EP1073900A1

EP1073900A1 - Method for amplifying the formation of ligand-receptor complexes and uses

Info

Publication number: EP1073900A1
Application number: EP99914611A
Authority: EP
Inventors: Jacques Benveniste; Didier Guillonnet
Original assignee: Digibio SA
Current assignee: Digibio SA
Priority date: 1998-04-20
Filing date: 1999-04-19
Publication date: 2001-02-07
Also published as: FR2777656A1; AU3336299A; WO1999054731A1; FR2777656B1

Abstract

The invention concerns a method for amplifying the formation of complexes between the two elements of a ligand/receptor pair and its uses for detecting the presence of a substance corresponding to one of the two elements of a ligand/receptor pair in a sample and the electromagnetic signal characteristic of a substance biological activity corresponding to one of the two elements of a ligand/receptor pair in an electromagnetic signal. Said amplification method consists in: contacting the two elements of the ligand/receptor pair in conditions ensuring their reaction; prior to, simultaneously with or subsequent to said contacting, applying to one and/or the other of said elements an electromagnetic signal characteristic of the biological activity of one and/or the other of said elements. The invention is applicable to biological diagnosis in human and veterinary medicine, bacteriological control in the pharmaceutical, cosmetic and food industry.

Description

1

METHOD FOR AMPLIFYING THE FORMATION OF LIGAND-RECEPTOR COMPLEXES AND USES THEREOF

The present invention relates to a method for amplifying the formation of complexes between the two elements of a ligand / receptor pair, to a method and to an apparatus for detecting the presence, in a sample (hereinafter "sample"). analytical "), of a substance corresponding to one of the two elements of a ligand / receptor pair, implementing this amplification method, to the applications of this detection method, as well as to a detection method of the presence, in an electromagnetic signal, of the electromagnetic signal characteristic of the biological activity of a substance corresponding to one of the two elements of a ligand / receptor pair, also implementing said amplification method.

To detect the presence of a substance in an analytical sample, numerous methods have been proposed based on the ability of this substance to specifically bind to and react with another substance.

In particular, the affinity properties of antibodies towards antigens are the basis of a large number of immunological detection methods which have in common the use of the formation of antigen-antibody complexes - the substance sought could be either the antigen or the antibody - and detect, or even quantify, the complexes thus formed.

Examples of immunological detection methods which are very frequently used include immunoprecipitation, agglutination reactions, equilibrium dialysis, fluorescence quenching, fluorescence polarization, immunoelectrophoresis. , counter-immunoelectrophoresis or electrosyneresis, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA) or immunofluorescence.

These immunological detection methods, although they undoubtedly have great qualities, are not entirely satisfactory, however. 2

First, their sensitivity (which is defined by the minimum concentration of the desired substance that these methods detect) is, in most cases, insufficient. Thus, BERZOFSKY and BERKOWER (Antigen-Antibody Interaction, In: WE Paul, Fundamental Immunology, RAVEN PRESS, New York, 1984, 595) have shown that, as regards for example the detection of antibodies, with the exception of phage neutralization tests with which it is possible to detect the presence of a single antibody molecule but whose use is extremely limited, very few methods have a sensitivity of less than 10 ng of antibody per ml of sample . It is therefore desirable to develop new techniques which make it possible to lower the detection threshold for a desired substance.

Furthermore, all of the immunological detection methods proposed to date include a step which consists in incubating a determined volume - which is generally at least 500 μl - of the sample to be analyzed with a specific reagent, for each substance sought. As a result, they have the drawback of requiring, as soon as the analysis of a sample relates to several substances - as is very often the case with medical analyzes for diagnostic purposes -, a sample of relatively large volume, which is not always well supported by patients, especially in the case of blood samples.

In addition, the fact that these detection methods require, for their implementation, to have the sample to be analyzed or, at least, a sample thereof, is not without presenting a certain number of constraints. Indeed: - on the one hand, it is frequent that samples which have given rise to analyzes must be kept so that the reliability of these analyzes can be checked later or that additional analyzes can be carried out. Thus, for example, blood transfusion centers, forensic services and tissue sampling centers keep samples of all the biological samples that they are called upon to perform. This 3

conservation, which is carried out by freezing said samples, in addition to having a non-negligible cost, requires suitable equipment and premises.

- on the other hand, it is also frequent that samples cannot be analyzed at the place where they were taken and that it is necessary to transport them to the laboratory responsible for carrying out the analysis. However, the transportation of biological samples, in addition to being never very easy to carry out due to the short shelf life of biological substances in the absence of freezing, poses a certain number of difficulties when these samples are potentially contaminating. In addition, the duration of such a routing differs all the more from obtaining the results of the analysis.

The problem therefore arises of providing a method which makes it possible to detect the presence of a substance in a sample with both very high sensitivity and high specificity, while offering the possibility of performing as many analyzes that are necessary from micro-samples, to overcome, moreover, the constraints of conservation, shipping and transport of samples presented by the methods currently used for the detection of a substance, and which can, in addition , be implemented easily and quickly without requiring heavy and expensive equipment.

However, in the context of their work on the transmission of a biological activity in the form of an electromagnetic signal, the inventors have found that the application, to one and / or the other of the elements of a couple ligand / receptor such as an antigen / antibody pair, of the electromagnetic signal characteristic of the biological activity of one and / or the other of these elements has, quite surprisingly, the effect of amplifying the formation complexes between the two elements of this couple when the latter are reacted together and this, in a very specific way, and had the idea of using this effect to detect on the one hand, the presence of a substance in an analytical sample and, on the other hand, the presence of the electromagnetic signal characteristic of the biological activity of a substance in electromagnetic radiation. 4

The present invention therefore has for its object a method for amplifying the formation of complexes between the two elements of a ligand / receptor pair by reaction of these two elements, which method is characterized in that it comprises:

- bringing the two elements of the ligand / receptor couple into contact under conditions suitable for their reaction, and

- Before, simultaneously or after this contacting, the application to one and / or the other of these elements of the electromagnetic signal characteristic of the biological activity of one and / or the other of said elements.

For the purposes of the present invention, the term “ligand / receptor couple” means any couple formed by two substances capable of recognizing each other specifically, of binding and reacting together by forming complexes. Thus, it may be an antigen / antibody or hapten / antibody pair in which the ligand (the antigen or the hapten) may be a biological compound (protein, enzyme, hormone, toxin, tumor marker, etc.). .), a chemical compound (active drug ingredient for example), or a cellular or particulate antigen (cell, bacteria, virus, fungus, ...), the receptor can be a soluble antibody or a membrane receptor. It can also be a couple formed by an enzyme and its specific substrate.

Furthermore, the term “electromagnetic signal characteristic of the biological activity” of an element means the electromagnetic signal picked up from a biologically active element such as a substance, a cell or a microorganism, etc. , or a material containing this element such as a purified preparation, a biological sample, an organ or a living being, as has been described in International Application WO 94/17406 in the name of J. BENVENISTE. The term “electromagnetic signal characteristic of the biological activity” of an element also means the signals derived from a signal as defined above by digitization and / or signal processing. Furthermore, in this expression, the term "characteristic" is used in the sense that the electromagnetic signal picked up contains information characterizing the fact that the material from which this signal is picked up exhibits the biological activity in question. The electromagnetic signal picked up from 5

a material containing a plurality of biologically active elements presents the biological activity of each of the elements which it contains.

According to a first preferred embodiment of the amplification method in accordance with the invention, the reaction between the ligand and the receptor is carried out using two reagents containing the ligand and the receptor respectively, and one is applied to one and / or the other of these reagents, an electromagnetic signal to be tested and suspected of comprising the electromagnetic signal characteristic of the biological activity of this ligand and / or of this receptor.

In the foregoing and in the following, the term “reagent” denotes any preparation whose composition is known, which contains the ligand or receptor in an amount also known and which is present either in a dry form such that a lyophilisate to be reconstituted in a solvent, either in a liquid form such as a solution or a suspension, the ligand or the receptor being able to be fixed on a solid phase (particles or beads of latex, glass or polystyrene, ... According to a first advantageous arrangement of this first embodiment, the application, to one and / or the other of the reagents, of the electromagnetic signal to be tested is carried out by exposure of a solution or of a suspension containing one and / or the other of these reagents, to this electromagnetic signal.

As a variant, the application, to one or the other of the reagents, of the electromagnetic signal to be tested is carried out by dilution of a solution or of a suspension comprising one and / or the other of these reagents , in a solvent having been previously exposed to this electromagnetic signal.

Thus, for example, when the reagents which one wishes to use are in solution or in suspension in a liquid phase, it is possible to apply to them the electromagnetic signal to be tested:

* either prior to their use:

By exposing one and / or the other of these reagents or aliquots of one and / or the other of these reagents to this electromagnetic signal, or

By diluting one and / or the other of said reagents or their aliquots in a volume of a solvent having been previously exposed to said electromagnetic signal, 6

* either during the implementation of the amplification process in accordance with the invention:

By exposing to this electromagnetic signal an aliquot of each of these reagents, after depositing these aliquots on a support (slide for example) but before bringing them into contact, or

• by mixing an aliquot of the first reagent with an aliquot of the second reagent on a support or in a tube, and by exposing this mixture to the electromagnetic signal, or else

• by mixing an aliquot of the first reagent with an aliquot of the second reagent on a support or in a tube and diluting this mixture in a volume of a solvent having been previously exposed to said electromagnetic signal.

According to another advantageous arrangement of this first embodiment, the application, to one and / or the other of the reagents, of the electromagnetic signal to be tested is carried out by dissolution or suspension of this or these reagents in a solvent having been previously exposed to this electromagnetic signal. This arrangement is of particular interest when the reagents which it is desired to use are in a dehydrated form such as a lyophilisate, since it is then possible to apply the electromagnetic signal to be tested to them simply by dissolving them or by suspending as appropriate, in a volume of a solvent having been previously exposed to this electromagnetic signal.

Advantageously, the electromagnetic signal to be tested is an electromagnetic signal picked up from a sample to be analyzed and suspected of containing this ligand and / or this receptor, this sample being able as well to come from a biological sample (blood, urine, milk, ...) than a non-biological sample (water, food product, pharmaceutical product, cosmetic product, ...).

As a variant, the electromagnetic signal to be tested can also be an electromagnetic signal radiated by a source of electromagnetic radiation, in particular a source suspected of emitting harmful radiation for living beings of the high-voltage line, transformer, electric motor, microwave oven type. waves, particle accelerator, X-ray source, ... Similarly, the 7

electromagnetic signal to be tested can come from the acquisition of a mechanical signal like vibrations, an electrostatic signal or other.

According to a second preferred embodiment of the amplification method in accordance with the invention, the reaction between the ligand and the receptor is carried out by bringing into contact a sample to be analyzed and suspected of containing the ligand and / or the receptor, with a reagent containing either the receptor or the ligand (depending on the substance suspected to be present in the analytical sample with which it is desired to react this reagent), and the characteristic electromagnetic signal is applied to this sample and / or to this reagent of the biological activity of said ligand and / or of said receptor.

According to a first advantageous arrangement of this second embodiment, the application, to the sample to be analyzed, of the electromagnetic signal characteristic of the biological activity of the ligand and / or of the receptor is carried out by exposure of this sample to this or these electromagnetic signals, or by diluting this sample in a solvent having been previously exposed to the said electromagnetic signal (s).

According to another advantageous arrangement of this second embodiment, the application, to the reagent intended to react with the sample to be analyzed, of the electromagnetic signal characteristic of the biological activity of the ligand and / or of the receptor is carried out by exposure of a solution or suspension containing this reagent to this or these electromagnetic signals, or by dilution of such a solution or suspension in a solvent having been previously exposed to this or these electromagnetic signals, or also by dissolution or suspension of this reagent in a solvent having been previously exposed to said electromagnetic signal (s).

As a variant, the electromagnetic signal characteristic of the biological activity of the ligand and / or the receptor is applied to the sample to be analyzed and to the reagent intended to react with it, by exposure of a solution or a suspension containing this sample and this reagent to this or these electro- 8

magnetic, or by dilution of such a solution or suspension in a solvent having been previously exposed to the said electromagnetic signal (s).

According to a particularly preferred arrangement of this second embodiment, there is applied to the sample to be analyzed and / or to the reagent intended to react with it, both the electromagnetic signal characteristic of the biological activity of the ligand and the electromagnetic signal characteristic of the biological activity of the receptor. Indeed, the inventors have found that, if it suffices to apply, to the elements of the ligand / receptor couple, the electromagnetic signal characteristic of the biological activity of only one of these elements to obtain an amplification of the complexes formed by their reaction, this amplification is higher when the electromagnetic signals characteristic of the biological activity of each of them are applied simultaneously to these elements.

Whatever the mode of implementation of the amplification process in accordance with the invention, the solvent having been exposed beforehand to the electromagnetic signal (s) is advantageously water or physiological solute.

The reagents capable of being used in the amplification process in accordance with the invention and containing the ligand on the one hand, and the receptor on the other hand, may also be ready-to-use reagents commercially available. as reagents specially designed and prepared for the implementation of this process. In addition to the fact that, as mentioned above, these reagents can be in different forms (dry, liquid, etc.), they can also be coupled to a marker such as a radioactive isotope, an enzyme, a fluorescent substance, a colored particle, biotin or an organometallic compound capable of detecting and / or measuring the ligand-receptor complexes resulting from the reaction between the ligand and the receptor.

The amplification method advantageously further comprises a step of acquiring the electromagnetic signal characteristic of the biological activity of one and / or the other of the elements of the ligand / receptor pair.

As previously indicated, the electromagnetic signal characteristic of the biological activity of one and / or the other of the elements of the couple 9

ligand / receptor can come either from a sample to be analyzed and suspected of containing this or these elements, or from a source of electromagnetic radiation or from the acquisition of a mechanical (vibration), electrostatic or other signal, or even from reagents containing the ligand or the receptor in solution or in suspension in a solvent, according to the embodiments of the amplification process in accordance with the invention.

In a particularly advantageous manner, the amplification method in accordance with the invention also comprises a step of recording and restoring information representative of the electromagnetic signal characteristic of the biological activity of one and / or the other elements of the ligand / receptor couple. Thus, the electromagnetic signal characteristic of the biological activity of an analytical sample, once recorded, can be kept indefinitely and used as many times as necessary. Similarly, the electromagnetic signals characteristic of the biological activity of the ligand and of the biological activity of the receptor, picked up from reagents, can be recorded once and for all and be used to carry out a plurality of reactions involving this ligand. and this receiver.

The amplification method advantageously further comprises a step of detecting the complexes resulting from the reaction between the ligand and the receptor and, optionally, of measuring these complexes. This step can, advantageously, be completed by the comparison of the results obtained with those observed for a reaction serving as a "control", that is to say a reaction carried out with the same ligand / receptor pair and under the same reaction conditions, but without application of an electromagnetic signal to the elements of this couple, whether it is before, simultaneously or after they are brought into contact.

The detection and / or measurement of ligand-receptor complexes can be carried out by all the methods conventionally used to reveal and quantify the formation of such complexes. Thus, in the case of antigen-antibody complexes, it is possible to use both a revelation by agglutination, by immunoprecipitation, by fluorescence quenching, by 10

fluorescence polarization than a radioimmunoassay, immunoenzymatic test or an immunofluorescence test.

According to a particularly preferred embodiment of the amplification method in accordance with the invention, the ligand is an antigen or a hapten, while the receptor is an antibody or a membrane receptor directed specifically against this ligand.

In a particularly advantageous manner, the reaction between this ligand and this receptor is a reaction revealed by agglutination, because of its simplicity and of its speed of execution. The present invention also relates to a method for detecting the presence of a substance corresponding to one of the two elements of a ligand / receptor pair in an analytical sample, characterized in that it comprises the implementation work of an amplification process as defined above.

According to a first particularly preferred embodiment of this detection method, it comprises:

contacting two reagents containing the ligand and the receptor respectively, under conditions suitable for allowing their reaction,

- before, simultaneously or after this contacting, the application, to one and / or the other of these reagents, of the electromagnetic signal characteristic of the biological activity of the analytical sample, and

- detecting and / or measuring the ligand-receptor complexes formed during the reaction between the two reagents.

Thus, obtaining an amplification of the formation of ligand-receptor complexes between the two reagents compared to a "control" reaction (as previously defined) reflects the presence, in the electromagnetic signal, of the biological activity of the 'sample to be analyzed, of the electromagnetic signal characteristic of the biological activity of the sought substance and, consequently, translates the presence, in this sample, of the sought substance.

In the case where such an amplification is obtained and where the analytical sample is likely to contain not only one of the two elements of the couple 11

ligand / receptor, but these two elements, the presence, in this sample, of the desired substance can be confirmed by comparing the results obtained with:

either those observed for a reaction carried out under the same reaction conditions but with application of both the electromagnetic signal characteristic of the biological activity of the sample to be analyzed and the electromagnetic signal characteristic of the biological activity of the ligand,

- or those observed for a reaction carried out under the same reaction conditions but with application of both the electromagnetic signal characteristic of the biological activity of the sample to be analyzed and the signal characteristic of the biological activity of the receptor.

Thus, if the simultaneous application of the electromagnetic signal characteristic of the biological activity of the analytical sample and the electromagnetic signal characteristic of the biological activity of the ligand results in an amplification of the formation of the ligand-receptor complexes with respect to the application of the only electromagnetic signal characteristic of the biological activity of said analytical sample, then this means that this sample does not contain a ligand and therefore only contains the receptor. The absence of an increase in the formation of ligand-receptor complexes, which sign the presence of the ligand in the analytical sample.

Similarly, if the simultaneous application of the electromagnetic signal characteristic of the biological activity of the analytical sample and the electromagnetic signal characteristic of the biological activity of the receptor results in an amplification of the formation of the ligand-receptor complexes relative the application of the only electromagnetic signal characteristic of the biological activity of said analytical sample, then it can be deduced therefrom that this sample does not contain a receptor and therefore only contains the ligand. The absence of an increase in the formation of ligand-receptor complexes, which sign the presence of the receptor in the sample. 12

In order to avoid obtaining false negative results, that is to say results which would not make it possible to reveal an amplification effect of the application of the electromagnetic signal characteristic of the activity of the analytical sample and this, although the latter actually contains the desired substance, the concentrations of the ligand and of the receptor reacted are advantageously chosen so as to be sufficient to lead to the production of ligand-receptor complexes detectable in the absence of the application of the electromagnetic signal characteristic of the biological activity of said sample, but lower than the concentrations likely to lead to saturation of the reaction between this ligand and this receptor. According to a second preferred embodiment of this detection method, it comprises:

- bringing the analytical sample into contact with a reagent containing either the receptor, if the substance sought in the sample is the ligand, or the ligand, if the substance sought in the sample is the receptor, under proper conditions to allow their reaction,

- before, simultaneously or after this contacting, the application, to this sample and / or this reagent, of the electromagnetic signal characteristic of the biological activity of the ligand and / or of the receptor, and

- Detection and / or measurement of ligand-receptor complexes possibly formed, in which case, obtaining ligand-receptor complexes reflects the presence of the substance sought in the analytical sample.

This second preferred embodiment is of particular interest for detecting the presence of substances in samples, which are known to be not detectable or only very difficult by the other available detection methods, because these substances are generally present at very low concentrations, even in trace amounts.

The method for detecting the presence of a substance in an analytical sample in accordance with the invention has many advantages.

On the one hand, it makes it possible to detect the presence of a desired substance with very high sensitivity and high specificity. Therefore, in the 13

case, for example, of a bacteriological analysis, it makes it possible to remove the need to isolate the various germs, to cultivate them, to carry out an antibiogram and to identify these germs by their biochemical, morphological and immunological characters, and authorizes obtaining results much faster than the immunological detection methods currently used in bacteriology.

On the other hand, insofar as it suffices to have a sample the size of a drop to be able to acquire and record the electromagnetic signal characteristic of the biological activity of this sample and where, this signal, once recorded can be restored on demand, this process offers the possibility of carrying out as many analyzes as desired from a micro sample.

Finally, the recording of an electromagnetic signal which can be kept indefinitely, for example in the form of a computer file capable of being kept on a simple floppy disk or a CD-Rom, and of being transmitted from a place to another by any means of transmitting digital data, this process makes it possible, in addition, to remove all the constraints of storage, dispatch and transport of the samples presented by the methods currently used for the detection of a substance.

This method can be used to detect any substance capable of specifically binding to and reacting with another substance, it being understood that the term "substance" as it is used here, denotes a biological compound as well, a chemical compound, a cell than a microorganism of the bacteria, virus or fungus type, knowing in particular that for any hapten, protein or protein complex, it is possible to find on the market or to have the corresponding antibodies produced. As such, this process finds, in particular, application in biological diagnosis, whether in human or veterinary medicine, or for bacteriological quality control in industries such as the pharmaceutical industry, the cosmetic industry, the productions and food industries.

The present invention also relates to a method for detecting the presence, in an electromagnetic signal to be tested, of a signal 14

electromagnetic characteristic of the biological activity of a substance corresponding to one of the two elements of a ligand / receptor pair, which method is characterized in that it comprises the implementation of an amplification method such as defined above. According to a preferred embodiment of this detection method, the electromagnetic signal to be tested is the electromagnetic signal radiated by a source of electromagnetic radiation.

The invention also relates to an apparatus for detecting the presence of a substance corresponding to one of the two elements of a ligand / receptor pair in an analytical sample, which apparatus is characterized in that it puts implementing a method according to the invention and in that it comprises: a) means for receiving the analytical sample and a reagent containing either the receptor or the ligand, enabling them to be brought into contact under proper conditions to allow their reaction; b) an electromagnetic signal source characteristic of the activity of the ligand and / or of the receptor; c) means for applying the signal delivered by said source of electromagnetic signal to the sample and / or the reagent; and d) means for detecting and / or measuring the ligand-receptor complexes formed during the reaction between the sample and the reagent.

The invention further relates to an apparatus for detecting the presence of a substance corresponding to one of the two elements of a ligand / receptor pair in an analytical sample, which apparatus is characterized in that it implements a method according to the invention and in that it comprises: a) means for receiving two reagents containing the ligand and the receptor respectively, allowing them to be brought into contact under conditions suitable for allowing their reaction; b) means for acquiring an electromagnetic signal from the analytical sample; 15

c) means for applying the signal delivered by said electromagnetic signal acquisition means to one and / or the other of the reagents; and d) means for detecting and / or measuring the ligand-receptor complexes formed during the reaction between the two reagents. According to an advantageous embodiment of these devices, the detection means comprise optical detection means.

Preferably, these devices include an enclosure provided with electrical and magnetic shielding surrounding said receiving means.

In addition to the foregoing provisions, the invention also comprises other provisions which will emerge from the additional description which follows, which relates to exemplary embodiments of signal acquisition, recording and application devices capable of be used according to the invention as well as to examples of experiments which have made it possible to validate the amplification process which is the subject of the present invention, and which refers to the appended drawings in which: - Figure 1 represents a diagram of a first embodiment of a signal acquisition device capable of being used according to the present invention;

- Figure 2 shows a diagram of a second embodiment of a signal acquisition device capable of being used according to the present invention;

- Figure 3 shows a diagram of a first embodiment of a signal recording device capable of being used according to the present invention;

- Figure 4 shows a diagram of a second embodiment of a signal recording device capable of being used according to the present invention;

- Figure 5 shows a diagram of an exemplary embodiment of a signal application device capable of being used in accordance with the invention;

- Figure 6 shows a black and white image of 320 pixels x 240 pixels of the agglutinates formed during an agglutination reaction between the antigen 16

polysaccharide of Escherichia coli Kl and an antibody directed against this antigen, after application of the electromagnetic signal characteristic of the biological activity of Streptococcus;

- Figure 7 shows a black and white image of 320 pixels x 240 pixels of agglutinates formed during an agglutination reaction between the polysaccharide antigen of Escherichia coli Kl and an antibody directed against this antigen, after application of the signal electromagnetic characteristic of the biological activity of Escherichia coli;

- Figure 8 shows a black and white image of 320 pixels x 240 pixels of the agglutinates formed during an agglutination reaction between the polysaccharide antigen of Escherichia coli Kl and an antibody directed against this antigen, after simultaneous application of the electromagnetic signals characteristic of the biological activity of Streptococcus and the biological activity of an antibody directed against Escherichia coli; - Figure 9 shows a black and white image of 320 pixels x 240 pixels of agglutinates also formed during an agglutination reaction between the polysaccharide antigen of Escherichia coli Kl and an antibody directed against this antigen, after simultaneous application electromagnetic signals characteristic of the biological activity of Escherichia coli and the biological activity of its specific antibody; and

- Figure 10 shows a diagram of an exemplary embodiment of a device for detecting and / or measuring ligand-receptor complexes capable of being used according to the present invention.

In Figures 1 to 5 and 10, the same references have been used to designate the same elements.

Furthermore, each image of Figures 6 to 9 corresponds to an area of approximately 2 mm x 1.5 mm of the support on which the agglutination reactions were carried out. 17

It should be understood, however, that these examples are given solely by way of illustration of the subject matter of the invention and do not in any way constitute a limitation thereof.

We first refer to Figures 1 to 5. In Figure 1, there is shown schematically a first embodiment of a device for acquiring the electromagnetic signal characteristic of the biological activity of a substance 1 arranged in a container 3, for example a test tube. A sensor 5, typically a coil of the "telephone sensor" type marketed with a view to being applied to a telephone earpiece and connected to a tape recorder, is applied against the container 3. The container 3 can also be formed by a biological wall, in particular the skin of a living being. In such a case, the acquisition of the electromagnetic signal is carried out in a non-invasive manner.

The signal collected by the coil 5 is advantageously amplified by an amplifier 7 and is available at an output terminal 9. Without this being in any way limiting in the example illustrated, a first end of the coil 5 is connected to the input of the amplifier-preamplifier 7, the opposite end being connected to a ground 11. In an exemplary embodiment, the coil 5 is a commercial telephone sensor having a length of 6 mm, an internal diameter of 6 mm containing a metal core, an external diameter of 16 mm and an impedance of 300 Ω.

In Figure 2, there is shown schematically the preferred embodiment of a device for acquiring the electromagnetic signal characteristic of the biological activity of a substance 1 contained in a container 3, in which the device preferably comprises , in an enclosure 13 provided with an electric and magnetic shielding, a transducer 15 for irradiating said substance 1 supplied by a generator 17. The transducer 15 comprises, for example, a coil, advantageously supplemented by waveguides, for example, an air gap (not shown) placed in contact with the outer walls of the container 3. 18

The generator 17 generates a low frequency sinusoidal signal, low frequency square signals, pink noise or, advantageously, white noise. The spectrum of the excitation signal supplying the coil 15 corresponds substantially to the spectrum of audible frequencies (20 Hz - 20,000 Hz). The generator 17 can be an analog signal generator of known type or, for example, a read-only memory (ROM, PROM, EPROM, EEPROM in English terminology) containing the digital signal of the desired noise and which is connected to a digital converter - analog, or the line output of a sound card from a multimedia microcomputer. However, the implementation of higher frequencies does not depart from the scope of the present invention.

The acquisition sensor 5 can comprise a coil similar to the coil 5 of the device of FIG. 1 or, advantageously, a coil of small diameter connected by an electromagnetic waveguide to the wall of the container 3. Advantageously, the signal received by the sensor 5 is available at an output terminal 9 of an amplifier-preamplifier 7.

The signal available on terminal 9 can be directly applied to the substance or substances to be irradiated, in particular to the ligand, to the receptor or to the ligand / receptor pair (in particular using the device illustrated in FIG. 5 and described below) . The signal can be recorded in analog by a signal recorder 19 (Figure 3), in particular on magnetic tape 21 adapted to the frequencies of the signal received. For acoustic frequencies, it is possible in particular to use a tape recorder. The output terminal 9 of the signal acquisition device of Figures 1 or 2 is connected to the microphone input or to the line input of such a tape recorder. During reading, the signal is collected at an output terminal 9 ′, in particular at the line output or at the speaker output of the tape recorder 19.

Advantageously, digital recording is carried out after analog-digital conversion of the signal. One uses, for example, a microcomputer 23 illustrated in Figure 4, provided with a signal acquisition card 25. It is for example a PC type computer, running under the operating system 19

WINDOWS ^® 95 from the company MICROSOFT and comprising, in addition to the acquisition card 25, a microprocessor 27, an input / output interface 29, a controller 31 of a mass memory 33 and a video interface 35 connected by one or several buses 37. The acquisition card 25 comprises an analog converter 39 preferably having a resolution greater than 10 bits, for example equal to 12 bits, as well as a sampling frequency twice the maximum frequency than the we want to be able to digitize for signal processing. In the acoustic frequencies, the sampling frequency is advantageously substantially equal to 44 kHz. For processing these types of signals, a sound card for a microcomputer is advantageously used, for example the Soundblaster 16 card or the Soundblaster 32 card sold by the CREATIVE LABS Company. The computer 23 provided with the reproduction acquisition card 25, in particular with a Soundblaster card 32 can advantageously replace the signal generator 17 of FIG. 2.

The output 9 of the signal acquisition devices of Figures 1 is connected to the input 9 of the analog-digital converter 39 of the card 25 of the computer 23; the signal is acquired for a period of time, for example, between 1 and 60 s and the digital file is recorded in a mass memory 33, for example in the form of a sound file in .WAV format. This file may possibly undergo digital processing, such as for example digital amplification for signal level calibration, filtering for the elimination of unwanted frequencies, or be transformed into its spectrum by a discrete FOURIER transform, preferably by l 'fast FOURIER transform algorithm (FTT in English terminology).

The sound reproduction time can be increased by repeating a fragment or the entire original sound file several times in a file.

On command, the file possibly processed is transformed by a digital-analog converter 41 of the card 25 (or of a separate card), which delivers on the output 9 'the analog electromagnetic signal characteristic of the biological activity to be applied, according to the amplification process in accordance with 20

the invention, for example to an aliquot 43 of a first reagent and to an aliquot 45 of a second reagent, as illustrated in FIG. 5.

Advantageously, the application of the signal to these aliquots is carried out prior to their mixing. The support on which these aliquots are deposited, for example, a blade 47 provided with a capillary 49 in the form of a serpentine, is arranged in an electromagnetic field radiated by a transducer 51, typically a coil of which a first end 9, 9 'is connected to the output 9 of an acquisition device of Figures 1 or 2 or to the output 9 'of a recording device of Figures 3 or 4. The end of the coil opposite to the connection terminal 9, 9 'is, for example, connected to ground 11.

Without this being of any limiting nature, the transducer 51 advantageously comprises a coil, of horizontal axis allowing the introduction of the blade 47. The coil has, for example, a length of 120 mm, an internal diameter of 25 mm, an outside diameter of 28 mm, has 631 turns of a wire with a diameter of 0.5 mm and a resistance of 4.7 Ω.

Advantageously, the electrical signal applied to this coil 51 will have an amplitude of 2 effective volts.

EXAMPLE 1 AMPLIFICATION OF THE FORMATION OF AGGLUTINATES BETWEEN THE POLYSACCHARIDE ANTIGEN WESCHEMCHIA COLI Kl AND ANTIBODIES DIRECTED AGAINST THIS ANTIGEN

The amplification process in accordance with the invention was validated by testing the effects, on an agglutination reaction between the polysaccharide antigen of Escherichia coli Kl and an antibody directed against this antigen:

- the application of the electromagnetic signal characteristic of the biological activity of an antigenic substance foreign to this reaction such as

Streptococcus,

- the application of the electromagnetic signal characteristic of the biological activity of Escherichia coli, 21

- the simultaneous application of the electromagnetic signal characteristic of the biological activity of Streptococcus and the electromagnetic signal characteristic of the biological activity of an antibody directed against Escherichia coli, and finally - the simultaneous application of the electromagnetic signal characteristic of l biological activity to! Escherichia coli and the electromagnetic signal characteristic of the biological activity of an antibody directed against this antigen.

1) Performing tests: a) Acquisition of electromagnetic signals:

The acquisition of electromagnetic signals characteristic of the biological activities of Streptococcus, Escherichia coli and its specific antibody was carried out using the recording equipment of Figure 2.

The acquisition of the electromagnetic signal characteristic of the biological activity of Streptococcus was carried out by placing in the center of the enclosure 13 a tube containing 1 ml of an aqueous suspension of previously formulated Streptococcus bacteria (6.10 ^δ bacteria / ml).

The electromagnetic signals characteristic of the biological activity of Escherichia coli and its specific antibody were acquired by operating in the same way, but using respectively:

- a tube containing 1 ml of an aqueous suspension of Escherichia coli bacteria previously formulated (6.10 ⁶ bacteria / ml); and

- a tube containing 1 ml of a suspension of particles of a latex sensitized with a monoclonal antibody specific for mouse of Escherichia coli Kl, from a kit PASTOREX ^® MENINGITIS (61709 Reference - Sanofi Diagnostics Pasteur), b) Preparation reagents for the agglutination reaction:

The tests were carried out using as reagents:

- on the one hand, a solution of Escherichia coli Kl polysaccharide antigen prepared by dissolving an antigenic extract from 22

a PASTOREX ^® MENINGITIS kit (Reference 61709 - SANOFI DIAGNOSTICS PASTEUR) in 1 ml of distilled and sterile water, then dilution 1/7, 1 / 7.5 or 1/8 in physiological saline; and

- on the other hand, the latex sensitized by a mouse monoclonal antibody specific for the antibody of Escherichia coli Kl present in this same kit, after dilution to 1/3 in physiological saline. c) Application of the electromagnetic signals to the agglutination reaction: For each of the tests, the following protocol was used:

- a transducer consisting of a coil measuring 120 mm long and 25 mm internal diameter, having 631 turns and a resistance of 4.7 Ω, is placed in an oven heated to 37 ° C, and connected to the converter output 9 ' digital-analog 41 of a Soundblaster card of a computer 23 reproducing the recording files constituted by the electromagnetic signals which it is desired to apply, the time necessary to bring this transducer to the temperature of 37 ° C;

- a drop (40 to 50 μl) of the antigen solution as described in point b) above, as well as a drop (also corresponding to a volume of 40 to 50 μl) of the latex sensitized by the antibody, taking care that these drops do not mix ;

- the desired electromagnetic signal (s) are applied to the two drops of reagents thus deposited by placing the slide in the center of the transducer for approximately 2 min and by restoring a sound file using the computer 23 of FIG. 4;

- The two drops of reagents are mixed for approximately 10 seconds and the reaction mixture is allowed to migrate into the capillary for approximately 13 minutes and the agglutination reaction takes place;

- the blade is then taken out of the oven and a reading of this agglutination is carried out. 23

As shown in Figure 10, this reading is performed by analysis using a software of analysis and image processing implemented on a PC-type computer 23 'running the WINDOWS ^® operating system 95

(MICROSOFT), of an image acquired using a video camera 53 positioned on an optical microscope 55 and connected to said computer by a video acquisition card 57. The camera 53 works in gray levels. A first treatment increasing the contrast, the threshold being adjusted so that the agglutinates appear in black, while the areas devoid of latex particles or agglutinates appear in white. From the analysis of the two-dimensional spatial distribution of the dark areas of the image, the computer determines an agglutination index (I) calculated according to the formula:

Surface occupied by agglutinates larger than 60 pixels 1 = Surface occupied by agglutinates equal to or smaller than 60 pixels

This agglutination index is higher the greater the size of the agglutinates formed during the agglutination reaction. Amplification is considered positive when, during an experiment, the application of the electromagnetic signals characteristic of the biological activity of Escherichia coli and / or the biological activity of its specific antibody leads to obtaining agglutination index at least 40% greater than the maximum agglutination index obtained, under the same conditions, and for example on 3 experiments, after application of the electromagnetic signal characteristic of the biological activity of Streptococcus.

2) Results:

Table 1 below presents the agglutination indexes (I) obtained in a first series of tests aimed at comparing the effects of the application of the electromagnetic signal characteristic of the biological activity of Escherichia coli with those observed after application, under the same reaction conditions, of the electromagnetic signal characteristic of the biological activity of Streptococcus, and this, for 24

3 different dilutions (1/7, 1 / 7.5 and 1/8) of the Escherichia coli Kl polysaccharide antigen solution used as a reagent in agglutination reactions.

TABLE 1

Agglutination index (I)

Dilution of E. coli Kl signal Streptococcus Signal E. coli antigen solution

11,173

1/7 6 52 16 154

58,141

1 / 7.5 32 117 12 107

10,113

1/8 6 37

8 21

Furthermore, FIGS. 6 and 7 show, by way of example, images of the agglutinates formed on the one hand, after application of the electromagnetic signal characteristic of the biological activity of Streptococcus (FIG. 6) and, on the other hand, after application of the electromagnetic signal characteristic of the biological activity of Escherichia coli (Figure 7). These images correspond respectively to the agglutination indexes of 32 and 117 which are reported in the 5th row of results in Table 1.

Table 2 below presents the agglutination index (I) obtained in a second series of experiments in which the effects of the simultaneous application of the electromagnetic signal characteristic of biological activity d 'Escherichia coli and the electromagnetic signal characteristic of the biological activity of the antibody directed against Escherichia coli, were compared with those of the simultaneous application, under the same reaction conditions, of the electromagnetic signal characteristic of the biological activity of Streptococcus and the electromagnetic signal characteristic of the activity 25

biological of the anticoφs directed against Escherichia coli and this, for 2 different dilutions (1/7 and 1 / 7.5) of the polysaccharide antigen solution of Escherichia coli Kl used as reagent.

TABLE 2

Agglutination index (I)

Dilution of Signal Streptococcus Signal E. coli antigen solution

E. coli Kl + +

Antibody signal Anti-E antibody signal. anti-E coli. coli

1/7 18 94 71 247

1 / 7.5 48 212 93 1141

Figures 8 and 9 show, also by way of example, images of the agglutinates which correspond respectively to the agglutination indexes of 71 and 247 reported in the second row of results in Table 2. All these results clearly show the aptitude presented by the electromagnetic signal characteristic of the biological activity of an element of a ligand / receptor pair, to amplify the formation of the complexes formed by the reaction between this ligand and this receptor, in a very specific way, since the electromagnetic signal characteristic of the biological activity of a biologically active element, but foreign to this reaction does not produce an amplification effect.

They also show that this amplification is all the more pronounced when we apply to the two elements of the ligand / receptor couple both the electromagnetic signal characteristic of the biological activity of this ligand and the electromagnetic signal characteristic of the biological activity of this receptor. 26

EXAMPLE 2 DETECTION OF THE PRESENCE OF ESCHERICHIA COLI IN A SAMPLE

The advantage of using the amplification method in accordance with the invention for the detection of a substance present in an analytical sample has been verified by carrying out a series of tests aimed at comparing the effects of the application, on a agglutination reaction between the polysaccharide antigen of Escherichia coli Kl and a mouse monoclonal anticoφs specific for this antigen identical to that used in Example 1 above, of the electromagnetic signal picked up from a sample d '' a food product, in this case an applesauce, previously contaminated with Escherichia coli bacteria, with those obtained during the application, under the same reaction conditions, of the electromagnetic signal picked up from a control sample, c i.e. uncontaminated, of the same food product. 1) Carrying out of the tests: The acquisition of the electromagnetic signals of the applesauce samples (control samples and contaminated samples) was carried out using the recording equipment of FIG. 2, by placing in the center of the enclosure 13:

- in the case of control samples, a tube containing 1 ml of compote previously diluted to 1/2 with physiological saline, and

- in the case of contaminated samples, a tube containing 1 ml of compote previously diluted to 1/2 with physiological and contaminated serum, by addition of Escherichia coli bacteria previously formulated, at the rate of 3.10 ⁶ bacteria per ml of diluted compote. The tests were carried out using as reagents:

on the one hand, a suspension containing Escherichia coli bacteria previously formulated in physiological saline, at a rate of 10 ⁷ bacteria / ml, and 27

- on the other hand, the latex sensitized by a mouse monoclonal anticoφs specific for the anticoφs of Escherichia coli Kl present in this same kit, after dilution to 1/3 in physiological serum, and by following an operating protocol identical to that described in paragraph c) of Example 1 above.

2 Results:

Table 3 below presents the agglutination indexes (I) obtained in three series of tests.

TABLE 3

Agglutination index (I)

Tests

Control samples Contaminated samples

10 42

Series 1 25 93 27 104

14 30

Series 2 17 153 46 40

Series 3 19 54

34,314

As shown in Table 3, the size of the agglutinates formed during the reaction between the polysaccharide antigen of Escherichia coli Kl and its specific anticoφs is substantially higher in the case where the electromagnetic signal applied during this reaction has been captured from a sample of applesauce contaminated with Escherichia coli bacteria.

These results show that the amplification method according to the invention can advantageously be used to detect the presence, in an analytical sample, of a biologically active substance such as a bacterium, even when this sample has a complex composition, c that is to say it 28

contains, as in the case of applesauce samples, many other biologically active substances.

As is apparent from the above, the invention is in no way limited to the embodiments which have just been described more explicitly; on the contrary, it embraces all the variants which may come to mind of the technician in the matter, without departing from the scope or the scope of the present invention.

Claims

29 CLAIMS

1. Method for amplifying a reaction between the two elements of a ligand / receptor pair, characterized in that it comprises:

2. Amplification method according to claim 1, characterized in that the reaction between the ligand and the receptor is carried out by bringing into contact two reagents respectively containing the ligand and the receptor, and one is applied to one and / or the other of these reagents, an electromagnetic signal to be tested and suspected of comprising the electromagnetic signal characteristic of the biological activity of this ligand and or of this receptor.

3. Amplification method according to claim 2, characterized in that the application, to one and / or the other of the reagents, of the electromagnetic signal to be tested is carried out by exposure of a solution or of a suspension containing either of these reagents to this electromagnetic signal.

4. Amplification method according to claim 2, characterized in that the application, to one and / or the other of the reagents, of the electromagnetic signal to be tested is carried out by diluting a solution or a suspension comprising one and / or the other of these reagents, in a solvent having been previously exposed to this electromagnetic signal.

5. Amplification method according to claim 2, characterized in that the application, to one and / or the other of the reagents, of the electromagnetic signal to be tested is carried out by dissolution or suspension of this or these reagents in a solvent having been previously exposed to this electromagnetic signal.

6. Amplification method according to claim 4 or claim 5, characterized in that the solvent having been previously exposed to 30

electromagnetic signal characteristic of the biological activity of the sample to be analyzed is water or physiological solute.

7. Amplification method according to any one of claims 2 to 6, characterized in that the electromagnetic signal to be tested is the electromagnetic signal picked up from a sample to be analyzed and suspected of containing the ligand and / or the receptor.

8. Amplification method according to any one of claims 2 to 6, characterized in that the electromagnetic signal to be tested is the electromagnetic signal radiated by a source of electromagnetic radiation.

9. Amplification method according to claim 1, characterized in that the reaction between the ligand and the receptor is carried out by bringing a sample to be analyzed and suspected of containing the ligand and or the receptor, with a reagent containing either the receptor , or the ligand, and the electromagnetic signal characteristic of the biological activity of said ligand and / or said receptor is applied to this sample and / or to this reagent.

10. Amplification method according to claim 9, characterized in that the application, to the sample to be analyzed, of the electromagnetic signal characteristic of the biological activity of the ligand and or of the receptor is carried out by exposure of this sample to this or these electromagnetic signals, or by diluting this sample in a solvent having been previously exposed to the said electromagnetic signal (s).

11. Amplification method according to claim 9 or claim 10, characterized in that the application, to the reagent intended to react with the sample to be analyzed, of the electromagnetic signal characteristic of the biological activity of the ligand and / or of the receiver is produced by exposure of a solution or suspension containing this reagent to this or these electromagnetic signals, or by dilution of such a solution or suspension in a solvent having been previously exposed to this or these electromagnetic signals, or by dissolving or suspending this reagent in a solvent having been previously exposed to the said electromagnetic signal (s). 31

12. Amplification method according to claim 9, characterized in that the electromagnetic signal characteristic of the biological activity of the ligand and / or of the receptor is applied to the sample to be analyzed and to the reagent intended to react with it. , by exposure of a solution or a suspension containing this sample and this reagent to this or these electromagnetic signals, or by dilution of such a solution or suspension in a solvent having been previously exposed to said said (s) electromagnetic signal (s).

13. Amplification method according to any one of claims 9 to 12, characterized in that one applies to the sample to be analyzed and / or to the reagent intended to react with it, both the electromagnetic signal characteristic of the biological activity of the ligand and the electromagnetic signal characteristic of the biological activity of the receptor.

14. Amplification method according to any one of claims 10 to 12, characterized in that the solvent having been exposed beforehand to the electromagnetic signal (s) is advantageously water or physiological solute.

15. Amplification method according to any one of claims 1 to 14, characterized in that it comprises a step of acquisition of the electromagnetic signal characteristic of the biological activity of one and / or the other of the elements of the ligand / receptor pair.

16. Amplification method according to claim 15, characterized in that it comprises a step of recording and restoring information representative of the electromagnetic signal characteristic of the biological activity of one and / or the other elements of the ligand / receptor pair.

17. Amplification method according to any one of claims 1 to 16, characterized in that it comprises a step of detecting and, optionally, measuring the complexes resulting from the reaction between the ligand and the receptor.

18. Amplification method according to any one of claims 1 to 17, characterized in that the ligand is an antigen or a hapten, 32

while the receptor is an anticoφs or a membrane receptor directed specifically against this ligand.

19. Amplification method according to claim 18, characterized in that the reaction between the antigen and the anticoφs or the hapten and the anticoφs is revealed by agglutination.

20. Method for detecting the presence of a substance corresponding to one of the two elements of a ligand / receptor pair in an analytical sample, characterized in that it comprises the implementation of an amplification method according to any one of claims 1 to 7 and 9 to 19.

21. Detection method according to claim 20, characterized in that it comprises:

- before, simultaneously or after this contacting, the application, to one or other of these reagents, of the electromagnetic signal characteristic of the biological activity of the analytical sample, and

22. Detection method according to claim 21, characterized in that the concentrations of the ligand and of the receptor are chosen so as to be sufficient to lead to the production of ligand-receptor complexes detectable in the absence of the application of the signal electromagnetic characteristic of the biological activity of said sample, but lower than the concentrations likely to lead to saturation of the reaction between this ligand and this receptor.

23. Detection method according to claim 20, characterized in that it comprises:

- bringing the analytical sample into contact with a reagent containing either the receptor, if the substance sought in the sample is the ligand, or the ligand, if the substance sought in the sample is the receptor, under proper conditions to allow their reaction, 33

- before, simultaneously with or after this contact, the application, to this sample and / or this reagent, of the electromagnetic signal characteristic of the biological activity of the ligand and or of the receptor, and

- detecting and / or measuring any ligand-receptor complexes that may have formed.

24. Apparatus for detecting the presence of a substance corresponding to one of the two elements of a ligand / receptor pair in an analytical sample, characterized in that it implements a method according to claim 20 and in that that it comprises: a) means (47) for receiving the analytical sample and a reagent containing either the receptor or the ligand, allowing them to be brought into contact under conditions suitable for allowing their reaction; b) a source (5, 9, 9 ', 19) of an electromagnetic signal characteristic of the activity of the ligand and / or of the receptor; c) means (51) for applying the signal delivered by said source

(5, 9, 9 ', 19) electromagnetic signal to the sample and / or the reagent; and d) means (53, 55, 57) for detecting and / or measuring the ligand-receptor complexes formed during the reaction between the sample and the reagent.

25. Apparatus for detecting the presence of a substance corresponding to one of the two elements of a ligand / receptor pair in an analytical sample, characterized in that it implements a method according to claim 20 and in that that it comprises: a) means (47) for receiving two reagents containing the ligand and the receptor respectively, allowing them to be brought into contact under conditions suitable for allowing their reaction; b) means for acquiring an electromagnetic signal from the analytical sample; 34

c) means (51) for applying the signal delivered by said acquisition means (5, 9, 9 ', 19) of the electromagnetic signal to one or the other of the reagents; and d) means (53, 55, 57) for detecting and / or measuring the ligand-receptor complexes formed during the reaction between the two reagents.

26. Apparatus according to claim 24 or claim 25, characterized in that the detection means comprise optical detection means.

27. Apparatus according to any one of claims 24 to 26 characterized in that it comprises an enclosure (13) provided with an electrical and magnetic shielding surrounding said receiving means (47).

28. Application of a method for detecting the presence of a substance in an analytical sample according to any one of claims 20 to 23 to biological diagnosis in human or veterinary medicine.

29. Application of a method for detecting the presence of a substance in an analytical sample according to any one of claims 20 to 23 to bacteriological control in the pharmaceutical industry, the cosmetic industry, the productions and the food industries.

30. Method for detecting the presence, in an electromagnetic signal to be tested, of an electromagnetic signal characteristic of the biological activity of a substance corresponding to one of the two elements of a ligand / receptor pair, characterized in that that it comprises the implementation of an amplification method according to any one of claims 1 to 8 and 15 to 19.

31. Detection method according to claim 30, characterized in that the electromagnetic signal is the electromagnetic signal radiated by a source of electromagnetic radiation.