Method for detecting molecular interactions

Abstract

The present invention relates to a method for detecting molecular interactions in a solution. In particular, the present invention relates to a method for detecting interactions between two substances that are likely to interact with one another. The present invention can be used in particular in the field of scientific research and in the field of medical analysis.

Claims

1. A method for detecting interactions in a solution comprising the following steps: a. introducing into a solution at least one first substance and at least one second substance that can interact with said first substance; b. introducing into the solution obtained in step a) at least two magnetic or magnetizable particles, said particles resting on a surface immersed in said solution, and said magnetic or magnetisable particles being free within the solution by not being bound to said first substance or said second substance; and c. determining an interaction of said first and second substances by application of an electric, magnetic or electromagnetic field designed to set said particles in motion relative to each other, the interaction between said first and second substances being detected when the mobility of said particles on said surface is changed.

2. The method according to claim 1, wherein said at least two magnetic or magnetizable particles are independently an electrically charged, magnetic or magnetizable particle or a particle covered with at least one magnetic or magnetizable layer.

3. The method according to claim 1, wherein said at least two particles are subjected to a pulsed electromagnetic field.

4. The method according to claim 1, wherein the change in the mobility is an acceleration of the movement of particles under the effect of said electric, magnetic or electromagnetic field.

5. The method according to claim 1, wherein the change in the mobility is a slowing of the movement of the particles under the effect of said electric, magnetic or electromagnetic field.

6. The method according to claim 1, wherein the change in the mobility is a change in trajectory of the particles under the effect of said electric, magnetic or electromagnetic field.

7. The method according to claim 1, wherein the interaction is detected by clustering of the particles under the effect of said electric, magnetic or electromagnetic field.

8. The method according to claim 1, wherein the interaction is detected by non-clustering of the particles under the effect of said electric, magnetic or electromagnetic field.

9. The method according to claim 1, wherein the interaction is detected by dispersion of the particles under the effect of said electric, magnetic or electromagnetic field.

10. The method according to claim 1, wherein said at least two particles are illuminated by means of a luminous source to detect their movement.

11. The method according to claim 1, wherein said at least two particles are signal generators.

12. The method according to claim 1, wherein the first substance is chosen in the group comprising eukaryotic cells, prokaryotic cells, membranes, viruses, proteins, antibodies, antigens, chemical molecules.

13. The method according to claim 1, wherein the second substance is chosen in the group comprising eukaryotic cells, prokaryotic cells, membranes, viruses, proteins, antibodies, antigens, chemical molecules.

14. The method according to claim 1, wherein the method includes, instead of step a), a prior step a′) of attaching said first substance onto a surface of a container; a″) introducing a solution into said container; and a′″) introducing at least one second substance that can interact with said first substance.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows photographs of the spots obtained with antibodies presenting (SwH) or not presenting (SwE) affinity to each other as the affinity substance. Photograph 1A was taken after 20 seconds of magnetizing, photograph 1B after 5 minutes of magnetizing.

(2) FIG. 2 shows photographs of the spots obtained with anti-blood group determinant antibodies and red blood cells as the affinity substance.

(3) FIG. 3 shows images of the spots obtained with anti-blood group determinant antibodies and red blood cells as the affinity substance.

EXAMPLES

Example 1: Detecting the Interaction of Antibodies with or without Affinity for Each Other

(4) In this example, two strips of 8 flat-bottom wells (Reference: MSW002B, BioFilm Control, France), respectively designated SwH and SwE were used. In each of the strips, 70 μl of an antibody solution were deposited in each of the wells obtained by mixing 15 μl of, respectively, either anti-human IgG antibodies (Reference BI2018, Paris Anticorps, France), or Anti-E. coli antibodies (Reference BP2298, Acris Antibodies, Germany), in 1.5 ml of PBS (8 g/l NaCl (Sigma Aldrich, USA), 200 mg/l KCl (Sigma Aldrich, USA)), 1.44 g/l Na.sub.2HPO.sub.4 (Sigma Aldrich, USA), 240 mg/l KH.sub.2PO.sub.4 (Sigma Aldrich, USA)) before placing them for 16 hours in a thermostatic oven (Reference BC240, Firelabo, France) stabilized at 37° C.

(5) Solutions designated, respectively, SIg0, SIgH and SIgE, containing 200 μl of a reagent for finding human anti-FY1 anti-red blood cell antibodies (IJB-IH Control 3, reference 108030357, Institut Jacques Boy, France), 2 μl of paramagnetic microbeads (Ton004N, BiofilmControl, France) and, respectively, either 15 μl of water for injection, or 200 μl of anti-human IgG antibodies (Reference BI2018, Paris Anticorps, France), or 200 μl of Anti-E. coli antibodies (Reference BP2298, Acris Antibodies GmbH, Germany) were prepared.

(6) The antibody solutions were then removed from the SwH and SwE strips, and then 150 μl of PBS were deposited and removed three times in each well.

(7) 100 μl of SIg0, SIgH and SIgE were respectively deposited in wells D, E and F of strips SwH and SwE. The strips were then placed for 20 minutes in a thermostatic oven (Reference BC240, Firelabo, France) stabilized at 37° C., then placed on a magnetized test block (BKT-MSW002 BioFilm Control, France) for 20 seconds. The strips were then placed in a document scanner (Perfection V-750 PRO, Epson, USA) with which a first image was taken with EpsonScan (Epson, USA) software. The magnetization/image capture cycle was reproduced a second time in order to have a second image after a total of 5 minutes of magnetization. The final images were obtained by subtracting the green component of the images from the red component of the color images obtained with the scanner by using ImageJ software (http://rsb.info.nih.gov.ij) and cutting out the images obtained with different contrast adjustments. The corresponding photographs are the images of FIG. 1.

(8) Rings of various diameters were obtained, corresponding to the accumulation of beads at the bottom of the well during their migration in the magnetic field. The ring diameter is considered as a measurement according to a decreasing law of magnetic mobility of the beads.

(9) As expected, the diameter of the ring observed in the presence of the expected affinity reaction between the immunoglobulins present in the anti-red blood cell antibody and anti-human Ig antibody research reagents (SwH strip well E) is larger than in the absence of affinity reactions between the immunoglobulins present in the research reagent for anti-red blood cell antibodies and anti-E. coli antibodies (SwH strip wells D and F) after 20 seconds of magnetization and in well E, after 5 minutes of magnetization (strips SwH and SwE wells E).

(10) As visible in FIG. 1, well E, this effect is amplified when the two affinity substances are simultaneously present in solution and adsorbed in the well, which greatly reduces the migration of the beads (strip SwH, well E) with regard to the situation, shown in FIG. 1, strip SwH, well D, where one of the substances is only present adsorbed on the surface.

(11) Moreover, when the two affinity substances, i.e., antibodies, are present in solution, if one of the substances is already adsorbed on the surface, the phenomenon of reducing the mobility of the beads is further amplified (wells E, strip SwH compared to SwE).

(12) Finally, when the affinity substance is not adsorbed on the surface, the occurrence of the affinity reaction in the solution is detectable by observing the diameter of the ring formed (strip SwE, well E, compared to wells D and F).

Example 2: Detecting the Interaction Between Anti-Blood Group Determinant Antibodies and Red Blood Cells

(13) In this example, three strips of 8 SBS flat-bottom wells (Reference: MSW002B, BioFilm Control, France), respectively designated SwA, SwB and SwAB were used. In each of the wells of the strips, 70 μl of antibody solution, respectively anti A (Reference 102010153, Institut Jacques Boy, France), anti B (102010253, Institut Jacques Boy, France) and anti AB (Reference 102010353, Institut Jacques Boy, France) were deposited.

(14) The strips were placed in a thermostatic oven (Reference BC240, Firelabo, France) stabilized at 37° C. for 10 minutes.

(15) Blood suspensions designated, respectively, hA, hB, hAB and hO, were prepared by diluting 1 ml of TS buffer comprised of 8 g/l NaCl (Sigma-Aldrich, USA) and 1 g/l of tryptone (Difco, USA) with, respectively, 100 μl of red blood cells of type A, B, AB, and O, respectively (IJB-IH Control 2, Reference 108020257, Institut Jacques Boy, France) and 6 μl of paramagnetic microbeads (Ton005N, BiofilmControl, France) and 1 μl of blue food coloring (Vahiné, France).

(16) The antibody solutions were removed from the wells before depositing 70 μl of preparation, hA, hB, hC and hO, respectively, in wells A and B, then C and D, then E and F, then G and H, respectively, of strips SwA, SwB and SwAB.

(17) The strips were placed in a thermostatic oven (Reference BC240, Firelabo, France) stabilized at 37° C. for 10 minutes, then placed on a magnetized test block (BKT-MSW002 BioFilm Control, France) for 1 minute. They were then placed in a document scanner (Perfection V-750 PRO, Epson, USA) with which an image was taken with EpsonScan (Epson, USA) software. The final image was obtained as in Example 1 in FIG. 2.

(18) As shown in FIG. 2 in wells C, D, G and H of strip SwA, in wells A, B, G and H of strip SwB and in wells G and H of strip SwAB, the formation of a dark disk was observed of a diameter of around 2 mm±1 mm which was attributed to the absence of affinity reaction between the antibodies bound to the well and the red blood cells. In the other wells, no dark shape is visible, demonstrating an affinity reaction between the antibodies bound to the well and the red blood cells. The observations are summarized in Table 1 below, where the absence of the disk is designated − and the presence of the disk is designated +:

(19) TABLE-US-00001 TABLE 1 observation of the wells of FIG. 2 A B C D E F G H (hA) (hA) (hB) (hB) (hAB) (hAB) (hO) (hO) SwA − − + + − − + + (anti A) SwB + + − − − − + + (anti B) SwAB − − − − − − + + (anti AB) Type A A B B AB AB O O deduced

(20) As demonstrated in this example, the results obtained clearly show the detection of affinity of the antibodies with the corresponding red blood cells. Furthermore, this example clearly shows that the method of the invention enables the serotype of a blood sample to be determined.

Example 3: Detection of the Interaction Between Anti-Determinant Antibodies of Anti-Red Blood Cell Antibodies

(21) In this example, two strips of 8 flat-bottom wells (Reference: MSW002B, BioFilm Control, France), designated SwD and SwK, were used. In each of the wells of the strips, 50 μl of a solution obtained by mixing 15 μl of anti-human IgG antibody (Reference BI2018, Paris Anticorps, France) in 1.5 ml of TS buffer (8 g/l NaCl (Sigma Aldrich, USA) and 1 g/l Tryptone (Difco, USA)) were deposited. The strips were then incubated for 16 hours in a thermostatic oven (Reference BC240, Firelabo, France) stabilized at 37° C.

(22) Solutions designated Sp and St, respectively, comprising 150 μl of buffer, respectively PBS and TS, and 0.75 μl of Ton005N were prepared. These solutions are control solutions without red blood cells.

(23) Suspensions designated Si, Sii and Siii, respectively, comprising 150 μl of a red blood cell suspension of type I, II and III (Reference ID-DiaCell I-II-III, Diamed, France) and 0.75 μl of paramagnetic microbeads (Ton005N, BiofilmControl, France) were also prepared.

(24) The antigens present on the surface of the red blood cells were verified by the supplier and are distributed according to Table 2 below:

(25) TABLE-US-00002 TABLE 2 Suspensions Red blood cells Duffy KEL Si Type I red blood cells + 0 Sii Type II red blood cells + 0 Siii Type III red blood cells 0 + +: presence of antigen, 0: absence of antigen.

(26) Suspensions designated SDi and SKi, SDii and SKii as well as SDiii and SKiii, respectively, comprising 100 μl of a red blood suspension of type I, II and III, respectively (Reference ID-DiaCell I-II-III, Diamed, France), 50 μl of serum, respectively anti FY1 (or anti Duffy) and anti KEL1 (IJB-IH Control 3, reference 108030357, Institut Jacques Boy, France) and 0.75 μl of paramagnetic microbeads (Ton005N, BiofilmControl, France) were also prepared (see Table 3). These suspensions are negative controls comprising red blood cells whose affinity sites are blocked by the antibodies contained in the serums, as well as red blood cell-antibody complexes that no longer have free affinity sites that can bind to the antibodies fixed to the bottom of the well.

(27) TABLE-US-00003 TABLE 3 Red blood cells Serum SDi I (D) Anti FY1 (anti D) SKi I (D) Anti KEL1 (anti K) SDii II (D) Anti FY1 (anti D) SKii II (D) Anti KEL1 (anti K) SDiii III (K) Anti FY1 (anti D) SKiii III (K) Anti KEL1 (anti K)

(28) The anti-human IgG antibody solution was then removed from strips SwD and SwK and 150 μl of TS buffer were deposited and removed twice. In each of the wells of strips SwD and SwK, 50 μl of anti-red blood cell antibody research reagents were deposited, respectively anti-FY1 (or anti Duffy) and antiKEL1 (IJB-IH Control 3, reference 108030357, Institut Jacques Boy, France). The strips were then placed in a thermostatic oven (Reference BC240, Firelabo, France) stabilized at 37° C. for 10 minutes.

(29) The solutions of anti-red blood cell antibody research reagents were then removed from strips SwD and SwK and 150 μl of TS buffer were deposited and removed twice.

(30) 70 μl of different suspensions were deposited as indicated in Table 4 below:

(31) TABLE-US-00004 TABLE 4 distribution of the deposition of the different suspensions A B C D E F G H SwD Si Sii Siii Sp St SDi SDii SDiii SwK Si Sii Siii Sp St SKi SKii SKiii

(32) The strips were then placed for 20 minutes in a thermostatic oven (Reference BC240, Firelabo, France) stabilized at 37° C., then placed on a magnetized test block (BKT-MSW002 BioFilm Control, France) for 30 seconds. The strips were then placed in a document scanner (Perfection V-750 PRO, Epson, USA) with which an image was taken with EpsonScan (Epson, USA) software. This image is used to analyse the SwK strip. The magnetization/image capture cycle was reproduced a second time so as to also have an image after a total of one minute of magnetization for the analysis of strip SwD. The final images were obtained as in Example 1 and are shown in FIG. 3.

(33) FIG. 3 shows that a perfectly defined ring is visible in control wells D and E, demonstrating a significant mobility of the paramagnetic beads, a similar ring is obtained in wells F, G and H, demonstrating that a significant mobility is conserved in the presence of red blood cells whose affinity sites are blocked by the antibodies contained in the serums.

(34) The presence of the following can be verified in wells A, B and C: either a ring comparable to those of wells F, G and H, indicating a significant mobility attributed to an absence of affinity bonds between the red blood cells and the antibodies bound to the bottom of the well, or a diffuse spot indicating a reduced mobility of the paramagnetic beads attributed to an affinity reaction between the antibodies bound to the well and the red blood cells.

(35) Rings and spots are visible according to Table 5 below:

(36) TABLE-US-00005 TABLE 5 images obtained A B C D E F G H SwD spot spot ring ring ring ring ring ring SwK ring ring spot ring ring ring ring ring

(37) The method of the invention therefore enables the molecular interaction between two substances to be detected.

Example 4: Detecting the Interaction Between Anti-Bacteria Antibodies Present in a Serum and Substances from Bacteria Cultures

(38) In this example, two strips of 8 flat-bottom wells (Reference: MSW002B, BioFilm Control, France), respectively designated SwH and SwE are used. In each of the strips, 70 μl of an antibody solution are deposited in each of the wells obtained by mixing 15 μl of, respectively, anti-human IgG antibodies (Reference BI2018, Paris Anticorps, France), and Anti-E. coli antibodies (Reference BP2298, Acris Antibodies, Germany), in 1.5 ml of PBS (8 g/l NaCl (Sigma Aldrich, USA), 200 mg/l KCl (Sigma Aldrich, USA)), 1.44 g/l Na.sub.2HPO.sub.4 (Sigma Aldrich, USA), 240 mg/l KH.sub.2PO.sub.4 (Sigma Aldrich, USA)) before placing them for 16 hours in a thermostatic oven (Reference BC240, Firelabo, France) stabilized at 37° C. (see Table 6).

(39) TABLE-US-00006 TABLE 6 A B C D E F G H SwH Anti- Anti- Anti- Anti- Anti- Anti- Anti- Anti- human human human human human human human human SwE Anti- Anti- Anti- Anti- Anti- Anti- Anti- Anti- E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli

(40) The IgG solution is then removed from strips SwH and SwE and 150 μl of TS buffer are deposited and removed twice. Deposited in each of the wells A, B, C, D, and E, F, G, H, respectively, of strips SwH and SwE, were 150 μl of human serum originating from, respectively, patients with a staphylococcus infection (Staph+serum) and controls without such an infection (Staph−serum) (see Table 7). The strips are then placed in a thermostatic oven (Reference BC240, Firelabo, France) stabilized at 37° C. for 10 minutes.

(41) TABLE-US-00007 TABLE 7 A B C D E F G H SwH Staph + Staph + Staph + Staph + Staph + Staph + Staph + Staph + serum/ serum/ serum/ serum/ serum/ serum/ serum/ serum/ Anti- Anti- Anti- Anti- Anti- Anti- Anti- Anti- human human human human human human human human SwE Staph + Staph + Staph + Staph + Staph + Staph + Staph + Staph + serum/ serum/ serum/ serum/ serum/ serum/ serum/ serum/ Anti- Anti- Anti- Anti- Anti- Anti- Anti- Anti- E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli

(42) 50 μl of reagent for detecting staphylococcus infection comprising substances from staphylococcus cultures and paramagnetic microbeads (Reference: RDS-01R, BioFilm Control, France), are deposited in the wells of the strips.

(43) The strips are then placed for 20 minutes in a thermostatic oven (Reference BC240, Firelabo, France) stabilized at 37° C., then placed on a magnetized test block (BKT-MSW002 BioFilm Control, France) for 10 seconds. The strips are then placed in a document scanner (Perfection V-750 PRO, Epson, USA) with which an image is captured with EpsonScan (Epson, USA) software. The magnetization/image capture cycle is reproduced a second time in order to also have an image after a total of 5 minutes of magnetization. The final images are obtained as in Example 1.

(44) The diameter of the ring observed in the presence of the expected affinity reaction between the immunoglobulins present in the serum of the infected patient and the substances contained in the reagent for detecting staphylococcus (wells A, B, C and D) is greater than in the absence of affinity reaction in the serum of uninfected patients (wells E, F, G and H).

LIST OF REFERENCES

(45) 1. U.S. Pat. No. 3,635,678 CLOT-TIMING SYSTEM AND METHOD, Seitz, L. J. and Bowen, J. G.; 2. WO 01/86255, METHOD AND APPARATUS FOR DETERMINING LOCAL VISCOELASTICITY, Cronin-Golomb, M. Shabtai, Y. Nemet, B.; 3. EP1544596, Method and device for determining viscosity, Kurowski D.; Schoen C.; Peters R.; Bartos H.; Yu Y.; 4. Catterall W A. (2000) Structure and regulation of voltage-gated Ca.sup.2+ channels. Annu. Rev. Cell. Dev. Biol. 16:521-55 5. Gerald Karp, Biologie cellulaire et moleculaire, Concepts et experiences [Molecular and Cellular Biology, Concepts and Experiments], 2.sup.nd edition 2004 6. Dick G M and Tune J D, Role of potassium channels in coronary vasodilation, Exp Biol Med (Maywood). 2010 January; 235(1):10-22 7. Mostallino M C et al. Plasticity and function of extrasynaptic GABA(A) receptors during pregnancy and after delivery, Psychoneuroendocrinology. 2009 December; 34 Suppl 1:S74-83