Chemistry: analytical and immunological testing – Optical result – With fluorescence or luminescence
Reexamination Certificate
1998-12-24
2001-05-22
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Optical result
With fluorescence or luminescence
C250S458100, C250S459100, C250S461100, C424S450000, C435S007100, C435S007200, C435S007920, C435S007940, C435S174000, C435S175000, C435S440000, C436S501000, C436S518000, C436S528000, C436S532000, C436S546000, C436S800000, C436S805000, C436S819000, C436S829000
Reexamination Certificate
active
06235535
ABSTRACT:
This is the U.S. National Stage Application of PCT/FI97/00419 filed Jun. 30, 1997.
This invention relates to a fluorescence-based immunoassay method for the detection of an analyte, or for the measurement of its concentration in a biological sample. The method is based on the ability of a multivalent analyte to induce aggregation of receptor molecules labeled with a fluorophore, which molecules are anchored to and are freely mobile on a lipid membrane, and thereby cause changes in the fluorescence.
Traditional fluorescence-based immunoassay methods exploit the principles of the conventional immunoassay—the analyte to be measured is immobilized on a solid substrate, whereafter it is detected e.g. by using a fluorescent antibody or fluorescence-generating enzymic reaction (Hemmilä, 1991, Kricka, 1994). Advantages of a fluorescence-based assay include high sensitivity, and independence of the use of radioisotopes. The fact that like most of the other conventional immunoassays, these fluorescence-based assays are of a heterogeneous nature, i.e. the unbound antibody or antigen is separated and the analyte is determined indirectly, can be considered as a disadvantage. These washing and separating steps increase the amount of work and costs.
Fluorescence resonance energy transfer (FRET) is a physical process, in which energy from a molecular chromophore (donor, D) excited to a high energy state is transferred to another chromophore (acceptor, A) via intermolecular dipole-dipole coupling (Clegg, 1995, Selvin, 1995). A necessary prerequisite for the energy transfer is that the distance between the molecules is short (10-100 Å), that the fluorescence spectrum of the donor and the absorption spectrum of the acceptor partially overlap, and that the quantum yield of the donor (&phgr;
D
), and the extinction coefficient of the acceptor (&egr;
A
) are high enough. FRET has been exploited to measure hybridization between nucleic acid molecules (Mergny et al., 1994) and to detect interactions between lipids and membrane proteins (Watts et al., 1986). The phenomenon has also been applied to immunoassay by labeling the antibody and antigen with fluorophores, which form a FRET donor-acceptor pair. The solution phase assay was based on the ability of the antigen (the substance to be measured) to displace the labeled antigen and thus prevent FRET phenomenon (Barnard and Walt, 1991).
Fluorescent labeling of proteins is known technology which is based on commercially available fluorophores and their derivatives (Waggoner, 1995). The fluorophores are either synthetic, relatively low-molecular organic compounds (e.g. fluorescein and rhodamine and derivatives thereof, and lanthanide chelates) or proteins (e.g. phycoerythrin and GFP, green fluorescent protein). Various parameters of the fluorescence emitted by the fluorophores can be measured: fluorescence intensity, life-time, polarization. These can also be used to monitor and quantitate the FRET phenomenon.
The Finnish patents No. 81680 and 93997 describe homogeneous immunoassay methods which use fluorescent labels. The first one of these describes a solid phase immunoassay, in which the fluorescence of the free label present in liquid phase is quenched by a light-absorbing substance, enabling the measurement of the bound label without separation steps. In the latter patent, a conventional quantitative immuno-fluorescence method is described, with the idea of achieving a sensitive assay by preventing short-lived background fluorescence. These methods have not, however, any relation to the FRET phenomenon.
Lipid bilayers are known to be structures that are spontaneously formed by phospholipids and some other polar lipids in water solution. In these, the polar heads of the lipid molecules are in contact with water (outwards), whereas the nonpolar hydrocarbon chains are oriented inwards. Planar lipid membranes can be prepared by using e.g. Langmuir-Blodgett technique (Arya et al., 1985, Zasadzinski et al., 1994) and transferred onto a solid substrate (Mrksich and Whitesides, 1995, Sackmann, 1996). Alternatively, vesicular closed lipid bilayer structures or liposomes can be prepared (New, 1992). A lipid membrane can be regarded as a two-dimensional liquid, in which the lipid molecules and the other molecules like proteins that are associated with it (natural membrane structures such as cell membranes) or have been attached on it, do move relatively freely on the plane of the membrane (lateral diffusion, Tamm, 1988, Glaser, 1993). There are applications which are based on the function of molecules which participate in specific binding and recognition reactions and are attached on lipid membranes (Odashima et al., 1991). This kind of functional lipid membranes have substantial potential as e.g. recognition surfaces in biosensors (Kiefer et al., 1991, Mrksich and Whitesides, 1995). Antibodies that can be generated by using known technology against almost any macromolecule and against numerous smaller molecules (Lerner et al., 1992, Nissim et al., 1994) are particularly interesting as recognition molecules.
We have developed a method for the attachment of bacterially-produced antibodies and other proteins on lipid membranes. The method is based on a so-called biosynthetic lipid-tagging technique (Laukkanen et al., 1993, Keinänen and Laukkanen, 1994), in which a lipid structure is attached on the N-terminal cysteine residue of a antibody-lipoprotein fusion during biosynthesis. This hydrophobic lipid anchors the antibody (protein) on the outer membrane of the bacterium. Likewise, the purified lipid-tagged antibody can be attached on the surface of a liposome, e.g. by using detergent dialysis method (Laukkanen et al., 1994).
Based on what has been described above, we have now developed a novel homogeneous immunoassay method, which enables simple and easy detection of a possible presence, or measurement of the concentration of an analyte in a sample. According to the invention antigens are preferably determined, in which case the receptor molecule to be attached on the lipid membrane is an antibody specific to the analyte.
Our invention originates from the idea that the binding reaction between the antigen in the liquid phase and the antibodies attached on the lipid membrane can be coupled to FRET phenomenon when the antigen is multivalent (FIG.
1
). The term “multivalent antigen” used herein refers to an antigen which carries more than one antibody-binding epitopes. The homogeneous immunoassay method according to the invention comprises two lipid-tagged antibody populations on a lipid membrane, with one population labeled with a FRET donor fluorophore and the other population with FRET acceptor fluorophore. When the antibody gets into contact with a multivalent antigen, a patching of the antibodies on the lipid membrane results. There the said antigen brings the acceptor and donor fluorophore-containing antibodies moving freely on the lipid membrane in close contact, in other words, a microaggregation takes place, in which case the energy transfer from the excited donor fluorophore to the acceptor fluorophore may occur (FRET phenomenon). The free lateral mobility of the antibody in immunoliposomes enables the FRET phenomenon. The mobility is achieved by the presence of the lipid anchor which attaches the antibody onto the lipid bilayer. As lipid membranes (e.g. planar membranes) can be attached on the surface of a solid substrate, e.g. by using the Langmuir-Blodgett technique (Mrksich and Whitesides, 1995, Sackmann, 1996), and liposomes can be attached e.g. by using biotin-streptavidin layer technique (Orellana et al., 1996), the invention enables a direct (“homogeneous”) immunoassay, which does not contain separate washing and separation steps but only the addition of the sample and the measurement of the signal.
In the measurement the fluorescence is excised in the donor (e.g. using a light source with a suitable wavelength), and the higher amount of the antibodies attached on the membrane are in close contact, the greater part of the excitation energy
Keinänen Kari
Laukkanen Marja-Leena
Söderlund Hans
Evenson, McKeown, Edwards & Lenahan P.L.L.C.
Le Long V.
Padmanabhan Kartic
Valtion teknillinen tutkimuskeskus
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