WO1991014181A1

WO1991014181A1 - Process for monitoring the dilution and contamination of highly dilute solutions

Info

Publication number: WO1991014181A1
Application number: PCT/FR1991/000205
Authority: WO
Inventors: Jacques Benveniste
Original assignee: Institut National De La Sante Et De La Recherche Medicale
Priority date: 1990-03-13
Filing date: 1991-03-13
Publication date: 1991-09-19
Also published as: FR2659740B1; FR2659740A1

Abstract

The object of the invention is a process for monitoring the dilution and contamination of a solution of given diluteness in which: before any dilution n, where n is a whole number greater than 0, a marker substance soluble in the solution and not interfering therewith is introduced into the solution of dilution n-1, while the marker substance has the property of disappearing between dilution n-1+m and n+m, where m is the number of dilutions in which the marker substance is present and is comprised particularly between 5 and 8; the marker substance is titred in at least once after dilution n; the concentration of the marker substance obtained is compared with that calculated after dilution. This process facilitates the qualitative and quantitative monitoring of the dilutions.

Description

METHOD FOR CONTROLLING THE DILUTION AND CONTAMINATION OF HIGH DILUTION SOLUTIONS

The subject of the invention is a process for controlling the dilution and the contamination of high dilution solutions, intended in particular for the preparation of homeopathic medicines.

By high dilution solution is meant those obtained from an initial substance diluted a sufficient number of times in a dilution solution for the active substance to be less than about 10 ^"10 moles / l, or no longer present.

To date, there is no process ensuring on the one hand that the dilution is correct, and on the other hand that there is no contamination during the preparation of the high dilution solutions.

However, the preparation of high dilution solutions is a particularly important stage in the manufacture of homeopathic medicines. Given the minute quantities of active substance used, it is essential to avoid the presence of any impurity or pollution. Indeed, if any impurity slipped during a dilution, it would gradually take on subsequent dilutions almost equal to the active substance itself and could then counteract its therapeutic effect. It is with this in mind that certain laboratories prepare dilutions in an enclosure with a strictly purified and controlled atmosphere. However, to date, the question has never been asked of controlling qualitatively and quantitatively the dilutions obtained from an initial solution as well as the possible presence of contaminants. Gold, the invention aims to pose the problem and provide a solution.

Assuming that the problem of quality control of dilutions has been posed (which has never been done), one might have thought of controlling dilutions by directly dosing the active substance, but, in this regard, it it should be remembered that the usual biochemical methods do not allow dosing of quantities less than 10 ^{* 6} ! *. By very sensitive systems and according to the active substances to be assayed, it may be possible to detect said active substances up to the concentration of 10 ^"12 M.

If a dosage up to 10 ^"12 M is possible for certain active substances, for example immunoglobulins for which antibodies are available, it is not for most substances used in therapy. In addition, direct dosing of the active substance would require the development and implementation of a specific dosage for each of the substances, which would imply a methodology almost impossible to implement on an industrial scale, since it would be necessary to directly dose 1000 or 2000 products.

The subject of the invention is a process for the qualitative control of high dilution solutions.

The invention also relates to a method for quantitative control of high dilution solutions.

The subject of the invention is a method for controlling the contamination of high dilution solutions.

These various aspects of the invention are carried out by a method for controlling the dilution and the contamination of a determined dilution solution originating from an initial solution containing an active substance, method in which the initial solution undergoes successive dilutions, the first dilution of the initial solution being obtained by taking a fraction or all of the initial solution, and mixing this fraction or all of the initial solution in a solution of dilution, which gives the first dilution solution, the second dilution of the initial solution being obtained by taking a fraction or all of the first dilution solution, and mixing this fraction or all of the solution of first dilution in a dilution solution, to give the solution of second dilution and so on, until the solution of the last dilution,

each solution ranging from the first dilution solution to the last dilution solution constituting a determined dilution solution,

- each determined dilution solution undergoes vigorous stirring,

the successive dilutions being such that at least one of the determined dilution solutions contains the active substance in an amount less than 10 ^"10 moles, advantageously less than 10 ^{" 12} moles / 1 and preferably less than ^10'14 moles / 1, or no longer contains any active substance, said determined dilution solution still having activity at dilutions greater than that at which the active substance has disappeared, characterized in that:

- Introducing, before at least any of the dilutions n, n being an integer greater than 0, in the dilution solution n-1 a control substance soluble in said solution and not interfering with the dilution solution n -1, and the control substance having the property of disappearing between the dilution n - l + m and n + m, m being the number of dilutions in which the control substance is present, and being in particular from 5 to 8,

- the control substance is assayed at least once after dilution n, preferably at least once in the interval from dilution n to dilution n - 1 + m and at least once in 1 • interval from dilution n + m at dilution n + m + y, y being the range of dilution free of control substance and advantageously being from 3 to 5,

- and from dilution n to dilution n - l + m, the value of the concentration of the control substance obtained and the value of the concentration of the control substance calculated according to the dilution are compared, which allows quantitative control dilutions, and

- from the dilution n + m to the dilution n + m + y, it is checked that there is no longer any control substance, which makes it possible to control on the one hand the quality of the dilutions and on the other hand the absence of contamination.

The invention lies in the use, as a control substance, of a substance on the one hand which is easily detectable and on the other hand which is detectable until it disappears. Advantageously, an enzyme detectable by its chromogenic activity is used.

The presence of the control substance in the first dilutions and its absence in the extreme dilutions (that is to say dilutions greater than the fourteenth dilution and in particular the twenty-third dilution) which are nevertheless active, affirm the relationship between the phenomenon observed and the mechanism of high dilutions. We start with an initial solution containing an active substance and dilute it a first time using a dilution solution, which leads to a first dilution solution, which in turn is diluted, to give a second dilution solution, and so on to give successive dilutions, up to the last dilution solution. Each of the dilutions leads to a determined dilution solution.

The method of the invention is such that it makes it possible to check at each dilution (which gives rise to a determined dilution solution), whether the dilution has been correctly made and if there is no contamination. An incorrect dilution could, for example, consist of forgetting a previous dilution or introducing a drop, containing for example the active substance at the last detected dilution, or using an incorrectly rinsed pipette , which at such dilutions may change everything.

The method is such that it makes it possible to control each of the dilutions and when the control substance is still present, to quantify the dilution by comparing the theoretical calculated quantity of control substance and the quantity actually found. When there is no longer any control substance, there is also no longer any active substance since the control substance disappears after the active substance. After a certain number of dilutions one should expect not only the absence of active substance, but also the absence of control substance.

As indicated above, the dilutions will be identified by first dilution, second dilution, third dilution, umpteenth dilution or even dilution 1, 2, 3, 4, ... n. Usually, the last dilutions are dilutions 30 or 40 (decimal).

These dilutions can be made on any scale, in particular decimal or centesimal.

In all of the above and in everything that follows, the figures correspond, unless otherwise indicated, to decimal dilutions. However, the term "dilution" can apply to centesimal dilutions. As and when dilutions, the diluted solution will be such that it contains 10 ^"10 moles / 1, then 10 ^{" 12} moles / 1, then an amount less than 10 ^"u moles / 1. Generally beyond this molar concentration per liter there are no more molecules in the solution, which does not prevent said dilute solution from still showing, and in an entirely unexpected way, an activity.

In the process of the invention, the control substance can be introduced before any dilution, that is to say in any determined dilution solution.

By "control substance which does not interfere with the dilution solution n - 1" is defined a control substance such that its presence has no effect on the possible activity of the dilution solution n - 1.

When the control substance has been introduced at dilution n - 1 and a first dilution of the dilution solution n - 1 containing the control substance is carried out, the dilution is then continued until it reaches a dilution at which the control substance disappears. The method of the invention involves at least one assay of the control substance after dilution n. This assay preferably takes place at least once in the interval from dilution n to dilution n - 1 + m, that is to say at least once from the first dilution of the control substance to dilution. at which the control substance disappears.

Over the interval from dilution n to dilution n - 1 + m of the control substance, it is possible in principle to make a quantitative assay since it is possible after each of the dilutions from n to n - 1 + m to dose the control substance and compare it with the corresponding theoretical calculated value. When the control substance is such that it is no longer present in any given dilution solution, it is also advantageous to make a determination, for example on the three dilutions which follow that from which the control substance has disappeared, dilution for to which no control substance is added.

This confirms that there has been no contamination compared to the dilution at which it is found that there is no longer any control substance. In this case, it is no longer a quantitative control but a qualitative control allowed by the absence of the control substance, the control substance then behaving like a negative control.

According to another preferred embodiment of the invention, the control substance is introduced at least twice, one of the two introduction of the control substance being carried out in the dilution solution n - 1, the control substance disappearing between the dilution n - l + m and n + m, the other introduction is carried out as soon as possible in the dilution solution n + m, and preferably in the dilution solution n + m + y, n being an integer greater than 0 , m being advantageously included from 5 to 8, being advantageously included there from 3 to 5.

This process corresponds to the fact that the control substance is introduced a first time into the dilution solution n - 1, diluted until a dilution solution in which the control substance has disappeared and without adding to new control substance before it has disappeared, otherwise the assay performed on each dilution of dilution solution n to the dilution in which the control substance has disappeared would no longer make sense.

We therefore wait until the control substance has disappeared before adding it again. We can add to again of the control substance at the dilution which immediately follows that for which the control substance has disappeared, but advantageously, the control substance is added again from 2 to 10 dilutions which follow the dilution at which the control substance has disappeared for the first time, and advantageously from the third, fourth or fifth dilution which follows the dilution corresponding to the disappearance of the control substance.

We can thus repeat the process of introducing the control substance whenever it has disappeared or while waiting for 3 dilutions to 5 dilutions after the dilution from which the control substance has disappeared.

In practice, according to the method of the invention, the control substance can be reintroduced at regular intervals, and it suffices for example to be able to detect the control substance over 3 or 4 dilutions, to show that the decrease in the control substance is regular ( therefore that the dilutions are correct) and that outside the areas where the control substance is detectable, there is no contamination.

According to one embodiment of the process of the invention, the control substance is introduced for the first time into the dilution solution n - 1, then the control substance is reintroduced into the dilution solution which follows that which corresponds to the disappearance of the control substance introduced in dilution n - 1, then the control substance is reintroduced a second time in the dilution solution which follows that which corresponds to the disappearance of the reintroduced control substance and so on.

According to another embodiment of the process of the invention, the control substance is introduced for the first time into the dilution solution n - 1, the control substance disappearing between the dilution n - l + m and n + m, m being included in particular from 5 to 8, the control substance is reintroduced into the dilution solution n + m + y, including being from 2 to 10, advantageously from 3 to 5, and and so on.

According to another embodiment, the control substance is introduced for the first time into the dilution solution n - 1, then all the m dilutions, m being comprised from 5 to 15, advantageously from 10 to 15, and advantageously still 10 or 15 .

According to an advantageous embodiment, the method of the invention is such that the initial solution undergoes successive dilutions, the first dilution of the initial solution being obtained by sampling a fraction or all of the initial solution, and the mixing this fraction or all of the initial solution in a dilution solution, which gives the first dilution solution, the second dilution of the initial solution being obtained by taking a fraction or all of the solution first dilution, and mixing this fraction or all of the first dilution solution in a dilution solution, to give the second dilution solution and so on, until the solution of the last dilution,

- each determined dilution solution undergoes vigorous stirring,

the successive dilutions being such that at least one of the determined dilution solutions contains the active substance in an amount less than ^10'10 moles, advantageously less than 10 ^"12 moles / 1 and preferably less than 10 ^{" 14} moles / 1, or does not contain no more active substance, said determined dilution solution still having activity at dilutions greater than that at which the active substance has disappeared, characterized in that:

- Introducing, before at least any of the dilutions n, n being an integer greater than 0, in the dilution solution n-1 a control substance soluble in said solution and not interfering with the dilution solution n -1,

* the control substance having the property of being detectable at dilutions greater than that from which the active substance is no longer detectable, and

* the control substance also having the property of disappearing between the dilution n - l + m and n + m, m being the number of dilutions in which the control substance is present and being included in particular from 5 to 8,

- the control substance is assayed at least once after dilution n, preferably at least once in the range from dilution n to dilution n - 1 + m and at least once in the range from dilution n + m at dilution n + m + y, y being the range of dilution free of control substance and advantageously being from 3 to 5,

- and from dilution n to dilution n - 1 + m we compare the value of the concentration of the control substance obtained and the value of the concentration of the control substance calculated according to the dilution, which makes it possible to quantitatively control the dilutions, and

- from the dilution n + m to the dilution n + m + y, it is checked that there is no longer any control substance, which makes it possible on the one hand to control the quality of the dilutions and on the other hand absence of contamination. The control substance is endowed with the property to be detected at higher dilutions than that from which the active compound is no longer detectable, that is to say if _T was introduced into the initial solution before first dilution of the starting solution it would disappear at a dilution greater than that at which the active substance is no longer detectable.

To fix the ideas, taking into account the technical means available to date, a substance is detectable by the usual biochemical means up to an amount of approximately 10 ^"6 moles / 1.

In some cases, the active substances can be detectable up to 10 ^{* 12} moles / 1. However, this implies very sensitive detection systems.

As for the control substance, in general, it is detectable up to approximately 3 × ¹⁰ -10 × 3 ⁻¹⁰ × ¹¹ moles / 1, which corresponds to dilution 7 when the initial concentration is approximately 0.1 approximately 10 mg / 1, especially around 1 mg / ml. The quantity in moles / 1, up to which the control substance is detectable corresponds to the limit from which it is considered that there is no longer any control substance.

However, the active substances which can be used in the process of the invention are detectable at concentrations of approximately 1x10 ³ to 1x10 ⁶ mol / l, that is to say up to approximately the third decimal dilution, for a initial concentration of about lxlO ^"3 moles / 1.

According to an advantageous embodiment of the process of the invention, the control substance is introduced at the dilution n - 1, and disappearing between the dilution n - l + m and n + m, it is reintroduced at the dilution n + m + y the interval m + y + 1 being such that. - over approximately one of the two halves of this interval the dilutions are such that the corresponding dilution solutions are not active and

- over approximately the other half of this interval, at least one of the corresponding dilution solutions is active.

Represented in Figure 1

- in dotted lines the variation in the activity of the dilution solution as a function of the dilution, in solid lines the variation in the initial concentration of the control substance added at the start as a function of the dilution, and

- in short dotted lines - long dotted lines, the variation in initial concentration of the active substance as a function of the dilution solution.

To fix this, the initial concentration of the control substance is approximately 1 mg / ml, and that of the active substance is approximately 1 mg / ml.

In this FIG. 1, given by way of nonlimiting illustration, the control substance disappears between dilution 7 and 8, the active substance disappears between dilution 4 and 5, the dilution interval over which the control substance is present is 0 at 8 dilutions. On the half of this interval, that is to say from 0 to 4 dilutions, we note that the solution has a certain activity while on the other half of the interval, that is to say of the fourth at the eighth dilution, the solution no longer shows activity. Over the interval from 0 to 4 dilutions, the control substance is such that there is activity in the corresponding dilution solution and over the interval from 4 to 8 dilutions the control substance is such that there is no only no activity of the corresponding dilution solutions.

According to another embodiment, the control substance has the property that if it is introduced into the initial solution before the first dilution of the starting solution and if the active substance disappears (is no longer detectable) between dilution p and dilution p + 1, the control substance disappears between dilution p + x and the dilution p + x + 1, x being understood in particular from 2 to 4, p being understood in particular from 3 to 6.

In the above definitions, m corresponds to p + x, when the control substance is introduced into the initial solution before the first dilution of the starting solution.

According to another embodiment of the invention, the method comprises the following steps:

the control substance is introduced into the initial solution containing the active substance before the first dilution of said initial solution,

the successive dilutions of the initial solution containing the active substance and the control substance are carried out, the first dilution of the initial solution being obtained by taking a fraction or all of the initial solution, and mixing this fraction or of the whole of the initial solution in a dilution solution, which gives the solution of first dilution, the second dilution of the initial solution being obtained by taking a fraction or all of the solution of first dilution, and the mixing this fraction or all of the first dilution solution in a dilution solution, to give the second dilution solution and so on, until the solution of the last dilution, the active substance disappears between dilution p and the dilution p + 1 and the control substance disappears between the dilution p + x and the dilution p + x + 1, p being between 3 and 6, x being between 2 to 4, the solution still having activity for at least one dilution greater than or equal to the dilution p + 1,

- After the first dilution, a sufficient quantity of solution is taken from the solution obtained at the end of said dilution for at least a given dilution, and preferably the control substance is dosed at each dilution from dilution 1 to dilution p + x and preferably at least once from dilution p + x + 1 to dilution 1 + p + x + y, being there advantageously from 2 to 10, advantageously from 3 to 5 , and advantageously also at each dilution from dilution p + x + 1 to dilution 1 + p + x + y,

- and from dilution 1 to dilution p + x, the value of the concentration of the control substance obtained and the value of the concentration of the control substance calculated according to the dilution are compared, which makes it possible to quantitatively control the dilutions and from the dilution p + x + 1 to the dilution 1 + p + x + y, it is checked that there is no longer any control substance, which makes it possible to check on the one hand that there is no contamination and on the other hand the quality of the dilutions.

According to another embodiment, the control substance is introduced for the first time before the first dilution, then the control substance is introduced for the second time into the dilution solution which follows that at which the control substance introduced for the second time disappears, then the control substance is introduced a third time into the dilution solution which follows that which corresponds to the disappearance of the control substance introduced the second time, and so on.

Advantageously, each introduction of the control substance takes place from 2 to 10, advantageously 3 to 5 dilutions after that which corresponds to the disappearance of the control substance previously introduced.

According to another embodiment, the control substance is introduced before the first dilution and all the m dilutions, m being comprised from 5 to 15, advantageously from 10 to 15, and advantageously still 10 or 15.

According to another embodiment, the control substance is introduced for the first time into the initial solution, then every ten dilutions starting from the initial solution.

According to another embodiment, the control substance is assayed advantageously every 10 dilutions, and preferably at each dilution.

According to another embodiment, the initial solution contains an active substance at a rate of approximately 1x10 ^-3 to approximately 1x10 ^-6 moles / 1.

According to another embodiment, the control substance consists of an enzyme, the presence of which is advantageously detectable by its chromogenic activity.

According to another embodiment, the enzyme is chosen from peroxidase, is in particular horseradish peroxidase, detectable in particular by its reactivity with the substrate D-phenylene dia ine in H ₂ 0 ₂ medium.

The initial solution and the dilution solution are aqueous solutions, of alcohol-water mixture, of pure alcohol, or of glycerine, and advantageously aqueous solutions.

The determined dilution solutions controlled as to their dilution and their contamination according to the process of the invention can be used as such as a finished medicament, or can be used as active solutions, for the manufacture of galenic homeopathic compositions. solid and manufacturing of any preparation at high dilution (beyond lxlO ^'10 M) for experimental and clinical pharmacological purposes (in animals and humans, but also in plant biology).

With regard to solid homeopathic pharmaceutical compositions, mention may be made of granules or globules, which are made active by impregnation in a determined dilution solution controlled as to its purity and their contamination according to the process of the invention.

The invention also relates to a process for obtaining a dilute solution of medium activity, characterized in that successive determined dilution solutions are mixed, each determined dilution solution being of different dilution, but corresponding to the same scale. dilution.

The determined dilution solutions can be used either individually, in particular for fundamental pharmacology studies, or after mixing them. By mixing several determined dilution solutions, for example from 2 to 20 successive dilution solutions (preferred number of mixed dilutions: 10), corresponding to the same dilution scale (i.e. only decimal or only centesimal, etc. ...), a dilute solution is obtained which will be designated hereinafter by "mixture" which, experimentally, has a medium activity. This process, consisting in using mixtures of successive determined dilution solutions, therefore makes it possible to overcome the drawback of using individual determined dilution solutions for which it cannot always be foreseen whether they will be active or not, in favor of an average activity, but more constant.

The invention also relates to a method for increasing the activity of a solution of determined dilution characterized in that a determined dilution solution is subjected to a single high dilution.

The invention also relates to a method for increasing the activity of a dilute solution (mixture) obtained as indicated above, characterized in that said mixture is subjected to a single high dilution.

A determined dilution solution or a mixture obtained as indicated above can be used as it is in experimental pharmacology or in therapy. However, the activity of this determined dilution solution or of this mixture can be considerably increased by carrying out a very high single dilution, that is to say a small amount of a determined dilution solution or of the above mixture, diluted all at once in a large volume of diluent liquid (water, water-alcohol, alcohol, etc.), for example 10 microliters in 10 ml with vigorous stirring. Maximum biological activity is thus obtained. The single dilution level should be adapted to the activity of the starting substance and to the nature of the diluted substance. The level of dilution is at least 100, but it can be diluted in one go without upper limit, this being indicated only by the material possibility of distributing a very small volume in a very large one. In practice, it can be from 100 to 10 ⁵ . For example, a factor of 1 x 10 ⁸ requires diluting one microliter in one hundred liters. The best dilution zone must therefore be determined in each case according to the sensitivity of the biological system and the nature of the substance to be diluted.

The mixtures of successive determined dilution solutions indicated above can be obtained from controlled determined dilution solutions as for their dilution and their contamination in accordance with the process described above.

The increase in activity of a determined dilution solution or of a mixture as described above can be carried out using determined dilution solutions controlled as to their dilution and their contamination according to the method described above. .

EXAMPLE I:

This example relates to controlling the activation and inhibition of achromasia of human basophils, using the method of the invention for controlling the dilution and contamination of a solution from an initial solution. , which undergoes successive dilutions.

More specifically, in this example, the effect of the high dilutions of an anti-IgE antibody on human basophils is verified, the high dilution solutions being controlled as to their dilution and their contamination according to the method of the invention. I- BLOOD SAMPLING:

It is performed in subjects with no recognized allergies, neither seropositive nor hepatitis-positive.

Twenty ml of blood from these donors is collected in two glycerin glass tubes, each containing 250 μl of anticoagulant thus prepared:

- Mix (1: 1) two solutions of EDTA-Na ₂ and EDTA-Na ₄ (Merck, Darmstadt, RFA) 0.2 M, pH 7.40.

* EDTA-Na ₂ (MW = 372.24): 3.7 g dissolved in 50 ml of heated distilled water,

* EDTA-Na ₄ (PM≈452.24): 4.5 g dissolved in 50 ml of cold distilled water.

- To 100 ml of the mixture, heparin without phenol (Choay, Paris, France) is added to the concentration final 40 U / ml (ie 4000 U in 100 ml of EDTA solution).

The tubes containing the anticoagulant (250 μl / tube) are prepared in advance and stored at +4 ^• C. II- PREPARATION OF THE BUFFERS:

There are two buffers:

- a washing buffer, not containing calcium, necessary for the preparation of the cells;

- a dilution buffer, containing calcium, necessary for the preparation of dilutions.

These two buffers are prepared extemporaneously from the following Tyrode buffer stock: 1. Tyrode buffer buffered with HEPES: "Tyrode- HEPES"

The products are dissolved hot by stirring in ultra-pure water (obtained after treatment by a reverse osmosis machine and filtration). The pH is adjusted to 7.40 with 5N NaOH and IN. The buffer is then filtered (2 μm filter, Costar, Cambridge, USA) in a sterile hood and stored at +4 ^β C for 10 days maximum. Source of products:

KCl, NaCl, Glucose, EDTA-Na ₄ are reagents for cell culture. Sigma Chemical Company, Saint Louis, Missouri, USA. HEPES, Seromed •, Biochro KG, Berlin, RDA.

2. The washing buffer:

It is the Tyrode-HEPES buffer brought to room temperature and adjusted to pH 7.40 extemporaneously.

3. The dilution buffer:

It is the preceding Tyrode's buffer, brought to ambient temperature and adjusted extemporaneously to pH 7.40 after addition of calcium. a) CaCl stock solution _? 220 mM:

1.62 g of CaCl ₂ 2H ₂ 0 in 50 ml of Tyrode-HEPES buffer at pH 7.40. Storage takes place at + 4 ° C. b) Dilution buffer:

The final concentration in the dilutions is llmM: 5 ml of CaCl ₂ 220 mM qs 100 ml of Tyrode-HEPES buffer. Adjust to pH 7.40 with NaOH IN or 0.1N. III- PREPARATION OF THE RANGES OF ANTI-IqE DILUTIONS (TEST), ANTI-IqG AND ULTRA-PURE DISTILLED WATER (CONTROLS):

The dilutions of ultra-pure distilled water are not systematically prepared and tested for all the experiments.

1. Anti-IqE and anti-IgG antisera:

It is a goat anti-human IgG antiserum (specific Fc, GAHu / IgG (Fc)) and a goat anti-human IgE antiserum (specific Fc, GAHu / IgE (Fc)) (Nordic Immunology , Tilburg, The Netherlands) with an antibody concentration of 1 mg / ml.

The lyophilized antisera are taken up in 1 ml of ultra-pure distilled water then aliquoted into eppendorf tubes (15 μl / tube) and stored at -20 "C. The antibody concentration is 1 mg / ml.

2. Dilution of antisera and ultra-pure distilled water:

The dilutions are made under the control of a researcher foreign to the laboratory and responsible for the statistical processing of the results (INSERM U292).

They are performed in a laminar flow hood on a Gilson 222-401E Programmable Logic Controller (Gilson Médical Electronics, France) using new sterile tubes drawn by lot of 5 ml in polypropylene (Greiner). The dilution buffer used is Tyrode-HEPES buffer containing 11 mM calcium and adjusted to pH 7.40.

First dilute the ultra-pure distilled water, then the anti-IgG, then the anti-IgE. A rinse cycle is programmed at the beginning and at the end of each of the dilution ranges. These comprise 29 tubes ranging from the 1 × 10 ² dilution to the 1 × 10 ³⁰ dilution of ultra-pure distilled water or of the starting anti-IgG or anti-IgE antiserum solution.

As an enzymatic tracer making it possible to judge the good practice of the dilutions, peroxidase (Sigma) is added at the same time as the distilled water or the anti-IgG or anti-IgE antiserum. The peroxidase solution was prepared at 1 mg / ml, aliquoted in eppendorf tubes (15 μl / tube) and stored at -20 ° C. Realization of a range of dilutions:

The 29 tubes of the range, empty and bearing the number corresponding to their dilution marked with felt, are placed on the rack of the Gilson automatic device.

In the first tube, corresponding to the dilution 1 x 10 ² , 10 μl of distilled water or anti-IgG or anti-IgE (1 mg / ml) and 10 μl of peroxidase (1 mg / ml) are added to 980 μl of Tyrode buffer containing 11 mM calcium (dilution buffer). The tube is closed and shaken for 30 sec on a Vortex.

The 1 x 10 ² dilution made manually is replaced on the rack and the automatic dilution program is then started. After a rinse cycle with the dilution buffer, a 500 μl syringe (stainless steel plunger) withdraws 100 μl of the 1 x 10 ² dilution and draws 400 μl of the dilution buffer. The whole is rejected in the following tube, corresponding to the dilution 1 x 10 ³ . Five hundred μl of dilution buffer are still aspirated and discharged into the 1 x 10 ³ dilution tube, which rinses the syringe. Stirring of the dilution is ensured by a suction-delivery of 500 μl of air, 5 times in succession, at the maximum delivery speed. The needle then takes 100 μl of the 1 x 10 ³ dilution, aspirates 400 μl then 500 μl of dilution buffer according to the same process as above to obtain the 1 x 10 ⁴ dilution and so on.

At a dilution of 1 x 10 ³⁰ , the device stops automatically and starts a rinsing cycle. A new range can then be implemented. Diagram of the automatic dilution process of the anti-IgE antiserum:

This diagram is the subject of FIG. 2, in which the automatic dilution method has been represented in which the stirring steps mentioned by "bubbling" are carried out in accordance with the method of the invention and in which the step of shaking following the first dilution is done manually on an eccentric shaking device.

3. Coding of the tubes of the dilutions of distilled water and of antisera:

During an "activation" experiment, we test: - 1 x 10 ² to 1 x 10 ⁴ weight dilutions of anti-IgE and of the anti-IgG and / or distilled water control;

- high dilutions 1 x 10 ²¹ to 1 x 10 ³⁰ (beyond the limit number of molecules calculated thanks to the number of Avogadro) of anti-IgE and of the anti-IgG and / or distilled water control. Any part of the dilution range beyond the limit given by the number of Avogadro can be used;

- the internal controls controlling the calcium sensitivity of basophils and corresponding to Tyrode buffers without calcium and Tyrode with calcium. a) Realization of the code: In this "activation" protocol, all the tubes are tested blind.

The researcher foreign to the laboratory and controlling the performance of the experiments assigns to each of the tubes, according to a randomization table: a number between 1 and 30 when the experiment compares the effectiveness of the dilutions of anti-IgE and anti IgG;

- a number between 1 and 43 when 1 ^• experience compares the efficacy of dilutions of distilled water, anti-IgG and anti-IgE.

To do this, the numbers corresponding to the dilutions and marked with felt on the tubes are erased with alcohol and labels with a code number are stuck on the tubes.

Example: in the case of a code between 1 and 30, the coded tubes will be:

- tubes of 1 x 10 ² to 1 x 10 ⁴ dilutions of anti-IgG (3 tubes) and anti-IgE (3 tubes);

- tubes of 1 x 10 ²¹ to 1 x 10 ³⁰ dilutions of anti-IgG (10 tubes) and anti-IgE (10 tubes); - the internal control tubes: 1) dilution buffer, Tyrode with calcium (2 tubes); 2) washing buffer, Tyrode without calcium (2 tubes). b) Split the coded range from 1 to 30 or from 1 to 43:

200 μl (Pipetman 200) are taken from each of the coded tubes, which are placed in 1 ml aggregameter tubes. The tubes are blocked and taken away by the person who made the code for a possible, subsequent and independent control assay, immunoglobulins which may be contained in the dilutions (assay by electrophoresis on polyacrylamide gel) or any other appropriate control (mass spectrometry etc ...). IV- CELLULAR PREPARATION:

Before proceeding to obtain a suspension enriched in basophils from the blood collected on the heparin-EDTA anticoagulant (paragraph I), the number of basophils present per mm ³ of whole blood of the subject is determined.

1. Coloring and counting of basophils on whole blood:

Basophils are counted thanks to their metachromatic coloring property with toluidine blue. a) Coloring solution: toluidine blue:

One hundred mg of toluidine blue (Toluidine Blue, CI N'52040 C ₁₅ H ₁₆ CIN ₃ S, PM = 305.84, Fluka, Mulhouse, France) are dissolved in 100 ml of 25% ethanol and adjusted to pH 3, 20-3.40 with 80-100 μl of glacial acetic acid. The solution is stored at room temperature in a tightly closed bottle and protected from light. b) Coloring: It is performed by mixing 90 μl of dye and 10 μl of whole blood in the round bottom well of a microtiter plate (Costar). The mixture is immediately and gently stirred by 5 to 6 aspirations and discharges using the pipette (Pipetman 200) used to deposit the dye. c) Basophil count:

Five to 10 minutes after mixing the blood and the dye, the latter is again gently agitated and immediately deposited in a Fuchs-Rosenthal chamber (3.2 mm ³ ) using a pipette (Pipetman 200) .

The slide is placed in a humid atmosphere (in a closed and humidified box) to prevent it from drying out. After 3 to 5 minutes corresponding to the time necessary for the colored suspension to be deposited in the chamber of the hemocytometer, the counting is carried out on an Olympus microscope at magnification G × 10 × 20.

Basophils are the only cells with a colored cytoplasm. They appear red and are very easily identified on a pale background. In case of doubt, it is necessary to modify the focusing so as to clearly distinguish the cytoplasms colored in pink-red of the basophils from those of the other cells which remain transparent. The nuclei of other leukocytes are slightly colored blue.

Generally, on whole blood, there are on average 7 to 15 basophils per Fuchs-Rosenthal chamber, their number possibly ranging from 2-3 to 30-35.

2. Obtaining a suspension enriched in basophils:

When the 10 μl necessary for counting basophils on whole blood are taken, Dextran T500 4,% (Pharmacia, Uppsala, Sweden) is added at the rate of one volume per five volumes of blood. The tubes are tilted and as the sedimentation takes place (approximately 20 min at 1 xg at room temperature), plasma rich in leukocytes is collected as well as red blood cells in suspension. The presence of the latter reinforces the coloration of basophils by toluidine blue (empirically observed in the laboratory during experimental tests).

The sedimentation is collected in 2 plastic tubes of 10 ml to which washing buffer is added (Tyrode-HEPES without calcium, pH 7.40). After centrifugation (150 x g, 10 min), the leukocyte pellets (+ red blood cells) are combined in a single tube, suspended in 10 ml of washing buffer and centrifuged again (150 x g, 10 min). The plasma rich in leukocytes is initially separated into 2 tubes in order to better wash the cells.

The pellet is finally taken up in an aliquot of the same buffer, between 400 and 900 μl approximately, depending on the number of wells to be deposited (10 μl of suspension x times the number of wells + 40 to 60 μl additional for losses on the walls of the tube).

FIG. 3 shows the diagram of cell preparation.

V- PROTOCOL FOR THE ACTIVATION OF HUMAN BASOPHILES BY ANTI-IGE (ANTI-IGG OR DISTILLED WATER FOR CONTROL):

After the preparation of the anti-IgG and anti-IgE antiserum dilutions (and, for fifteen experiments, dilutions of distilled water) and their coding, after obtaining the suspension enriched in basophils, the test is carried out properly. says, under laminar flow hood. The process is identical for the 30-tube code and the 43-tube code. 1. The protocol:

- Ten μl of washing buffer (Tyrode without calcium) are deposited at the bottom of 30 (or 43) round bottom wells of a sterile microtiter plate (Costar), avoiding the peripheral wells where the risks of contamination and evaporation are larger,

- twenty μl of each of the coded dilutions (code from 1 to 30 or from 1 to 43) are then deposited at the bottom of these same wells,

- the plate is preincubated for 5 min at 37 ° C., under tape and cover to avoid the evaporation of the contents of the wells,

- ten μl of enriched suspension are then added,

- The plate is then gently agitated by slow rotational movements in order to homogenize the content of each of the wells; it is important that this agitation is gentle in order to avoid any contamination from one well to another,

- the plate is then incubated for 15 min at 37 ° C, under tape and cover to avoid any evaporation,

- after incubation, 90 μl of toluidine blue are added to each of the wells and immediately agitated by 5 to 6 aspirations and discharges with a multichannel pipette. We change cones between each row of wells,

- after staining, the plate is taped and kept overnight at +4 ^β C before reading. On cells, washed, the coloration is more homogeneous after several hours.

2. Plate diagram:

Example: in the case of a code from 1 to 30:

t 10 μl washing buffer (Tyrode without Ca ⁺⁺ )

+ 20 μl coded dilutions (1 to 30)

PREINCUBATION 5 min at 37 ^β C

+ 10 μl suspension rich in basophils (+ red blood cells)

INCUBATION 15 min at 37 * C

+ 90 μl toluidine blue

CONSERVATION one night at +4 ^β C then READING

3. Reading under an optical microscope. Basophil count:

Basophils are counted the next day 1 • experiment by the same person who prepared the cells the day before. The technique is identical to that of counting basophils in whole blood (paragraph IV-1-c).

When the number of basophils is large (greater than 100 over an entire Fuchs-Rosenthal chamber), it is possible to read (for all wells) only a half-chamber. If more than 150 basophils appear on a half-chamber of Fuchs-Rosenthal, it is preferable to deposit the contents of the wells in the chamber of a hemocytometer of Malassez (1 mm ³ ).

It takes three to five minutes to count the basophil content of a hemocytometer chamber. A trained experimenter can prepare 3 to 4 hemocytometers at the same time provided they are stored in a box moistened to prevent them from drying out. In case of doubt about an account, it is possible to redeposit the contents of a well in a room of Fuchs-Rosenthal to proceed to a new account. But it is recommended not to repeat this operation more than twice for the same well because it seems that repeated withdrawals with agitation can damage the sample and then lead to erratic results.

The numbers of basophils are given in a table like the diagram on the plate.

4. Quality control of dilutions of anti-IgG and anti-IgE antisera: Peroxidase assay:

The peroxidase is assayed on the same day of the experiment, independently, by the second person taking part in the protocol and who has not experienced it on that day. Its purpose is to control the dilution process and to detect a possible contamination of high dilutions by weight concentrations of antiserum, contamination which would then be responsible for the biological activity observed at high dilution.

It is an assay by spectrocolorimetry at 490 nm based on the reactivity of the peroxidase with the substrate O-Phenylene-Diamine in H ₂ 0 medium. a) Preparation of the buffers and solutions:

- Citrate buffer pH 5.0

It is a mixture of 20.5 ml of a 0.1M solution of citric acid (MW = 210.1) and 29.5 ml of a 0.1M solution of sodium citrate (MW = 294, 1).

- O-Phenylene-Diamine solution (OPD):

Eight mg of OPD (Sigma) are dissolved extemporaneously in 10 ml of citrate buffer pH 5.0. The solution is stored protected from light under aluminum foil. b) The assay: In the wells of a 96-well microtiter plate with a flat bottom (Costar), successively:

- 50 μl of each of the coded dilutions (from 1 to 30 or from 1 to 43) and non-coded dilutions (lx 10 ⁵ to 1 x 10 ²⁰ ) of the ranges of distilled water, anti-IgG and anti -IgE (Pipetman 200).

- 50 μl of 8 mg / 10 ml OPD (eppendorf dispensing pipette).

- 10 μl of H ₂ 0 ₂ 30 vol. (eppendorf dispensing pipette).

There is a yellow-orange coloration of the wells corresponding to the most concentrated dilutions.

Leave the reaction to take place for 10 minutes, protected from light, under aluminum.

Add 50 μl (eppendorf dispensing pipette) of H ₂ S0 ₄ 9% (H ₂ S0 ₄ concentrated, diluted 10 times, 3% final in the well). The previously observed coloration is accentuated (ocher-orange coloration).

Read immediately at 490 nm with an automatic plate reader spectrophotometer (Dynatech Laboratories). The results are automatically saved and printed. Plate diagram: Example in the case of a code from 1 to 30:

5) The "activation" results:

The results (numbers of basophils + peroxidase assay) are given every day to the person who made the codes.

The tubes corresponding to each experiment are enclosed in a sealed and dated envelope and kept at + 4 * C, for the purpose of subsequent checks, a) Interpretation of the results:

It will be done after an independent statistical analysis, on the experiences selected according to the following criteria:

1. Number of basophils in the controls greater than 35. The controls correspond to the dilutions of distilled water, anti-IgG and to the internal controls (Tyrode buffer with and without Ca ⁺⁺ ).

2. Anti-IgE activity at a weight dose greater than 40% achromasia compared to the respective weight dilutions of distilled water or anti-IgG. (Anti-IgG by weight dose can theoretically lead to achromasia of basophils compared to the same dilutions of distilled water or compared to the "Tyrode with calcium" controls. One can invoke a recognition of the light chains of 1 • IgE by 1 anti-IgG or an anaphylactic IgG-dependent reaction).

3. In the presence of calcium alone, that is to say without the addition of anti-IgE, the number of basophils should not vary by more than 25%. This is verified by comparing the number of basophils placed in the presence of Tyrode-calciu buffer with that of the basophils placed in the presence of Tyrode buffer without calcium.

The achromasia is thus determined: Mb basos of the control well - Nb basos of the test well _κinn

No. basos of basos control well = basophils For each experience selected according to the criteria:

1) calculation of the difference between the average number of basophils counted in the wells containing the anti-IgE solution and the average the number of basophils counted in the wells containing the control solution (distilled water or anti-IgG), for all dilutions between 1 x 10 ²¹ and

1 x 10 ³⁰ .

2) also, for high dilutions of anti-IgE, research for the presence of at least one achromasia peak of 3 to 4 significant successive points according to the data in the chart (paragraph VI-2). b) Representation of results:

It is made after opening the codes, after 18 interpretable activation experiences (i.e. meeting the selection criteria).

The number of experiments required was determined after statistical analysis of the results provided by preliminary experiments.

The results are represented as follows:

Logarithmic dilutions (anti-IgE, anti-IgG or distilled water) are plotted on the abscissa while the number of basophils is plotted on the ordinate.

Each graph corresponds to an experience. It comprises :

1) a curve which represents the variations in the number of basophils based on dilutions of ^"anti-IgE;

2) one (or two) control curve (s) showing the variations in the number of basophils as a function of the dilutions of distilled water and / or of anti-IgG.

Depending on the average number of basophils obtained for the control dilutions of distilled water and for the "Tyrode with calcium" control, a significance limit is defined corresponding to the number below which the effect of anti-IgE (or anti-IgG) will be considered significant. This limit most often corresponds to around 20% of achromasia. It is given by an abacus (see Figure 4) and is shown in dotted lines on the graphs.

The lower the number of basophils compared to the average of the controls, the more significant the effect observed.

FIG. 4 shows the abacus to determine the significance of the achromasia of human basophils.

The chart indicates the significance (p <0.05) of the achromasia observed for basophils. Example: when 70 basophils are counted in the control well, at most 56 basophils must be counted in the test well for the achromasia to be significant.

VI- EXPERIMENTAL PROTOCOL OF THE MODULATION OF BASOPHILIC ACHROMASIS BY APIS MELLIFICA:

This protocol is practiced either at the same time as the "activation" protocol when the number of basophils in the donor's whole blood is sufficient (greater than 15 on a Fuchs-Rosenthal chamber), or independently of the "activation" protocol, on the blood from another donor.

1) Principle:

The modulating effect of dilutions of Apis mellifica from 15 to 20CH (Hahnemannian Centesimal) is tested compared to the corresponding control, NaCl 137 mM 20CH on 1 * achromasia of basophils in the presence of anti-IgE weight dilutions. The effect of Apis mellifica and of 137 mM NaCl is tested, as a control, on basophils put in the presence of the only anti-IgE dilution buffer, without anti-IgE. 2) Dilutions of Apis mellifica and 137 mM NaCl:

They are supplied by Boiron-L.H.F. (Lyon, France) in sterile 1 ml ampoules, in 137 mM NaCl. The contents of the ampoules are transferred to new sterile 5 ml polypropylene tubes, under a laminar flow hood. The tubes are closed progressively and agitated for 30 seconds on a Vortex.

Identical bulbs are sent to an outside laboratory in order to control the quality of the products by mass spectrometry.

3) Coding of the dilutions of Apis mellifica and of 137 mM NaCl:

In this protocol, we studied the modulating effect of the 15 to 20CH dilutions of Apis mellifica compared to a control, the 20CH dilution of 137 mM NaCl.

All these dilutions are tested blind. An arbitrary code number between 1 and 8 is randomly assigned to each dilution (6 dilutions 15 to 20CH of Apis mellifica and 2 dilutions 20CH of 137 mM NaCl) by the researcher foreign to the laboratory which controls the process and is responsible statistical interpretation of the results.

To do this, a label with a code number is stuck on each of the tubes containing the corresponding dilution. The code is changed with each new experience, new labels bearing a new number replacing the previous ones.

The coded tubes are stored from one experiment to another at +4 ^β C under aluminum foil for 2 weeks. After this time, a new procedure (transfer of the ampoules to the tubes and coding) is carried out throughout the duration of the experimental protocol.

4) The dilutions by weight of anti-IgE:

The dilutions (1 x 10 ² to 1 x 10 ⁴ ) are prepared manually in dilution buffer (Tyrode-HEPES + Ca ** 11 mM final buffer, pH 7.40), under a laminar flow hood, in sterile tubes of 5ml in polypropylene, from goat anti-human IgE antiserum (1 mg / ml of antibody) aliquot in eppendorf tubes and stored at -20 ^β C.

Ten μl of anti-IgE (1 mg / ml) are added to 990 μl of dilution buffer. The tube is closed and stirred for 30 seconds on a Vortex: the dilution 1 x 10 ² is obtained.

One hundred μl are taken from it and added to 900 μl of dilution buffer contained in a second tube. This is blocked and agitated in turn for 30 seconds on the Vortex. The 1 x 10 ³ dilution is thus obtained and the same is done for the 1 x 10 ⁴ dilution.

5) The protocol itself:

At first, therefore, were prepared:

- the dilutions of Apis mellifica,

- anti-IgE dilutions,

- the cell suspension enriched in basophils (paragraph IV).

The test :

- Ten μl of each of the 8 coded dilutions are deposited at the bottom of the round bottom wells of a sterile microtiter plate (Costar). Each dilution is deposited as many times as doses of anti-IgE to be inhibited and once for the dilution buffer. In this protocol, the coded dilutions of Apis mellifica and of 137 mM NaCl are therefore deposited 4 times (anti-IgE 1 × 10 ² , 1 × 10 ³ , 1 × 10 ⁴ ; dilution buffer). - Ten μl of suspension rich in basophils are then deposited in each of the wells.

- The plate is gently agitated by very gentle rotation in order to homogenize the content of the wells and left for 30 minutes at room temperature, under adhesive tape and cover to avoid any evaporation.

- After this preincubation time for basophils with 20 μl anti-IgE products at dilutions

1 x 10 ² , 1 x 10 ³ , 1 x 10 ⁴ and 20 μl of Tyrode-HEPES buffer containing 11 mM calcium (and without anti-IgE) are added to the wells for each of the coded dilutions of Apis mellifica and 137 mM NaCl.

The plate is gently shaken to homogenize the contents of the wells and placed for 15 minutes at 37 ° C. under adhesive tape and cover in order to avoid evaporation in the wells.

- After incubation, 90 μl of toluidine blue are added to each of the wells and immediately stirred by suction and delivery, using a multichannel pipette. The cones are changed between each row of wells.

- After coloring, the plate is covered with an adhesive tape and kept overnight at +4 ^β C before reading. The coloration of the washed cells is more homogeneous after several hours. Plate diagram:

1 2 3 4 5 6 7 8 “• - • corresponding code no.

STORAGE 1 night + 4 ° C READING

The "Tyrode without Ca **" (*) controls are added, in the same way as in the "activation" protocol, to control the sensitivity of basophils to calciua.

6) Reading under the microscope and counting basophils:

The principle is identical to that described for the activation of basophils (paragraph V-3).

7) The results:

The basophil counts are reported in a table like the diagram on the plate and are given for each experiment to the person who did the code. a) Interpretation of results:

Experiments, after decoding, are only retained for statistical interpretation if they meet 3 criteria:

REPLACEMENT SHEET 1. Number of basophils in the controls greater than 35 (basophils preincubated with 137 mM NaCl or with Apis mellifica and not having been put in the presence of anti-IgE).

2. Spontaneous sensitivity of basophils to calcium alone less than 25% achromasia by comparing on the one hand the basophils which, preincubated with 137 mM NaCl, are, in the absence of any anti-IgE, placed in the presence of buffer of Tyrode-calcium with, on the other hand, those placed in the presence of Tyrode buffer without calcium.

3. Presence of at least one dose. anti-IgE with achromasia of between 40 and 60% of basophils which, preincubated with 137 mM NaCl, are brought into contact with anti-IgE 1 × 10 ² to 1 × 10 ⁴ compared to those brought into contact with Tyrode-Ca ** buffer without anti-IgE. This is based on preliminary studies which have shown that below 40%, the basophile achromasia is too low for the inhibition study to be carried out. Above 60%, it is too strong to be able to be significantly modulated by high dilution agonists.

The basophile achromasia is thus determined: Nb basos of the control well - Nb basos of the test well _vinn

Nb basos of the control well basos = basophils b) Representation of the results: Three types of results (in number of basophils) are obtained and must be compared:

- the wells corresponding to the basophils placed in the presence of Apis mellifica or of 137 mM NaCl but without anti-IgE give the maximum number of basophils. These are the reference witnesses.

- wells corresponding to basophils in the presence of 137 mM NaCl and anti-IgE without Apis mellifica give the minimum number of basophils (maximum achromasia).

- the wells corresponding to the basophils placed in the presence of Apis mellifica and of anti-IgE give a number of basophils on which is evaluated 1 • possible modulating effect of the product. The closer this number is to the maximum number of basophils, the greater the inhibitory effect. Conversely, the closer this number is to the minimum number of basophils, the weaker or zero the inhibitory effect will be. If it becomes less than this minimum number, the effect is activating.

The graphic representation:

For each dose of anti-IgE tested, the dilutions of Apis mellifica and the control dilutions of 137 mM NaCl are plotted on the abscissa while the number of basophils is plotted on the ordinate.

Each graph includes:

1) a curve which represents the variations in the number of basophils in the presence of anti-IgE as a function of the dilutions of Apis mellifica and of 137 mM NaCl;

2) a curve which represents the variations in the number of basophils in the presence of Tyrode-Ca ⁺⁺ buffer without anti-IgE as a function of the dilutions of Apis mellifica and of 137 mM NaCl. c) Statistical analysis of the results: The modulating effect of the dilutions of APis mellifica is studied statistically by a Whitney-ilcoxon rank test after a series of fifteen independent experiments. For each dilution of APis mellifica, the number of basophils in the presence of a dose of anti-IgE will be compared with the number of basophils preincubated with 137 M NaCl and in the presence of the same dose of anti-IgE. IgE. These studies are carried out independently by the persons responsible for monitoring the experiments and for the statistical interpretation of the results.

Claims

CLAIMS 1. Process for controlling the dilution and contamination of a determined dilution solution originating from an initial solution containing an active substance, process in which: the initial solution undergoes successive dilutions, the first dilution of the initial solution being obtained by taking a fraction or all of the initial solution, and mixing this fraction or all of the initial solution in a dilution solution, which gives the solution of first dilution, the second dilution of the initial solution being obtained by taking a fraction or all of the first dilution solution, and mixing this fraction or all of the first dilution solution in a dilution solution, to give the second solution dilution and so on, until the solution of the last dilution,

- each determined dilution solution undergoes vigorous stirring,

the successive dilutions being such that at least one of the determined dilution solutions contains the active substance in an amount less than 10 ^"10 moles, advantageously less than 10 ^{" 12} moles / 1 and preferably less than ^10'14 moles / 1, or no longer contains any active substance, said determined dilution solution still having activity at dilutions greater than that at laguelle the active substance has disappeared, characterized in that: - Introducing, before at least any of the dilutions n, n being an integer greater than 0, in the dilution solution n-1 a control substance soluble in said solution and not interfering with the dilution solution n -1, and the control substance having the property of disappearing between the dilution n - l + m and n + m, m being the number of dilutions in which the control substance is present, and being comprised in particular from 5 to 8,

- the control substance is assayed at least once after dilution n, preferably at least once in the interval from dilution n to dilution n - 1 + m and at least once in the interval from dilution n + m at dilution n + m + y, y being the range of dilution free of control substance and advantageously being from 3 to 5,

- and from dilution n to dilution n - 1 + m, the value of the concentration of the control substance obtained and the value of the concentration of the control substance calculated according to the dilution are compared, which allows quantitative control dilutions, and

2. Method for controlling the dilution and the contamination of a determined dilution solution originating from an initial solution containing an active substance, process in which: the initial solution undergoes successive dilutions, the first dilution of the initial solution being obtained by taking a fraction or all of the initial solution, and mixing this fraction or all of the initial solution in a dilution solution, which gives the first dilution solution, the second dilution of the initial solution being obtained by taking a fraction or all of the first dilution solution, and mixing this fraction or all of the first dilution solution in a dilution solution, to give the second dilution solution and so on, until the last dilution solution,

- each determined dilution solution undergoes vigorous stirring,

the successive dilutions being such that at least one of the determined dilution solutions contains the active substance in an amount less than 10 ⁻¹⁰ moles, advantageously less than 10 ^{* 12} moles / 1 and preferably less than ^10'14 moles / 1, or no longer contains any active substance, said determined dilution solution still having activity at dilutions greater than that at which the active substance has disappeared, characterized in that:

* the control substance also having the property of disappearing between the dilution n - l + m and n + m, m being the number of dilutions where is presents the control substance and being understood in particular from 5 to 8,

3. Method according to one of claims 1 or 2, wherein the control substance has the property that if it is introduced into the initial solution before the first dilution of the starting solution and if the active substance disappears (is more detectable) between the dilution p and the dilution p + l, the control substance disappears between the dilution p + x and the dilution p + x + 1, x being understood in particular from 2 to 4, p being understood in particular from 3 to 6 .

4. Method according to one of claims 1 or 2, wherein the control substance is introduced for the first time into the dilution solution n - 1, then the control substance is reintroduced into the dilution solution which follows that which corresponds to the disappearance of the control substance introduced in the dilution n - 1, then the control substance is reintroduced a second time in the dilution solution which follows that which corresponds to the disappearance of the reintroduced control substance and so on.

5. Method according to claim 4, in which the control substance is introduced for the first time into the dilution solution n-1, the control substance disappearing between the dilution n-1 + m and n + m, the control substance is reintroduced in the dilution solution n + m + y, including 2 to 10, advantageously 3 to 5, and so on.

6. Method according to one of claims 1 or 2, in which the control substance is introduced for the first time into the dilution solution n - 1, then all the m dilutions, m being comprised from 5 to 15, advantageously from 10 to 15, and advantageously still 10 or 15.

7. Method according to one of claims 1 or 2, wherein

the successive dilutions of the initial solution containing the active substance and the control substance are carried out, the first dilution of the initial solution being obtained by taking a fraction or all of the initial solution, and mixing this fraction or of the whole of the initial solution in a dilution solution, which gives the solution of first dilution, the second dilution of the initial solution being obtained by taking a fraction or all of the solution of first dilution, and the mixing this fraction or all of the first dilution solution in a dilution solution, to give the second dilution solution and so on, until the solution of the last dilution, the active substance disappears between dilution p and dilution p + 1 and the control substance disappears between dilution p + x and dilution p + x + 1, p being between 3 and 6, x being between 2 and 4, the solution still having activity for at least one dilution greater than or equal to the dilution p + 1,

8. The method of claim 1, wherein the control substance is introduced for the first time before the first dilution, then the control substance is introduced for the second time into the dilution solution which follows that to which the control substance introduced for the second time disappears, then the control substance is introduced a third time into the following dilution solution that which corresponds to the disappearance of the control substance introduced the second time, and so on, and advantageously, each introduction of the control substance takes place from 2 to 10, advantageously 3 to 5 dilutions after that which corresponds to the disappearance of the control substance previously introduced.

9. Method according to one of claims 1 or 2 wherein the control substance is introduced before the first dilution and all the m dilutions, m being comprised from 5 to 15, advantageously from 10 to 15, and advantageously still 10 or 15.

10. The method of claim 2 wherein the control substance is introduced at dilution n - 1, and disappearing between dilution n - l + m and n + m, it is reintroduced at dilution n + m + y the interval m + y + 1 being such that,

- over approximately one of the two halves of this interval the dilutions are such that the corresponding dilution solutions are not active and

11. Method according to any one of the preceding claims, characterized in that the control substance is diluted, advantageously every 10 dilutions, and more preferably at each dilution.

12. Method according to any one of the preceding claims, characterized in that the initial solution contains an active substance in an amount of about lxlO ^'3 to about lxlO ^"6 moles / 1.

13. Method according to any one of the preceding claims, characterized in that the control substance consists of an enzyme, of which the presence is advantageously detectable by its chromogenic activity.

14. Method according to any one of the preceding claims, characterized in that the enzyme is chosen from peroxidase, is in particular horseradish peroxidase, detectable in particular by its reactivity with the substrate D-phenylene diamine in H ₂ 0 ₂ medium .

15. Method for obtaining a dilute solution (mixture) of average activity, characterized in that successive determined dilution solutions are mixed, each determined dilution solution being of different dilution, but corresponding to the same scale of dilution.

16. Method for increasing the activity of a determined dilution solution, characterized in that a determined dilution solution is subjected to a single high dilution.

17. A method of increasing the activity of a dilute solution (mixture) obtained according to claim 15, characterized in that said mixture is subjected to a single high dilution.

18. A method of obtaining a diluted solution according to claim 15, in which the determined dilution solutions used are checked as to their dilution and their contamination according to the method of any one of claims 1 to 14.

19. Method for increasing the activity of a determined dilution solution according to claim 16, in which the determined dilution solutions used are checked as to their dilution and their contamination according to the method of any one of claims 1 to 14.

20. Method for increasing the activity of a dilute solution (mixture) according to claim 17, in which the determined dilution solutions are checked as to their dilution and their contamination in accordance with the process of any one of claims 1 to 14.