Fluorescence immunoassay based on fluorescent quenching.

p0001The present invention relates to a method for fluorescence immunoassay, wherein the extent of quenching of fluorescence of an antigen or antibody by an externally added quencher and can be measured after complex formation, and in which from the change of the erase efficiency as a result of the binding process to the concentration of antigen or antibody can be closed in an unknown sample.

p0002Immunoassay methods have proven to be extremely useful for the qualitative and quantitative determination of biological substances as immunological reactions proceed with high specificity and can be detected with high sensitivity. Recently, however, the classical radioimmunoassay (RIA) has been increasingly by the fluorescence immunoassay (FIA) replaced, as the use of radioactively labeled substances is associated with various disadvantages. Although FIA methods usually are not as sensitive as the RIA, they have experienced a rapid development since the fluorescence spectrometry is one of the most versatile methods of analysis. The following review articles are representative: GC Visor and SG Schulman, J. Pharm. Sci. 70, 469 (1981); RP Ekins and S. Dakubu, "The Development of High Sensitivity Pulsed Light, Time-Resolved FIA", Pure & Appl. Chem 57, 473 (1985). TS Smith, M. Hassan and RD Nargessy, "Principles and Practice of FIA Procedures", in: Modern Fluorescence Spectroscopy, vol. 3 (EL Wehry, editor), Plenum Press, New York, 1981; NJ Seare, "Immunoassay Techniques", in: Chemical sensor (TE Edmonds, editor), Chapman and Hall, New York, 1988; I. Karube, "Novel immunosensor", in the biosensor 2, 343 (1986). JS Woodhead and I. Weeks, "Chemiluminescence Immunoassay", Pure & Appl. Chem. 57, 523 (1985). MJ Grayeski, "Chemiluminescence Immunoassay", Anal. Chem. 59, 1250A (1987).

p0003In all these methods, an antibody (Ab) or antigen (Ag) is labeled with a fluorescent molecule and investigated the change in fluorescence properties of the fluorescent label as a result of binding of the antibody to the antigen. A distinction is made between homogeneous and heterogeneous FIA. The homogeneous FIAs have the advantage that no separation step is required to separate bound from unbound protein. A distinction is further depending on the nature of the spectroscopic method between several types of FIAs:

1) FIA based on intensity measurements

p0004These methods are based on an observed reduction or an increase in the fluorescence intensity of a labeled ligand to the binding by the antibody. The cause of this change in intensity is not known exactly but it is believed that there is a change in the electronic structure of the fluorescent dye. Changes in the electronic distribution of bound labeled ligand may enhance the radiationless deactivation of the excited molecules. As another interpretation, the possibility is contemplated that the polarity of the environment of the fluorescent marker is altered by the binding so that the quantum yield of the fluorophore characteristic changes (Solvatochromism). This may lead to both an increase and a decrease in fluorescence. A prediction, in which direction this change to go, is not possible.

2) energy transfer - FIA

p0005(Acceptor called) overlaps If the absorption band of a fluorophore with fluorescence of another fluorophore (called the donor), there may be an electronic energy transfer (ET) from the donor to the acceptor come. The best known example is the pair of fluorescein and rhodamine. it stimulates in a mixture of both the fluorescein on, one observes the fluorescence of rhodamine. The efficiency of ET depends inversely on the 6th power of the distance between the two fluorophores. So as long as z. B. the antigen (rhodamine-labeled) and the antibody (fluorescein-labeled) are not yet available bonded to each other, no ET takes place. Are they bound, ET is observed. A typical application example is by Fisher et al. in Clin. Chem. 26, 987 (1980) described ben.

p0006In another form, the energy acceptor may not be fluorescent and as long as its absorption spectrum matches the emission spectrum of the donor. This method is just as awkward as the previous one, since they require the marking of both Ab and Ag. A typical example is described by Velich et al. in Proc. Natl. Acad. Sci. 46, 1470 (1969).

3) Fluorescence Polarization

p0007While the fluorescence emission of a small and rapidly rotating molecule is practically not polarized, because rotate during the lifetime of the excited state all molecules in a random manner and emit so in all directions and at all levels, this is not in a complex of antigen and antibody more the case, since this is extremely large molecules with a small rotational speed. Thus it is observed that the degree increase of fluorescence polarization in the degree to which a fluorescently labeled antibody is bound by an antigen, or vice versa. The degree of polarization P can assume values ​​between +1/2 and -1/3. From the fact that P varies depending on the extent of binding of a labeled ligand, one can set up a calibration curve, by means of which a homogeneous immunoassay may be performed by application of P against the concentration of unlabeled ligand. A typical example is described by Lu et al Steffes. in Clin. Chem. 28, 2278 (1982). This principle is applied in various devices commercially available. In US Patent 4,451,149 such a method in combination with fiber optic cables will be described.

4) Time-resolved FIA

p0008Just as changes the fluorescence intensity and polarization, a fluorophore by forming an Ag / Ab complex, also the fluorescence decay time changes. By measuring the change in the decay time of the fluorescent label as a result of the binding process can be concluded that the amount of antigen. A typical example is known from DE-OS 2,628,158, where a fluorescent marker is used with very long decay. A detailed description can be found in an article by Hemmila et al. in Anal. Biochem. 137, 335 (1984).

p0009The method has the advantage that it can suppress background radiation well (z. B. by Raman and Rayleigh scattering). However, this requires a complicated electronic configuration to separate the decay of the complex from that of the free ligand, and the use of various additional Reagentiensätze.

5) Enzymatic FIAs

p0010If an enzyme linked to an antibody, it is like in solution capable of cleaving enzyme substrates. From non-fluorescent substrates so formed fluorescent hydrolysis products. But if the Ab / Ag complex formed, the enzyme for the substrate is no longer accessible, and this can not be digested so quickly. The reduced hydrolysis rate is therefore a measure of the amount of formed Ab / Ag complex. A typical application is the determination of IgM, described by Warah et al. in Clin. Chem. 27, 673 (1981).

p0011A relatively complicated immunoassay based on the fluorescence quenching is in the Eur. Appl. EP 104.926 described: Thereafter, for the detection of an immunogenic substance such as a medicament, this first bound to a large protein, then isolated a corresponding antibody, and this used together with a fluorescently labeled hapten in the immunoassay. The process is time consuming and labor intensive.

p0012In addition to the methods discussed so far, there are heterogeneous FIAs, which are characterized by increased effort and better sensitivity. In these methods, the antibody-antigen complex is first separated from the free ligand and then fed to a first quantification. The advantage is that thus the background fluorescence is practically completely eliminated, resulting in a considerable increase in sensitivity.

p0013The present invention is based on the principle of fluorescence quenching by an externally added quencher. It is known that various fluorophores are weakened upon addition of so-called quencher molecules in their fluorescence intensity. This, depending on the mechanism referred to as static or dynamic fluorescence quenching phenomenon based on the fact that the added quencher, the quantum yield and, in the case of dynamic quenching also may reduce the average life time of the fluorophore.

p0014Cause of the static quenching is the formation of a non-fluorescent quencher fluorophore complex in the electronic ground state. Cause of the dynamic deletion is usually an electron transfer in the first excited state, which leads to the formation of a non-fluorescent molecule. Typical fluorophore quencher combinations are summarized in the following table.<tables id="tabl0001" num="0001"><table frame="all"><title>Tab.1.</title><tgroup cols="4" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="39.37mm" /><colspec colnum="2" colname="col2" colwidth="39.37mm" /><colspec colnum="3" colname="col3" colwidth="39.37mm" /><colspec colnum="4" colname="col4" colwidth="39.37mm" /><thead valign="top"><row><entry namest="col1" nameend="col4" align="center">Typical fluorophore-quencher combinations and Stern-Volmer quenching constants in water at room temperature.</entry></row><row><entry namest="col1" nameend="col1" align="left">fluorophore</entry><entry namest="col2" nameend="col2" align="left">extinguisher</entry><entry namest="col3" nameend="col3" align="left">K</entry><entry namest="col4" nameend="col4" /></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">Quinine cation</entry><entry namest="col2" nameend="col2" align="left">chloride</entry><entry namest="col3" nameend="col3" align="right">115</entry><entry namest="col4" nameend="col4" align="left">M⁻¹</entry></row><row><entry namest="col1" nameend="col1" align="left">N-Methyl</entry><entry namest="col2" nameend="col2" align="left">Bromide, iodide</entry><entry namest="col3" nameend="col3" align="right">290, 385</entry><entry namest="col4" nameend="col4" align="left">M⁻¹</entry></row><row><entry namest="col1" nameend="col1" align="left">6-Methoxychinolinium</entry><entry namest="col2" nameend="col2" align="left">Iodide, Ag (I), resorcinol</entry><entry namest="col3" nameend="col3" align="right">255, -</entry><entry namest="col4" nameend="col4" align="left">M⁻¹</entry></row><row><entry namest="col1" nameend="col1" align="left">pyrene</entry><entry namest="col2" nameend="col2" align="left">oxygen</entry><entry namest="col3" nameend="col3" align="right">0.08</entry><entry namest="col4" nameend="col4" align="left">Torr⁻¹</entry></row><row><entry namest="col1" nameend="col1" align="left">anthracenes</entry><entry namest="col2" nameend="col2" align="left">O₂, SO₂, (sulfite)</entry><entry namest="col3" nameend="col3" align="right">12:05, 0010</entry><entry namest="col4" nameend="col4" align="left">Torr⁻¹</entry></row><row><entry namest="col1" nameend="col1" align="left">perylene</entry><entry namest="col2" nameend="col2" align="left">Ag (I), Pb (II)</entry><entry namest="col3" nameend="col3" align="right">-</entry><entry namest="col4" nameend="col4" /></row><row><entry namest="col1" nameend="col1" align="left">Fluorscein, rhodamine</entry><entry namest="col2" nameend="col2" align="left">Iodide, azide</entry><entry namest="col3" nameend="col3" align="right">170, 7</entry><entry namest="col4" nameend="col4" align="left">M⁻¹</entry></row><row><entry namest="col1" nameend="col1" align="left">tryptophan</entry><entry namest="col2" nameend="col2" align="left">Pyridine, acrylamide</entry><entry namest="col3" nameend="col3" align="right">about 6x10⁻⁴</entry><entry namest="col4" nameend="col4" align="left">M⁻¹</entry></row><row><entry namest="col1" nameend="col1" align="left">tryptophan</entry><entry namest="col2" nameend="col2" align="left">Bromide, iodide</entry><entry namest="col3" nameend="col3" align="right">-</entry><entry namest="col4" nameend="col4" /></row><row><entry namest="col1" nameend="col1" align="left">porphyrin</entry><entry namest="col2" nameend="col2" align="left">nitroaromatics</entry><entry namest="col3" nameend="col3" align="right">-</entry><entry namest="col4" nameend="col4" /></row><row><entry namest="col1" nameend="col1" align="left">heterocycles</entry><entry namest="col2" nameend="col2" align="left">Azides, isocyanates</entry><entry namest="col3" nameend="col3" align="right">20-330</entry><entry namest="col4" nameend="col4" align="left">M⁻¹</entry></row><row><entry namest="col1" nameend="col1" align="left">Indoles, carbazoles</entry><entry namest="col2" nameend="col2" align="left">pyridine</entry><entry namest="col3" nameend="col3" align="right">about 10⁵</entry><entry namest="col4" nameend="col4" align="left">M⁻¹</entry></row><row><entry namest="col1" nameend="col1" align="left">quinine</entry><entry namest="col2" nameend="col2" align="left">nicotinamide</entry><entry namest="col3" nameend="col3" align="right">-</entry><entry namest="col4" nameend="col4" /></row><row><entry namest="col1" nameend="col1" align="left">APTS^a</entry><entry namest="col2" nameend="col2" align="left">Thiamine (vitamin B₁)</entry><entry namest="col3" nameend="col3" align="right">about 445</entry><entry namest="col4" nameend="col4" align="left">M⁻¹</entry></row><row><entry namest="col1" nameend="col1" align="left">9-amino acridine</entry><entry namest="col2" nameend="col2" align="left">various. purines</entry><entry namest="col3" nameend="col3" align="right">-</entry><entry namest="col4" nameend="col4" /></row><row><entry namest="col1" nameend="col1" align="left">Flavin</entry><entry namest="col2" nameend="col2" align="left">adenine</entry><entry namest="col3" nameend="col3" align="right">-</entry><entry namest="col4" nameend="col4" /></row><row><entry namest="col1" nameend="col1" align="left">AOPTS^b</entry><entry namest="col2" nameend="col2" align="left">cationic detergents</entry><entry namest="col3" nameend="col3" align="right">-</entry><entry namest="col4" nameend="col4" /></row></tbody></tgroup><tgroup cols="4" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="39.37mm" /><colspec colnum="2" colname="col2" colwidth="39.37mm" /><colspec colnum="3" colname="col3" colwidth="39.37mm" /><colspec colnum="4" colname="col4" colwidth="39.37mm" /><tbody valign="top"><row><entry namest="col1" nameend="col4" align="justify">^a 1-aminopyrene-3,6,8-trisulfonate,</entry></row><row><entry namest="col1" nameend="col4" align="justify">^b 1-Alkoxypyren-3,6,8-trisulfonate</entry></row></tbody></tgroup></table></tables>

p0015After Stern and Volmer exists between the fluorescence intensity of the fluorophore in the presence and absence of a quencher, and the concentration of the quencher, the following relationship I_O/ I = 1 + K [Q] (1) wherein I_O and I mean fluorescence intensity of the fluorophore in the absence or presence of a quencher, which is present in a concentration [Q]. K is the extinction constant, which is specific for each pair of the fluorophore and the quencher and, inter alia, on the temperature, the viscosity and solvent dependent.

p0016Instead of the intensity I in the case of the dynamic erase the lifetime of the fluorophore are used, also because t_O/ T = 1 + K [Q] (2) t_O and t is the decay of the fluorophore in the absence or presence of an extinguisher in concentration [Q].

p0017The FIA ​​invention is based on that one examines the quenching of the fluorescence of an antibody or an antigen by an external quencher before and after the formation of the Ag / Ab complex. One can distinguish between two types of fluorescent antibodies and antigens: the one hand, those having an intrinsic fluorescence, on the other hand those which are labeled with a fluorophore. Autofluorescence is observed, are when the molecule tyrosine (Tyr) or tryptophan modules (Trp). The fluorescence can be excited at 280 and 295 nm and has a maximum at 300 to 350 nm. More common is the second case, in which an antigen or antibody with a synthetic fluorophore is selected.

p0018A method for labeling proteins or antigen and antibodies with fluorescent markers are to be considered as prior art. For descriptions of the methods may be found in: RP Haugland, "Covalent Fluorescent Probes" in the book of RF Steiner: "Excited States of Biopolymers", Plenum Press, New York, 1983. The most common are reactions with fluorescent isothiocyanates (which with the terminal amino groups of proteins react) with sulfonic acid chlorides (which react with amines and hydroxyl), and with the Iodacetami and maleimides (which may be associated with SH groups for reaction).

p0019An essential feature of the method according to the invention is the fact that the extinguisher is in the solvent and not either-fluorescent or at most only weakly fluorescent (as in the case of resorcinol) is. But if it is fluorescent, so is its fluorescence so that it can not come to an energy transfer. Typical quencher molecules are listed in Tab. 1, but without levy thus intended to be comprehensive. A distinction is made between ionic quenchers such. As the halides, and neutral quenchers such. As oxygen, acrylamide, pyridine and resorcinol. Until now the binding partner is free in solution, its fluorescence is greatly reduced by the quencher molecules present in the solvent. After the formation of Ag / Ab complex, however, the fluorophore is sterically shielded by the quencher molecules, so that there is a smaller number of collisions (and deletions). As a result, one observes an increase in the fluorescence intensity or the decay time as a result of the binding process.

p0020The following figures illustrate the invention and highlight its advantages over previous methods.<ul><li>Fig. 1 schematically shows a typical Y-shaped antibody (Ab) before (1a) and after bonding (1b) to an antigen (Ag). It is obvious that a solution in existing quencher (Q) can the protein contained in the fluorescent tyrosine or tryptophan (indicated by an asterisk) with much greater efficiency to delete as long as the antigen is not bound. The advantage of this method is that the Ab or Ag need not be marked and that in a precisely defined concentration can already admit the solvent the extinguisher.</li><li>Fig. 2 shows schematically a fluorescence-labeled antibody (Ab) before (2a) and after bonding (2b) to an antigen (Ag). The fluorophore, indicated by an asterisk, is covalently bound to the antibody. Again it is apparent that a solution in existing quencher (Q) able to clear the fluorophore with much greater efficiency, as long as the antigen is not bound. The advantage of this method is that only the Ab or Ag need to be highlighted and that in a precisely defined concentration already the solvent can (and not the other binding partner) reflect the Löscher.</li><li>Fig. 3 shows the corresponding representation of a fluorescent-labeled antigen before (3a) and after bonding (3b) to an antibody.</li><li>Fig. 4 shows a typical curve as from plots of relative fluorescence intensity [I in Eq. (1)] or relative cooldown [t in Eq. (2) was obtained given in nano-seconds] of an antigen against an added amount of antibody. The measurement is thus reduced to an intensity or Abklingzeitbestimmung. There are no polarizers required, and separation of the Ab / Ag complex from the remainder of the sample can (but need not) be dispensed with.</li><li>Fig. 5 diagrammatically shows an experimental arrangement for measuring the fluorescence intensity of an immobilized on an optically transparent carrier or the end of a fiber optic light guide antigen or antibody. The antibody may be immobilized either directly or at the distal side end of the light guide, or he is first on a support (eg. As a polymer membrane) attached and this is then fixed at the end of the light guide. Such support membranes are very cheap to manufacture plastic materials. Thus, tests are carried out with the aid once usable and disposable carrier materials.</li></ul>

p0021Using the fiber optic light guide, it is finally also possible to perform such immunological measurements also invasive.<ul><li>Fig. 6 shows an arrangement for measuring the fluorescence of labeled Ab or Ag by means of total internal reflection. It is known that light n₁ in total reflection at an interface between two media with refractive index n₂ and not directly reflected at the interface, but that the electromagnetic field a few nm in the second phase penetrated ( "evanesziert"). It can excite fluorescence there, which can be conducted away from the optical waveguide. The advantage of this method is that one has a very defined penetration depth (which depends on the refractive indexes and the wavelength of light), so that on the sample (in this case, an Ag or Ab) is an optically non-transparent phase (eg. , blood) can be located, without causing a disturbance occurs.</li></ul>

p0022A typical process will now be explained with reference to the determination of immunoglobulin G (IgG) in the serum. It is used, for. Example, anti-human IgG (from sheep) which with fluorescein isothiocyanate (FITC) is reacted in 4 ml of bicarbonate buffer pH 9.5 for 24 hrs. At 22 ° C (50 mg and 4 mg of IgG FITC). The FITC-labeled protein is separated from the unreacted FITC on a Sephadex G-25 column (10 cm x 2 cm). The labeled protein is at a concentration of typically 0.1 - released 1.0 mg / 1 in a 0.1 normal solution of potassium bromide in water with 0.1% emulsifier. Instead of sodium may also sodium iodide or azide used.

p0023Iodide, azide or bromide are strong quencher of fluorescence of FITC. As long as the surface-bound anti-IgG fluorescein free solution is exposed, it is strongly quenched by the present halide in its fluorescence intensity or in its fluorescence decay according to equation (2) decreases.

p0024but using this solution, increasing amounts of serum which contains IgG, as a complex from IgG and anti-IgG will form, whereby the FITC antibody is screened off from the solution, so that it is less easily deleted in its fluorescence. Up to that point at which all anti-IgG is bound, is thus observed a continuous increase in the fluorescence. This consists of (a) largely erased fluorescence of unbound FITC / anti-IgG and (b) practically quicklime fluorescence of FITC in the antibody-antigen complex.

p0025This results in a calibration curve as shown in Fig. 4. By comparing the fluorescence intensity of the pure FITC-Anti-IgG solution to the Fluoreszenzintensiät the serum-added solution one has the possibility to carry out a quantitative determination of IgG with reference to the calibration curve.

p0026In addition to the FITC used in this example, a number of other fluorophores used as marker in question. Particularly noteworthy is the relatively specific deletion of acridinium ion and 6-Methoxychinolinium ion by chloride, bromide and iodide. The extinguisher chloride comes in human blood in a concentration of about 100 mmol / l, so that chloride can be used in in-vivo measurements as quenchers.

p0027The deletion of the intrinsic fluorescence of the proteins (due to the presence of Tyr and Trp) is possible, for example with pyridine, acrylamide, bromide, iodide, or heavy metal salts.

p0028In a particular embodiment of the method, the labeled antigen or the labeled antibody at the surface of an optically transparent polymeric support is immobilized, eg. As at the surface of a glass plate or a fiber optic light guide. It keeps fluorescence or their changes through the optically transparent carrier, for. Example, also by means of evanescent waves at the total internal reflection spectroscopy (Fig. 5). Such optical methods are described by JF Place et al. in an article "Opto-Electronic immunosensor: A Review of Optical immunoassays at Continuous Surfaces" in Biosensors, 1, 321 ff (1985) and in U.S. Patent No. 4,447,546, wherein a method for the immunological determination in a very defined sample volume with Help the evanescent art will be described. The inventors enter but the possibility of dynamic fluorescence quenching not and do not recognize their special advantages in this particular case. A related principle is described in U.S. Patent 4,582,809.

p0029Even with the use of optical fibers followed one the change in fluorescence signal as a result of the deletion prevented by an externally added Löscher after successful bond process. The advantage of the process by means of evanescent waves is that they also strongly colored solutions, such. As blood, can investigate because the evanescent wave at the total internal reflection at an interface penetrates only a few nanometers in the protein phase. This is quite a selective excitation of fluorescence of Tyr or Trp in the Ab or Ag permits and the background fluorescence of the biological sample material can be kept small.

p0030If the antibody or antigen immobilized on the end of a fiber optic light guide, so you taking advantage of the fact that the possibilities in the blood quencher are (z. B. chloride), the opportunity in hand, immunological tests with a fiber optic catheter directly in the blood can carry. A compilation of the most important immunological methods using optical waveguides can be found in the article by JF Place et al.

p0031When externally added quencher are in particular those in question, which are to be introduced in a precisely defined concentration in the test sample. These are, for. Example, the halides and pseudohalides, pyridines, acrylamide, hydroquinone, as well as transition metal cations, preferably those which have no inherent color. Also, molecular oxygen and sulfur dioxide (or sulfite) are known strong quencher of fluorescence, such as aromatic hydrocarbons, but is a defined concentration of z. B. oxygen much more difficult to prepare in aqueous solution, since its solubility may vary with the protein content.

p0032Unlike the FIAs described in the energy transfer methods under 2), in the method according to the invention, the double labeling omitted, and any of extinguishers are used, regardless of whether their absorption spectrum coincides with the fluorescence of the first marker. Also, the erasing mechanism is quite different, as evidenced by the different mathematical treatment of the two situations: energy-transfer quenching takes place according to the theory of Förster with an efficiency which depends on R⁻⁶ (R is the distance between the two species). Fluorescence quenching by added Löscher obeys the Stern-Volmer equation (Eq. 1).