Flow assay method for an object of interest

Abstract

The present invention relates to a flow assay method in a liquid medium for an object (or element) of interest via the formation of aggregates of particles that are surface-functionalized by at least one functionalizing molecule, or receptor, specific for said object of interest.

Claims

1. A method for quantifying in a liquid medium at least one object of interest, comprising: mixing particles surface functionalized with at least one receptor specific for said object of interest to be assayed with the object of interest, for an initial time (t.sub.1) to form a first mixture; immediately sampling a volume (v) of the first mixture and counting the number N1 of singlets in said volume (v) by flow measurement; incubating the first mixture for a second time (t.sub.2) sufficient to allow the formation of aggregates, thereby forming a second mixture; sampling a volume of the second mixture and counting the number N2 corresponding to both the singlets and the aggregates contained in the volume (v) by the same flow measurement technique as used previously; determining a calculated degree of aggregation using the formula DA=(N1N2)/N1 (calculated DA) and quantifying said object of interest by comparison of the calculated DA with a standard range (DA=f([C]) previously produced by measuring and calculating the degree of aggregation obtained using the same flow measurement technique with the object of interest at predetermined concentrations ([C]) of said object of interest.

2. The method according to claim 1, wherein said object of interest is a protein, an antibody, a nucleic acid, a cell, a cell fragment, a microorganism, a microorganism fragment or a chemical molecule.

3. The method according to claim 1, wherein the object of interest is in a biological fluid, a tissue extract, purification plant waste, or water intended for consumption.

4. The method according to claim 3, wherein said biological fluid is blood, serum, plasma, saliva, urine or cerebrospinal fluid.

5. The method according to claim 3, wherein the object of interest is in a biological fluid or a tissue extract.

6. The method according to claim 3, wherein said tissue extract is bone marrow.

7. The method according to claim 1, wherein said receptor is a peptide, a protein, a nucleic acid, a saccharide, a lipid, or a hormone.

8. The method according to claim 1, wherein the aggregates have a size between 5 and 10,000 nm.

9. The method according to claim 8, wherein the aggregates have a size between 100 and 1000 nm.

10. The method according to claim 1, wherein the functionalized particle is a magnetic particle.

11. The method according to claim 1, wherein the initial time (t.sub.1) is less than three minutes.

12. The method according to claim 11, wherein the initial time (t.sub.1) is at most 2 minutes.

13. The method according to claim 1, further comprising increasing the aggregate collision frequency during the second time (t.sub.2).

14. The method according to claim 13, wherein the frequency of the collisions is increased by a magnetic or electric field or ultrasound.

15. The method according to claim 1, wherein the second time (t.sub.2) is between 5 seconds and 3 hours.

16. The method according to claim 15, wherein the second time (t.sub.2) is between 5 minutes and 60 minutes.

17. The method according to claim 1, wherein the flow measurement is carried out in flow mode.

18. The method according to claim 17, wherein the measurement is carried out by flow cytometry, capillary electrophoresis, or flow in a microfluidic channel.

19. The method according to claim 18, wherein the measurement is carried out by flow cytometry.

Description

(1) FIG. 1 is a diagrammatic representation of the principle of the method according to the invention with, on the left part of the diagram, a representation of the functionalized particles free of any linkage and the objects of interest bound to a single functionalized particle (singlets). It is the number N1 of singlets which is measured in the first step of the method according to the invention. On the right part of the diagram are represented, on the one hand, the singlets still present after aggregation (n), or the functionalized particles involved in aggregates (n), the sum n+n corresponding to the number N2 of singlets and of aggregates after aggregation. It is thus understood that N1N2 (106=4) corresponds to the number of linkages in the n (3) aggregates formed and that, in this theoretical case, the degree of aggregation (DA) is equal to (N1N2)/N1=(106)/10=0.4.

(2) FIG. 2 shows the results of the detection by the automated device for an aggregation in presence of 1 pM of CRP. The continuous-line curve represents the pulses detected for 30 seconds by the automated measuring device before aggregation. The sum of these pulses corresponds to N1. The dashed-line curve represents the pulses detected for 30 seconds by the automated measuring device after aggregation. The sum of these pulsed corresponds to N2.

(3) FIG. 3 shows the standard curve obtained by measuring the degree of aggregation of particles of 500 nm for various CRP concentrations (0 to 5 pM).

(4) FIG. 4 shows the standard curve obtained by measuring the degree of aggregation of particles of 200 nm for various CRP concentrations (0 to 15 pM).

(5) FIG. 4 shows the standard curve obtained by measuring the degree of aggregation of particles of 200 nm for various concentrations of biotinylated bovine serum albumin (0 to 500 pM).

(6) Other subjects, characteristics and advantages of the invention may emerge from the examples which follow.

EXAMPLE 1: ASSAYING OF CRP (C-REACTIVE PROTEIN) USING SUPER-PARAMAGNETIC PARTICLES 500 nm IN DIAMETER

(7) Anti-CRP polyclonal antibodies (L66616G, Meridian Life Science) (approximately 10 g of antibodies per mg of beads) were grafted onto superparamagnetic particles 500 nm in diameter (MasterBeads Carboxylic Acid 0215, Ademtech).

(8) The assays were carried out in a 30 mM glycine buffer, pH 8.5, containing variable concentrations of CRP (ABX Pentra CRP cal, Horiba Medical).

(9) In order to limit the formation of linkages between particles in the absence of CRP, taurocholic acid (T4009, Sigma-Aldrich) was added to the medium at a final concentration of 3 mM in the reaction mixture.

(10) The final concentration of particles in the medium is approximately 0.6 pM. The flow analysis is carried out by illuminating the particles with a laser working at 488 nm. The scattering at 90 is measured for each object passing through the measuring cell using a photomultiplier of the Hamamatsu brand, model H9307-02.

(11) After the various reagents have been brought into contact, the medium is left to incubate for 2 min. During these 2 min, a volume (V1) of 53 l of the mixture is sampled and injected into the flow analyzer. The latter dilutes the volume V1 to 1/100 and performs a count for 30 s on a volume V2 of 35.5 l of this mixture. The number of singlets present in the suspension at the beginning of the reaction (N1) in said volume V2 is then determined.

(12) After 2 min, the medium undergoes 5 field cycles, composed of 30 s under 10 mT, then 300 s under 3 mT and, finally, 30 s of relaxation under 0 mT.

(13) After the magnetization cycle, a second volume (V1) of the mixture, identical to the volume previously sampled (53 l), is sampled and analyzed by the flow analyzer according to the same preparation cycle. This measurement makes it possible to determine the number of aggregates formed (N2).

(14) FIG. 2 shows the results of the detection by the automated device for an aggregation in the presence of 1 pM of CRP. It represents the number of elements detected in the volume (V2) for 30 seconds as a function of the height of the pulse which is associated with said elements, itself a function of the size of the object. It is noted that, before aggregation (continuous line), the suspension has a complex distribution with numerous sizes of particles present. After aggregation (dashed line), no population clearly associated with the particle aggregates is apparent; however, the total number of objects detected (area under the curve) has clearly decreased.

(15) Establishment of the Standard Curve:

(16) The degree of aggregation was determined for various CRP concentrations (from 0 to 5 pM) according to the protocol previously described. The results are presented in FIG. 3.

(17) It is noted that the degree of aggregation actually varies with the CRP concentration in the medium. On the bases of the standard deviation measured on several repetitions in the absence of CRP, the detection limit of this system could be evaluated at 25 fM of CRP, for an analysis time of approximately 35 min.

(18) Assaying of the CRP concentration in an unknown solution: A serum sample, with an unknown CRP concentration, was diluted 10 000-fold in a 30 mM glycine buffer, pH 8.5. 27.6 l of this mixture were added to a 30 mM glycine buffer containing the same functionalized superparamagnetic particles 500 nm in diameter (MasterBeads Carboxylic Acid 0215, Ademtech) as previously used, and also taurocholic acid. The final volume of the medium is 600 l, with a final concentration of particles of 0.6 pM and of taurocholic acid of 3 mM.

(19) After the various reagents have been brought into contact, the Medium is left to incubate for 2 min. During these 2 min, a volume (V1) of 53 l of the mixture is sampled and injected into the flow analyzer. The latter dilutes the volume V1 to 1/100 and performs a count for 30 s on a volume (V2) of 35.5 l of this mixture. The number of singlets present in the suspension at the beginning of the reaction (N1) in said volume V2 is then determined.

(20) After 2 minutes, the medium undergoes 5 field cycles, composed of 30 s under 10 mT, 30 s under 3 mT and 30 s of relaxation under 0 mT.

(21) After the magnetization cycle, a second volume (V1) of the mixture, identical to the volume previously sampled (53 l), is sampled and analyzed in a manner identical to the first sampling in order to determine the value of N2.

(22) According to the protocol previously described, it was thus possible to determine, for the unknown sample, a degree of agglutination DA=0.23. Relating this to the calibration curve makes it possible to determine a CRP concentration in the reaction medium equal to 0.17 pM, i.e. a CRP concentration in the unknown serum of 37 nM.

EXAMPLE 2: ASSAYING OF CRP USING SUPERPARAMAGNETIC PARTICLES OF 200 nm

(23) Establishment of the Standard Curve:

(24) Approximately 35 g of anti-CRP polyclonal antibodies (L66616G, Meridian Life Science) per mg of particles were grafted onto magnetic particles 200 nm in diameter (Carboxyl Adembeads, 0212, Ademtech).

(25) The assays were carried out in a 30 mM glycine buffer, pH 8.5, containing variable concentrations of CRP (ABX Pentra CRP cal, Horiba Medical).

(26) In order to limit the formation of linkages between particles in the absence of CRP, saponin (30502-42, Nacalai Tesque) was added to the medium at 0.08% by weight in the reaction mixture as detergent.

(27) The final concentration of particles in the medium is approximately 3 pM.

(28) After the various reagents have been brought into contact, the medium is left to incubate for 2 min. During these 2 minutes, a volume (V1) of 53 l of the mixture is sampled and injected into the flow analyzer. The latter dilutes the volume V1 to 1/1200 and performs a count for 30 s on a volume (V2) of 35.5 l of this mixture. The number of singlets present in the suspension at the beginning of the reaction (N1) in said volume V2 is then determined.

(29) After 2 minutes, the medium undergoes 2 magnetic field cycles, composed of 30 s under 50 mT, 300 s under 20 mT and 30 s of relaxation under 0 mT.

(30) After the magnetization cycle, a second volume (V1) of the mixture, identical to the volume previously sampled (53 l), is sampled and analyzed in a manner identical to the first sampling in order to determine the value of N2. This measurement makes it possible to determine the number of aggregates formed (N2).

(31) The degree of aggregation was determined for various CRP concentrations (from 0 to 16 pM) according to the protocol previously described. The standard curve obtained is given in FIG. 4.

(32) On the basis of the repetitions in the absence of CRP, the detection limit was evaluated at 100 fM of CRP, for an analysis time of approximately 15 min.

(33) Assaying of the CRP Concentration in an Unknown Solution:

(34) A serum sample, with an unknown CRP concentration, was diluted 100-fold in a 30 mM glycine buffer, pH 8.5. 2 l of this mixture were added to a 30 mM glycine buffer containing the same functionalized magnetic particles 200 nm in diameter, and also saponin. The final volume of the medium is 600 l, with a final concentration of particles of 3 pM and of saponin of 0.08% (weight/volume).

(35) After the various reagents have been brought into contact, the medium is left to incubate for 2 min. During these 2 min, a volume (V1) of 53 l of the mixture is sampled and injected into the flow analyzer. The latter dilutes the volume V1 to 1/1200 and performs a count for 30 s on a volume (V2) of 35.5 l of this mixture. The number of singlets present in the suspension at the beginning of the reaction (N1) in said volume V2 is then determined.

(36) After 2 min, the medium undergoes 2 field cycles, composed of 30 s under 50 mT, 300 s under 20 mT and 30 s of relaxation under 0 mT.

(37) After the magnetization cycle, a second volume (V1) of the mixture, identical to the volume previously sampled (53 l), is sampled and analyzed in a manner identical to the first sampling in order to determine the value of N2. This measurement makes it possible to determine the number of aggregates formed (N2).

(38) It was thus possible to determine, for the unknown sample, a degree of agglutination DA=0.55. Relating this to the calibration curve makes it possible to determine a CRP concentration in the reaction medium equal to 6.3 pM, i.e. a CRP concentration in the unknown serum of 189 nM.

EXAMPLE 3: ASSAYING OF BIOTIN USING SUPERPARAMAGNETIC PARTICLES of 200 nm

(39) Establishment of the Standard Curve:

(40) Streptavidin-covered particles 200 nm in diameter were used (Bio-Adembeads Streptavidin 0312, Ademtech).

(41) The assays were carried out in a 30 mM glycine buffer, pH 8.5, containing 0.5% of bovine serum albumin (BSA Protease Free, ID Bio) and also variable concentrations of biotinylated bovine serum albumin (BSAb) (A8549, Sigma-Aldrich). The final concentration of particles in the medium is approximately 6 pM.

(42) After the various reagents have been brought into contact, the medium is left to incubate for 2 min. During these 2 min, a volume (V1) of 53 l of the mixture is sampled and injected into the flow analyzer. The latter dilutes the volume V1 to 1/2400 and performs a count for 30 s on a volume (V2) of 35.5 l of this mixture. The number of singlets present in the suspension at the beginning of the reaction (N1) in said volume V2 is then determined.

(43) After 2 min, the medium undergoes 2 field cycles, composed of 30 s under 50 mT, 300 s under 20 mT and 30 s of relaxation under 0 mT.

(44) After the magnetization cycle, a second volume (V) of the mixture, identical to the volume previously sampled (53 l), is sampled and analyzed in a manner identical to the first sampling in order to determine the value of N2. This measurement makes it possible to determine the number of aggregates formed (N2).

(45) The degree of aggregation was determined for various biotin concentrations (from 0 to 500 pM) according to the protocol previously described. The standard curve obtained is given in FIG. 5. On the basis of the repetitions in the absence of biotinylated BSA, the detection limit was evaluated. The experimental conditions were not optimized for this assay, and a high detection limit, of about 7 pM of biotinylated BSA, is found for an analysis time of approximately 15 min.

(46) Assaying of the Biotin Concentration in an Unknown Solution:

(47) A sample with an unknown BSAb concentration was diluted 10-fold in a 5% BSA solution. 60 l of this mixture were added to a 30 mM glycine buffer containing the same functionalized magnetic particles 200 nm in diameter, The final volume of the medium is 600 l, with a final concentration of particles of 6 pM and of BSA of 0.5% (weight/volume).

(48) After the various reagents have been brought into contact, the medium is left to incubate for 2 min. During these 2 min, a volume (V1) of 53 l of the mixture is sampled and injected into the flow analyzer. The latter dilutes the volume V1 to 1/2400 and performs a count for 30 s on a volume (V2) of 35.5 L of this mixture. The number of singlets present in the suspension at the beginning of the reaction (N1) in said volume V2 is then determined.

(49) After 2 min, the medium undergoes 2 field cycles, composed of 30 s under 50 mT, 300 s under 20 mT and 30 s of relaxation under 0 mT.

(50) After the magnetization cycle, a second volume (V1) of the mixture, identical to the volume previously sampled (53 l), is sampled and analyzed in a manner identical to the first sampling in order to determine the value of N2. This measurement makes it possible to determine the number of aggregates formed (N2).

(51) It was thus possible to determine, for the unknown sample, a degree of agglutination DA=0.33. Relating this to the calibration curve makes it possible to determine a BSAb concentration in the reaction medium equal to 61.4 pM, i.e. a BSAb concentration in the unknown sample of 614 pM.