Folic Acid-Anchored PEGgylated Phospholipid Bioconjugate and Its

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Anal. Chem. 2009, 81, 5671–5677

Folic Acid-Anchored PEGgylated Phospholipid Bioconjugate and Its Application in a Liposomal Immunodiagnostic Assay for Folic Acid Ja-an Annie Ho,*,† Chi-Hsiang Hung,† Li-Chen Wu,‡ and Ming-Yuan Liao§ BioAnalytical Lab, Department of Chemistry, National Tsing Hua University, Hsin-chu, 300 Taiwan, Biochemistry, Department of Applied Chemistry, National Chi Nan University, Puli, 545 Taiwan, and Department of Chemistry, National Chung Hsing University, Taichung, 402 Taiwan A folic acid-anchored, poly(ethylene glycol)-linked (PEGgylated) phospholipid and an immunoaffinity chromatographic column were prepared and employed to develop a liposomal immunodiagnostic assay for the direct determination of folic acid (FA) in this study. Distearoylphosphatidylethanolamine-poly(ethylene glycol)2000-folic acid (DSPE-PEG2000-FA) was synthesized through carbodiimide-mediated coupling of FA and DSPE-PEG2000amine and characterized using thin layer chromatography, 1H nuclear magnetic resonance spectroscopy, and electrospray ionization-mass spectrometry. Liposomal biolabels were constructed using the synthesized DSPE-PEG2000-FA in conjunction with other phospholipids. A stationary phase having affinity for FA was prepared by covalently linking purified anti-FA monoclonal antibodies onto N-hydroxysuccinimide-activated Sepharose beads, which were subsequently packed into a 1.9 cm diameter polypropylene column. The calibration curve for FA had a linear range from 10-8 to 10-4 M. The limit of detection was 6.8 ng (equivalent to 500 µL of 3.1 × 10-8 M FA). The elution buffer (35% methanol in Tris buffered saline containing 0.1% Tween 20) also served as the regeneration buffer, which allowed the same column to be used for up to 50 times without any observable loss of reactivity. The immunoaffinity chromatographic column was reusable and capable of concentrating analytes from sample solution; in conjunction with folic acid-sensitized liposomal biolabels, however, they hold great potential as sensitive immunoaffinity assays for the determination for FA. To confirm the feasibility of using this system in the analysis of real samples, the folic acid contents of three over-the-counter vitamin supplements were tested. The recoveries of folic acid of 90-112% for these three samples were obtained, suggesting contents that were consistent with the information obtained from their nutritional facts panels. * Corresponding author. Fax: +886-3-571-1082. E-mail: [email protected]. † National Tsing Hua University. ‡ National Chi Nan University. § National Chung Hsing University. 10.1021/ac900402v CCC: $40.75  2009 American Chemical Society Published on Web 06/11/2009

Folic acid (FA) was originally extracted from yeast by Willis1 in 1931; it was initially called vitamin M or vitamin Bc. Technically, the name “folic acid” refers to the manmade type of B vitamin that is used in vitamin supplements or added to certain foods (socalled enriched or fortified foods) and “folate” refers to the natural type of B vitamin, which is already present in some foods. Nowadays, however, folate is a generic term that refers to all derivatives of folic acid. Biologically, FA is essential for transferring one-carbon units involved in phospholipid, DNA, protein, and neurotransmitter syntheses.2 Folate is an essential dietary component for the formation of red and white blood cells and the epithelial cells in the digestive tract.3 At present, adequate folate nutriture is encouraged because of the possible correlation between folate intake and the occurrence of pregnancy neural tube defects and occlusive vascular disease, where an increased concentration of homocysteine in the blood4,5 has been implicated as a risk factor.6,7 Therefore, sufficient folate nutriture is believed to reduce the incidence of cardiovascular disease by lowering homocysteine concentrations in plasma and serum. Furthermore, Selhub and Rosenberg (1996)2 observed a close relationship between folate and choline in the reactions of the methyl cycle. Moreover, folate intake appears to be inversely related to the risk of colorectal cancer.8 In light of these health benefits, in January 1998, the U.S. Food and Drug Administration mandated9 the fortification of cereal grain products with FA at a level of 140 mg per 100 g, which is expected to yield a reduction in the incidence of folate-associated diseases in the U.S.10 The average folate intake of adult men and (1) Willis, S. L. Br. Med. J. 1931, 1, 1059–1064. (2) Selhub, J.; Rosenberg, I. H. Folic Acid. In Present Knowledge in Nutrition, 7th ed.; Ziegler, E. E., Filer L. J., Jr., Eds.; International Life Sciences Institute Press: Washington, DC, 1996; pp 206-219. (3) Gregory, J. F.; Ristow, K. A.; Sartain, D. B.; Damron, B. L. J. Agric. Food Chem. 1984, 32, 1337–1342. (4) Wald, N. Lancet 1991, 338, 131–137. (5) Boushey, C. J.; Beresford, S. A. A.; Omenn, G. S.; Motulsky, A. G. JAMA 1995, 274, 1049–1057. (6) Hankey, G. J.; Eikelboom, J. W. Lancet 1999, 354, 407–413. (7) Refsum, H.; Ueland, P. M.; Nygard, O.; Vollset, S. E. Annu. Rev. Med. 1998, 49, 31–62. (8) Johnson, I. T.; Lund, E. K. Aliment. Pharmacol. Ther. 2007, 26, 161–181. (9) Food and Drug Administration (FDA). Food Standards: Amendment of Standards of Identity for Enriched Grain Products to Require Addition of Folic Acid; Final Rule (21 CFR Parts 136, 137, and 139). Fed. Regist. 1996, 61, 8781-8797. (10) Tucker, K. L.; Mahnken, B.; Wilson, P. W. F.; Jacques, P.; Selhub, J. JAMA 1996, 276, 1879–1885.

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women in various European countries ranges from 150 to 400 µg/day.11,12 The existence of folate in many different chemical forms (with various stabilities) in foods makes it considerably difficult to characterize the vitamin and establish accurate data regarding its levels. Over the past four decades, many techniques have been employed in the analysis of folate, including microbiological,13-16 radiobinding,17 radiometric,18 electrochemical,19-21 and highperformance liquid chromatography-based13,21-25 assays, immunoassays,26,27 and optical biosensing devices based on surface plasmon resonance (SPR).28-30 Microbiological assays (MBAs) are used most widely; they are considered among the most versatile approaches to determining food folate levels. The principle of MBAs is based on the quantitative relationship between folate content and the growth of certain microorganisms, including Lactobacillus casei (L. casei, ATCC 7469), Streptococcus faecium (ATCC 8043), and Leuconostoc citrovorum (ATCC 8081).13,14,16 Among these microorganisms, L. casei is used most widely for determining food folate levels because it responds almost equally to the widest variety of folate derivatives. Despite their popular use, MBAs are particularly laborious and timeconsuming, making it difficult to be established as reliable routine laboratory methods. Although liquid chromatography/mass spectrometry-based analysis has been developed in recent years to determine food folate levels, it remains expensive and not accessible to all research laboratories. The relatively low accuracies of the established methods and their complicated sample preparation requirements, often including extraction, deconjugation, and purification, mean that none of them have attained official status as a reference method for the measurement of natural folate in food.31-33 The past decade has witnessed rapid growth in the development of immunoassays for the screening of biologically and environmentally important target molecules. Because of their high (11) de Bree, A.; van Dusseldorp, M.; Brouwer, I. A.; van het Hof, K. H.; SteegersTheunissen, R. P. Eur. J. Clin. Nutr. 1997, 51, 643–660. (12) Hawkes, J. G.; Villota, R. Crit. Rev. Food Sci. Nutr. 1989, 28, 439–538. (13) Tamura, T. J. Nutr. Biochem. 1998, 9, 285–293. (14) Horne, D. W. Methods Enzymol. 1997, 281, 38–43. (15) Finglas, P. M.; Faure, U.; Southgate, D. A. T. Food Chem. 1993, 46, 199– 213. (16) Horne, D. W.; Patterson, D. Clin. Chem. 1988, 34, 2357–2359. (17) Desouza, S.; Eitenmiller, R. J. Micronutr. Anal. 1990, 7, 37–57. (18) Chen, M. F.; Hill, J. W.; Mcintyre, P. A. J. Nutr. 1983, 113, 2192–2196. (19) Shin, H. C.; Shimoda, M.; Kokue, E. J. Chromatogr., B: Biomed. Sci Appl. 1994, 661, 237–244. (20) White, D. R.; Lee, H. S.; Kruger, R. E. J. Agric. Food Chem. 1991, 39, 714–717. (21) Bagley, P. J.; Selhub, J. Clin. Chem. 2000, 46, 404–411. (22) Osseyi, E. S.; Wehling, R. L.; Albrecht, J. A. J. Chromatogr., A 1998, 826, 235–240. (23) Vahteristo, L. T.; Ollilainen, V.; Koivistoinen, P. E.; Varo, P. J. Agric. Food Chem. 1996, 44, 477–482. (24) Gounelle, J. C.; Ladjimi, H.; Prognon, P. Anal. Biochem. 1989, 176, 406– 411. (25) Konings, E. J. M. J. AOAC Int. 1999, 82, 119–127. (26) Das Sarma, J.; Duttagupta, C.; Ali, E.; Dhar, T. K. J. Immunol. Methods 1995, 184 (1), 1–6. (27) Lermo, A.; Fabiano, S.; Hernandez, S.; Galve, R.; Marco, M. P.; Alegret, S.; Pividori, M. I. Biosens. Bioelectron. 2009, 24 (7), 2057–2063. (28) Caselunghe, M. C. B.; Lindeberg, J. Food Chem. 2000, 70 (4), 523–532. (29) Gao, Y.; Guo, F.; Gokavi, S.; Chow, A.; Sheng, Q.; Guo, M. Food Chem. 2008, 110 (3), 769–776. (30) Indyk, H. E.; Evans, E. A.; Caselunghe, M. C. B.; Persson, B. S.; Finglas, P. M.; Woollard, D. C.; Filonzi, E. L. J. AOAC Int. 2000, 83 (5), 1141– 1148.

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sensitivity, rapidity, and simplicity of operation, immunoassays are among the most powerful tools for selectively detecting various physiological, biological, and environmental substances. Because the demand for food folate determination is increasing gradually, there is an ongoing need for simpler, faster, less expensive, and more reliable and user-friendly analytical tools. In this paper, we present a simple method for preparing FA-anchored, poly(ethylene glycol)-linked (PEGgylated) phospholipid bioconjugates, which were subsequently incorporated into the formation of FA-anchored liposomes. We have also developed a simple yet novel alternative method for the detection of FA, one that combines an immunoaffinity chromatographic (IAC) assay with FA-anchored liposomal fluorescent biolabels, encapsulating carboxyfluorescein (CF) as signal amplifiers, that compete with FA for a limited number of immobilized antibody-binding sites on the stationary phase of an IAC column (Figure 1). IAC columns are often used to concentrate analytes; in conjunction with liposomal biolabels, however, they hold great potential as sensitive immunoaffinity assays for the determination for FA. We found that the number of liposomes bound to the anti-FA antibodies was inversely proportional to the concentration of FA in the sample solution. We observed competition between the liposomal biolabels and FA for the immobilized antibody’s binding sites; we quantified the level of FA by measuring the fluorescence intensity of CF after lysis of the bound liposomes with elution buffer. The antibody-binding activity was regenerated after washing with 35% methanol (MeOH) to elute the bound FA off the column, which we reconditioned by passing a buffer through the system prior to performing the next measurement. EXPERIMENTAL SECTION Reagents, Materials, and Apparatus. All inorganic chemicals and organic solvents were of analytical grade or the highest purity commercially available; they were used as received. FA, dihydrofolic acid, folinic acid (calcium salt), anti-FA monoclonal antibody (Mab) produced in mouse (clone VP-52, ascites fluid), cholesterol, 5(6)-carboxyfluorescein, Tween 20, sucrose, tris(hydroxymethyl) aminomethane (Trizma base, Tris), dicyclohexylcarbodiimide (DCC), octanoic acid, ammonium sulfate, sodium hydroxide, sodium azide, sodium bicarbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride, sodium acetate, molybdenum blue spray reagent, isopropyl ether, chloroform, MeOH, dimethylsulfoxide (DMSO), pyridine, acetic acid, hydrochloric acid (HCl), and Sephadex G-7550 were purchased from Sigma (St. Louis, MO). N-Hydroxysuccinimide (NHS)-activated Sepharose 4 Fast Flow slurry was purchased from Pharmacia Biotech (Uppsala, Sweden). Disposable polypropylene columns were purchased from Pierce Chemical (Rockford, IL). 1,2-Distearoyl-sn-glycero-3-phosphoethanolamineN-[amino(polyethylene glycol)2000] ammonium salt (DSPEPEG2000-amine), dipalmitoylphosphatidylcholine (DPPC), and dipalmitoylphosphatidylglycerol (DPPG) were obtained from Avanti Polar Lipids (Alabaster, AL). Dialysis bags [molecular (31) Martin, C. A. BNF Nutr. Bull. 1995, 20, 8–15. (32) Vahteristo, L.; Finglas, P. M. Chromatographic Determination of Folates. In Modern Chromatographic Analysis of Vitamins; De Leenheer, A. P.; Lambert, W. E.; Van Bocxlaer, J. F., Eds.; Marcel Dekker: New York, 2000. (33) Vahteristo, L.; Kariluoto, S. M.; Ba¨rlund, S.; Ka¨rkka¨inin, M.; Lamberg-Allardt, C.; Salovaara, H.; Piironen, V. Eur. J. Nutr. 2002, 41, 271–278.

Figure 1. Schematic representation of the operation of a liposomal immunodiagnostic assay for the detection of folic acid.

weight cutoffs (MWCO): 12 000-14 000 and 6000-8000 Da] were purchased from Spectrum Laboratories (Rancho Dominguez, CA). A protein assay kit was purchased from Bio-Rad Laboratories (Hercules, CA). All solutions were prepared with deionized water having a resistivity of no less than 18 MΩ cm-1 (MilliQ, Bedford, MA). Nuclear magnetic resonance (NMR) spectra were acquired on a Unity Inova 600 MHz spectrometer (Varian, Walnut Creek, CA). Mass spectroscopic analysis was performed by HPLC equipped with an electrospray ionization-mass spectrometer (Finnigan LCQ spectrometer, Thermo, San Jose, CA). The size distribution and ζ potential of the liposomes were measured using a Brookhaven 90Plus nanoparticle size analyzer and a ZetaPlus zeta potential analyzer, respectively (Brookhaven Instruments Corporation, Holtsville, NY). Distearoylphosphatidylethanolamine-Poly(ethylene glycol)2000-Folic acid (DSPE-PEG2000-FA) Bioconjugate. DSPEPEG2000-FA bioconjugate was synthesized on the basis of a modification of methods reported by Gabizon et al.34 and Saul et al.35 Briefly, DSPE-PEG2000-amine (33.6 mg), DCC (15.3 mg), and pyridine (167 µL) were added to a solution of FA (16.7 mg) in DMSO (667 mL), and the resulting solution was then left to react for 4 h at room temperature. After the pyridine was removed under rotary evaporation, water (4.2 mL) was added to the solution. The product was dialyzed with a 12 000-14 000 MWCO dialysis bag: twice against 50 mM sodium chloride (1 L) and three times against water (1 L). The solution was lyophilized to yield the final product (33.6 mg, 86%). 1H NMR (δ): 0.87 (t, 6H, CH3), 1.25 (s, 56H, CH2), 1.53-1.65 (m, 4H, CH2CH2CO), 2.21-2.39 (m, 8H, CH2CH2CO and CH2 of Glu), 3.40-3.49 (m, 4H, CH2CH2N), 3.64 (s, ca. 180H, PEG2000), 4.35-4.37 (m, 1H, R-CH), 4.55 (d, 2H, 9-CH2N),

5.20 (m, 1H, PO4CH2CH), 6.64 (d, 2H, 3′,5′-ArH), 7.68 (d, 2H, 2′,6′-ArH), 8.77 (s, 1H, C7-H). Liposomal Biolabels (FA-Anchored, CF-Encapsulated Liposomes). The preparation of liposomal biolabels was described previously.36,37 In short, aqueous 150 mM CF (1 mL) was added to a solution of DPPC, cholesterol, DPPG, and DSPEPEG2000-folic acid (10:10:1:0.01 molar ratio) in a mixture of chloroform, isopropyl ether, and MeOH (6:6:1 v/v/v, 4 mL). After sonication for 5 min, the organic solvent was evaporated from the mixture under reduced pressure, leaving the liposomes as an orange gel. Another aliquot of CF was added to this gel, followed by an additional 5 min period of sonication and vortexing at 45 °C. The liposome size was regulated by extruding them at least 20 times through 1 and 0.4 µm polycarbonate filters and then by subjecting them to gel filtration and dialysis (12 000-14 000 MWCO) to remove any free, nonencapsulated CF. This suspension of liposomes was stored in 0.01 M phosphate buffered saline (PBS, pH 7.4) containing 0.15 M NaCl and 0.12 M sucrose (osmolarity: 430 mmol/kg). IAC Column. Purified monoclonal anti-FA antibodies were used to generate the IAC columns. The immunosorbent was produced on the basis of the modification of the methods reported by vanSommeren et al. (1993)38 and Ho and Hung (2008).39 NHSactivated Sepharose 4 Fast Flow slurry was washed on a sintered glass filter with 1 mM HCl. The gel was then suspended in 0.2 M NaHCO3 containing 0.5 M NaCl (pH 8.3). The gel solution was subsequently pipetted into a reaction vial and left to react with the anti-FA antibody solution for 9 h under shaking (150 rpm, 4 °C). After the coupling reaction was complete, unreacted NHS groups on the Sepharose medium were blocked through

(34) Gabizon, A.; Horowitz, A. T.; Goren, D.; Tzemach, D.; Mandelbaum-Shavit, F.; Qazen, M. M.; Zalipsky, S. Bioconjugate Chem. 1999, 10, 289–298. (35) Saul, J. M.; Annapragada, A.; Natarajan, J. V.; Bellamkonda, R. V. J. Controlled Release 2003, 92, 49–67.

(36) Ho, J. A. A.; Durst, R. A. Anal. Chim. Acta 2000, 414, 51–60. (37) Ho, J. A. A.; Hsu, H. W. Anal. Chem. 2003, 75, 4330–4334. (38) vanSommeren, A. P. G.; Machielsen, P. A. G. M.; Gribnau, T. C. J. J. Chromatogr. 1993, 639, 23–31. (39) Ho, J. A. A.; Hung, C. H. Anal. Chem. 2008, 80, 6405–6409.

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Scheme 1. Synthesis of DSPE-PEG2000-FA

reaction with 0.1 M Tris-HCl (pH 8.5) at 4 °C overnight. The resulting immunosorbent was then packed into a disposable polypropylene column (1.9 × 10.7 cm). Noncovalently bound anti-FA antibodies retained in the IAC column were washed alternately with three gel volumes of 0.1 M Tris-HCl buffer (pH 8.5) and three gel volumes of 0.1 M acetate buffer containing 0.5 M NaCl (pH 4.5) for at least five cycles. The effluents containing the washed off anti-FA antibody were collected individually and subjected to protein analysis. The coupling efficiency was evaluated by monitoring the original antibody solution (prior to coupling) and the collected effluent solutions (unbound antibody) spectrophotometrically at 595 nm. Finally, the IAC column was stored at 4 °C in PBS prior to use. Real Sample Preparations. Stresstabs, Jamieson, and Health Diary brands of vitamin B complex tablets were purchased locally; they contained 400 µg (per 1.00 g tablet), 400 µg (per 0.660 g tablet), and 800 µg (per 0.400 g tablet) of folic acid, respectively. After the individual tablets were finely powdered in a porcelain mortar, a portion of the powder was weighed and then dissolved in appropriate volumes of 10 mM PBS to form concentrations corresponding to 10-5 M FA. Each solution was stirred vigorously for 15 min to ensure complete dissolution. The pH of the tested stock solutions was adjusted to pH 7-8 and were stored at -20 °C prior to use. The working solutions were prepared through 100-fold dilution of the stock solutions to form concentrations corresponding to 10-7 M FA. Assay Procedure. The assay procedure was initiated by equilibrating the IAC column with 10 mM phosphate buffer (pH 7.4) containing 0.15 M NaCl and 0.12 M sucrose. The FA sample (500 µL) was loaded onto the IAC column and then rinsed with the same buffer to wash off any unbound FA. The liposome solution (500 µL) was introduced to occupy the remaining available antibody binding sites. The IAC column was rinsed again to flush out any excess unbound liposomes. The elution buffer (35% MeOH 5674

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in Tris buffered saline (TBS) containing 0.1% Tween 20) was then passed through the column, causing the rupture of liposomes and the release of the encapsulated CF dye molecules. A portion of the effluent (360 µL) was collected, and its fluorescence intensity was measured spectrophotometrically (excitation, 494 nm; emission, 517 nm). Finally, the antibody’s binding sites were regenerated through continuous flushing of the column with the elution buffer. The column was reconditioned with PBS prior to subsequent analyses. RESULTS AND DISCUSSION Characterization of DSPE-PEG2000-FA. Scheme 1 depicts our synthetic approach toward DSPE-PEG2000-FA. We performed thin-layer chromatography (TLC) analysis on 10 × 20 cm silica gel plates, eluting with CHCl3/MeOH/H2O (50:50:6, v/v/v). A new spot having an Rf of 0.97, due to DSPE-PEG2000-FA, appeared under UV examination (the corresponding Rf of folic acid was 0). We confirmed the presence of phospholipids by resolving the plate with a molybdenum blue spray, which revealed blue spots having Rf values of 0.90 and 0.97 for DSPEPEG2000-amine and DSPE-PEG2000-FA, respectively. The 1H NMR spectrum of DSPE-PEG2000-FA revealed signals at 0.87 and 8.77 that are characteristic of its methyl group on DSPE and of the proton on the pteridine ring of folic acid, confirming the successful conjugation of DSPE-PEG2000 to folic acid. LC/MS analysis of DSPE-PEG2000-FA revealed bell-shaped distributions at m/z 1088.3 (charge ) +3) and m/z 1631.7 (charge ) +2), suggesting that the molecular weight of the product was ca. 3264 Da. This molecular mass is consistent with the proposed structure. Characterization of Liposome Biolabels. The size homogeneity of the liposomal biolabels is the key aspect affecting the development of reproducible and reliable liposome-based diagnostic applications. In this study, we extruded the liposome

Figure 2. Fluorescence signals (λex, 490 nm; λem, 520 nm) generated at various volumes of added liposome biolabels.

preparations through polycarbonate filters to decrease their size heterogeneity. Using a nanoparticle size analyzer, we determined an average diameter for the liposomes of 249 ± 37 nm, suggesting that the average volume of a single liposome was ca. 8.1 × 10-12 µL, with an entrapped volume (assuming a bilayer thickness of 4 nm)40 of ca. 7.3 × 10-12 µL. Assuming that the CF concentration inside the liposomes was equal to that in the original solution and comparing the fluorescence of the lysed liposomes with that of standard CF solutions, we calculated that there were ca. 9.4 × 1012 liposomes/mL and that each liposome contained 6.6 × 105 molecules of CF. Because the average surface areas of DPPC and cholesterol molecules are ca. 71 and 19 Å2, respectively,40 we estimated that ca. 103 molecules of FA were present on the outer surface of each liposome, given that 0.005 mol % of DSPE-PEG2000-FA was successfully incorporated during the formation of liposomes. The average ζ potential of the liposomal biolabels was -11.86 ± 0.76 mV, consistent with the existence of negatively charged DPPG units in the liposome structure. Optimization of Parameters. Figure 2 depicts the effect of the amount of liposome on the performance of the IAC assay for FA. We diluted various volumes of the liposome concentrate to 500 µL and found that the optimal added amount was 100 µL; this sample contained ca. 9.4 × 1011 liposomes and encapsulated ca. 1.0 × 10-6 mol of CF dye. We also investigated the optimal amount of anti-FA antibody for use in the manufacture of the IAC column. Accordingly, we manufactured IAC columns containing 0.1 and 0.4 mg of anti-FA antibody, respectively. Figure 3 reveals a much more noticeable competition between 0 and 10-3 M FA (analyte) solutions when using the IAC column incorporating 0.4 mg of the anti-FA antibody. This IAC column offered a minimal, but sufficient, number of antibody binding sites; we chose it for our subsequent studies. In contrast, we observed poorer competition when using the column incorporating 0.1 mg of the antibody, presumably because of the paucity of antibody binding sites. Table 1 lists the coupling efficiencies of the two IAC columns. We calculated the amount of anti-FA antibody coupled by (40) Israelachvili, J. N.; Mitchell, D. J. Biochim. Biophys. Acta 1975, 389, 13– 19.

Figure 3. Effect of the amount of anti-FA antibody used to prepare IAC columns on the competition between FA-anchored liposomal biolabels and FA.

subtracting the total amount of anti-FA found in all of the wash fractions from the amount of anti-FA offered. The coupling efficiency was determined by measuring values of A595, using a Bio-Rad protein assay, based on the method of Bradford.41 We observed almost no residual anti-FA antibody in the wash fractions, indicating almost quantitative coupling. Assay Performance. For each analysis, we loaded 500 µL of FA onto the IAC column. These FA molecules bound to the antibodies, occupying a fraction of the total number of antibody binding sites in proportion to their concentration. An aliquot of liposomes (500 µL, appropriately diluted) was then added onto the column, resulting in binding to the unoccupied binding sites of the antibody. We transformed the collected data into a binding ratio using the equation binding ratio ) F/F0 × 100% where F is the fluorescence signal generated at a given concentration of FA and F0 is the fluorescence signal in the absence of FA. For our IAC column prepared with 0.4 mg of the anti-FA antibodies, Figure 4 displays the dose-response curve that we obtained after plotting F/F0 (%) against the concentration of the FA standards. The inset reveals a correlation coefficient (R2) of 0.9853 for the linearized portion of the curve between 10-8 and 10-4 M (a wide dynamic range of at least 4 orders of magnitude). The standard curve was, therefore, fitted to a fourparameter logistic equation according to the formula y ) {(A2 - A1)/[1 + 10 exp((log (X0 - X)) × p)]} + A1, where A1 and A2 are the maximal and minimal fluorescence signals, respectively, while X0 is the concentration producing half of the maximal fluorescence signal, X is the FA concentration, and p is the slope at the inflection point of the sigmoid curve.27 We define the operational detection limit after subtracting 3 times the standard deviation of the control (free of FA) from its (41) Bradford, M. M. Anal. Biochem. 1976, 72 (1-2), 248–254.

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Table 1. Efficiency of Coupling Anti-FA Monoclonal Antibodies to Sepharose Gel Beads amount of anti-FA offered (mg)

amount of unbound anti-FA (from protein assay; mg)

amount of anti-FA coupled (mg)

coupling efficiency (amount of anti-FA coupled/ amount of anti-FA offered; %)

0.10 0.40

0 0

0.10 0.40

100 100

average value. Accordingly, the limit of detection (LOD) for FA detected by the IAC column was 3.1 × 10-8 M with a 99.7% level of confidence. Our elution buffer (35% MeOH in TBS containing 0.1% Tween 20) was capable not only of releasing the liposomal CF dye molecules but also of regenerating the antibody’s activity, which allowed the immunoreactor to be used for at least 50 sample injections without any observable loss of reactivity (data not shown). Cross-Reactivity of Anti-FA Mab-Containing IAC Column. Figure 5 reveals that the anti-FA Mab IAC column did not display significant cross-reactivity toward either of the FA derivatives dihydrofolic acid or folinic acid (calcium salt). After loading solutions (10-4 M) of FA, dihydrofolic acid, and folinic acid (calcium salt) into the IAC column and defining the binding reactivity of FA toward anti-FA Mab as 100%, we determined that the binding reactivities of these FA derivatives toward antiFA Mab on the Sepharose beads were 3.1 and 3.7%, respectively. Real Sample Analysis. We analyzed real samples to investigate the performance our IAC assays for the determination of FA. Thus, we analyzed samples from three brands of vitamin B complex tablets (Stresstabs, Jamieson, and Health Diary) for their FA contents. We diluted these samples to desired concentrations prior to analysis so that they would fit into the linear portion of our dose-response curve. We then loaded 500 µL of each sample solution directly onto the IAC column and performed the assay using the same procedure as described above for the stock solutions. Table 2 summarizes the quantitative data obtained from the IAC; the FA contents correlate well with the values listed on

Figure 4. Dose-response curves obtained from IAC columns coated with 0.4 mg of anti-FA; error bars, (1 standard deviation. Sensitivity is defined as the capability of responding reliably and measurably to changes in folic acid concentration and is represented as the slope of calibration curve. Inset: linear portion of the main curve. 5676

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Table 2. Recoveries of FA from the IAC Analyses of Real Samples vitamin brand Stresstabs Health Diary Jamieson

labeled amount found amount recovery (µg/tablet) (µg) (found/labeled; %) 400 800 400

449 ± 62 764 ± 72 362 ± 36

112 95.5 90.5

the nutrition facts labels provided with each vitamin brand. Less than 30 min is needed to run a single assay. Therefore, our IAC assay appears to be suitable for the rapid and simple analysis of real samples without the need for complicated sample preconcentration or purification techniques. CONCLUSION We have developed a simple, yet sensitive, immunodiagnostic column assay for the determination of an important water-soluble vitamin, FA, via liposomal amplification. The combination of CFencapsulated, FA-anchored, PEGgylated liposomes and an IAC column packed with anti-FA Mab-immobilized Sepharose beads enables the construction of an IAC for determining FA levels in real samples. The use of fluorescent liposomal biolabels amplified the signals when testing low concentration of FA; in addition, the IAC column served as a concentrator to extract FA efficiently from the sample solutions. This approach offers acceptable sensitivity [LOD for FA: 6.8 ng (equivalent to 500 µL of 3.1 × 10-8 M FA)], comparable with those reported previously, with a wide dynamic range (4 orders of magnitude). With its low LOD, high accuracy, and short analysis times (