a fluoroimmunoassay based on immunoliposomes

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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

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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


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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.

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a fluoroimmunoassay based on immunoliposomes

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