Anal. Chem.

1997, 69, 1295-1298

A Fluoroimmunoassay Based on Immunoliposomes Containing Genetically Engineered Lipid-Tagged Antibody

Eiry Kobatake, Hiroyuki Sasakura, Tetsuya Haruyama, Marja-Leena Laukkanen, Kari Keina 1 nen, and Masuo Aizawa,*,

Department of Bioengineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226, Japan, and VTT Biotechnology and Food Research, P.O. Box 1500, FIN-02044 VTT, Espoo, Finland

Immunoliposomes were prepared by using biosynthetically lipid-tagged anti-2-phenyloxazolone single-chain antibody. Carboxyfluorescein as a fluorescent marker was encapsulated in the immunoliposomes. Some conditions for fluoroimmunoassay using the immunoliposomes were optimized by binding assays with hapten-coated microtiter wells. A competitive fluoroimmunoassay for the caproic acid conjugate of 2-phenyloxazolone as a model antigen was performed with the immunoliposomes. In the optimized assay conditions, antigen could be determined in the concentration range from 10-7 to 10-9 M.
Immunoliposomes bearing antibody molecules on their surface have been used in several biotechnological applications such as drug delivery systems,1,2 transfection of cells,3,4 and immunoassays.5,6 To incorporate soluble antibody molecules stably on the surface of liposomes, it is necessary to introduce hydrophobic moieties to antibody molecules, e.g., by directly coupling antibody molecules to lipids. So far, incorporation of antibody molecules to the surface of liposomes has been performed by chemical coupling. In this procedure, fatty acyl groups in lipids are coupled to appropriately exposed sulfhydryl and amino acid groups in the protein molecule with a bifunctional reagent.7-9 However, in such chemical coupling procedures, the conjugate often forms a heterogeneous complex in terms of number and location of lipid moieties; as a result, this treatment may lead to a loss or decrease in antigen-binding properties. In recent years, much attention has been focused on genetically fused proteins because of the easy stoichiometric control of the conjugation.10,11 We have constructed some fusion proteins as
Tokyo Institute of Technology. VTT Technology and Food Research. (1) Hughes, B. J.; Kennel, S.; Lee, R.; Huang, L. Cancer Res. 1989, 49, 62146220. (2) Ahmad, I.; Longenecker, M.; Samuel, J.; Allen, T. M. Cancer Res. 1993, 53, 1484-1488. (3) Holmberg, E. G.; Reuer, Q. R.; Geisert, E. E.; Qwens, J. L. Biochem. Biophys. Res. Commun. 1994, 201, 888-893. (4) Wang, C.-Y.; Huang, L. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 7851-7855. (5) Ho, R. J. Y.; Huang, L. J. Immunol. 1985, 134, 4035-4040. (6) Ishimori, Y.; Rokugawa, K. Clin. Chem. 1993, 39, 1439-1443. (7) Huang, A.; Huang, L.; Kennel, S. J. J. Biol. Chem. 1980, 255, 8015-8018. (8) Loughrey, H. C.; Choi, L. S.; Cullis, P. R.; Bally, M. B. J. Immunol. Methods 1990, 132, 25-35. (9) Martin, F. J.; Hubbell, W. L.; Papahadjopoulos, D. Biochemistry 1981, 20, 4229-4238. (10) Bu low, L. Eur. J. Biochem. 1987, 163, 4443-448. (11) Bu low, L.; Mosbach, K. Trends Biotechnol. 1991, 9, 226-231.

reagents for enzyme immunoassay by genetic engineering.12,13 Through the use of this method, it is possible to form a homogeneous conjugation between two kinds of proteins in a sitespecific manner. Therefore, gene fusion may be applied to a new method to conjugate between antibody and lipid molecules for the construction of stable and functional immunoliposomes. Recent techniques in bacterial expression of functional antibodies14,15 also prompted us to use genetic engineering to convert antibodies into membrane-bound molecules for immunoliposome applications. Recombinant Fv fragments, which are the smallest functional unit of an antibody, have been successfully produced in Escherichia coli.16,17 For stabilization of Fv fragments, VH and VL domains have linked together with linker peptide and been expressed as a single-chain antibody.18,19 This form of antibody has many advantages for genetic modification because of its simplicity of handling. To construct a stable and functional conjugate between antibody and lipid molecules by gene fusion, we have exploited the major lipoprotein (lpp) of E. coli, which contains a specific lipid modification at its amino terminus to anchor the bacterial membrane. The determinants for the biosynthetic lipid modification are contained within a signal peptide of 20 amino acid residues and nine amino-terminal amino acid residues of the lpp.20 We reported a production of lipid-tagged single-chain antibody by fusion of genes for a single-chain anti-2-phenyloxazolone antibody and the essential part of the lpp of E. coli required for lipid modification.21 The resulting lipid-tagged antibody carries a single covalently bound glycerolipid anchor at the amino-terminal cysteinyl residue which is separated from the variable region of the immunoglobulin heavy chain by a linker peptide (Figure 1A). The genetically prepared single-chain antibody modified with lipid
(12) Kobatake, E.; Nishimori, Y.; Ikariyama, Y.; Aizawa, M.; Kato, S. Anal. Biochem. 1990, 186, 14-18. (13) Kobatake, E.; Iwai, T.; Ikariyama, Y.; Aizawa, M. Anal. Biochem. 1993, 208, 300-305. (14) Ward, E. S.; Gussow, D.; Griffiths, A. D.; Jones, P. T.; Winter, G. Nature 1989, 341, 544-546. (15) Skerra, A. Curr. Opin. Immunol. 1993, 5, 256-262. (16) Huston, J. S.; Levison, D.; Mudgett-Hunter, M.; Tai, M.-S.; Novotny, J.; Margolies, M. N.; Ridge, R. J.; Bruccoleri, R. E.; Haber, E.; Crea, R.; Oppermann, H. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 5879-5883. (17) Field, H.; Yarranton, G. T.; Rees, A. R. Protein Eng. 1989, 3, 641-647. (18) Bird, R. E.; Hardman, K. D.; Jacobson, J. W.; Johnson, S.; Kaufman, B. M.; Lee, S.-M.; Lee, T.; Pope, S. H.; Riordan, G. S.; Whitlow, M. Science 1988, 242, 423-426. (19) Skerra, A.; Plu ckthun, A. Science 1988, 240, 1038-1043. (20) Ghrayeb, J.; Inouye, M. J. Biol. Chem. 1984, 259, 463-467. (21) Laukkanen, M.-L.; Teeri, T. T.; Keina nen, K. Protein Eng. 1993, 6, 449454.

S0003-2700(96)01162-6 CCC: $14.00

1997 American Chemical Society

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Figure 1. (A) Schematic drawing of the lipid-tagged antibody. The fusion protein consists of a 20 amino acid signal peptide, N-terminal nine amino acids of lpp, VH and VL domain joined by a linker peptide, and a hexahistidinyl tail. The relevant N-terminal nine amino acid sequence of lpp is shown. (B) Schematic drawing of carboxyfluorescein (CF)-encapsulated immunoliposome.

molecules retained its antigen-binding activity. The antibodies were expected to be incorporated stably to liposomes with high orientation. The immunoliposome consisting of the lipid-tagged antibody which was prepared by a detergent dialysis method could be demonstrated as a possibility for the application of immunoassay by surface plasmon resonance22 and time-resolved fluoroimmunoassay.23 In the present study, we describe the preparation of carboxyfluorescein-encapsulated immunoliposome containing biosynthetically lipid-tagged anti-2-phenyloxazolone single-chain antibody in a simplified manner (Figure 1B). Furthermore, application of the immunoliposome to a simple fluoroimmunoassay is demonstrated. EXPERIMENTAL SECTION Materials. Phosphatidylcholine (PC) was purchased from Sigma (St. Louis, MO), and 5 (and 6)-carboxyfluorescein (CF) was from Wako Pure Chemicals (Osaka, Japan). Bovine serum albumin (BSA) conjugated with approximate 21 molecules of 2-phenyloxazolone (Ox21BSA) was synthesized as described previously.24 The caproic acid derivative of 2-phenyloxazolone (Ox(22) Laukkanen, M.-L.; Alfthan, K.; Keina nen, K. Biochemistry 1994, 33, 1166411670. (23) Laukkanen, M.-L.; Orellana, A.; Keina nen, K. J. Immunol. Methods 1995, 185, 95-102. (24) Ma kela , O.; Kaartinen, M.; Pelkonen, J. L. T.; Karjalainen, K. J. Exp. Med. 1978, 148, 1644-1660.

CA) was synthesized and used as a soluble hapten. All other chemicals were of analytical grade. Expression and Purification of Lipid-Tagged Antibody. The expression plasmid for the lipid-tagged antibody, pML3.7H, is encoding the signal peptide and nine N-terminal amino acid residues of lpp fused to the anti-2-phenyloxazolone single-chain Fv fragment with a hexahistidinyl tail.21 Expression and purification of the lipid-tagged antibody were described before.21 Briefly, E. coli strain HB101 was transformed with the plasmid pML3.7H and cultured in LB medium with 100 g/mL of ampicillin at 37 C. After induction with IPTG, the cells were cultured another 12 h at 30 C and harvested by centrifugation. The cells from 1 L of culture were suspended in 50 mL of lysis buffer (10 mM HEPES, pH 7.4, 1 mM EDTA, 0.5 M NaCl, 0.1 mM PMSF, and 0.1 mg/mL lysozyme) and lysed by sonication. The cell envelopes were collected by ultracentrifugation (150000g, 1 h, 4 C), and the pellet was suspended in buffer A (10 mM HEPES, pH 7.4, 1 M NaCl, 10% (v/v) glycerol, and 0.1 mM PMSF) containing 1% (w/v) Triton X-100. The sample was applied to a chelating Sepharose fast flow column (Pharmacia Biotech, Uppsala, Sweden) with Ni2+ to purify the lipid-tagged antibody having a hexahistidinyl tail. The fraction eluting in 100 mM imidazole was used for further experiments. Preparation of Immunoliposome. Ten milligrams of PC was dissolved in 1 mL of chloroform in a test tube. After being dried well under a stream of nitrogen, the PC was suspended in 1 mL of 50 mM CF in 20 mM HEPES buffer solution (pH 7.4) and sonicated for 10 min. Unencapsulated CF was removed by repeated centrifugation at 30000g for 20 min, and the final pellet of liposomes was suspended in 1 mL of HEPES buffer. The solution of purified lipid-tagged antibody was then added to the resulting liposome solution with stirring at 4 C. Fluoroimmunoassay. Binding properties of the immunoliposomes were characterized by using a binding assay in microtiter plates (Becton Dickinson, Rutherford, NJ). In routine experiments, the wells were coated with 100 L of Ox21BSA (0.25 mg/mL) for 2 h at 37 C, followed by incubation with 1% (w/v) BSA to block the sites for remaining nonspecific adsorption. The immunoliposomes were then added to each well. After thorough washing with PBS, the bound immunoliposomes were disrupted by adding 150 L of ethanol, and the fluorescence of released CF was determined by an FP-777 spectrofluorometer (Jasco, Tokyo, Japan) with excitation at 460 nm and emission at 520 nm. Fluoroimmunoassay for the determination of analytes by using the immunoliposomes was performed as follows. The immunoliposome-entrapped CF was incubated with various concentrations of Ox-CA as a soluble hapten in a final volume of 100 L. After incubation for 1 h, the reaction mixture was poured into a well coated with Ox21BSA and reacted for 4 h at 37 C. The fluorescence from bound liposome on each well was then determined as described above. RESULTS AND DISCUSSION Preparation of Immunoliposomes. About 1 mg of purified lipid-tagged antibody as a 30 kDa protein was obtained from 1 L of culture. The affinity constant (Ka) of the single-chain antibody for a soluble hapten, Ox-CA, was in the micromolar range, corresponding to the Ka of the parental monoclonal antibody as

1296 Analytical Chemistry, Vol. 69, No. 7, April 1, 1997

Figure 2. Relationship between fluorescent intensity and amount of antibody for immunoliposome preparation. The immunoliposomes were prepared with 10 mg of PC and varying amounts of purified lipid-tagged antibody. The binding of immunoliposomes to the Ox21BSA on microtiter wells was analyzed by fluorescence measurement.

Figure 3. Reaction time of immunoliposomes with immobilized antigen in binding assay. The immunoliposomes were reacted with Ox21BSA immobilized on microtiter wells for a prolonged period of time. The binding of immunoliposomes was determined by fluorescence measurement.

described elsewhere.25 Furthermore, the hapten-binding activity of the single-chain antibody was retained even after lipid-tagging.21 To optimize the conditions for fluoroimmunoassay using immunoliposomes, a binding assay for hapten-conjugated BSA (Ox21BSA) was performed. First, the effect of the amount of lipidtagged antibody incorporated in immunoliposome was investigated. In the present study, immunoliposomes were prepared by a simplified manner, only adding the lipid-tagged antibody to liposomes, as compared with the detergent dialysis method described previously.22 The immunoliposomes were prepared with 10 mg of PC and various amounts of purified lipid-tagged antibody as described in the Experimental Section. The immunoliposomes were then incubated for 4 h in Ox21BSA (0.25 mg/ mL)-coated microtiter wells for adsorption. After washing of the immunoliposomes with PBS, fluorescence from the bound liposomes by disruption with ethanol was determined. The fluorescence intensity of each well was plotted against the amount of antibody used for preparation of immunoliposomes (Figure 2). The fluorescence increased with the amount of antibody and reached a constant value when 50 g of antibody was used. When the liposomes were prepared without antibody, no fluorescence was observed, indicating that nonspecific adsorption of the liposomes to Ox21BSA-coated microtiter wells was negligible. In our previous study, all the antibody molecules used for preparation of immunoliposomes were efficiently incorporated into liposomes by a dialysis method when 80 g of the antibody/100 mg of lipid was used.22 The present result shows that the incorporation of antibody in excess of 50 g/10 mg of lipid does not lead to further improvement in binding, although the incorporation of antibody into liposomes is not saturated. Hence, the binding of immunoliposomes on hapten-coated microtiter wells may be saturated at this amount of antibody used for immunoliposomes preparation. Time of Reaction of Immunoliposomes to Immobilized Antigen. Next, the required incubation time of immunoliposomes and antigen on a microtiter well was investigated. The immunoliposomes were incubated in Ox21BSA (0.25 mg/mL)-coated
(25) Takkinen, K.; Laukkanen, M.-L.; Sizmann, D.; Alfthan, K.; Immonen, T.; Vanne, L.; Kaartinen, M.; Knowles, J. K. C.; Teeri, T. T. Protein Eng. 1991, 4, 837-841.

Figure 4. Binding of immunoliposomes to immobilized Ox21BSA. The immunoliposomes were reacted with varying amounts of Ox21BSA immobilized on microtiter wells. The binding was determined by fluorescence intensity.

microtiter wells for a prolonged period of time at 37 C. As shown in Figure 3, the fluorescence intensity from bound liposomes increased in a time-dependent manner. It seems to take a relative longer time to reach steady state, in comparison with general immunosorbent assay systems. We dont know the reason for this, but it is probably due to the steric hindrance between bulky immunoliposomes and the solid phase. As a sufficient fluorescence intensity could be obtained after 4 h of reaction, we used that time for further experiments, although 1 h of reaction may be sufficient to determine the fluorescence intensities for a more rapid assay. Binding Assay. To evaluate the usefulness of the immunoliposomes in a fluoroimmunoassay, a binding assay was performed for hapten-conjugated BSA on a well of microtiter plate. Various concentrations of Ox21BSA as a model antigen were adsorbed on a well of microtiter plate for 2 h at 37 C. A constant volume (100 L) of CF-containing immunoliposomes prepared from 10 mg of PC and 50 g of lipid-tagged antibody was then added into each well, followed by incubation for 4 h at 37 C. After washing of the solution with PBS to remove nonspecifically bound immuAnalytical Chemistry, Vol. 69, No. 7, April 1, 1997
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ically by the preincubation with Ox-CA in a concentrationdependent manner. The concentration range from 10-7 to 10-9 M Ox-CA was determined by taking the CF-encapsulated immunoliposome. In the previous work, a competitive assay using immunoliposomes and surface plasmon resonance, detection of almost the same concentration range of hapten as in the present work was reported.22 However, the assay method described here is very simple, and no special instruments are needed. CONCLUSIONS In the present study, we demonstrated the feasibility of using immunoliposomes containing biosynthetic lipid-tagged antibody in a fluoroimmunoassay. Under optimized assay conditions, the concentration of Ox-CA as a model hapten with low molecular weight could be determined in a simple manner. This approach should be applicable to immunoassays for any antigen but with different antibody. In particular, the recent technology of phage display has dramatically simplified the isolation and modification of antibodies.26,27 Not only in the application to immunoassays, the use of lipidtagged antibodies will open a new field for the development of biosensors, because oriented and high-density assembly of protein molecules to a lipid bilayer will be possible by this method. ACKNOWLEDGMENT This work was supported in part by the Monbusho International Scientific Research Program (No. 07044134), funded by the Ministry of Education, Science, Sports, and Culture of Japan. K.K. and M.-L.L. are grateful for financial support from the Technology Development Centre of Finland (TEKES).

Figure 5. Competitive fluoroimmunoassay for Ox-CA. The immunoliposomes were preincubated in the presence of varying amounts of Ox-CA as a soluble hapten. The reaction mixture was incubated in Ox21BSA-immobilized microtiter wells, and the binding of immunoliposomes was determined by fluorescence measurement.

noliposomes on the surface, the fluorescence of each well was determined and plotted against the concentration of adsorbed antigen (Figure 4). The fluorescence intensities depend on the antigen concentrations as a sigmoidal curve. Saturated binding of immunoliposomes was observed over 0.25 mg/mL of Ox21BSA. Fluoroimmunoassay. Finally, competitive fluoroimmunoassay for Ox-CA as a soluble hapten was performed under the optimized conditions as clarified above. The immunoliposomes were perincubated with various concentrations of Ox-CA solutions for 1 h. The hapten-bound immunoliposome was then reacted with the Ox21BSA adsorbed on the microtiter wells. The standard curve for Ox-CA is shown in Figure 5. The binding of immunoliposomes to hapten-coated microtiter wells was inhibited specif(26) Hoess, R. H. Curr. Opin. Struct. Biol. 1993, 3, 572-579. (27) Hoogenboom, H. R.; Marks, J. D.; Griffiths, A. D.; WInter, G. Immunol. Rev. 1992, 130, 41-68.

Received for review November 15, 1996. January 23, 1997.X

AC961162+
X

Accepted

Abstract published in Advance ACS Abstracts, March 1, 1997.

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