FR2777656A1 - Amplification of ligand-receptor reaction, especially for increasing sensitivity of agglutination assays - Google Patents

Abstract

Amplifying a reaction between a ligand and receptor comprises applying an electromagnetic signal characteristic of the biological activity of the ligand and/or receptor to the ligand and/or receptor before, while or after contacting the ligand with the receptor.

Description

METHOD FOR AMPLIFYING THE FORMATION OF COMPLEXES
LIGANDS-RECEIVERS AND ITS USES
The present invention relates to a method of amplifying the formation of complexes between the two elements of a ligand / receptor pair, to a method for detecting the presence, in a sample (hereinafter "analytical sample"), 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 method for detecting the presence, in an electromagnetic signal, 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, a large number of methods have been proposed based on the ability of that substance to specifically bind to and react with another substance.

 In particular, the affinity properties of antibodies against antigens are at the basis of a large number of immunological detection methods which have in common the use of the formation of antigen-antibody complexes - the desired substance being able to 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), immunoenzymatic assays (ELISA) or immunofluorescence.

 These immunological methods of detection, although they undeniably have great qualities, do not, however, give complete satisfaction.

In the first place, their sensitivity (which is defined by the minimum concentration of 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, for example, for 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 less than 10 ng of antibody per ml of sample.

 It is therefore desirable to develop new techniques that make it possible to lower the detection threshold of a desired substance.

 Furthermore, all 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 desired substance. As a result, they have the disadvantage of requiring, as soon as the analysis of a sample relates to several substances - as is very often the case for diagnostic medical tests - a sample of a relatively large volume. which is not always well supported by patients, especially in the case of blood samples.

Moreover, the fact that these detection methods require, for their implementation, to dispose of the sample to be analyzed or, at the very least, of a sample thereof, is not without presenting a certain number of constraints. Indeed:
- On the one hand, samples that have been analyzed must be kept in such a way that the reliability of these analyzes can be checked later or that additional analyzes can be carried out. For example, blood transfusion centers, forensic services and tissue sampling centers keep samples of all the biological samples they are required to make. This preservation, which is carried out by freezing said samples, besides having a significant cost, requires appropriate equipment and premises.

 - On the other hand, it is also common that samples can not be analyzed at the place where they were made and that it is necessary to send them to the laboratory responsible for carrying out the analysis. However, the delivery of biological samples, besides it is never very easy to implement because of the low shelf life of biological substances in the absence of freezing, poses a number of difficulties when these samples are potentially contaminants. In addition, the duration of such a routing differs all the way to 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, at the same time, a very high sensitivity and a high specificity, while offering the possibility of carrying out as much analysis of the need for microprocessing, to overcome, moreover, the constraints of conservation, shipping and transport of samples that present methods currently used for the detection of a substance, and which can, in addition , be implemented easily and quickly without requiring heavy equipment and expensive.

 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 other of the elements of a couple ligand / receptor such as an antigen / antibody pair, 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 pair when they are reacted together and this, very specifically, and had the idea to use 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.

The subject of the present invention is therefore a method of amplifying the formation of complexes between the two elements of a ligand / receptor pair by reaction of these two elements, which process is characterized in that it comprises:
bringing the two elements of the ligand / receptor pair into contact under conditions suitable for allowing their reaction, and
beforehand, simultaneously or after this contact, 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 pair" means any pair formed by two substances capable of recognizing each other specifically, of bonding and of reacting together to form complexes. Thus, it may be an antigen / antibody or hapten / antibody pair in which the ligand (the antigen or hapten) may be a biological compound (protein, enzyme, hormone, toxin, tumor marker, etc.). .), a chemical compound (medicinal active principle for example), or a cellular or particulate antigen (cell, bacterium, virus, fungus, etc.), the receptor possibly being 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, the electromagnetic signal captured from a biologically active element such as a substance, a cell or a microorganism, ... 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 the
International Application WO 94/17406 in the name of J. BENVENISTE. The term "electromagnetic signal characteristic of the biological activity" of an element is also understood to mean 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 captured electromagnetic signal contains information characterizing the fact that the material from which this signal is captured has the biological activity in question. The electromagnetic signal picked up from a material containing a plurality of biologically active elements has the biological activity of each of the elements it contains.

 According to a first preferred embodiment of the amplification method according to 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 of them is applied to one and / or the other of these reagents, an electromagnetic signal to be tested and suspected to include the electromagnetic signal characteristic of the biological activity of this ligand and / or this receptor.

 In the foregoing and in what follows, the term "reagent" denotes any preparation whose composition is known, which contains the ligand or the receptor in an equally known quantity and is either in a dry form such that a lyophilizate to be reconstituted in a solvent, either in a liquid form such as a solution or a suspension, the ligand or the receptor can be fixed on a solid phase (particles or beads of latex, glass or polystyrene, .. .).

 According to a first advantageous disposition 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 suspension containing one and / or the other of these reagents, to this electromagnetic signal.

 In a variant, 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.

Thus, for example, when the reagents that 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:
* prior to their use: by exposing one or both of these reagents or aliquots of one and / or the other of
these reagents to this electromagnetic signal, or e by diluting one and / or the other of said reagents or their aliquots in a volume of one
solvent having previously been exposed to said electromagnetic signal,
when carrying out the amplification process according to the invention: by exposing to this electromagnetic signal an aliquot of each of these reagents,
after depositing these aliquots on a support (blade for example) but beforehand
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 exposing this mixture to the signal
electromagnetic, or 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 one
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 dissolving or suspending this or these reagents in a solvent having been previously exposed to this electromagnetic signal. This provision is of particular interest when the reagents that one wishes to use, are in a dehydrated form such as a lyophilizate, since it is then possible to apply the electromagnetic signal to be tested simply by dissolving them or by suspending, as the case may be, in a volume of a solvent having previously been 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 to contain this ligand and / or this receptor, this sample possibly being 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 may also be an electromagnetic signal radiated by a source of electromagnetic radiation, in particular a source suspected to emit harmful radiation for living beings of the high voltage line, transformer, electric motor, microwave oven type. waves, particle accelerator, X-ray source, ... Similarly, the electromagnetic signal to be tested may come from the acquisition of a mechanical signal such as vibration, an electrostatic signal or other.

 According to a second preferred embodiment of the amplification method according to the invention, the reaction between the ligand and the receptor is carried out by contacting a sample to be analyzed and suspected to contain the ligand and / or the receptor, with a reagent containing either the receptor or the ligand (depending on the substance suspected of being present in the analytical sample with which it is desired to react this reagent), and applying to this sample and / or reagent the characteristic electromagnetic signal the biological activity of said ligand and / or 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 the receptor is carried out by exposing this sample to this sample. or these electromagnetic signals, or by dilution of this sample in a solvent having been previously exposed to 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 the receptor is carried out by exposure a solution or a suspension containing this reagent to this or these electromagnetic signals, or by dilution of such a solution or suspension in a solvent that has been previously exposed to this or these electromagnetic signals, or by dissolution or suspension of this reagent in a solvent having previously been 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 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 (s) signal (s) electromagnetic (s).

 According to a particularly preferred arrangement of this second embodiment, both the sample to be analyzed and / or the reagent intended to react with it are applied to 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 is sufficient to apply to the elements of the ligand / receptor pair, 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 applying to these elements simultaneously the electromagnetic signals characteristic of the biological activity of each of them.

 Whatever the mode of implementation of the amplification method according to the invention, the solvent having been previously exposed to the (s) electromagnetic (s) signal (s) is preferably water or physiological saline.

 The reagents that may be used in the amplification process according to the invention and containing the ligand on the one hand, and the receptor on the other hand, can be both ready-to-use reagents commercially available. reagents specially designed and prepared for carrying out this process. In addition to the fact that, as mentioned above, these reagents can be in various forms (dry, liquid, ...), they can, moreover, 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 allowing the detection and / or measurement of the ligand-receptor complexes resulting from the reaction between the ligand and the receptor.

 The amplification method advantageously also 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 ligand / receptor pair can come either from a sample to be analyzed and suspected to contain this or these elements, or from a source of electromagnetic radiation or
The acquisition of a mechanical signal (vibrations), electrostatic or other, or else of reagents containing the ligand or the receptor in solution or in suspension in a solvent, according to the modes of implementation of the amplification method according to the 'invention.

 In a particularly advantageous manner, the amplification method according to the invention also comprises a step of recording and reproducing information representative of the electromagnetic signal characteristic of the biological activity of the one and / or the other elements of the ligand / receptor pair. Thus, the electromagnetic signal characteristic of the biological activity of an analytical sample, once recorded, can be retained indefinitely and used as many times as necessary. Similarly, the electromagnetic signals characteristic of the biological activity of the ligand and the biological activity of the receptor captured from reagents can be recorded once and for all and be used to perform a plurality of reactions involving this ligand. and this receiver.

 The amplification method advantageously also comprises a step of detecting the complexes resulting from the reaction between the ligand and the receptor and, optionally, measuring these complexes. This step may advantageously be completed by comparing the results obtained with those observed for a "control" reaction, that is to say a reaction conducted with the same ligand / receptor pair and under the same reaction conditions, but without applying an electromagnetic signal to the elements of this pair, whether previously, simultaneously or after contacting them.

 Detection and / or measurement of ligand-receptor complexes are likely to be performed by all 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 revelation by agglutination, by immunoprecipitation, by fluorescence quenching, by fluorescence polarization, or by radioimmunoassay, immunoenzymatic assay or else immunofluorescence test.

 According to a particularly preferred embodiment of the amplification method according to 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.

 Particularly advantageously, the reaction between this ligand and this receptor is a reaction revealed by agglutination, because of its simplicity and 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 of an amplification method as defined above.

According to a first particularly preferred embodiment of this detection method, this method comprises
contacting two reagents respectively containing the ligand and the receptor, under conditions suitable for allowing their reaction,
- previously, simultaneously or after this contact, the application. to one and / or the other of these reagents, the electromagnetic signal characteristic of the biological activity of the analytical sample, and
the detection and / or measurement of 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 reactants with respect to a "control" reaction (as defined above) reflects the presence, in the electromagnetic signal, of the biological activity of the sample to be analyzed, the electromagnetic signal characteristic of the biological activity of the desired substance and, consequently, reflects the presence in this sample. of the desired substance.

In the case where such amplification is obtained and the analytical sample is likely to contain not only one of the two elements of the pair
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 conducted under the same reaction conditions but with an 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 an 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 of 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 contains only the receptor. The absence of an increase in the formation of ligand-receptor complexes signifies 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 ligand-receptor complexes relative to to the application of the only electromagnetic signal characteristic of the biological activity of said analytical sample, then it can be deduced that this sample does not contain a receptor and therefore contains only the ligand. The absence of an increase in the formation of ligand-receptor complexes signifies the presence of the receptor in the sample.

In order to avoid false-negative results, ie results which would not reveal an amplification effect of
The application of the electromagnetic signal characteristic of the activity of. the analytical sample, even though the latter actually contains the desired substance, the concentrations of the ligand and of the receptor which are reacted are advantageously chosen so as to be sufficient to lead to the production of ligand-receptor complexes detectable by absence of the application of the electromagnetic signal characteristic of the biological activity of said sample. but lower than the concentrations likely to result in a saturation of the reaction between this ligand and this receptor.

According to a second preferred embodiment of this detection method, this method comprises
contacting the analytical sample 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 conditions specific to allow their reaction,
beforehand, 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 the receptor, and
the detection and / or measurement of the ligand-receptor complexes that may be formed, in which case the production of ligand-receptor complexes reflects the presence of the desired substance 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 undetectable or very difficult by the other available detection methods, because of the fact that these substances are generally present at very low concentrations, even at trace levels.

 The method of detecting the presence of a substance in an analytical sample according to the invention has many advantages.

 Indeed, on the one hand, it can detect the presence of a desired substance with a very high sensitivity and high specificity. For example, in the case of, for example, a bacteriological analysis, it makes it possible to eliminate the need to isolate the different germs, to cultivate them, to carry out an antibiogram and to identify these germs by their biochemical characteristics, morphological and immunological, and allows to obtain results much faster than the immunological detection methods currently used in bacteriology.

 On the other hand, to the extent that it is sufficient to have a sample of 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 method offers the possibility of performing as many analyzes as desired from a micropremise.

 Finally, the recording of an electromagnetic signal can be kept indefinitely. for example in the form of a computer file likely to be kept on a simple diskette or a CD-ROM, and to be transmitted from one place to another by any means of digital data transmission, this method allows, in addition, to remove all the constraints of conservation, shipping and transport of samples that are present methods used for the detection of a substance.

 This method is capable of being used to detect and react with any substance capable of specific binding with another substance, it being understood that the term "substance" as used herein refers both to a biological compound, a chemical compound, a cell that a microorganism of the bacterium, virus or fungus type, especially knowing that for any hapten. protein or protein complex, it is possible to find on the market or have the corresponding antibodies made. As such, this method finds, in particular, application in biological diagnosis, whether in human or veterinary medicine, or for the control of bacteriological quality in industries such as the undue is characterized in that it includes the implementation implementation of an amplification method 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.

In addition to the foregoing, the invention also comprises other arrangements which will emerge from the following additional description which relates to exemplary embodiments of recording acquisition and signal application devices which may be used according to the invention as well as to examples of experiments having made it possible to validate the amplification method which is the subject of the present invention and which refers to the attached drawings in which
- Figure 1 shows a diagram of a first embodiment of a signal acquisition device may be used according to the present invention;
FIG. 2 represents a diagram of a second exemplary embodiment of a signal acquisition device that can be used according to the present invention;
FIG. 3 represents a diagram of a first exemplary embodiment of a signal recording device that can be used according to the present invention
FIG. 4 represents a diagram of a second exemplary embodiment of a signal recording device that can be used according to the present invention;
FIG. 5 represents a diagram of an exemplary embodiment of a signal application device that can be used in accordance with the invention;
FIG. 6 shows a 320 x 240 pixel black and white image of the agglutinates formed during an agglutination reaction between the polysaccharide antigen of Escherichia coli K1 and an antibody directed against this antigen, after application of the signal. electromagnetic characteristic of the biological activity of Streptococcus;
FIG. 7 shows a 320 x 240 pixel black and white image of the agglutinates formed during an agglutination reaction between the polysaccharide antigen of Escherichia coli K1 and an antibody directed against this antigen, after application of the signal. electromagnetic characteristic of the biological activity of Escherichia coli;
FIG. 8 shows a black and white image of 320 pixels × 240 pixels of the agglutinates formed during an agglutination reaction between the polysaccharide antigen of Escherichia coli K1 and an antibody directed against this antigen, after simultaneous application of electromagnetic signals characteristic of the biological activity of Streptococcus and the biological activity of an antibody to Escherichia colt; and
FIG. 9 shows a 320 × 240 pixel black and white image of the agglutinates also formed during an agglutination reaction between the polysaccharide antigen of Escherichia coli K1 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.

 In Figures I to 5, the same references were used to designate the same elements.

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

 It should be understood, however, that these examples are given solely as illustrations of the subject of the invention and in no way constitute a limitation thereof.

 Reference is first made 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 disposed in a container 3, for example a test tube. A sensor 5, typically a coil of the "telephone sensor" type marketed for application to a telephone earphone and connected to a tape recorder, is applied against the container 3. The container 3 can also be constituted by a biological wall, in particular the skin of a living being.

In such a case, the acquisition of the electromagnetic signal takes place non-invasively.

 The signal collected by the coil 5 is advantageously amplified by an amplifier 7 and is available at an output terminal 9. Without this having any limiting character of the illustrated example, 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 outer diameter of 16 mm and an impedance of 300 Q.

 FIG. 2 diagrammatically shows the preferred embodiment of an electromagnetic signal acquisition device 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 electrical and magnetic shielding, an irradiation transducer 15 of said substance 1 supplied by a generator 17. The transducer 15 comprises, for example, a coil, advantageously completed by waveguides, for example, an air gap (not shown) placed in contact with the outer walls of the container 3.

 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 substantially corresponds to the spectrum of audible frequencies (20 Hz - 20 000 Hz). The generator 17 may 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 an analogue digital converter , or the line output of a sound card from a multimedia microcomputer.

However, the implementation of higher frequencies is not beyond the scope of the present invention.

 The acquisition sensor 5 may 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 picked up 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 recording of the signal can be carried out in analog form by a signal recorder 19 (FIG. 3), in particular on a magnetic tape 21 adapted to the frequencies of the signal picked up. For the acoustic frequencies, it is possible in particular to use a tape recorder. The output terminal 9 of the signal acquisition device
Figures 1 or 2 is connected to the microphone input or the line input of such a tape recorder. During the reading, the signal is collected at an output terminal 9 ', in particular at the line output or the loudspeaker output of the tape recorder 19.

 Advantageously, a digital recording is performed after analog-digital conversion of the signal. For example, a microcomputer 23 illustrated in FIG. 4 is used, provided with a signal acquisition card 25. It is for example a PC-type computer running on the WINDOWS 95 operating system. 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 more buses 37. The acquisition card 25 comprises an analog converter 39 having, preferably, a resolution greater than 10 bits, for example equal to 12 bits, and a double sampling frequency of the maximum frequency that can be digitized. for signal processing. In acoustic frequencies, the sampling frequency is advantageously substantially equal to 44 kHz. For the processing of these types of signals, a microcomputer sound card is advantageously used, for example the Soundblaster 16 card or the Soundblaster 32 card sold by CREATIVE LABS. The computer 23 equipped with the reproduction acquisition card 25, in particular a soundblaster card 32, can advantageously replace the signal generator 17 of FIG. 2.

The output 9 of the signal acquisition devices of the
Figures 1 at the input 9 of the analog-digital converter 39 of the card 25 of the computer 23; the signal is acquired for a duration of, for example, between 1 and 60 seconds, 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 can optionally undergo a digital processing, such as a digital amplification for signal level calibration, a filtering for the elimination of unwanted frequencies, or be transformed into its spectrum by a transformation of
FOURIER discrete, preferably by the fast FOURIER transform algorithm (FTT in English terminology).

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

 On command, the file optionally processed is converted by a digital-analog converter 41 of the card 25 (or 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 method according to the invention, for example an aliquot 43 of a first reagent and an aliquot 45 of a second reagent, as illustrated in FIG.

Advantageously, the application of the signal to these aliquots is performed prior to mixing them. The support on which are deposited these aliquots, for example, a blade 47 provided with a capillary 49 in the form of a coil, is disposed in an electromagnetic field radiated by a transducer 51, typically a coil whose first end 9, 9 'is connected to output 9 of a device for acquiring
Figures 1 or 2 or the output 9 'of a recording device of Figures 3 or 4.

The end of the coil opposite 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 outer diameter of 28 mm, has 631 turns of a 0.5mm diameter wire and a resistance of 4.7 Q.

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

EXAMPLE: AMPLIFICATION OF AGGLUCTIN FORMATION
BETWEEN POLYSACCHARIDE ANTIGEN OF ESCHERICHM COLI K1
AND ANTIBODY DIRECTED AGAINST THIS ANTIGEN
The amplification method according to the invention and its use for the detection of a substance present in a sample were validated by testing the effects, on an agglutination reaction between the polysaccharide antigen of Escherichia coli K1 and a antibody against this antigen:
the application of the electromagnetic signal characteristic of the biological activity of an antigenic substance foreign to this reaction such that
Streptococcus
the application of the electromagnetic signal characteristic of the biological activity of Escherichia coli.

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 the biological activity of Escherichia coli and of the electromagnetic signal characteristic of the biological activity of an antibody directed against this antigen.

1) Realization of tests a) Acquisition of electromagnetic signals
The acquisition of the electromagnetic signals characteristic of the biological activities of Streptococcus, Escherichia coli and its specific antibody was performed using the recording material 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 chamber 13 a tube containing 10 ml of an aqueous suspension of Streptococcus bacteria previously formolated (6.times.10 6 cfu / ml).

The acquisition of the electromagnetic signals characteristic of the biological activity of Escherichia coli and of its specific antibody was carried out by operating in the same way, but using respectively:
a tube containing 10 ml of an aqueous suspension of Escherichia coli bacteria previously formolated (6.1 cfu / ml); and
a tube containing 10 ml of a suspension of particles of a latex sensitized with a mouse monoclonal antibody specific for Escherichia coli K1, originating from a PASTOREXs MENINGITIS kit (Reference 61709 - SANOFI
PASTEUR DIAGNOSTICS). b) Preparation of reagents of the agglutination rection
The tests were carried out using as reagents
on the one hand, a solution of polysaccharide antigen of Escherichia coli K1 prepared by dissolving an antigenic extract from a PASTOREX MENINGITIS kit (Reference 61709 - SANOFI
PASTEUR DIAGNOSTICS) in 1 ml of distilled and sterile water, followed by dilution to 1/7, 1/7 or 1/8 in saline; and
on the other hand, the latex sensitized with a mouse monoclonal antibody specific for the Escherichia coli K1 antibody 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 test, the following protocol was used
a transducer constituted by a coil measuring 120 mm in length and 25 mm in internal diameter, having 631 turns and a resistance of 4.7 Ω, and connected to the output 9 'of the converter, is placed in an oven heated to 37 ° C. digital-analog 41 of a soundblaster card of a computer 23 restoring the recording files constituted by the electromagnetic signals that it is desired to apply, the time required to bring this transducer to the temperature of 37 "C;
- is deposited on a blade provided with a coil-shaped capillary (of the type provided in kits PASTOREX MENINGITIS), a short distance from the opening of the latter, a drop (or 40 to 50 pLl) of the antigenic 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 ;
- Apply to the two drops of reagents thus deposited. the desired electromagnetic signal or signals by placing the blade in the center of the transducer for about 2 minutes and restoring a sound file using the computer 23 of Figure 4;
the two drops of reagent are mixed for about 10 seconds and the reaction mixture is allowed to migrate into the capillary for about 13 minutes and the agglutination reaction occurs.
- Then we take out the blade of the oven and we proceed to a reading of this agglutination.

 This reading is performed by analysis, using an analysis and image processing software implemented on a PC type computer running the WINDOWSt '95 operating system (MICROSOFT), an image acquired at using a video camera positioned on an optical microscope and connected to said computer by a video acquisition card. The camera works in grayscale.

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 index of agglutination (I) calculated according to the formula
Area occupied by agglutinates larger than 60 pixels
I = ------------------------------------------------ ---------------------------------------
Area occupied by agglutinates of size 60 pixels or less
This agglutination index is all the higher as the size of the agglutinates formed during the agglutination reaction is greater.

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 the obtaining of agglutination at least 40% higher than the maximum agglutination index obtained, under the same conditions, and for example 3 experiments, after application of the electromagnetic signal characteristic of the biological activity of Streptococcus.

2) Results:
Table I below shows the agglutination indices obtained in a first series of tests for comparing the effects of the application of the electromagnetic signal characteristic of the biological activity of Escherichia coli to those observed after application, in the same reaction conditions, of the electromagnetic signal characteristic of the biological activity of Streptococcus, and this, for 3 different dilutions (1/7, 1 / 7.5 and 1/8) of the solution of Escherichia coli antigen polysaccharide used as a reagent in agglutination reactions.

TABLE 1

<tb><SEP><SEP> Agglutination Index <SEP> (I)
<tb><SEP> Dilution <SEP> of <SEP> the <SEP> solution
<tb> antigen <SEP> E. <SEP> coli <SEP> K1
<tb><SEP> Signal <SEP> Streptococcus <SEP> Signal <SEP> E. <SEP> coli
<tb><SEP> 11 <SEP> 173
<tb> 1/7 <SEP> 6 <SEP> 52
<tb><SEP> 16 <SEP> 154
<tb><SEP> 58 <SEP> 141
<tb><SEP> 1 / 7.5 <SEP> 32 <SEP> 117
<tb><SEP> 12 <SEP> 107
<tb><SEP> 10 <SEP> 113
<tb><SEP> 1/8 <SEP> 6 <SEP> 37
<tb><SEP> 8 <SEP> 21
<Tb>
Moreover, FIGS. 6 and 7 show, by way of examples, 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 indices of 32 and 117 which are related to the 5th row of results in Table 1.

Table 2 below presents, for its part, the agglutination indices obtained in a second series of experiments in the context of which the effects of the simultaneous application of the electromagnetic signal characteristic of the biological activity of Escherichia coli and the electromagnetic signal characteristic of the biological activity of the antibody directed against
Escherichia colt, 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 biological activity of the antibody directed against Escherichia coli and this, for 2 different dilutions (1/7 and 1 / 7.5) of the Escherichia coli K1 polysaccharide antigen solution used as reagent.

TABLE 2

<tb><SEP><SEP> Agglutination Index <SEP> (I)
<tb><SEP> Dilution <SEP> of <SEP> la
<tb><SEP> solution of antigen <SEP><SEP> Signal <SEP> Streptococcus <SEP> Signal <SEP> E. <SEP> coli <SEP>
<tb><SEP> E. <SEP> coli <SEP> K1 <SEP> | <SEP> |
<tb><SEP> Signal <SEP> Antibody <SEP> Signal <SEP> Antibody
<tb><SEP> anti-E. <SEP> coli <SEP> anti-E. <SEP> coli
<tb><SEP> 1/7 <SEP> 18 <SEP> 94
<tb><SEP> 71 <SEP> 247
<tb><SEP> 1 / 7.5 <SEP> j <SEP> 48 <SEP> 212 <SEP>
<tb><SEP> 93 <SEP> 1141
<Tb>
Figures 8 and 9 also show, by way of examples, images of the agglutinates which respectively correspond to the agglutination indices of 71 and 247 reported in the second row of results in Table 2.

 All these results clearly demonstrate the ability of 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 and this, very specifically. 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 the two elements of the ligand / receptor pair are applied to both the electromagnetic signal characteristic of the biological activity of this ligand and the electromagnetic signal characteristic of the ligand / receptor pair. biological activity of this receptor.

As is apparent from the above, the invention is not limited to the embodiments that have just been described more explicitly; on the contrary, it embraces all the variants that may come to the mind of the technician in the field, without departing from the scope or scope of this
Invention.

Claims (27)

 A method for amplifying a reaction between the two elements of a ligand / receptor pair, characterized in that it comprises:  bringing the two elements of the ligand / receptor pair into contact under conditions suitable for allowing their reaction, and  beforehand, simultaneously or after this contact, 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.  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 containing respectively the ligand and the receptor, and is applied to one and / or the other of these reagents, an electromagnetic signal to be tested and suspected to include the electromagnetic signal characteristic of the biological activity of this ligand and / or this receptor.  3. amplification method according to claim 2, characterized in that the application to one and / or the other of the reagents, the electromagnetic signal to be tested is performed by exposure of a solution or a suspension containing one or the other 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, the electromagnetic signal to be tested is carried out by dilution of 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, the electromagnetic signal to be tested is performed 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 the electromagnetic signal characteristic of the biological activity of the sample to be analyzed is water or physiological saline.  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 to contain 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 contacting a sample to be analyzed and suspected to contain the ligand and / or the receptor, with a reagent containing either the receptor, or the ligand, and applied to this sample and / or reagent, the electromagnetic signal characteristic of the biological activity of said ligand and / or said receptor.  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 the receptor is achieved by exposure of this sample to this or these electromagnetic signals, or by dilution of this sample in a solvent having been previously exposed to said (s) signal (s) electromagnetic.  11. amplification method according to claim 9 or claim 10, characterized in that the application, to the reagent for reacting with the sample to be analyzed, electromagnetic signal characteristic of the biological activity of the ligand and / or the receptor is performed by exposing 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 that has been previously exposed to this or these electromagnetic signals, or by dissolving or suspending this reagent in a solvent having been previously exposed to said electromagnetic signal (s).  12. The amplification method as claimed in claim 9, wherein 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 exposing a solution or a suspension containing this sample and this reagent to this or these electromagnetic signals, or by diluting such a solution or suspension in a solvent that has been previously exposed to said (s) electromagnetic signal (s).  13. amplification method according to any one of claims 9 to 12, characterized in that applied to the sample to be analyzed and / or the reagent for reacting 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 previously exposed to the (s) signal (s) electromagnetic (s) is preferably water or physiological saline.  15. Amplification method according to any one of claims 1 to 14, characterized in that it comprises a step of acquiring the electromagnetic signal characteristic of the biological activity of one and / or the other elements. of the ligand / receptor pair.  16. amplification method according to claim 15, characterized in that it comprises a step of recording and retrieval of 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, while the receptor is an antibody or a membrane receptor specifically directed against this ligand.  19. Amplification method according to claim 18, characterized in that the reaction between the antigen and the antibody or hapten and the antibody is revealed by agglutination.  20. 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 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:  contacting two reagents respectively containing the ligand and the receptor, under conditions suitable for allowing their reaction,  prior to, simultaneously with or subsequent to 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  the detection and / or measurement of the ligand-receptor complexes formed during the reaction between the two reagents.  22. The detection method as claimed in 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 detectable ligand-receptor complexes 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 result in a saturation of the reaction between this ligand and this receptor.  23. The detection method according to claim 20, characterized in that it comprises:  contacting the analytical sample 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 clean conditions to allow their reaction,  prior to, simultaneously or after this contacting, the application to this sample and / or reagent of the electromagnetic signal characteristic of the biological activity of the ligand and / or the receptor, and  the detection and / or measurement of the ligand-receptor complexes that may be formed.  24. 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 the biological diagnosis in human or veterinary medicine.  25. 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 cosmetics industry, food products and industries.  26. A 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 it comprises the implementation of an amplification method according to any one of claims 1 to 8 and 15 to 19.  27. The detection method according to claim 26, characterized in that the electromagnetic signal is the electromagnetic signal radiated by a source of electromagnetic radiation.