DE102011086568A1 - Amplifying signals in heterogeneous binding assays based on donor or receptor complex, comprises adding particle of first particle class and particle of second particle class to donor or receptor complex and optionally repeating steps - Google Patents

Abstract

Amplifying signals in heterogeneous binding assays based on a donor or receptor complex (1) made of a receptor molecule (10) and a donor molecule (20), which is bound by the receptor molecule, comprises: (a) forming donor or receptor complex; (b) adding at least one particle of a first particle class to the donor or receptor complex, where at least one particle of first particle class (n) is coupled with the donor or receptor complex, the particles of the first particle class further comprises binding site; and (d) optionally repeating the steps, preferably the step (b) and (c) at least once. Amplifying signals in heterogeneous binding assays based on a donor or receptor complex (1) made of a receptor molecule (10) and a donor molecule (20), which is bound by the receptor molecule, comprises: (a) forming donor or receptor complex; (b) adding at least one particle of a first particle class to the donor or receptor complex, where at least one particle of first particle class (n) is coupled with the donor or receptor complex, the particles of the first particle class further comprises binding site, which can couple to at least one particle of a second particle class (m), where n is a natural number, which is >= 2, and the particle of a second particle class comprises binding site, which can couple to at least one particle of the first particle class, where m is a natural number, which is >= 2; (c) adding at least one particle of a second particle class; and (d) optionally repeating the steps, preferably the step (b) and (c) at least once.

Description

The present invention relates to a method for amplifying signals in heterogeneous binding assays based on donor / receptor complexes, in particular ligand-receptor complexes.

Heterogeneous binding assays are very widespread in the form of enzyme-linked immunosorbent assays (ELISA). In them, conjugates consisting of a detection antibody and an enzyme, preferably horseradish peroxididase or alkaline phosphatase are used in order to obtain a colorimetrically, fluorimetrically or luminometrically well detectable signal after addition of a substrate. The main advantage of this technique is the good sensitivity which, for example, allows for protein detection limits in the range of a few pg / ml. Their main disadvantage is the susceptibility, in particular the strong temperature dependence of the enzyme reaction and the need to store the required reagents (conjugates, substrates) at 4 ° C. These disadvantages can be avoided if a dye conjugate is used instead of the enzyme conjugate. In particular, in the form of gold-particle antibodies or color-particle-antibody conjugates, it is thus possible to produce simple and easily stored heterogeneous test systems. Among the best known types can be found in US-A-6,485,982 disclosed lateral flow test strips or test columns WO-A-92/05442 . WO-A-94/09365 or WO 95/19569 be counted. A disadvantage of these formats is their low sensitivity, which only allows detection limits in the range of about 1 ng / ml for proteins.

The invention is based on the object of specifying a method with which it is possible to increase the sensitivity of the analysis with heterogeneous assays, without having to use an enzymatic amplification reaction.

• – in einem ersten Schritt ein Donator/Rezeptorkomplex 1, gebildet wird,
• – danach in einem zweiten Schritt mindestens ein Partikel einer ersten Partikelklasse zu dem gebildeten Donator/Rezeptorkomplex 1 gegeben wird,
• – der mindestens eine Partikel der ersten Partikelklasse an den Donator/Rezeptorkomplex 1 koppelt und
• – der mindestens eine an den Donator/Rezeptorkomplex 1 gekoppelte Partikel der ersten Partikelklasse n ≥ 2, wobei n eine natürliche Zahl ist, weitere Bindungsstelle aufweist, an die mindestens ein Partikel einer zweiten Partikelklasse koppeln kann,
• – der Partikel der zweiten Partikelklasse seinerseits mindestens m ≥ 2, wobei m eine natürliche Zahl ist, Bindungsstellen aufweist an die mindestens ein Partikel der ersten Partikelklasse koppeln kann,
• – in einem dritten Schritt mindestens ein Partikel der zweiten Partikelklasse zugegeben wird und
• – gegebenenfalls die Schritte, insbesondere der zweite und dritte Schritt, mindestens einmal wiederholt werden.

This object is achieved according to the invention by a method for amplifying signals in heterogeneous binding assays based on donor / receptor complexes, wherein

• - In a first step, a donor / receptor complex 1 , is formed,
• - Then in a second step, at least one particle of a first particle class to the donor / receptor complex formed 1 is given
• - The at least one particle of the first particle class to the donor / receptor complex 1 couples and
• - the at least one to the donor / receptor complex 1 coupled particles of the first particle class n ≥ 2, where n is a natural number, has a further binding site to which at least one particle of a second particle class can couple,
• The particle of the second particle class is in turn at least m ≥ 2, where m is a natural number, has binding sites to which at least one particle of the first particle class can couple,
• - In a third step, at least one particle of the second particle class is added and
• If appropriate, the steps, in particular the second and third steps, are repeated at least once.

According to the invention, the term donor is understood to mean a chemical entity, in particular a molecule or molecular structure, which interacts strongly with a receptor, which is likewise a chemical entity, in particular a molecule or a group of molecules. This interaction leads to the formation of a coupling between donor and receptor. The coupling is a specific interaction whose specificity can be detected in particular by the variables used for the characterization of chemical complexes. This is, for example, the dissociation constant K _D (reciprocal to the association constant), which results from the law of mass action. Generally, the smaller K _D , the stronger the binding between the complex-forming components donor and receptor. Typical dissociation constants that occur upon binding of IgG-type antibodies are in the range of 10 ^-4 -10 ^-11 M, for the donor-receptor pair biotin / avidin, the interaction is in the range of 10 ^-14 and 10 ^-15 M. This can be z. As by electrostatic interactions or those caused by polarization (hydrogen bonds), but also by so-called van der Waals attractions are taught. Often, these interactions are also made possible by complementary structural elements on the surface of the respective molecules or molecular assemblies. Depending on the viewpoint, the donor may also be a receptor of the receptor molecule. This is clearly shown in the comparison of the donor / receptor pairs avidin / biotin, in which one of the partners acts as a donor for the other, then the receptor-representing part. Usually, a low molecular weight donor in complexes with very large molecules, for example, enzymes or neuroreceptors, is called a ligand.

In particular, the method according to the invention uses an immune complex as donor / receptor complex 1 , Among them is a complex of a ligand, which may be a hapten or antigen and a molecule involved in the immune response of an immune system in the detection of molecular Structures, in particular epitopes, for example, antibodies regardless of their class, or fragment of these antibodies, understood. In one embodiment of the method according to the invention, at least one particle of a third particle class different from the particles of the first particle class may be added to the addition of the at least one particle of the second particle class in the third step. This has the advantage that a competition of the particles of the first particle classes is excluded.

The particles have at least one binding site which can form a donor-receptor complex with another binding site of another particle class. For this purpose, the particles, unless they are inherently such binding sites, modified by chemical reactions that bind the corresponding donor or receptor molecules on the surface of the particles of different particle class. Thus, for example, avidin can be arranged on the surface of the particle class 1, which then binds with the immune complex, provided that it is biotinylated, whereas the particles of particle class 2 are then provided with biotin molecules on their surface, which then adhere to the particles of particle class 1 can bind.

Methods for binding the appropriate donor and receptor moieties to the particles of the particular particle class depend on the nature of the particle and the nature of the donor / receptor molecules to be coupled. The skilled person has at his disposal an arsenal of various possibilities in order to find suitable solutions for the respective problem. If the chemical nature of the donor / receptor molecules allows this, they can already be coupled directly to the surface of the particles, but it is also possible to bind them via so-called spacers on the surface of the particles. If the particles are chemically more or less inert, the surface of the particles can be chemically treated in upstream steps in order to allow bonds with the particular organic molecules of the donor / receptor type, or the chemically inert particles can be provided with modifiable coatings.

According to the invention, in one embodiment, n and m can each be> 2, in particular 3-10. At n, m = 2 a linear amplification is formed and at n, m = 3 already a planar amplification.

In another embodiment of the invention, the particles of the first and second particle classes have approximately the same size or different size. Typically, nanoparticles or biopolymers can be used. The particle size depends on the particular type used and / or on the conditions underlying the respective assay and can be adapted and selected by the person skilled in the art by means of his average knowledge. Typically, the particles may have a mean size of 5 nm to 5 μm, assuming an approximately spherical shape. If the particles have a very irregular shape, these values should be regarded as their largest extension in a spatial direction. According to the invention, particles of the particle class may be organic or inorganic particles or combinations thereof.

As inorganic particles in the process according to the invention, in particular gold particles, magnetic particles, or as organic particles, particles of polymers and biopolymers, for. As latex, proteins, in particular fluorescent proteins such as phycoerythrin, phycocyanin, or cellulose derivatives, dextran derivatives are used.

Typical particles are gold particles with mean diameters of 5-50 nm, in particular 10-20 nm, latex particles with dye or fluorescent dye in a polymer matrix with average diameters of 10 nm-4 .mu.m, in particular 100 nm-400 nm), dye dispersions with particles having medium Diameters of 100 nm-500 nm, as in WO 97/19354 Magnetic particles having a mean diameter in a size of 10 nm-4 .mu.m, in particular 40 nm-400 nm or polymers, in particular biopolymers such as proteins, in particular fluorescent proteins such as phycoerythrin, phycocyanin, or cellulose derivatives, dextran derivatives.

In a further embodiment of the method according to the invention, the particles of the first, second and / or third particle class are detectable.

In one embodiment of the method according to the invention, the free binding sites of the first and second particle classes can be mutually formed by a ligand / receptor pair as donor / receptor pair. Thus, for example, as mutual ligand / receptor pairs avidin / streptavin, Protein A / IgG, especially the F _C portion of IgG, lectin / oligosaccharide, polymer binding domain / biopolymer, anti hapten antibody / hapten or combinations thereof.

The detectable particles are detected in particular by optical methods such as transmitted light photometry, reflectometry, fluorimetry, luminometry, determination of the refractive index and birefringence, by imaging methods, by electrical methods such as conductometry, potentiometry, or magnetic methods such as linear and nonlinear susceptometry and relaxometry based on Giant Magnetic Resistors, Hall Sensors, Flux Gate Sensors, Squids or Induction Coils.

1A shows schematically the formation of an immune complex of immobilized on a solid phase antibody and a ligand.

1B Figure 3 shows schematically the binding of a class 1 particle to the immune complex. The particle of class 1 has specificities against the analyte (n1 = 4) as well as particles of particle class 2 (n2 = 4).

1C schematically shows the situation that exists when Class 2 particles (m = 8) to the aggregate according to 1B have tied. 1D schematically shows the situation after repeated passage through the method according to the invention.

The 2A -D show the training of reinforcement, according to 1 has been described generally for an embodiment in which avidin is immobilized on the class 1 particles and biotin on the class 2 particles.

The 3A D show an embodiment according to the embodiment 2A - 2D analogous performance of the amplification reaction, wherein the particles of class 2 as protein molecules 52 (here BSA) are executed.

The 4A -D show the training of reinforcement, according to 1 has been generally described, has been for an embodiment in which antibodies against FITC and antibodies against the analyte to be determined on the particles of class 1 and FITC are immobilized on the particles of class 2.

The 5A -D show the formation of the amplification, in an indirect serological assay for the detection of IgG for an embodiment in which three particle classes are used. Antibodies to the F _C region of an antibody that binds the analyte and biotin to the particles of class 1, avidin to particles of class 2 and biotin to the particles of class 3 are immobilized.

The 6A -D show the formation of reinforcement in a competition assay.

The 7A -D show histograms of the increase of the signals as a function of performed cycles.

Detailed description of the invention

1A D shows in diagrammatic form an embodiment of the present invention. The associated principle illuminates the structure of the method according to the invention and its result. On a carrier 15 are receptor molecules 10 immobilized with a donor molecule 20 a donor / receptor complex 1 can train. This receptor / donor complex 1 consists of the receptor 10 and donor 20 , Thereafter, in a second step, a particle 40 on its surface also receptor molecules 41 immobilized with the donor 20 can bind, offset. The particles 40 of particle class 1 thus bind to the donor / Rezeportkomplex. The particles 40 the particle class 1 have next to the receptor molecules 10 groups 16 on that with complementary groups 18 on particles 50 Particle class 2 can interact. The on the particle 40 Particle class 1 arranged additional receptor molecules 10 that are complementary to the particle 50 Class 2 immobilized molecules, quasi donor molecules 20 , interact, ensure a lasting interaction between the particles 40 class 1 and particles 50 class 2. Thereafter provides the further addition of particles 40 Class 1, with free donor molecules 18 on the particles 50 Particle class 2 interacts to further enhance the formed multiparticulate complex. The molecules act here 18 Particle class 2 practically as receptors for the previous step as receptors on the particles 40 Particle class 2, but in this step as donors 20 acting molecules.

Overall, this methodology can be used to amplify a signal if appropriately labeled particles are used. The label can then be read by suitable detection methods.

Variants of the method according to the invention can be found in the 2 - 4 be followed. For the sake of simplicity, the principle is based on the formation of immune complexes 1 explained in more detail. In this case, the skilled person can easily recognize and abstract by replacing the term "immune complex" by "donor / receptor complex", the general principle behind the assay according to the present invention.

First, therefore, the example of 1 The system is explained in more detail on the basis of the concretisation of an immune complex.

The 1A shows a typical immune complex 1 in which a capture antibody 10 an antigen 20 has tied. The catcher antibody 10 is on a carrier 15 immobilized. This complex 1 points, presented by the antigen 20 , a binding site for particles 40 Class 1.

In the second step accordingly 1B bind particles 40 Class 1 on its antigenic specificity 41 to the immune complex 1 , In addition, the particles own 40 Class 1 has a specificity 16 against particles 50 Class 2. In a third step accordingly 1C bind particles 50 Class 2 particles 40 Class 1, by interaction of specificity 16 of the particle 40 with the specificity 18 of the particle 50 , Depending on the stoichiometry and spatial formation of the binding partners, a multiparticulate complex is formed. The addition of labels, in particular Class 1 colored particles and Class 2 colored particles, makes the complex 1 enlarged accordingly (see 1D ).

The process can be continued several times including washing steps. When using color particles, an increase in color intensity is observed after each addition step. The detection method is particularly simple and advantageous to carry out when immobilizing the capture antibody 10 three-dimensional porous carrier 15 how filters or sintered materials are used and the individual steps are carried out in the flow in a column or on a lateral flow membrane. Typical residence or incubation times for the individual steps are in the range of 10 seconds to 10 minutes (preferably 30 seconds to 2 minutes).

In the variant according to 2A - 2D be the steps in 1A - 1D already described varied. Again, a catcher antibody is first 10 who is on a carrier 15 is immobilized, contacted with an analyte-containing sample, followed by an immune complex 1 between the capture antibody 10 and the analyte 20 formed. In a subsequent step, the mixture becomes an intermediary, in this case a detector antibody 30 that with biotin 31 is added, added.

At the formed immune complex 1 - consisting of immobilized capture antibody 10 , Analyte 20 , Detector antibody 30 with biotin 31 - then binds a particle 40 Class 1, which with avidin 41 is provided.

It is advantageous in this embodiment that the particles of the particle class 1 and the particles of the particle class 2 each only have to have a specificity to cause the gain, since the class 1 particle via a mediator, in this case a biotinylated antibody 19 to which immune complex binds. In general, the mediator must be a molecule that has at least two different binding sites, one to the analyte 20 and on the other to the particle 40 Class 1, has.

2C shows the situation after addition of a particle 50 the particle class 2, which in turn with biotin 31 is provided. Due to the biotin label 31 of the particle 50 Particle class 2 binds the particle 50 to the already existing particles 40 of class 1, with the complementary avidin 41 are provided, which then already leads to a first amplification of the immobilized on the carrier signal.

2D now shows in a further step, the formation of an even more complex system, after repeating the first step in the particle 40 Particle class 1, in turn, with avidin 41 are marked arises. The re-addition of the particles 40 the first particle class containing avidin 41 are marked, now leads to a further amplification of the signal by these particles 40 of particle class 1 to free biotin binding sites 31 in the previous step according to 2C added particles 50 Particle class 2 tie. Ultimately, there is an amplification of the amplification caused amplification of the signal and thus increase the detection sensitivity.

The 3A - 3D illustrate a variant of the embodiment according to 2A - 2D in which instead of the particles designed as particles 50 Particle class 2 is a protein 52 is used, with a specificity to the particle 40 Particle class 2 is equipped. In this case, it is a biotinylated protein 52 , in this case BSA.

Another variant presents in the 4A - 4D shown procedure, in the antibody 10 as a capture antibody on a support 15 are immobilized. An analyte 20 is from the capture antibody 10 bound, leaving the immune complex 1 can train. Unlike in 2A not described another antibody, for example, with biotin 31 can be labeled and as a detection antibody 30 acts, added, but it becomes a particle 40 Particle class 1 added, on the surface of two different antibodies 45 . 48 are bound, with the first antibody 45 that is attached to the particle 40 Particle class 1 is bound to the immune complex 1 binds. The particle 40 Particle class 1 also has an antibody 48 on that with surface markings 55 on the particle 50 the second particle class interacts bindingly. In this case, the antibody 48 for example against FITC 55 be directed, whereas the antibody 45 against the analyte 20 not at the same binding site as the capture antibody 10 binds. After training of in 4C The multiparticulate complex shown can then in a further step, as in 4D shown, in turn, a variety of particles 40 Particle class 1, as in 4B clarified, added. The antibody 48 then binds to vacancies 55 of the particle 50 the second particle class, resulting in a further amplification of the signal.

5 shows another executable variant using particles belonging to three different classes, first according to 5A on a carrier 15 an immobilized antigen 20 is presented to which an antibody 25 from the sample to be examined. It has an immune complex this time 1 from immobilized ligand 20 and antibodies 20 formed from the sample.

According to 5B binds to this immune complex 1 from antibodies 25 , Ligand 20 who is on the carrier 15 immobilized, a particle 40 Particle class 1. This carries antibodies on its surface 60 against the F _C region of IgG antibodies raised with the antibody 25 of the immune complex 1 react and beyond, such as biotin 31 , By the antibody 60 mediates, binds the particle 40 the particle class 1 at the on the carrier 15 immobilized immune complex 1 , The biotin labels 31 serve to bind the particles 50 Particle class 2, which are added in a next step (see 5C ). The previously described particles 50 Particle class 2 carry avidin molecules on their surface that interact with the biotin molecules 31 the particle 40 Particle class 1 form a bond and form an adherent network of particles. According to 5D can then further amplify the signal a particle class 70 be added to the surface in turn Biotinmolküle 31 attach and with free avidin molecules on the surface of the particles 50 interact.

According to 5D the enormous amplification of the signal is immediately recognizable. This exemplary embodiment can be used in serological tests in which correspondingly indicative antibodies must be detected.

6A D shows the principle of a competition assay which can be carried out according to the invention.

In this embodiment, the concentration of mycotoxin zearalenone (Zea) from cereal extracts is determined based on an immunological competition assay. In 6A For example, a cereal extract contaminated with "free" Zea is mixed with zea-biotin. Subsequently, the mixture is applied to a sintered matrix 16 made of polyethylene. There the "free" Zea competes 20 with the zea-biotin around the binding site on the immobilized antibody. In principle, two immune complexes can form. Immune complex A consists of antibody and zea-biotin ( 10 + 31 ). This complex forms when no mycotoxin is present in the original sample. Immune complex B consists of antibodies 10 and "free" Zea 20 , This complex forms when mycotoxin is present in the original sample. In the following steps according to 6B -D only the immune complexes A1 are amplified. After abandonment of the particle 40 Class 1 binds this via its streptavidin 41 to the coupled zea-biotin 10 + 31 , Then it binds with biotin 31 labeled biopolymer BSA-biotin 52 to the bound particles 40 Class 1. Finally, Class 1 particles are returned and bind to coupled BSA-biotin 52 , Depending on the desired signal amplification or number of cycles, BSA-biotin is alternately 52 and particles 40 abandoned the class 1. It should be noted that in this embodiment, the concentration of zearalenone in the Output sample is inversely proportional to signal strength. This means that the more mycotoxin is present in the cereal, the lower the measurable or amplified signal.

The 7A -D relate to the graphical representation of the table results of the respective examples 1-4.

example 1

Detection of staphylococcal enterotoxin B (SEB)

The following procedure describes how to achieve specific signal enhancement on an immunologically functionalized surface. The basis for this is a PE-based 3-dimensional cylindrical sintered matrix ("frit") on whose surface antibodies are immobilized. These antibodies bind specifically from a solution SEB (staphylococcal enterotoxin B). Subsequently, class 1 particles are added to this immune complex. Class 1 particles have specificity (antibodies) to SEB and thus bind to the immune complex. In addition, they also have specificity against haptens immobilized on class 2 particles. Thus, in a subsequent step, particles of class 2 bind to particles of class 1. An oligomeric particle complex is formed. This is increased by mutual addition (cycling) of both particle classes. Both particle classes have a self-absorption at 525 nm. This can be measured photometrically. The optical density increases with increasing number of cycles and produces a specific coloration of the filter. Execution: No. step volume reagent Time Measurement OD [] 1 sample application 750 μl Sample [50 ng / ml SEB or 0 ng / ml SEB] 6 minutes 2 Cycling 500 μl Class 1 particles 6 minutes 3 750 μl wash buffer Yes 4 500 μl Class 2 particles 6 minutes 5 750 μl wash buffer optional 6 Repeat from step 2 (1) 750 μl of the sample added with SEB is pipetted onto the column where it binds specifically to the immobilized antibody. The incubation period of 6 minutes is awaited.
(2) 500 μl Class 1 particles are given up. The incubation period of 6 minutes is awaited.
(3) Wash 750 μl of wash buffer with excess particles.
(4) 500 μl Class 2 particles are added and bind to Class 1 particles. The incubation period of 6 minutes is awaited.
(5) With 750 μl wash buffer, excess particles are washed down again.
(6) Depending on the desired number of cycles, step 2 is started again. Results OD cycles SEB [ng / ml] 1 2 3 4 5 50 0.358 0.664 0,952 1,236 1,407 0 0.047 0,052 0,061 0.078 0.09

Please refer 7A

Example 2

Detection of BoNT / B

Similarly, the method described above was applied to the detection of BoNT / B. Results Table BoNT-B with specific antibodies OD cycles BONT / B [ng / ml] 1 2 3 4 5 10 0.2 0.413 0.68 0.9 1.3 0 0.04 0,055 0.06 0.063 0.064

Please refer 7B

Example 3

Detection of tetanus IgG (horse) from serum according to embodiment 3

In this embodiment, the titre is determined from tetanus IgG from horse serum. The basis is a 3-dimensional sintered matrix made of polyethylene. Tetanus toxoid is immobilized on its surface. The tetanus-specific IgG from the sample bind to the immobilized antigen to form an immune complex. Particles of class 1 bind to this immune complex by their specificity against the Fc portion of the bound horse IgG. In addition, the class 1 particles still have a biotin label. Class 2 particles have an avidin label and bind to the biotin label of the class 1 particles. A multiparticulate aggregate is formed. This can be measured photometrically. To enhance this aggregate, particles of class 3 are added. These particles have a biotin label and bind to the avidin of class 2 particles. By interacting class 2 particles and class 3 particles, a specific oligomeric particle aggregate is formed, which is measured photometrically. Here, the intensity of the measured signal correlates with the tetanus IgG titer from the horse serum. execution No. step volume reagent Time Measurement OD [] 1 sample application 750 μl Sample [0.01 IU / ml tetanus IgG or 0 IU / ml tetanus IgG] 6 minutes 2 initial detection 500 μl Class 1 particles 6 minutes 3 to wash 750 μl wash buffer 4 Cycling 500 μl Class 2 particles 6 minutes 5 750 μl wash buffer Yes 6 500 μl Class 3 particles 6 minutes 7 750 μl wash buffer optional 8th Repeat from step 4 (1) 750 μl horse serum are given. Tetanus IgG bind specifically to immobilized tetanus toxoid.
(2) 500 μl Class 1 particles are added and bind specifically to bound tetanus IgG.
(3) 750 μl wash buffer is added to remove excess Class 1 particles.
(4) 500 μl Class 2 particles are added and bind to class 1 particles.
(5) 750 μl of wash buffer are added to remove excess Class 2 particles. The optical density [OD 525 nm] is measured.
(6) 500 μl Class 3 particles are added and bind to class 2 particles.
(7) 750 μl of wash buffer are added to remove excess Class 3 particles.
(8) Depending on the desired degree of reinforcement, the steps from No. 4 are repeated (Cycling) results OD cycles Tetanus IgG horse [IE / ml] 1 2 3 4 5 0.01 0.13 0.26 0.58 0.92 1.26 0 0.03 0,036 0.04 0.048 0.054

Please refer 7C

Example 4

Detection of zearalenone from cereal extracts

execution No. step volume reagent Time Measurement OD [λ] 1 preincubation 600 μl 300 μl sample (free Zea) + 300 μl Zea-biotin 6 minutes 2 sample application 600 μl Task Pre-incubate on column 6 minutes 3 initial detection 500 μl Class 1 particles 6 minutes 4 to wash 750 μl wash buffer 5 Cycling 500 μl BSA biotin (biopolymer) 6 minutes 6 750 μl wash buffer Yes 7 500 μl Class 1 particles 6 minutes 8th 750 μl wash buffer optional 9 Repeat from step 5 (1) 300 μl of a sample of free zearalenone is mixed with 300 μl of zea-biotin
(2) 600 μl of mixture (pre-incubate) are pipetted onto the filter. ("Free" Zea from sample competes with Zea-biotin for binding site on immobilized antibody
(3) Class 1 particles are abandoned and bind specifically to the immune complex of antibody and zea-biotin
(4) Excess Class 1 particles are washed down
(5) BSA biotin is abandoned and binds specifically to class 1 particles
(6) Excess BSA-biotin is washed down.
(7) Class 1 particles are abandoned and bind to BSA-biotin
(8) Excess Class 1 particles are washed down
(9) Depending on the desired number of cycles / amplification, the steps from no. 5 are repeated. Results OD cycles Zearalenone [ng / ml] 1 2 3 4 5 200 0.15 0.17 0.24 0.39 0.55 0 1.1 1.5 1.7 1.9 2

Results in 7D illustrates

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• US 6485982 A [0002]
• WO 92/05442 A [0002]
• WO 94/09365 A [0002]
• WO 95/19569 A [0002]
• WO 97/19354 A [0012]

Claims (13)

Method for amplifying signals in heterogeneous binding assays on the basis of a donor / receptor complex ( 1 ) from a receptor molecule ( 10 ) and a donor molecule ( 20 ) derived from the receptor molecule ( 10 in which, in a first step, the donor / receptor complex ( 1 ), - then in a second step at least one particle of a first particle class to the donor / receptor complex formed ( 1 ), - the at least one particle of the first particle class to the donor / receptor complex ( 1 ) and - the at least one to the donor / receptor complex ( 1 ) coupled particles of the first particle class n ≥ 2, where n is a natural number, further binding site to which at least one particle of a second particle class can couple, - the particles of the second particle class in turn at least m ≥ 2, where m is a natural number , Binding sites has to which at least one particle of the first particle class can couple, - added in a third step, at least one particle of the second particle class and - optionally the steps, in particular the second and third steps, at least once repeated. The method of claim 1, wherein after the addition of the at least one particle of the second particle class in the third step of claim 1, at least one particle of a third, different from the particles of the first particle class particle class is added. Method according to claim 1 or 2, wherein n and m are each> 2, in particular 3, 4, 5. Method according to at least one of the preceding claims, wherein the particles of the first and second particle class have approximately the same size or different size. Method according to at least one of the preceding claims, wherein the particle size have an average size of 5 nm to 5 microns. Method according to at least one of the preceding claims, wherein particles of the particle class organic or inorganic particles or combinations thereof are used. A method according to claim 5, wherein as inorganic particles gold particles magnetic particles, or as organic particles particles of polymers and biopolymers, such as latex, proteins, in particular fluorescent proteins such as phycoerythrin, phycocyanin, or cellulose derivatives, dextran derivatives are used. Method according to at least one of claims 3 to 6, wherein gold particles having an average diameter of 5-50 nm, in particular 10-20 nm, latex particles with dye or fluorescent dye in a polymer matrix having an average diameter of 10 nm-4 .mu.m, in particular 100 nm -400 nm, dye dispersions with particles having a mean diameter of 100 nm-500 nm, magnetic particles with a mean diameter of 10 nm-4 .mu.m, in particular 40 nm-400 nm or polymers, in particular biopolymers such as proteins, in particular fluorescent proteins such as phycoerythrin, phycocyanin , or cellulose derivatives, dextran derivatives are used. Method according to at least one of the preceding claims, wherein the particles of the first, second and / or third particle class are detectable. Method according to at least one of the preceding claims, wherein the free binding sites of the first and second particle classes are mutually formed by a ligand / receptor pair. The method of claim 10, wherein the mutual ligand / receptor pair is formed by avidin / streptavin, protein A / IgG, especially the F _c portion of IgG, lectin / oligosaccharide, polymer binding domain / biopolymer, anti hapten antibody / hapten or combinations thereof. The method of claim 9, wherein the detection of the detectable particles by spectroscopic methods, such as transmitted light, reflectometry, fluorometry, luminometry, refractive index and birefringence, by imaging methods, by electrical methods such as conductometry, potentiometry, or magnetic methods such as linear and non-linear susceptometry and relaxometry based on Giant Magnetic Resistors, Hall Sensors, Flux Gate Sensors, Squids or Induction Coils. Method according to at least one of the preceding claims, wherein the donor / receptor complex ( 1 ) is an immune complex.

Images