing p-HPA and H 2O 2.
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the results are shown in Fig. 3. A good linear relationship was observed between the fluorescence intensity and the AFP concentration in the range of 0.1–100 ng/mL. The detection limit (3/S) for AFP was calculated from the standard deviation of blank (n ⫽ 12) to be 0.04 ng/mL. The correlation coefficient was 0.996 (n ⫽ 7). Immunoassay of AFP in Human Blood Sera
FIG. 5. Calibration curve for the determination of HBsAg. In a sandwich immunoassay, the standard HBsAg first reacted with the antibody immobilized on P(NIPAAm-co-MAA), then the pH of solution was adjusted below the polymer’s LCSP to precipitate the polymer–immune complex, and the polymer–immune complex precipitate was separated and redissolved by the adjustment of pH. The resultant precipitate was further reacted with antibody–HRP, the final immune complex, P(NIPAAm-co-MAA)–MAb–HBsAg–antibody–HRP, was separated by controlling the pH, and finally the HRP-labeled AFP in the immune complex was quantified by measuring the fluorescence produced in a solution containing p-HPA and H 2O 2.
ume of P(NIPAAm-co-MAA)–MAb was studied, and 50 L of 500 ng/mL P(NIPAAm-co-MAA)–MAb in the system was shown to be optimal. The results also showed that the immune reaction of P(NIPAAm-co-MAA)–MAb and antigen would reach equilibrium within 4 min, which is much shorter than that of solid immunoreaction, and thermal-sensitive polymers modulated immuoreactions (8 –11). Calibration Graph for AFP The calibration curve for the determination of AFP was constructed under the optimal conditions, and
The AFP levels in colon carcinoma patient sera were determined by the proposed assay, the results are summarized in Table 1. The relative standard deviation within a batch was below 7% (n ⫽ 12). To evaluate the accuracy of the assay, a standard solution of AFP (20 ng) was added to 50 L of each serum sample solution containing different AFP concentrations. After measurement of the concentrations of AFP in sera samples and in sera samples with standard AFP, the recoveries of the AFP added were calculated. The results in Table 1 show that the recoveries are satisfactory, and the reproducibility of the determination is good. The AFP concentrations in 25 human sera were determined with both the pH phase-separating immunoassay method and the ELISA method. Correlation of the two methods is showed in Fig. 4. The correlation coefficient of AFP determination was 0.990. Calibration Graph for HBsAg The calibration graph for HBsAg was constructed under optimum conditions and the result are shown in Fig. 5. A good linear relationship was observed between the fluorescence intensity and the HBsAg con-
TABLE 2
Determination of HBsAg in Human Serum HBsAg levels Sample No. Healthy serum c 1 2 patient serum d 3 4 5 6 7 8 9 a
This method (ng/mL) a
ELISA b
RSD (%)
Added (ng)
Recovery (%)
0 0
⫺ ⫺
— —
— —
— —
3224 1785 2173 3574 2563 2736 1475
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
8.8 7.4 9.2 6.9 7.2 8.3 4.2
100 100 100 100 100 100 100
107 104 107 96 106 94 109
Mean of six determinations. Results supplied by the Hospital of Xiamen University. c The sample was not diluted. d The original sera were diluted by 10-fold. b
FLUORESCENCE IMMUNOASSAY SYSTEM USING pH-SENSITIVE POLYMER
centration in the range of 0.5–250 ng/mL. The detection limit (3/S) calculated from the standard deviation of the blank determination (n ⫽ 9) was found to be 0.2 ng/mL HBsAg. The correlation coefficient was 0.991. To evaluate the accuracy of the assay, a standard solution of HBsAg (100 ng) was added to 50 L of each serum sample solution containing different HBsAg concentrations. After measurement of the concentrations of HBsAg in sera samples and in sera samples with standard HBsAg, the recoveries of the HBsAg added were calculated. The results in Table 2 show that the recoveries are satisfactory, and the reproducibility of the determination is good. Immunoassay of HBsAg in Blood Sample The HBsAg levels in healthy human serum and hepatitis B patient sera were determined by the proposed assay. These samples were also analyzed in the hospital of Xiamen University by the conventional ELISA method. Results obtained by these two methods are summarized in Table 2. The relative standard deviation within a batch was below 10% (n ⫽ 6) for the determination of HBsAg in human serum. The possibility of using the proposed method for the analysis of samples was further confirmed by determining the recovery of known amounts of HBsAg added to the sample. The results in Table 2 show that the proposed method is feasible in practice.
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ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of Fujian Province (No. 9810004) and the Research Fund for the Doctoral Program of Higher Education.
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