Method and device for the detection of very small quantities of particles

European).

8060/07

Method and device for detecting very small amounts of particles

The present invention provides a method and apparatus for detecting very small amounts of particles by detecting reaction products of antigen-antibodies in the femto- and attomolar range.

Various methods for detecting small amounts of particles are known in the art, for example nephelometric and turbimetric methods, as well as methods known as dynamic-light-scattering (DLS).

For nephelometric and turbimetric methods, the Tyndall effect is used, in which the lighting of a small particle releases scattered light from many angles. The scattering of light can be determined either by measuring the reduction in the intensity of the incident light beam after passing through the scattering medium, or by determining the intensity of the light deflected sideways. In the first case we are talking about the turbimetric method or extinction measurement, and in the second case the proper nephelometry or tyndalometry.

Another approach is implemented in methods that are referred to as dynamic light scattering (DLS). In these methods, only one (or few) points on the light sphere surrounding the particle are observed, and additionally the modulation of illumination caused by Brownian molecular movements is assessed. By collecting to a small observation space, attempts are made to reduce the disruptive overlap of scattered light of many particles. As a result, the particle runs very quickly through a small illuminated space so that the analyzing optoelectronics must recognize significant frequency fluctuations. During complex signal processing it is delivered

- a lot of data so that these systems can only conditionally be used for quantitative analysis.

Methods in which particle aggregation by antigen-antibody binding is used to measure the product being analyzed are known, for example, from US Patent Nos. 5534441 and US 5100805. Optical methods for detecting aggregates formed therefrom are also known from US Patent 5534441 and include dispersion light as well as measurement of transmission value.

There are also known methods that determine the size of a particle in solution by diffusion time in a small light cone (Eigen M. et al., "Sorting single molecules: Application to diagnostics and evolutionary biotechnology", Proceedings of the National Academy of Sciences of USA, National Academy of Science, Washington, USA, vol. 91, June 1994 (1994-06), pp. 5740-5747).

The state of the art detection methods also have other disadvantages. It should be noted that although the detection sensitivity using the disclosed methods in is increasing, there is still a need in various fields for detection methods with higher sensitivity. In addition, prior art detection methods still require relatively large samples, which, in particular in medical technology, may be additional to the test subject.

The object of the present invention is to provide a method for detecting small amounts of particles, which method would have a higher sensitivity than the prior art methods. In addition, such a method should require less sample dilution and / or a smaller minimum sample amount, should be suitable for larger-scale sample testing, and should ensure that employees with elementary training can perform without special experience. Furthermore, it is an object of the invention to provide a device for detecting small amounts of particles.

This task solves the method of detecting small amounts of particles by detecting antigen-antibody precipitation products in the femto- and attomolar range, in which way: a fluid sample is prepared that essentially contains particles of a given maximum particle size, wherein the particles exhibit at least two antibody binding sites; preparing a fluid containing antibodies that generally contains particles of a given maximum particle size; contacting the fluid sample with the antibody-containing fluid to form a reaction fluid, where antibodies with the presence of a particle with at least two antibody binding sites can form an antigen-antibody precipitation product; a ray of light is directed through the reaction fluid; the signal is detected by means of a photodetector by measuring extinction at the light-dark border of the light cone, which is formed when the light generated by the laser passes through a measuring chamber containing the reaction fluid, the signal intensity depends on the size and number of precipitated antigen antibodies produced, and the number and size the particle is determined by measuring the passage time.

The invention furthermore provides a kit for the qualitative and / or quantitative detection of a specific, detectable particle, wherein the specific particle has at least two antibody binding sites, the kit comprising: at least one antibody that can specifically bind to the specific particle, and at least one suitable fluid for receiving the sample, and a device for detecting small amounts of particles, which includes: laser, measuring chamber and photo detector, which is suitable for extinction measurement

- 4 at the light-dark border of the light cone, which is formed when the light generated by the laser passes through a measuring chamber containing particles in the liquid, the photodetector is located relative to the laser so that the laser beam passes near the photodetector.

Short description of the drawings

Figures 1a-d schematically illustrate processes occurring during the formation of antigen-antibody precipitation products using a bivalent antibody.

Fig. 2a shows the result of the method of the invention, testing the particle-containing sample fluid after filtering through a 200 nm filter (before adding the antibody). The signals correspond to particles whose diameter is smaller than the filter size used. The x-axis represents the particle size and the y-axis represents the number of particles.

Fig. 2b shows the result of a procedure in which the reaction mixture is obtained, which is obtained after the simultaneous separate injection of filter solutions with a pore size of 200 nm of sample fluid and fluid containing antibodies. In this reaction mixture, a reaction occurs and micro precipitations are formed, which have a diameter larger than the filter pore size used. The x-axis represents the particle size and the y-axis represents the number of particles.

Fig. 3 shows a schematic drawing of the device for carrying out the method according to the invention.

During numerous attempts directed at the present invention, it has been possible to find a method whose sensitivity exceeds 1000 times the sensitivity of comparable methods from the prior art. This significant increase in sensitivity results from a modified physical procedure in which the signal is captured by means of a photo detector by measuring extinction at the light-dark border of the light cone, which is created when the light generated by the laser passes through the measuring chamber containing the reaction fluid in the set with the analytical preparation of the sample adapted to this.

Furthermore, the invention allows a 30 to 50-fold reduction in the volume of the measuring chamber. While the methods of the prior art use a volume of the measuring chamber of, for example, 1.6 ml, the method of the invention can use measuring chambers with a volume in the microliter range (e.g. 40 μΐ). This is a significant advantage because in order to obtain a homogeneous matrix, any sample that has, for example, the same transparency, viscosity, etc., must be diluted, and in the method of the invention compared to the methods of the prior art, the sample must be diluted to a much lesser degree .

In addition, it is possible with the method of the invention to reduce the minimum amount of sample required. While the prior art methods require more than 100 μΙ, in the method according to the invention several times smaller amounts of sample (for example 3.5 μΙ) are sufficient.

The term "particle" in this specification means any three-dimensional entity that has a different refractive index than the carrier environment.

The term "precipitation" in the present description describes a process in which during the reaction of soluble antigens with specific antibodies an antigen-antibody complex is formed which exhibits less solubility in the solvent used than the antigen used or the antibody used, which first leads to turbidity of the reaction mixture, and then for the deposition of this antigen-antibody complex.

The method of the invention allows the detection of small amounts of particles. For example, in the case of low molecular weight substances, i.e. substances with a molecular weight of less than 500 g / mol, using the method according to the invention, a limit of detection in the range of femto and atomic grams per liter can be obtained, whereas the current limit of detection was usually in the range of micro -, nano- or picograms per liter. For substances with a molecular weight in the range above 500 g / mol, the detection limit is higher, for example for substances with a molecular weight of 150,000 g / mol (e.g. IgG antibodies) the detection limit is about 300 femtograms / liter. This means that the sample fluid may contain particles in the order of femto- or atto-moles per liter.

In the first step of the process of the invention, a sample fluid is prepared which contains particles of a given maximum particle size. This can be achieved, for example, in two ways. According to the first variant, a fluid is first prepared which essentially contains only particles of a given maximum particle size, and then a sample is introduced into the fluid that essentially contains particles of a given maximum particle size. According to the second variant, a fluid sample is obtained by first supplying the fluid and then introducing the sample into the fluid and further separating the particles that exceed a certain particle size.

The maximum particle size in the sample liquid or other fluids that generally only contain particles with a particular maximum particle size can be selected depending on the desired application. When using many known antibodies

Particles that are larger than 20-450 nm, preferably larger than 100-300 nm, especially larger than 200 nm can be separated. Such separation can be carried out, for example, by means of filters with an appropriate, appropriate pore size of 20-450 nm, preferably 100-300 nm, especially 200 nm, or by other methods known to those skilled in the art. If agglutination is considered approximately a plane, then dividing the filter in half by the size affects the number of molecules that provide detectable reaction products in a square proportion. For example, if a 100 nm filter is used instead of a 200 nm filter, only about a quarter of the antigen-antibody molecules needed for the 200 nm filter must be reacted and must react with each other to produce a detectable result. In addition, when using a 25 nm filter, it is possible, for example, to detect antigen-antibody-antigen trimers. When using filters with such a small pore size, however, attention should be paid to the exact way of working, because very few particles lead to a detectable reaction.

In order for the cross-linking reaction to take place, the particles contained in the sample liquid must contain at least two antibody binding sites and can therefore act as an antigen. Figures 1a to 1d show schematically that substances or particles foreign to the body, such as bacteria, viruses, toxins, proteins, play the role of antigens and react with the antibody according to the "key-lock" rule (Fig. 1b). A bivalent antibody, such as, for example, an IgG antibody, binds to two antigens (Fig. 1c). Since one antigen can bind several antibodies, crosslinking (precipitation) occurs which is detected in the method of the invention (Fig. 1d). In the case of antibodies with a higher binding capacity, precipitation occurs in an analogous manner. If the antigen used has a higher number of antibody binding sites, it is preferred that the cross-linking reaction may be more reproducible.

The method of the invention is applicable to any antigen as long as it contains at least two antibody binding sites. Preferably, the tested particles should be larger than about 10 nanometers and at the same time should be smaller than the selected maximum particle size. Particles smaller than 10 nm can act as haptens. Haptens are incomplete antigens, i.e. their particle size is insufficient to elicit an immune response or agglutination. Although these low molecular weight substances are highly specific at the paratopic antibody binding site, they do not protrude far enough from the binding site for the second antibody to bind to it. They block a specific antibody, preventing cross-linking. Larger antigens, such as bacteria, must be chemically or physically destroyed before measurement, which can be obtained by various methods known to those skilled in the art, such as by treating with ultrasound, acids, bases, and surfactants. This has the advantage of getting dozens of debris from just one bacterium, and it is not necessary to agglutinate individual bacteria. The fragments thus obtained, for example surface proteins , are again smaller antigens and can, with a suitable antibody, lead to a measurable reaction.

In addition, the antigen should be substantially soluble in the buffer used and should show only a slight tendency to adsorb on the walls of the devices and filters used.

The method of the invention further comprises producing a fluid containing the antibodies.

In principle, any desired antibodies may be used in the method of the invention. Antibodies that have proved to be particularly advantageous for use in the method of the invention are, for example, divalent immunoglobulin G (IgG) or decavalent immunoglobulin M (IgM). According to a method known to those skilled in the art, antibodies with specific specificities can be obtained. In addition, depending on the type of particles detected, other antibodies that belong to a different class of antibodies may also be used. Antibodies may be monoclonal or polyclonal antibodies. When using monoclonal antibodies, two monoclonal antibodies directed to different antigens can be used to obtain one precipitation. As stated above with respect to the antigen, the antibody should be substantially soluble in the buffer used and should have a slight tendency to adsorb onto the walls of the devices and filters used.

In the process of the invention, undesired, excessively high antigen or antibody excess can be avoided in a variety of ways, for example by carrying out an appropriate series of dilutions. Such too strong excess of antigen or antibody could otherwise lead to inhibition of precipitation (to the so-called "prozone effect"), because with a strong excess of antibody each epitope (antibody binding site) binds only one single antibody monovalently and cross-linking is no longer possible, or because trimmers consisting of one molecule of an antibody and two molecules of antigen are formed in large amounts with a strong excess of antigen.

As a fluid, both for the production of a sample of the fluid and for the production of the fluid containing the antibodies, essentially any gas or liquid may be used. Preferably the fluid is liquid. Often the liquid is water or known buffer solutions such as PBS (phosphate buffered saline), especially when analytical methods are based on a biochemical reaction. In principle, other transparent liquids can also be considered for liquids, for example liquid hydrocarbons, acids or bases.

In the method of the invention, in a next step, the sample fluid is contacted with the fluid containing the antibody, wherein the antibody in the presence of the antigen can form an antigen-antibody precipitation product, which can now be determined, for example, with the device according to the invention.

In addition, however, it is interesting to note the presence of haptens in the sample. Haptens are incomplete antigens, that is, they are too small to bind more than one antibody. In particular, haptens may be pharmaceutical substances, drugs, pesticides, environmental poisons, steroid hormones or mycotoxins. Haptens bind specifically to antibodies. However, they only block the paratopic antibody binding site, and the chain reaction cannot occur. The smallest size of immunogens (complete antigens) is 5 to 10 kDa, i.e.> 30 amino acid residues, or length> 3 nm, since from this size at least two antibody molecules can conjugate with these antigens with appropriately occurring epitope binding sites and can trigger chain reaction, which often leads to precipitation.

Determination of haptens can be carried out, for example, with hydrophilic macromolecular multispaces (hmM) or with related compounds known to those skilled in the art. Hydrophilic macromolecular multispaces are known in the art and include a hydrophilic macromolecule such as, for example, albumin. The same hapten molecules or different hapten molecules are chemically coupled to this hydrophilic macromolecule together with the spacers known in the art. If the hydrophilic macromolecular multispacer contains at least two identical hapten molecules, it can be used for precipitation. Such a molecule can be used for antibody detection tests based on substitution reactions. In the case of haptens, therefore, there is a measurement principle based on the substitution reaction, described below, for example, where only the negative peaks show reaction peaks, i.e. only the negative samples detect reaction products.

If a blood or saliva sample is to be tested for hapten, e.g. cocaine, an anti-cocaine antibody is added to the diluted droplet of blood or saliva. After one minute, a small amount of the synthetically obtained cocaine-hmM substance, i.e. a hydrophilic macromolecule, is coupled with the cocaine molecules.

If cocaine is present in the sample, it acts like hapten and blocks antibody binding sites. The next addition of the substance cocainahmM has no effect. However, when there was no cocaine in the sample, the antibody binding sites are not blocked and the addition of cocaine-MHM leads to chain formation, which as the growth of the molecule can be measured by the method of the invention, for example using the Q-MAP device described below.

In the method of the invention, a ray of light, especially coherent light, is passed through the test liquid, such as for example

- 12 laser beam. The method according to the invention is described below with regard to the laser light beam, but this does not exclude that other suitable sources of light known to those skilled in the art can also be used in the method according to the invention. For example, the laser beam may come from the device described in detail below which is also referred to as the Q-MAP (quantitative measurement of attomolar precipitation-products) device. However, other devices may also be used, based on the solution of the present description, which may be developed by those skilled in the art, based on the knowledge of the present invention.

The laser sends a ray through the liquid under test and is used to determine the number of particles there and their size. This is done by measuring extinction at the light-dark border of the light cone, which is formed when passing through a laser generated mesh by a measuring chamber containing the reaction fluid, using a photodetector (where the photodetector can be equipped with adjustable signal amplification and an adjustable working point), that the device acts as a "molecular light barrier". The Q-MAP device, as described below, can for example determine particle sizes between 20 nm and 5 μm, however, based on this measurement, the structure or composition of the measured particles cannot be inferred. The laser and photodetector are located approximately coaxially. The laser beam passes very close to the photodetector. The previously diffused light forms a cone at whose brightness-darkness the photodetector sets.

Each particle that is in the liquid in the measuring chamber and hits the laser beam produces a signal, with the number of signals

- 13 corresponds to the statistical distribution of particles in the measuring chamber. When the particles, also the smallest particles, wander through the light cone in the laser focus, they create an obstacle to light as if casting a shadow. The change in original brightness (without the shadow of particles) is measured. A fast computer, such as a Pentium computer, can distinguish up to 10,000 particle transitions per second. Small particles move faster than large ones, so the transit time is a direct measure of the particle size.

If a series of particles are simultaneously in the laser beam, only the largest is measured. Based on the different velocities at which the particles pass through the laser beam, the particle size can be determined using the Stokes-Einstein equation known to those skilled in the art. In this measurement, the absolute particle size is less important than the change in particle size (caused by the increase in the particle). The particular filter used serves as a measure for determining the onset of molecule growth. The larger the filter pores, the larger the deposits must be to distinguish themselves from "background noise". For 100-200 nm filters, very few particles are enough to obtain a measurable signal. Because the particles are distributed more statistically, also in the laser focus, which is inside the measuring chamber, there is always a growth of particles.

The speed at which the particles pass through the laser beam is determined by measuring the time at which the molecule passes through the beam (from the beginning of the brightness change to its end).

It can be seen here that the greater the purity of the solution, the more likely it is that the particles occur individually in the laser beam, while the more contaminated the solution, the more signals overlap, which leads to a decrease in sensitivity.

The signal strength depends on the size and number of antigen-antibody products precipitated.

At constant antibody concentration, the loss of the measurement signal is directly related to the antigen concentration.

According to the invention, for example, one procedure may be as follows.

All tested fluids are injected into the measuring chamber through a filter with a specific pore size, so that during one test of sample fluid or fluid containing antibodies, only one signal is present at a single determination which shows particles smaller than the specified particle size. In the method of the invention, both the antibodies used and the antigens used have a size below a certain particle size limit, so they are smaller than the pore size of the filter used for separation (e.g., less than 200 nm), so that they are not removed during separation.

An example of the image recorded during the analysis of the sample fluid based on NaCl solution is shown in Fig. 2a. The images show the particle size along the x axis and the number of particles along the y axis. As can be seen from Fig. 2a, only particles are present up to a certain maximum particle size, the smaller the number of particles, the larger the particle size. An analogous result is obtained for a filtered fluid containing antibodies (not shown), i.e. containing essentially only particles of a given particle size.

After simultaneous, separate injection of sample fluid or fluid containing antibodies in the measuring chamber, micro-precipitation occurs and detection of the size and number of antigen-antibody products formed.

Such a measurement result is shown, for example, in Fig. 2b, with the presence of particles that are larger than a defined particle size limit of, for example, 200 nm is shown. The formation of larger particles therefore indicates that a reaction has occurred. On the other hand, if there is no such reaction, it means that the tested liquid lacks the appropriate antigen.

Measurements can be started at any time after the reaction chamber has been filled with sample fluid and fluid containing antibodies. According to one embodiment of the invention, measurements only start, for example, 60 seconds after filling the measuring chamber, as slight variations in injection speed can lead to convective differences in the measuring chamber. After a further 60 seconds, for example, a maximum precipitation is reached. The subsequent reduction of the measurement signal enables an approximate quantitative determination or estimation of different antigen concentrations. The initial concentration of both reaction components plays a decisive role here. The higher these starting concentrations are, the longer the time until the measurable reduction in precipitation maximum is.

Using the method according to the invention, it is preferable to use fluids of the highest purity possible, previously filtered through a filter with a suitable pore size, e.g. 200 nm, to eliminate or minimize background noise before measurement. Furthermore, the materials of the devices used in the method according to the invention should give off as few particles as possible. For example, the measuring chamber can be made of PTFE (polytetrafluoroethylene). To prevent prolonged particle release from the materials used (e.g. measuring chamber), it is advisable to keep the measurement time as short as possible.

In addition, the concentrations of the antigen or antibody solutions used should be so low that the antigen does not cause too much precipitation, or that the precipitation is not too high, because then many molecules can be in radius at the same time and the transition time is difficult determination. The highest sensitivity of the method according to the invention is in the femto- and atto-molar range.

Particularly preferably, the method according to the invention allows quantitative or semi-quantitative determinations.

For example, two methods can be used to quantify. For the first method of determination, the fields (F) under the measurement curves are determined at time point t1 and t2 (e.g. at time point t1 = 120 seconds and t2 = 180 seconds), and from the formula

FN = (Ft1 + Ft2): 2 a good approximate value of FN is obtained for the field, from which it is then possible to obtain a quantitative value as to the number of particles. At these extremely low concentrations, particle growth is linear, i.e., for example, doubling the concentration leads to a double measurement signal field.

An alternative and / or complementary method for obtaining quantitative determination is that the solutions are diluted until the kinetic limit of the two-molecular reaction is reached. At concentrations below 100 attomolar the average free paths of the reactant particles are too large (> 100 μm) and no measurable precipitation occurs in the given measurement time. At concentrations above 10 nanomolar, a steric hindrance begins to occur due to too many and too large reaction products, and the "prosonic effect" explained above occurs. For example, in a 10 micromolar solution, the free path length of a particle is only 100 nm.

The specific degree of dilution at this physical endpoint is now only dependent on temperature and convection, since viscosity at high dilutions e.g. in PBS (phosphate buffered saline) becomes irrelevant. If these two parameters are kept constant, the dilution of the stock solutions is a direct measure of the concentration of the solutions tested.

Accurate quantification of the sample concentration is possible by the method of the invention using a comparison solution with a known concentration of a given protein, using procedures known to those skilled in the art.

According to a further implementation of the method, continuous measurements and / or samples of a fluid randomly selected in the measuring chamber may be carried out. In particular, it is possible to set the end point of the reaction and to create mean values ​​of the determinations. Preferably, the measurement is essentially free of particles, with a particle size above a certain value, a carrier fluid before the addition of the antibody and / or before the addition of the sample and the result of this measurement is taken as the reference value for measuring precipitation.

The method according to the invention uses relatively small amounts of fluid, whereby the effort put into removing from the fluid used substances whose particle size exceeds a given value can be reduced. For the removal of such substances, the fluid used can, for example, also be passed through a valve assembly through a bypass element with a filter that filters out the said substances. Filtering of the fluid used can be carried out for a predetermined period of time before sample introduction to ensure that

- 18 there are no more interfering substances. Substances whose particle size exceeds a certain value can be removed from the sample, in particular by means of filters that are integrated in or positioned in front of them. Also, after the sample has been introduced, the fluid sample thus obtained may be filtered over a period of time to remove interfering substances that may have entered the fluid with the sample or from the delivery system.

According to a further embodiment of the method of the invention, the carrier fluid after the addition of the antibody, which led to or did not detect the antigen, is again filtered and then another antibody is added. In this way, substances that could interfere with the measurement can be removed in the meantime. In addition, it is possible to remove the detected reaction product from the fluid used to be able to analyze any further sample components.

The method of the invention is suitable for a variety of applications in the field of water analytics (detection of ingredients and harmful substances), food technology (detection of microorganisms, harmful ingredients), or in the case of biological or medical tests (e.g. detection of specific DNA or RNA sequences, bacteria or allergens (allergy tests)). In addition, it is possible to use in the field of hydrophilic macromolecular multispaces, DNA branching probes or quantitative PCR. In particular, the method of the invention may also be used to detect infectious PrP Prion proteins^sc in BSE tests. Since extremely small amounts of these prion proteins can be detected in the method of the invention, the method of the invention is also suitable for detecting infectious PrP prion proteins^sc in blood.

In addition, the Q-MAP measurement method according to the invention provides a quick and simple possibility to test the adsorptive behavior of various proteins on various surfaces. Highly polished surfaces can be tested free of damage for "protein adsorption values", with deviations from the setpoint indicating a surface defect. In this way, for example, vaporized thin metallic surfaces can easily and quickly detect surface defects.

In addition, it is also possible to study the completeness of immobilization of specific proteins on the surfaces tested, which is for example of great importance in the field of DNA analytics and biochip technology.

Another possibility of using the method according to the invention is that the effect of chemicals on the surface can be determined. In particular, for example, prior / follow-up tests for detecting changes due to surface treatment are considered, since the etched plane has a larger surface area than the non-etched one, and therefore a larger amount of antigenic material, e.g. proteins, can be adsorbed on such surface. The method can, for example, determine the amount of proteins that remain free in solution.

Yet another application option is that the condition of the inner surfaces of hoses and pipes can be checked by the method of the invention.

After completing the procedure of the invention, the antigen-antibody deposits can be dissolved, for example, with protease K for substances that are not degradable by protease K (e.g., prions and substances without amino acids) and subjected to further processing or testing.

Another important advantage of the method according to the invention is, as outlined above, the possibility of quantification using the method. In addition, this method can be fully automated, which leads to clear savings in labor input, so that the method provided is comparatively economically advantageous.

In addition, the method of the invention allows the detection of very small amounts of disease pathogens in fluids or secretions from the body, especially in the blood. This is a very important advantage, because small amounts of blood, for example a small droplet of blood (about 10-20 μΐ), can be taken from the fingertip or earlobe using a capillary tube, without the help of a doctor or a trained nurse. For example, such tests can easily be carried out in a pharmacy or in an old people's home or in rehabilitation rooms, and they provide simple and quick information about whether there is an infection with a particular bacterium. It is particularly advantageous that both sampling and carrying out the method according to the invention using the device according to the invention do not require medically trained or highly qualified personnel. Of course, other substances, such as drugs or medications, can be determined in the blood using these tests and with the use of specific antibodies.

While in the case of commercially available measuring devices, the ratio of antigen to antibody must not exceed a factor of 2-3, using Q-MAP measurements are possible even when the ratio of antigen and antibody deviates from the ideal mixing ratio by a factor of 100. (With a constant the level of antibody concentration, the ideal concentration of antigen decreases to 1% and increases to 10,000%. The same, vice versa, also applies to situations with a constant level of antigen). This interesting deviation from the "Heidelberg-21 ger curve" in the femto- and attomolar range, which is caused by smaller steric obstacles to the molecules, makes time-consuming serial dilutions unnecessary.

The method according to the invention is particularly suitable for testing collected samples, which is of particular interest for testing conserved blood (e.g. for HIV or hepatitis) and for BSE testing. About 50 measurements per hour can be carried out with the method according to the invention, which means that at least 400 tests can be carried out in 8-10 hours a day, over 80,000 measurements per year. Thus, with 10-100 collected samples per measurement, 800,000-800,00000 samples can be tested per year with just one measuring instrument.

For example, you can add 10 different antibodies (10 different diseases) to your blood at once in a conserved blood test to see if the conserved blood is suitable. In a positive reaction, conserved blood should be rejected because it does not matter if it is infected, for example, by HIV or by hepatitis pathogens. However, if it is of interest as to which pathogen triggers a positive reaction, antibodies can be added individually. For example, if 100 conserved blood samples are collected and 10 different antibodies are simultaneously used, then with just one measurement that lasts about 60 seconds, you can determine if all 100 samples are usable.

A test in which such a small amount of product is sufficient and which has a sufficiently high sensitivity up to the femto or attomolar range is not known in the prior art and represents an important advance in the art.

In addition, the invention includes a computer program product that includes program code stored on a computer readable medium to enable the method of the invention to be carried out when the computer program product is running on a computer, network device or device, in particular a detection analytical device . Furthermore, the invention also relates to a computer program product that includes program code loaded from a server to enable the method of the invention to be performed when a computer program product is run on a computer, network device or device, in particular a detection analytical device (e.g., one of the devices for designation presented in this description).

The invention further includes a device for detecting small amounts of particles, which is also referred to as a Q-MAP (quantitative measurement of attomolar precipitation products) device.

The device includes a light source, a laser being preferably used as the light source.

The measuring chamber of such a device should be of a material that essentially does not release any particles and which allows the passage of a light beam, especially a laser beam. Such materials are known to those skilled in the art. The measuring chamber can for example be made of PTFE (polytetrafluoroethylene). The measuring chamber has a capacity of less than 100 μΙ, preferably 30-50 μΙ, especially 40 μΙ.

The device according to the invention comprises in particular a photodetector equipped with adjustable signal amplification and an adjustable working point.

The photodetector may for example be selected from the group consisting of thermal detectors, photodiodes, in particular photoconductive detectors, photovoltaic detectors, avalanche diodes, diode systems, photomultipliers.

In particular, the detection device according to the invention enables the determination of particles between 20 nm and 5 μm.

In addition, the present invention provides a kit for the qualitative and / or quantitative detection of specific assayed particles, for example proteins or hormones, wherein the provided particles have at least two antibody binding sites. The kit includes a detection device as described above, at least one antibody that can specifically bind to a particular particle, and at least one suitable fluid for sampling. Such a kit can be adapted specifically to the user's requirements and contains successively arranged ingredients and an appropriate description for the user, who with this kit can immediately and very easily carry out the determinations according to the invention. For example, such a kit can be used to determine a particular disease pathogen in a small amount of blood, or for the above-described surface test applications.

Detailed description of Fig. 3

The invention is explained below with reference to the attached exemplary schematic drawing of the system for carrying out the method according to the invention, which drawing does not limit the scope of the invention.

Sample changer 1 can introduce both different antigen solutions (e.g. blood samples) and different antibody solutions into the respective mixers 2 and 3 to test the sample in turn for different possible pathogens. Mixers 2 and 3 for antigen solutions

24 and the antibodies can serve not only to dilute each solution, but also to flush the measuring chamber with buffer (PBS) or antibody solution.

Filters 4 are replaceable and have, for example, a pore size of 200 nm. The valve 5 can be switched so that the solutions can flow individually or together into the measuring chamber 6, where the determination is carried out continuously, singly or at random.

Pump 7 is e.g. a small vacuum pump that sucks all solutions. It is combined with valve 5 and after the measurement it pumps the contents of the measuring chamber into a waste container

8. This container can, for example, be provided with a disinfecting liquid or killing microorganisms to immediately dispose of all possible hazardous substances.

In the event that the introduced antibody reacts with the antigen from the sample to form the antigen-antibody precipitate, particles are formed that exceed a certain value. These precipitates generate a signal that is clearly different from the signals of the particles possibly still present in the carrier fluid, with a particle size below a certain value. Therefore, these reaction products are clearly detected by the assay device. This determination indicates that the substance in question is contained in the sample and, if appropriate, the composition can also be determined according to the methods described above.

If, after the injection of the first antibody solution, the assay device does not show the presence of particles larger than the specified particle size, or if no further antigens are specifically predicted to react with the antibody used, then another antibody solution may be introduced. Before the next test, if necessary, any previously detected deposits should be flushed out of the measuring chamber.

The following examples serve to explain the present invention. However, the scope of the invention is not limited to the subject matter of these examples.

Examples

Example 1. Blood testing for BSE

The cattle population in Germany is around 15 million. Annually, 2-3 million BSE tests (ELISA and Western blot) are carried out in Germany on the brains of dead animals. The BSE rapid test (ELISA or Western blot) currently lasts 6-8 hours. By the method of the invention BSE pathogens can be detected on a live animal using only one drop of blood in 2 minutes, the costs are only a fraction of the previous costs.

Example 2. Examination of conserved blood for a new variant of Creutzfeld-Jakob disease (nvCJD)

To protect the public against the possible risk of blood transfer of nvCJD, various measures are used. The most promising step would be to test every donated blood for infection, as with hepatitis and AIDS. However, this pathogen occurs in patients only in very small quantities. There is no reliable test in the prior art. The method of the invention is able to detect these pathogens in the blood or in blood products.

Example 3. Applications in the food industry

Mycotoxins are highly toxic products derived from specific fungi. Mycotoxins are haptens, and therefore they can

- be detected qualitatively and quantitatively by the methods of measurement described above.

For screening tests, for example for whole transports of coffee, tea, flour or nuts, a qualitative test that can be carried out on site in about 2 minutes is sufficient.

To determine if an animal was illegally fed hormones, a meat sample must be tested for approximately 15 different hormones considered. Hormones are also haptens and can be tested qualitatively and quantitatively by the method of the invention. Hormone tests are also used, for example, in pregnancy tests and thyroid tests.

Since larger slaughterhouses process 2000-3000 cattle per month, about 50-100 random samples are needed to detect illegal fattening. The price for one hormone test (for 15 different hormones) is sometimes EUR 600 per piece of meat. Therefore, slaughterhouses only carry out 5-10 random tests per month, which may not be enough to detect illegal fattening. During talks with large slaughterhouses, interest was shown to carry out 10 times more tests that would be cost-effective (around 60-70 euros per piece of meat) based on the method of the invention. Typical hormonal tests can never reach that price.

Example 4. Detection of plant protection products

Tubulin monomers are single small protein spheres that, in reaction with the GTP energy vector, grow into long chains.

Inhibitors and other toxins impede or prevent this chain growth. Individual chains loop into rope-like structures, so-called protofilaments. These protofilaments, in turn, bind to

- 27 microtubules that play a decisive role in cell division. By preventing chain formation by monomers, you can affect cell division and kill pests.

Conversely, this method also allows the detection of plant protection product residues in foodstuffs, which provides increased consumer protection. The chain growth described above and the prevention of this growth can be directly measured by the method of the invention.