View
220
Download
0
Category
Preview:
Citation preview
8/13/2019 A Fluoroimmunoassay Based on Immunoliposomes
1/4
A Fluoroimmunoassay Based on ImmunoliposomesContaining Genetically Engineered Lipid-TaggedAntibody
Eiry Kobatake, Hiroyuki Sasakura, Tetsuya Haruyama, Marja-Leena Laukkanen,
Kari Keina1
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 biosyntheti-
cally lipid-tagged anti-2-phenyloxazolonesingle-chain an-
tibody. Carboxyfluorescein as a fluorescent marker was
encapsulatedin theimmunoliposomes. Someconditions
for fluoroimmunoassayusingtheimmunoliposomes were
optimizedbybindingassayswith 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 opti-
mized 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 immuno-
assays.5,6 To incorporate soluble antibody molecules stably on
the surface of liposomes, it i s necessary to introduce hydrophobic
moietiesto 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 chemicalcoupling. 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 hasbeen focused on genetically
fused proteins because of the easy stoichiometric control of the
conjugation.10,11 We have constructed some fusion proteins as
reagents for enzyme immunoassay by genetic engineeri ng.12,13
Through the use of this method, it is possible to form a
homogeneous conjugation between two kinds of proteins in asite-
specific 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 antibod-
ies14,15 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 modifica-
tion are contained within a signal peptideof 20amino 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 l pp of E. coli required for lipid
modification.21 The resulting li pid-tagged antibody carries a single
covalently bound glycerolipid anchor at the amino-terminal cys-
teinyl 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
Tokyo Institute of Technology.
VTT Technology and Food Research.(1) Hughes, B. J.; Kennel, S.; Lee, R.; Huang, L. Cancer Res. 1989, 49, 6214-
6220.
(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, 20 1, 888-893.
(4) Wang, C.-Y.; H uang, L. Proc. Natl.Acad. Sci. U.S.A. 1987,84, 7851-7855.
(5) Ho, R. J. Y.; Huang, L. J. Immunol. 1985, 13 4, 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, 25 5, 8015-8018.
(8) Loughrey, H. C.; Choi, L. S.; Cullis, P. R.; Bally, M. B. J.Immunol. M ethods
1990, 13 2, 25-35.
(9) Martin, F. J.; Hubbell, W. L.; Papahadjopoulos, D. Biochemistry1981, 20,
4229-4238.
(10) Bulow, L. E ur. J. B iochem. 1987, 16 3, 4443-448.
(11) Bulow, L .; M osbach, K . Trends Biotechnol. 1991, 9, 226-231.
(12) Kobatake, E.; N ishimori, Y.; I kariyama, Y.; Aizawa, M.; Kato, S. Anal.
Biochem. 1990, 18 6, 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, 34 1, 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.; Br uccoleri, R. E.; H aber, 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. Science1988,
242, 423-426.
(19) Skerra, A.; Pluckth un, A. Science1988, 24 0, 1038-1043.
(20) Ghrayeb, J.; Inouye, M. J. Biol. Chem. 1984, 25 9, 463-467.
(21) Laukkanen, M .-L.; Teeri, T. T.; Keinanen, K . Protein Eng. 1993, 6, 449-
454.
Anal. Chem. 1997, 69, 1295-1298
S0003-2700(96)01162-6 CCC: $14.00 1997 American Chemical Society Analytical Chemistry, Vol. 69, No. 7, April 1, 1997 1295
8/13/2019 A Fluoroimmunoassay Based on Immunoliposomes
2/4
molecules retained its antigen-binding activity. The antibodieswere 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 immuno-
assay by surface plasmon resonance22 and time-resolved fluoro-
immunoassay.23
In the present study, we describe the preparation of carboxy-
fluorescein-encapsulated immunoliposome containing biosyntheti-
cally 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 previ-
ously.24 The caproic acid derivative of 2-phenyloxazolone (Ox-
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 pM L3.7H and cultured in LB medium with 100g/ mL of ampicillin at 37 C. After induction with IPTG, the cells
were cultured another 12 h at 30 C and harvested by centrifuga-
tion. 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,
0.1mM PMSF, and 0.1 mg/ mL lysozyme) and lysed by sonication.
The cell envelopes were collected by ul tracentri fugation ( 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 N i2+ to purify
the lipid-tagged antibody having ahexahistidinyl tail. The fractioneluting 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 30000gfor 20 min, and the final pellet
of l iposomes 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 immuno-
liposomes were characterized by using a binding assay in
microtiter plates (Becton Dickinson, Rutherford, NJ). In r outine
experiments, the wells were coated with 100L of Ox21BSA (0.25
mg/ mL) for 2 h at 37 C, followed by i ncubation with 1%(w/ v)
BSA to block the sites for remaining nonspecific adsorption. The
immunoliposomes were then added to each well. After t horough
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 perfor med as follows. The immuno-
liposome-entrapped CF was incubated with various concentrations
of Ox-CA as a soluble hapten in a final volume of 100 L. Afterincubation 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 ofImmunoliposomes. 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,
corr esponding to the Kaof the parental monoclonal antibody as
(22) Laukkanen, M.-L.; Alfthan, K.; Keinanen, K. Biochemistry1994, 33, 11664-
11670.
(23) Laukkanen, M.-L.; Orellana, A.; Keinanen, K. J. Immunol. M ethods1995,
185, 95-102.
(24) Makela, O.; Kaartinen, M.; Pelkonen, J. L. T.; Karjalainen, K. J. E xp. M ed.
1978, 14 8, 1644-1660.
Figure 1. (A) Schematic drawing of the lipid-tagged antibody. Thefusion protein consists of a 20 amino acid signal peptide, N-terminal
nine amino acids of lpp, VHand VLdomain 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 carboxyfluo-rescein (CF)-encapsulated immunoliposome.
1296 Analytical Chemistry, Vol. 69, No. 7, April 1, 1997
8/13/2019 A Fluoroimmunoassay Based on Immunoliposomes
3/4
described elsewhere.25 Furthermore, the hapten-binding activity
of the single-chain antibody was retained even after li pid-tagging.21
To optimize the conditions for fluoroimmunoassay using
immunoli posomes, a bi nding assay for hapten-conjugated BSA
(Ox21BSA) was performed. First, the effect of the amount of lipid-
tagged antibody incorporated in immunoliposome was investi-
gated. In the present study, immunoli posomes 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 descri bed in the Experimental Section. The immu-
noliposomes were then incubated for 4 h in Ox 21BSA (0.25 mg/
mL)-coated microtit er wells for adsorption. After washing of the
immunoliposomes with PBS, fluorescence from the bound lipo-
somes by disruption with ethanol was determined. The fluores-
cence 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 50g 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 previousstudy, all the antibody molecules used for preparation
of immunoliposomes were efficiently incorporated into liposomes
by a dialysis method when 80g of the antibody/ 100 mg of lipid
was used.22
The present result shows that the incorporation ofantibody i n excess of 50g/ 10 mg of lipid does not lead to further
improvement in binding, although the incorporation of antibody
into l iposomes is not saturated. Hence, the binding of i mmuno-
liposomes on hapten-coated microtiter wells may be saturated at
this amount of antibody used for immunoliposomes preparation.
Time of Reaction of Immunoliposomes to I mmobilized
Antigen. Next, the required incubation time of immunoliposomes
and antigen on a microti ter well was investigated. The immuno-
liposomes were incubated in Ox21BSA (0.25 mg/ mL)-coated
microtiter wells for a prolonged period of time at 37 C. As shown
in Figure 3, the fluorescence intensity from bound liposomes
increased in atime-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 r eason for
this, but it is probably due to the steric hindrance between bulky
immunoliposomes and the solid phase. As a sufficient fl uores-
cence intensity could be obtained after 4 h of reaction, we usedthat 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 immuno-
liposomes in a fluoroimmunoassay, a binding assay was performed
for hapten-conjugated BSA on a well of microti ter 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 li pid-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 immu-
(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 2. Relationship between fluorescent intensity and amount
of antibody for immunoliposome preparation. The immunoliposomeswere prepared with 10 mg of PC and varying amounts of purified
lipid-tagged antibody. The binding of immunoliposomes to the Ox21-BSA on microtiter wells was analyzed by fluorescence measurement.
Figure 3. Reaction time of immunoliposomes with immobilized
antigen in binding assay. The immunoliposomes were reacted withOx21BSA immobilized on microtiter wells for a prolonged period of
time. The binding of immunoliposomes was determined by fluores-
cence measurement.
Figure 4. Binding of immunoliposomes to immobilized Ox21BSA.
The immunoliposomes were reacted with varying amounts of Ox21-BSA immobilized on microtiter wells. The binding was determined
by fluorescence intensity.
Analytical Chemistry, Vol. 69, No. 7, April 1, 1997 1297
8/13/2019 A Fluoroimmunoassay Based on Immunoliposomes
4/4
Recommended