Patent ID: 12241894

EXAMPLES

Example 1: Red Blood Cell Typing and Phenotyping in the In Vitro Device With Direct and Indirect Coupling of Anti-Red Blood Cells Antibodies on Beads Via Anti-Immunoglobulins IgM and IgG

1. Protocol for Preparing the In Vitro Device That Comprises on the Surface of the Reaction Area the Anti-Red Blood Cells Antibodies Directly Spotted on the Membrane

1.1. Materials for the Preparation of the In Vitro Device

Membrane “POREX” 0.6 mm hydrophobic with 9 to 12 μm porosity; Cotton;Cleanis;Hydrophilization buffer (1% triton, 1% Green dye, phosphate buffered saline).Wash Buffer (TLA composition);Dilution buffer (Chromasolcoombs);Antibodies (Anti-ABO01, Anti-ABO02, Anti-ABO03, Anti-RH1, Anti-RH2, Anti-RH3, Anti-RH4, Anti-RHS, Anti-KEL1, Negative Control).

1.2. Preparation Mode of the Reaction Medium

Deposit 1 μl of the hydrophilization solution in each well;Drying 16 hours at 37° C. and 10% humidity;Deposit 2 μl of antibody per well;Drying 72 hours at 37° C. and 10% humidity.

Materials for the realization of the test are the reaction support, wash buffer and dilution buffer

1.3. Test Embodiment

Dilute the pellet of red blood cells to 20% in the dilution buffer;Deposit 20 μl of the suspension in the wells;Incubate for 3 min at room temperature;Deposit 80 μl of washing buffer;Incubate 1 min at room temperature;Deposit 80 μl of washing buffer.

2. Protocol for Preparing The In Vitro Device With Direct and Indirect Coupling of Anti-Red Blood Cells Antibodies on Beads Via Anti-Immunoglobulins IgM and IgG

2.1. Protocol for Direct Coupling of the anti-ABO01 (Anti-A), Anti-ABO02 (Anti-B) and Anti-ABO03 (Anti-AB) Antibodies to Latex Beads

On 1 ml of homogenized 4% beads (Thermo Fisher latex Aldehyde/Sulfate beads 9 μm 4% w/v), 100 μl of 20× phosphate buffered saline are added and 2 ml of concentrated antibodies are dissolved. The volume is completed up to 4 ml with demineralized water. The solution is end over end mixed overnight to ensure coupling.

The excess active groups are blocked by suspending in the bead antibody solution 150 μl of 1M ethanolamine followed by a mixing for 2 hours at room temperature. The coupled beads are then washed extensively 2 times with 3 ml of conservation buffer (centrifugation 5 min at 2500 g before and after washes). Finally the bead pellet is resuspended in the conservation buffer to get back the introduced bead solution weight.

2.2. Anti-RH1, Anti-RH2, Anti-RH3, Anti-RH4, Anti-RH5 and Anti-KEL1 Antibodies Indirect Coupling Protocol

On 1 ml of homogenized 4% beads, 200 μl of 20× phosphate buffered saline are added and 250 μl of 6D4 (anti human IgM, Diagast clone) at 1 mg/ml or 100 μl of C5-1 (anti human IgG, Diagast clone) at 3.69 mg/ml are dissolved. The volume is completed up to 4 ml with demineralized water. The solution is end over end mixed overnight to ensure coupling.

The excess active groups are blocked by suspending in the bead antibody solution 150 μl of 1M ethanolamine followed by a mixing for 2 hours at room temperature. The Anti-Human Globulin (AHG) coupled beads are then washed extensively 2 times with 3 ml of conservation buffer (centrifugation 5 min at 2500 g before and after washes). The bead pellet is resuspended with 2 ml of concentrated antibody plus 2 ml of phosphate buffered saline. The solution is end over end mixed overnight to ensure coupling.

The resulting beads are then washed extensively 2 times with 3 ml of conservation buffer (centrifugation 5 min at 2500 g before and after washes). Finally the bead pellet is resuspended in the conservation buffer to get back the introduced bead solution weight. The negative control beads are directly saturated with ethanolamine.

The antibodies clones used in the Examples are shown in Table 4 below:

TABLE 4Antibody clones used in the Examples.CloneAntigenIsSpecieCoupling type9113D1ABO01IgmouseDirect9621ABO02IgmouseDirect152D12ABO03IgmouseDirectP3X255RH2Ighumanindirect throughRH3RH3Ighumanindirect throughRH4RH4Ighumanindirect throughP3GD5RH5Ighumanindirect throughK601KEL1Ighumanindirect throughP3X212RH1Ighumanindirect through6D4AHGIgmouseDirectC51AHGIgmouseDirect

The antibody clones in Table 4 are clones performed and sold by the Applicant (Diagast, France).

2.3. Reaction Device Preparation and Detection Procedure

The device is a moulded plastic cassette in which a Porex HDPE hydrophobic membrane of 0.6 mm thickness and porosity between 9 to 12 μm, a draining pad (NA7150 PES from Subrenat) and an absorbent pad (Cleanis) are assembled.

The porous membrane is hydrophilized with 0.5 μl of a surfactant solution (phosphate buffered saline supplemented with 0.5% Triton X-100; 0.5% green dye and 0.13M saccharose) and potentially dried for 1 hour at 37° C., 10% humidity. 5 μl of antibody or negative control coupled beads at 4% are then spotted and the device is dried for 1 hour at 37° C., 10% humidity before immediate use or extended conservation.

A test is initiated by diluting red blood cells at 5% in Chromasolcoombs (Diagast formulation) and 10 μl are introduced in each well followed by two 30 μl washes (phosphate buffered saline supplemented with 0.2% Tween20 and 0.54% green dye).

In this example, the Applicant also compared the in vitro device containing anti-red blood cells antibodies coupled to beads for the blood grouping and phenotyping prepared by the above described protocol to an in vitro device in which the anti-red blood cells antibodies are directly spotted on the porous membrane (similar to those described in WO2013/186482).

3. Results

The obtained comparative results are shown onFIG.2, wherein the presence of a dark gray spot (corresponding to red spot in the color version) indicates an immune complex formation, and therefore a positive reaction (presence of the antigen at the red blood cell surface), while the presence of a colourless spot reflects a negative reaction (absence of the antigen at the red blood cell surface).

It appears fromFIG.2that antibodies of blood groups anti-ABO01, anti-ABO02, anti-ABO03, anti-RH1, anti RH2, anti-RH3 anti-RH4 anti RH5 and anti-KEL1 fixed directly or indirectly on the bead surface bind the corresponding antigens present on the surface of red blood cells of the tested samples in specific manner. Indeed, the surface of the spot where the beads were deposited remains well colored in red which indicates that the antibodies of red blood cells are not adsorbed by the porous membrane which allows specific and sensitive detection of red blood cells antigens (FIG.2b).

It can be seen fromFIG.2athat in the in vitro device without beads the reactions with some antibodies, particularly Anti-RH1, Anti-RH3, Anti-RH4, Anti-RH5 and Anti-KEL1 antibodies have low sensitiveness (very low intensity of dark grey).

When comparing the results onFIG.2bobtained with the in vitro device of the invention to those ofFIG.2aobtained with the in vitro device without bead, it appears that the greys pots for antibodies Anti-RH1, Anti-RH3, Anti-RH4, Anti-RH5 and Anti-KEL1KEL1 are much more visible onFIG.2athan those ofFIG.2a, which demonstrates that the in vitro device of the invention allows to obtain a more sensitive detection of the most anti-red blood cells antigens.

Example 2: Indirect Coupling of Red Blood Cells Antibodies Via Affinity Proteins for Antibodies

1. Indirect Antibodies Coupling

On 1 ml of homogenized 4% latex beads (Thermo Fisher latex Aldehyde/Sulfate 9 μm 4% w/v), 5 or 2.5 μg of Protein AGL are dissolved in 3 ml PBS1× and are added. The solution is end over end mixed overnight to ensure coupling.

On 1 ml of centrifugated 4% beads (Thermo Fisher latex Aldehyde/Sulfate 9 μm 4% w/v) coupled with protein AGL, 3 ml of 1× phosphate buffered saline are added and 1 ml of Anti-ABO01 antibody (91 13 D10 Diagast clone) or Anti-ABO02 antibody (96 21 A8 Diagast clone) or Anti-ABO03 (152D12 Diagast clone) are dissolved. The solution is end over end mixed overnight to ensure coupling.

The coupled beads are then centrifuged 5 min at 2500 g. Finally the bead pellet is resuspended in the conservation buffer (phosphate buffered saline supplemented with 0.5% bovine serum albumin and 0.25M saccharose) to get back the introduced bead solution weight.

2. Reaction Device Preparation and Detection Procedure

The device is a moulded plastic cassette in which a Porex HDPE hydrophobic membrane of 0.6 mm thickness and porosity between 9 to 12 μm, a draining pad (9610HY) and an absorbent pad (Cleanis) are assembled.

The porous membrane is hydrophilized with 0.5 μl of a surfactant solution (phosphate buffered saline supplemented with 2% Triton X-100 and 1% of green dye). 2 μl of antibody coupled beads at 2%, are then spotted. The device is dried for 15 min at 37° C., 20% humidity before use.

The detection procedure is initiated by introducing in each well 50 μl of red blood cells diluted at 1.5% in PBS1× supplemented with 0.00625% Tween20.

A first image before washes is acquired. The reaction zone is then washed with 30 μl of phosphate buffered saline supplemented with 0.2% Tween-20. An image is acquired after wash.

3. Results

FIG.3shows the resulting image on which is observed the presence of dark grey spots (corresponding to the red spot in color image) indicating an immune complex formation and therefore a positive reaction, while the presence of a colorless spot reflects a negative reaction.

Example 3: Coupling of Antibodies of Red Blood Cells on Beads Having Different Chemical Functional Groups on Their Surface and Different Size in Different Reactional Conditions

1. Chloromethyl Group

1.2. Antibodies Coupling

On 500 μl of homogenized chloromethyl beads (Thermo Fisher chloromethyl latex beads 4% w/v, 2.0 μm) 1.5 ml of phosphate buffer is added and the obtained solution is centrifuged at 3000 g for 20 min. The supernatant is discarded and the bead pellet is then resuspended with a mix of 950 μl of phosphate buffered saline and 50 μl of concentrated CPC51 or CP6D4 (Diagast AHG). The antibody bead solution is incubated overnight at room temperature on a tube rocker.

After AHG chemical coupling, the bead solution is centrifuged at 3000 g for 15 min and the supernatant is discarded. The beads are washed with 1.5 ml of phosphate buffered saline and the solution is centrifuged at 3000 g for 10 min. The supernatant is discarded and the excess active groups are blocked by adding 1 ml of 1M ethanolamine before mixing for 20 min at room temperature. The AHG coupled bead solution is centrifuged at 3000 g for 20 min before washing with 1.5 ml of phosphate buffer saline. 1 ml of concentrated anti-A (Anti-ABO1) antibodies (clones IgM 91 13 D10 or IgG 162 47 (3) E6 both Diagast clones) is dissolved on the bead pellet and the solution is mixed for two hours at room temperature before centrifugation and wash with 1.5 ml of phosphate buffered saline. Finally the bead pellet is resuspended in the conservation buffer to get back the introduced bead solution weight.

1.2. Reaction Device Preparation and Detection Procedure

The device is a moulded plastic cassette in which a Porex HDPE hydrophobic membrane of 0.6 mm thickness and porosity between 9 to 12 μm, a draining pad (cotton wool from Alan&co) and an absorbent pad (Cleanis) are assembled.

The porous membrane is hydrophilized with 2.5 μl of a surfactant solution (filtered water supplemented with 1% Triton X-100) and 20 μl (FIGS.4A and4C) or 10 μl (FIGS.4B and4D) of antibody coupled beads at 4% are then spotted. The device is let to stand 30 min at room temperature before use.

The detection procedure is initiated by introducing in each well 13 μl of a mix of 30 μl of red blood cells at 10% in chromasolcoombs (Diagast formulation) and 10 μl of an hexadimethrine bromide solution.

The reaction zone is washed twice with 25 μl of phosphate buffered saline supplemented with 0.1% Tween-20.

1.3. Results

FIG.4shows the resulting image on which is observed the presence of dark gray spot (corresponding to a red spot in color image) indicating an immune complex formation (group A red blood cells), and therefore a positive reaction, while the presence of a colourless spot reflects a negative reaction (group B red blood cells).

2. Epoxide Group

2.1. Antibodies Coupling

0.5 g of epoxide resin (Biorad Profinity epoxide resin, supplied as a dry powder, particle size 45-90 microns) are weighted out and swelled in 50 ml of phosphate buffered saline for 30 min under gentle agitation. Beads expand to 5 ml which give a solution at approximately 10%.

Beads are centrifuged at 2500 g for 5 min, the supernatant is discarded and the pellet is resuspended in 4.5 ml of phosphate buffered saline then centrifuged again.

360 μl of concentrated anti IgM AHG (CP6D4 Diagast) are dissolved in 12 ml of phosphate buffered saline and the mix is added on the bead pellet for coupling. The antibody bead solution is incubated 1H30 at room temperature on a rotary shaker.

After AHG chemical coupling, the bead solution is centrifuged at 2500 g for 5 min and the supernatant discarded. The beads are washed with 4.5 ml of phosphate buffered saline and the solution is centrifuged at 2500 g for 5 min. The supernatant is discarded and the excess active groups are blocked by adding 3 ml of 1M ethanolamine before mixing for 45 min at room temperature. The AHG coupled bead solution is centrifuged at 2500 g for 5 min before washing with 4.5 ml of phosphate buffered saline.

300 μl of anti-RH2, anti-RH3, anti-RH4 or anti-RH5 (respectively P3X255 13 G8 Diagast clone, RH3 906 Diagast clone, MS33 Millipore clone and P3GD C512 Diagast clone) are diluted in 2200 μl of phosphate buffered saline and added on the AHG coupled beads. For anti-Kell antibody 1500 μl are diluted in 1000 μl of phosphate buffered saline and for negative control 2500 μl of phosphate buffered saline. Obtained solution is mixed for 45 min at room temperature before centrifugation and wash. Finally the bead pellet is resuspended in phosphate buffered saline supplemented with conservatives to get back a 50% solution of coupled beads.

2.2. Reaction Device Preparation and Detection Procedure

The device is a moulded plastic cassette in which a Porex HDPE hydrophobic membrane of 0.6 mm thickness and porosity between 9 to 12 μm, a draining pad (cotton wool from Alan&co) and an absorbent pad (Cleanis) are assembled.

The porous membrane is hydrophilized with 1 μl of a surfactant solution (filtered water supplemented with 1% Triton X-100) and dried overnight at room temperature. 10 μl of antibody coupled beads at 50% are then spotted. The device is let to stand 30 min at room temperature before use.

The detection procedure is initiated by introducing in each well 10 μl of undiluted red blood cells. The reaction zone is washed twice with 40 μl of a phosphate buffered saline supplemented with 0.2% Tween-20 and preservatives.

2.3. Results

FIG.5shows the resulting image on which is observed the presence of a dark gray spot (corresponding to a red spot in the color image) indicating an immune complex formation, and therefore a positive reaction, while the presence of a colourless spot reflects a negative reaction. Whatever the red blood cell phenotype, the negative control results in a colourless spot.

3. N-Hydroxysuccinimide (NHS) Group

3.1. Antibodies Coupling

1 ml of NHS Beads (Pierce™ NHS-Activated Agarose Slurry) are diluted with 5 ml of phosphate buffered saline and centrifuged at 2500 g for 5 min, the supernatant is discarded and the pellet is resuspended in 4.5 ml of phosphate buffered saline for 3 additional washes.

2 ml of concentrated anti IgG AHG (C51 Diagast) are dissolved in 3 ml of phosphate buffered saline and the mix is added on the bead pellet for coupling. The antibody bead solution is incubated one hour and a half at room temperature on a rotary shaker.

After AHG chemical coupling, the bead solution is centrifuged at 2500 g for 5 min and the supernatant discarded. The beads are washed with 4.5 ml of phosphate buffered saline and the solution is centrifuged at 2500 g for 5 min. The supernatant is discarded and the excess active groups are blocked by adding 3 ml of 1M ethanolamine before mixing for 45 min at room temperature. The AHG coupled bead solution is centrifuged at 2500 g for 5 min before washing with 4.5 ml of phosphate buffered saline.

10 ml of anti-RH1 and anti-FY1 antibodies (respectively HM16 and F655 Diagast clones) are added on the AHG coupled beads. Obtained solution is mixed for 1 hour at room temperature before centrifugation and washing. Finally the bead pellet is resuspended in phosphate buffered saline supplemented with conservatives to get a 50% solution of coupled beads.

3.2. Reaction Device Preparation and Detection Procedure

The device presented onFIGS.6(A, B and C) is a moulded plastic cassette in which a Porex HDPE hydrophobic membrane of 0.6 mm thickness and porosity between 9 to 12 μm, a draining pad (cotton wool from Alan&co) and an absorbent pad (Cleanis) are assembled.

The porous membrane is hydrophilized with 2.5 μl of a surfactant solution (filtered water supplemented with 1% Triton X-100) and dried overnight at 37° C. 20 μl of antibody coupled beads at 50% are then spotted. The device is let to stand 30 min at room temperature before use.

FIG.6A: The detection procedure is initiated by introducing in each well 7 μl of red blood cells diluted at 25% in an 0.25% hexadimethrine bromide solution. The reaction zone is washed with 20 μl+30 μl of phosphate buffered saline supplemented with 0.01% Tween-20.

FIG.6B: The detection procedure is initiated by introducing in each well 5 μl of red blood cells diluted at 25% in an 0.3% hexadimethrine bromide solution. The reaction zone is washed with 20 μl+30 μl of phosphate buffered saline supplemented with 0.01% Tween-20.

FIG.6C: The detection procedure is initiated by introducing in each well 5 μl of red blood cells diluted at 25% in an 0.4% hexadimethrine bromide solution. The reaction zone is washed with 20 μl+30 μl of phosphate buffered saline supplemented with 0.01% Tween-20.

The device presented onFIG.6Dis a moulded plastic cassette in which a Lydall UHMWPE hydrophobic membrane of 105 μm thickness and porosity of 12 μm (Solupor 70M01A), a draining pad (cotton wool from Alan&co) and an absorbent pad (Cleanis) are assembled.

The porous membrane is hydrophilized with 2 μl of a surfactant solution (filtered water supplemented with 1% Triton X-100) and dried for 1 hr 30 min at 37° C. 20 μl of antibody coupled beads at 50% are then spotted. The device is let to stand 30 min at room temperature before use.

The detection procedure is initiated by introducing in each well 7 μl of red blood cells diluted at 25% in an 0.25% hexadimethrine bromide solution. The reaction zone is washed with 30 μl+50 μl of phosphate buffered saline supplemented with 0.01% Tween-20 and additional 20 μl of phosphate buffered saline supplemented with 0.1% Tween-20 and 0.007% Triton X-100.

3.3. Results

OnFIG.6from the resulting image is observed the presence of a dark gray spot (corresponding to a red spot in color image) indicating an immune complex formation, and therefore a positive reaction (detection of RH1 or FY1 antigen), while the presence of a colourless spot reflects a negative reaction.

Example 4: Concentration of the Beads

The aim of this experience is to test different concentrations of beads to be deposited on the porous membrane.

1. Antibodies Coupling

On 1 ml of homogenized 4% beads (Thermo Fisher latex Aldehyde/Sulfate 9 μm 4% w/v), 100 μl of 20× phosphate buffered saline are added and 2 ml of affinity purified anti-ABO02 antibody (96 21 A8 Diagast clone) are dissolved. The volume is completed up to 4 ml with demineralized water. The solution is end over end mixed overnight to ensure coupling.

The excess active groups are blocked by suspending in the bead antibody solution 150 μl of 1M ethanolamine and mixed for 2 hours at room temperature. The coupled beads are then washed extensively 2 times with 3 ml of conservation buffer (centrifugation 5 min at 2500 g before and after washes). Finally the bead pellet is resuspended in the conservation buffer to get back the introduced bead solution weight.

2. Reaction Device Preparation and Detection Procedure

The device is a moulded plastic cassette in which a Porex HDPE hydrophobic membrane of 0.6 mm thickness and porosity between 9 to 12 μm, a draining pad (cotton wool from Alan&co) and an absorbent pad (Cleanis) are assembled.

The porous membrane is hydrophilized with 1 μl of a surfactant solution (phosphate buffered saline supplemented with 1% Triton X-100 and 1% of green dye). 10 μl of antibody coupled beads, concentration between 1 to 5%, are then spotted. The device is dried for 15 min at 37° C., 20% humidity before use.

The detection procedure is initiated by introducing in each well 50 μl of red blood cells diluted at 0.15% in Chromasolcoombs supplemented with 0.00625% Tween20. The reaction zone is washed one or two times with 30 μl of phosphate buffered saline supplemented with 0.2% Tween-20.

3. Results

FIG.7shows the resulting image on which is observed the presence of dark gray spots (corresponding to the red spot in color image) indicating an immune complex formation, and therefore a positive reaction (group ABO02 red blood cells), while the presence of a colourless spot reflects a negative reaction. The specificity and sensitivity are respected for a bead concentration going from 1 to 5%, after one or two washes. Mixed field blood cells are detectable through a decrease in intensity compared to the signal of a 100% positive population.

Example 5: Thickness of Porous Hydrophobic Membrane

1. Antibodies Coupling

1.1. Anti-ABO01 Coupling

On 1 ml of homogenized 4% beads (Thermofisher latex Aldehyde/Sulfate 9 μm 4% w/v), 100 μl of 20× phosphate buffer are added and 2 ml of affinity purified anti-ABO01 (25 21 B8 Diagast clone) antibody are dissolved. The volume is completed up to 4 ml with demineralized water. The solution is end over end mixed overnight to ensure coupling.

The excess active groups are blocked by suspending in the bead antibody solution 150 μl of 1M ethanolamine and mixed for 2 hours at room temperature. The coupled beads are then washed extensively 2 times with 3 ml of conservation buffer (centrifugation 5 min at 2500 g before and after washes). Finally the bead pellet is resuspended in the conservation buffer (phosphate buffered saline supplemented with 0.5% bovine serum albumin and 0.25M saccharose) to get back the introduced bead solution weight.

1.2. Anti-RH2, Anti-RH5 and Anti-KEL1 Coupling

On 1 ml of homogenized 4% beads, 200 μl of PBS20× are added and 250 μl of 6D4 (anti human IgM) for anti-RH2 and anti-RH5 (respectively P3x255 13 G8 and P3GD C512 Diagast clones) at 1 mg/ml or 100 μl of C5-1 (anti human IgG) for anti-KEL1 (601 Diagast clone) at 3.5 mg/ml are dissolved. The volume is completed up to 4 ml with demineralized water. The solution is end over end mixed overnight to ensure coupling.

The excess active groups are blocked by suspending in the bead antibody solution 150 μl of 1M ethanolamine and mixed for 2 hours at room temperature. The AHG coupled beads are then washed extensively 2 times with 3 ml of conservation buffer (centrifugation 5 min at 2500 g before and after washes). The bead pellet is resuspended with 2 ml of concentrated antibody plus 2 ml of phosphate buffer. The solution is end over end mixed overnight to ensure coupling.

The coupled beads are then washed twice with 3 ml of conservation buffer (centrifugation 5 min at 2500 g before and after washes). Finally the bead pellet is resuspended in the conservation buffer (phosphate buffered saline supplemented with 0.5% bovine serum albumin and 0.25M saccharose) to get back the introduced bead solution weight.

1.3. Reaction Device Preparation and Detection Procedure

The device shown onFIG.8ais a moulded plastic cassette in which a Porex HDPE hydrophobic membrane of 1.5 mm thickness and porosity between 7 to 12 μm, a draining pad (cotton wool from Alan&co) and an absorbent pad (Cleanis) are assembled.

The porous membrane is hydrophilized with 1 μl of a surfactant solution (phosphate buffered saline supplemented with 3% Triton X-100, 0.26M Saccharose and 1% of green dye). The device is dried a first time for 1 hour at 37° C., 10% humidity. 5 μl of antibody coupled beads, concentration between 0.125 to 4%, are then spotted. The device is dried a second time for 1 hour at 37° C., 10% humidity before use.

The detection procedure is initiated by introducing in each well 10 μl of red blood cells diluted at 5% in physiological water. The reaction zone is washed with 100 μl phosphate buffered saline supplemented with 0.1% Tween-20 and 0.007% Triton X-100.

The device shown onFIG.8bis a moulded plastic cassette in which a Porex HDPE hydrophobic membrane of 0.6 mm thickness and porosity between 9 to 12 μm, a draining pad (NA7300PES from Subrenat) and an absorbent pad (Cleanis) are assembled.

The porous membrane is hydrophilized with 0.5 μl of a surfactant solution (phosphate buffered saline supplemented with 0.5% Triton X-100, 0.13M Saccharose and 0.5% of green dye). The device is dried a first time for 1 hour at 37° C., 10% humidity. 5 μl of antibody coupled beads at 4%, are then spotted. The device is dried a second time for 1 hour at 37° C., 10% humidity before use.

The detection procedure is initiated by introducing in each well 10 μl of red blood cells diluted at 5% in physiological water. The reaction zone is washed with 100 μl of phosphate buffered saline supplemented with 0.1% Tween-20 and 0.007% Triton X-100.

1.4. Results

The presence of a dark grey spot (a red spot) onFIG.8aindicates an immune complex formation and therefore a positive reaction. The sensitivity is respected for a bead concentration going from 4 to 0.125% with a decrease in intensity.

The presence of a dark grey spot (a red spot) onFIG.8bindicates an immune complex formation, and therefore a positive reaction, while the presence of a colourless spot reflects a negative reaction.

Example 6: Type and Concentration of Surfactant Used For Hydrophilizing the Porous Membrane

1. Anti-ABO01 Coupling

On 1 ml of homogenized 4% beads (Thermo Fisher latex Aldehyde/Sulfate 9 μm 4% w/v), 100 μl of 20× phosphate buffered saline are added and 1 ml of affinity anti-ABO01 antibody (25 21 B8 Diagast clone) is dissolved. The volume is completed up to 4 ml with demineralized water. The solution is end over end mixed overnight to ensure coupling.

The coupled beads are then centrifuged 5 min at 2500 g. Finally the bead pellet is resuspended in the conservation buffer (phosphate buffered saline supplemented with 0.5% bovine serum albumin and 0.25M saccharose) to get back the introduced bead solution weight.

2. Reaction Device Preparation And Detection Procedure

The device is a moulded plastic cassette in which a Porex HDPE hydrophobic membrane of 0.6 mm thickness and porosity between 9 to 12 μm, a draining pad (cotton wool from Alan&co) and an absorbent pad (Cleanis) are assembled.

The porous membrane is hydrophilized with 1 μl of a surfactant solution listed abovea. 1.5% Triton X-100b. Polyoxyethylene 10%c. Nonyl-β-D glucoside 50 mM

10 μl of antibody coupled beads, concentration at 3% are then spotted. The detection procedure is initiated by introducing in each well 50 μl of red blood cells diluted at 1.5% in phosphate buffered saline supplemented with 0.00625% Tween-20. The reaction zone is washed with 30 μl phosphate buffered saline supplemented with 0.2% Tween-20.

3. Results

FIG.9shows the resulting image on which is observed the presence of dark grey spots (corresponding to the red spot in color image) indicating an immune complex formation and therefore a positive reaction, while the presence of a colourless spot reflects a negative reaction. The specificity is respected whatever the surfactant used.

Example 7: Absorbing and Draining Material Used in the Device of the Invention

1. Absorbing Material

1.1. Antibodies Coupling

On 1 ml of homogenized 4% beads, 200 μl of 20× phosphate buffered saline are added and 250 μl of 6D4 (anti human IgM Diagast clone) are dissolved. The volume is completed up to 4 ml with demineralized water. The solution is end over end mixed overnight to ensure coupling.

The excess active groups are blocked by suspending in the bead antibody solution 150 μl of 1M ethanolamine and mixed for 2 hours at room temperature. The AHG coupled beads are then washed extensively 2 times with 3 ml of conservation buffer (centrifugation 5 min at 2500 g before and after washes). The bead pellet is resuspended with 2 ml of concentrated anti RH2 antibody (P3X255 13 G8 Diagast clone) plus 2 ml of phosphate buffered saline. The solution is end over end mixed overnight to ensure coupling.

The coupled beads are then washed extensively 2 times with 3 ml of conservation buffer (centrifugation 5 min at 2500 g before and after washes). Finally the bead pellet is resuspended in the conservation buffer to get back the introduced bead solution weight.

1.2. Reaction Device Preparation and Detection Procedure

The device is a moulded plastic cassette in which a Porex HDPE hydrophobic membrane of 0.6 mm thickness and porosity between 9 to 12 μm, a draining pad (NA7150 PES from Subrenat) and an absorbent pad (references from Mc Arlaid below) are assembled.

The used adsorbing material is as follows:T-499 or SCP-300 TCF (FIG.10a)T-099 or T-499 (FIG.10b)T-499 or T-183-5 (FIG.10c)T-499 or SCP-200-TCF (FIG.10d)

The porous membrane is hydrophilized with 1 μl of a surfactant solution (phosphate buffered saline supplemented with 0.5% Triton X-100; 1% green dye). 10 μl of antibody or negative control coupled beads at 1% are then spotted and the device is dried for 15 min at 37° C., 20% humidity before immediate use or extended conservation.

A test is initiated by diluting red blood cells at 2.5% in chromasolcoombs (Diagast formulation) and 50 μl are introduced in each well followed by two times 30 μl of washing buffer (phosphate buffered saline supplemented with 0.2% Tween20).

1.3. Results

It appears fromFIG.10a, b, canddthat any of the tested absorbent layer could guarantee the detection of RH2 positive red blood cell (presence of a red spot, visible as dark gray spots). The specificity is respected; an uncolored spot reflects an RH2 negative red blood cell.

2. Draining Materials

2.1. Antibodies Coupling

On 1 ml of homogenized 4% beads, 1 ml of concentrated anti-AB antibody (152 D12 Diagast clone) is dissolved. The volume is completed up to 4 ml with phosphate buffered saline. The solution is end over end mixed overnight to ensure coupling.

The coupled beads are then washed extensively 2 times with 3 ml of conservation buffer (centrifugation 5 min at 2500 g before and after washes). Finally the bead pellet is resuspended in the conservation buffer to get back the introduced bead solution weight.

2.2. Reaction Device Preparation and Detection Procedure

The device is a moulded plastic cassette in which a Porex HDPE hydrophobic membrane of 0.6 mm thickness and porosity between 9 to 12 μm, a draining pad (references below) and an absorbent pad (Cleanis) are assembled.

The draining material is as follows:cotton wool (from Alan &co), shown onFIG.11aNT 9750 HY (from Subrenat), shown onFIG.11b,NT 9610 HY (from Subrenat), shown onFIG.11c

The porous membrane is hydrophilized with 1 μl of a surfactant solution (phosphate buffered saline supplemented with 1% Triton X-100; 1% green dye). 10 μl of antibody or negative control coupled beads at 1% are then spotted and the device is dried for 15 min at 37° C., 10% humidity before immediate use or extended conservation.

A test is initiated by diluting red blood cells at 2.5% in chromasolcoombs (Diagast formulation) and 50 μl are introduced in each well followed by two 30 μl washes (phosphate buffered saline supplemented with 0.2% Tween20).

2.3. Results

FIG.11shows that any of the draining pad could guarantee the detection of B positive red blood cell (presence of a red spot visible as dark gray spot on the figure). The specificity is respected; an uncolored spot is obtained with O red blood cells.

Example 8: Grouping and Detection of Mixed Field Population Using the Device of the Invention

1. Antibodies Coupling

On 1 ml of homogenized 4% beads (Thermo Fisher latex Aldehyde/Sulfate 9 μm 4% w/v), 100 μl of 20× phosphate buffered saline are added and 2 ml of anti-ABO01 antibody (91 13 D10 Diagast clone) or anti-ABO02 antibody (96 21 A8 Diagast clone) are dissolved. The volume is completed up to 4 ml with demineralized water. The solution is end over end mixed overnight to ensure coupling.

The excess active groups are blocked by suspending in the bead antibody solution 150 μl of 1M ethanolamine and mixed for 2 hours at room temperature. The coupled beads are then washed extensively 2 times with 3 ml of conservation buffer (centrifugation 5 min at 2500 g before and after washes). Finally the bead pellet is resuspended in the conservation buffer to get back the introduced bead solution weight.

2. Reaction Device Preparation and Detection Procedure

The device is a moulded plastic cassette in which a Porex HDPE hydrophobic membrane of 0.6 mm thickness and porosity between 9 to 12 μm, a draining pad (cotton wool from Alan&co) and an absorbent pad (Cleanis) are assembled.

The porous membrane is hydrophilized with 1 μl of a surfactant solution (phosphate buffered saline supplemented with 1% Triton X-100 and 1% of green dye). 10 μl of antibody coupled beads at 3%, are then spotted. The device is dried for 15 min at 37° C., 20% humidity before use.

The detection procedure is initiated by introducing in each well 50 μl of red blood cells diluted at 0.15% in Chromasolcoombs supplemented with 0.00625% Tween20.

Mixed field A and B populations are generated by mixing respectively A and O or B and O red blood cells pre diluted at 0.15% in Chromasolcoombs at desired concentrations (30% O with 70% A or B red blood cells or 50% O with 50% A or B red blood cells).

A first image before washes is acquired. The reaction zone is then washed twice with 30 μl of phosphate buffered saline supplemented with 0.2% Tween-20. An image is acquired after each wash.

3. Results

As shown onFIGS.12aandc, before washes, all the spots appear in dark gray (or in red). Specificity is reached after the second wash: the presence of a red spot (visible as a dark gray spot) indicates an immune complex formation, and therefore a positive reaction, while the presence of a colourless spot reflects a negative reaction.

FIGS.12banddshows that the discrimination between native and mixed field populations is met after intensity interpretation of the acquired images (the signal before and after two washes is subtracted).

Example 9: Detection of Weak RH1 Antigens and Extended Phenotyping With Indirect Coupling of Red Blood Cells Antibodies to Beads Via Affinity Proteins for Antibodies

1. Protein AGL Coupling

On 1 ml of homogenized 4% beads, 50 μg of Protein AGL are dissolved in 3 ml PBS1× and are added. The solution is end over end mixed overnight to ensure coupling.

The coupled beads are then centrifuged 5 min at 2500 g. Finally the bead pellet is resuspended in the conservation buffer (phosphate buffered saline supplemented with 0.5% bovine serum albumin and 0.25M saccharose) to get back the introduced bead solution weight.

2. Reaction Device Preparation and Detection Procedure

The device is a moulded plastic cassette in which a Porex HDPE hydrophobic membrane of 0.6 mm thickness and porosity between 9 to 12 μm, a draining pad (9610HY) and an absorbent pad (Cleanis) are assembled.

The porous membrane is hydrophilized with 1 μl of a surfactant solution with AGL coupled beads (1.5% Triton X-100; phosphate buffered saline supplemented with 0.5% bovine serum albumin; 0.25M saccharose and 5.1% beads). The device is dried for 15 min at 37° C., 20% humidity before use.

The detection procedure is initiated by introducing in each well 50 μl of reactive (Weak RH1 or Extended phenotyping); add 50 μl of red blood cells diluted at 1.5% in PBS1× supplemented with 0.00625% Tween20.

A first image before washes is acquired. The reaction zone is then washed with 30 μl of phosphate buffered saline supplemented with 0.2% Tween-20. An image is acquired after wash.

3 Results

FIG.13ashows the weak RH1 resulting image on which is observed the presence of dark grey spots (corresponding to the red spot in color image) indicating an immune complex formation and therefore a positive reaction, while the presence of a colorless spot reflects a negative reaction.

FIG.13bshows the extending phenotyping resulting image on which is observed the presence of dark grey spots (corresponding to the red spot in color image) indicating an immune complex formation and therefore a positive reaction, while the presence of a colorless spot reflects a negative reaction.

Example 10: Reverse Grouping Via 6D4 Antibody Coupled to Beads

1. Reverse Grouping

1.1. Antibodies Coupling

On 1 ml of homogenized 4% beads (Thermo Fisher latex Aldehyde/Sulfate 9 μm 4% w/v), 3 ml of 1× phosphate buffered saline are added and 100 μg of CP6D4 antibody are dissolved. The solution is end over end mixed overnight to ensure coupling.

The excess active groups are blocked by suspending in the bead antibody solution 150 μl of 1M ethanolamine and mixed for 2 hours at room temperature. The coupled beads are then washed extensively 2 times with 3 ml of conservation buffer (centrifugation 5 min at 2500 g before and after washes). Finally the bead pellet is resuspended in the conservation buffer to get back the introduced bead solution weight.

1.2. Reaction Device Preparation And Detection Procedure

The device is a moulded plastic cassette in which a Porex HDPE hydrophobic membrane of 0.6 mm thickness and porosity between 9 to 12 μm, a draining pad (Subrenat 9610 HY) and an absorbent pad (Cleanis) are assembled.

The porous membrane is hydrophilized with 0.5 μl of a surfactant solution (phosphate buffered saline supplemented with 1.5% Triton X-100; 0.5% green dye). 5 μl of antibody coupled beads at 1% are spotted and the device is dried 15 min at 37° C., 10% humidity before immediate use or extended conservation.

A test is initiated by incubating 30 μl of plasma and 20 μl red blood cells (A1; B; A2; O) during 5 min. 25 μl are introduced in each well followed by a 30 μl of wash buffer (phosphate buffered saline supplemented with 0.2% Tween20).

1.3. Results

FIG.14shows the presence of dark gray spots (corresponding to red spots) which indicate a capture of IgM sensitized red blood cells, and therefore a positive reverse grouping reaction, while the presence of a colorless spot reflects a negative reaction (non-sensitized red blood cells).

Example 11: DAT (Direct Coombs Test) Test Using the Device of the Invention

1. Red Blood Cell Pre Sensitized With Antibodies

1.1. Antibodies Coupling

On 3 ml of homogenized 4% beads (Thermo Fisher latex Aldehyde/Sulfate 9 μm 4% w/v), a 3 ml solution of anti-human globulin is dissolved. The AHG studied are SA6532 (polyclonal rabbit antibodies directed against human IgGs), C5-1 (Diagast clone, mouse IgG directed against human IgGs) and 188 33 (Diagast clone, mouse IgM directed against human IgGs). The solution is end over end mixed one hour to ensure coupling.

The bead suspension is centrifuged 5 min at 2500 g. The excess active groups are blocked by resuspending the bead pellet in 6 ml of an ethanolamine solution at 50 mM, followed by a 30 min mixing. The coupled beads are then washed extensively 2 times with 9 ml of conservation buffer (centrifugation 5 min at 2500 g before and after washes). Finally the bead pellet is resuspended in the conservation buffer to get back the introduced bead solution weight.

1.2. Reaction Device Preparation and Detection Procedure

The device is a moulded plastic cassette in which a Porex HDPE hydrophobic membrane of 0.6 mm thickness and porosity between 9 to 12 μm, a draining pad (Subrenat 9610 HY) and an absorbent pad (Cleanis) are assembled.

The porous membrane is hydrophilized with 0.5 μl of a surfactant solution (phosphate buffered saline supplemented with 1.5% Triton X-100; 0.5% green dye). 5 μl of antibody coupled beads at 3% are spotted and the device is dried 15 min at 37° C., 10% humidity before immediate use or extended conservation.

Red blood cells are in vitro pre sensitized with FY, KEL1KEL1 or RH1 IgG antibodies to simulate direct coombs positive red blood cells.

A test is initiated by diluting red blood cells (coombs negative or positive) at 0.25% in chromasolcoombs (Diagast formulation) supplemented with 0.00625% Tween20. 50 μl are introduced in each well followed by a 30 μl of wash buffer (phosphate buffered saline supplemented with 0.2% Tween20).

2. Direct Antiglobulin Test With Anti-IgG and Anti-C3d

2.1. Antibodies Coupling

On 1 ml of homogenized 4% beads (Thermo Fisher latex Aldehyde/Sulfate 9 μm 4% w/v), 3 ml of 1× phosphate buffered saline are added and 100 μg of SA6532 or C7610 antibody are dissolved. The solution is end over end mixed overnight to ensure coupling. The coupled beads are then centrifuged 5 min at 2500 g. Finally the bead pellet is resuspended in the conservation buffer to get back the introduced bead solution weight.

2.2. Reaction Device Preparation and Detection Procedure

The device is a moulded plastic cassette in which a Porex HDPE hydrophobic membrane of 0.6 mm thickness and porosity between 9 to 12 μm, a draining pad (Subrenat 9610 HY) and an absorbent pad (Cleanis) are assembled.

The porous membrane is hydrophilized with 0.5 μl of a surfactant solution (phosphate buffered saline supplemented with 1.5% Triton X-100; 0.5% green dye). 5 μl of antibody coupled beads at 4% are spotted and the device is dried 15 min at 37° C., 10% humidity before immediate use or extended conservation.

A test is initiated by diluting red blood cells (DAT negative or positive) at 1.5% in phosphate buffered saline supplemented with 0.00625% Tween20. 50 μl are introduced in each well followed by a 30 μl of wash buffer (phosphate buffered saline supplemented with 0.2% Tween20).

3. DAT Via Affinity Proteins for Antibodies

3.1. Protein AGL Coupling

On 1 ml of homogenized 4% beads, 50 μg of Protein AGL are dissolved in 3 ml PBS1× and are added. The solution is end over end mixed overnight to ensure coupling.

The coupled beads are then centrifuged 5 min at 2500 g. Finally the bead pellet is resuspended in the conservation buffer (phosphate buffered saline supplemented with 0.5% bovine serum albumin and 0.25M saccharose) to get back the introduced bead solution weight.

3.2. Reaction Device Preparation And Detection Procedure

The device is a moulded plastic cassette in which a Porex HDPE hydrophobic membrane of 0.6 mm thickness and porosity between 9 to 12 μm, a draining pad (9610HY) and an absorbent pad (Cleanis) are assembled.

The porous membrane is hydrophilized with 1 μl of a surfactant solution with AGL coupled beads (1.5% Triton X-100; phosphate buffered saline supplemented with 0.5% bovine serum albumin; 0.25M saccharose and 5.1% beads). The device is dried for 15 min at 37° C., 20% humidity before use.

The detection procedure is initiated by incubation in well 20 μl of reactive (DAT); add 50 μl of red blood cells diluted at 3% in PBS1× supplemented with 0.00625% Tween20 in well; add 20 μl IAT solution 2 buffer. Transfer 120 μl of mix in cartridge well.

A first image before washes is acquired. The reaction zone is then washed with 40 μl of IAT solution 2 buffer. An image is acquired after wash.

4. Results

FIG.15ashows the presence of dark gray spots (corresponding to red spots) which indicate a capture of IgG sensitized red blood cells, and therefore a positive direct coombs reaction, while the presence of a colourless spot reflects a negative reaction (non-sensitized red blood cells).

FIG.15bshows the presence of dark gray spots (corresponding to red spots) which indicate a capture of IgG or/and C3D sensitized red blood cells, and therefore a positive reaction, while the presence of a colorless spot reflects a negative reaction (non-sensitized red blood cells).

FIG.15cshows the resulting image on which is observed the presence of dark grey spots (corresponding to the red spot in color image) indicating an immune complex formation and therefore a positive reaction, while the presence of a colorless spot reflects a negative reaction.

Example 12: Detection of Antibodies Specific for Malaria Antigen Using the Device of Invention

1. Antigen Coupling

On 1 ml of homogenized 4% beads, 100 μg of Antigen Mal003 are dissolved in 3 ml PBS1× and are added. The solution is end over end mixed overnight to ensure coupling.

The coupled beads are then centrifuged 5 min at 2500 g. Finally the bead pellet is resuspended in the conservation buffer (phosphate buffered saline supplemented with 0.5% bovine serum albumin and 0.25M saccharose) to get back the introduced bead solution weight.

2. Antibodies Coupling for Detection

On 1 ml of homogenized 4% beads 1 μm, 200 μg of Antibodies 6D4 are dissolved in 3 ml PBS1× and are added. The solution is end over end mixed overnight to ensure coupling.

The coupled beads are then centrifuged 5 min at 5000 g. Finally the bead pellet is resuspended in the conservation buffer (phosphate buffered saline supplemented with 0.5% bovine serum albumin and 0.25M saccharose) to get back the introduced bead solution weight.

3. Reaction Device Preparation and Detection Procedure With HRP

The device is a moulded plastic cassette in which a Porex HDPE hydrophobic membrane of 0.6 mm thickness and porosity between 9 to 12 μm, a draining pad (9610HY) and an absorbent pad (Cleanis) are assembled.

The porous membrane is hydrophilized with 0.5 μl of a surfactant solution (phosphate buffered saline supplemented with 2% Triton X-100 and 1% of green dye). 2 μl of antigen Mal003 coupled beads at 4%, are then spotted. The device is dried for 15 min at 37° C., 20% humidity before use.

The detection procedure is initiated by introducing in each well 100 μl of sample (kit ab178649). Add 100 μl of antibody Anti-IgG and/or Anti-IgM HRP conjugate (kit ab178649). A first image before washes is acquired. The reaction zone is then washed with 30 μl of phosphate buffered saline supplemented with 0.2% Tween-20. The reaction zone is then washed with 30 μl of revelation buffer (TMB Substrate (kit ab178649)). An image is acquired after 15 min.

4. Reaction Device Preparation and Detection Procedure With Beads

The device is a moulded plastic cassette in which a Porex HDPE hydrophobic membrane of 0.6 mm thickness and porosity between 9 to 12 μm, a draining pad (9610HY) and an absorbent pad (Cleanis) are assembled.

The porous membrane is hydrophilized with 0.5 μl of a surfactant solution (phosphate buffered saline supplemented with 2% Triton X-100 and 1% of green dye). 2 μl of antigen Mal003 coupled beads at 4%, are then spotted. The device is dried for 15 min at 37° C., 20% humidity before use.

The detection procedure is initiated by introducing in each well 100 μl of sample (Kit ab178649). Add 30 μl of antibody 6D4 beads 1 μm at 0.1%. A first image before washes is acquired. The reaction zone is then washed with 120 μl of phosphate buffered saline supplemented with 0.2% Tween-20. An image is acquired after wash.

5. Reaction Device Preparation and Detection Procedure With Colloidal Gold Nanoparticles

The device is a moulded plastic cassette in which a Porex HDPE hydrophobic membrane of 0.6 mm thickness and porosity between 9 to 12 μm, a draining pad (9610HY) and an absorbent pad (Cleanis) are assembled.

The porous membrane is hydrophilized with 0.5 μl of a surfactant solution (phosphate buffered saline supplemented with 2% Triton X-100 and 1% of green dye). 5 μl of antigen Mal003 coupled beads at 4%, are then spotted. The device is dried for 15 min at 37° C., 20% humidity before use.

The detection procedure is initiated by introducing in each well 50 μl of sample (Kit ab178649). Add 10 μl of Anti-Human IgA, IgG, IgM 60 nm gold conjugate (AC-60-14-10). A first image before washes is acquired. The reaction zone is then washed with 50 μl of phosphate buffered saline supplemented with 0.2% Tween-20. An image is acquired after wash.

6. Results

FIG.16ashows the resulting image on which is observed the presence of dark grey spots indicating an immune complex formation and therefore a positive reaction, while the presence of a colorless spot reflects a negative reaction and graphic with intensity of grey color.

FIG.16bshows the resulting image on which is observed the presence of dark grey spots indicating an immune complex formation and therefore a positive reaction, while the presence of a colorless spot reflects a negative reaction and graphic with intensity of grey color.

FIG.16cshows the resulting image on which is observed the presence of dark grey spots indicating an immune complex formation and therefore a positive reaction, while the presence of a colorless spot reflects a negative reaction and graphic with intensity of grey color.

Example 13: Detection of Antibodies Specific forT. PallidumAntigens Using the Device of Invention With 1 μm-Red Colored Beads as Detection Method

1. Antigen Coupling

On 1 ml of homogenized 4% beads, 100 μg of p15, p17, p47T. Pallidum antigens(30-AT76) are dissolved in 3 ml PBS1× and are added. The solution is end over end mixed overnight to ensure coupling.

The coupled beads are then centrifuged 5 min at 2500 g. Finally the bead pellet is resuspended in the conservation buffer (phosphate buffered saline supplemented with 0.5% bovine serum albumin and 0.25M saccharose) to get back the introduced bead solution weight.

2. Antibodies Coupling for Detection

On 1 ml of homogenized 4% beads 1 μm, 200 μg of AGL protein are dissolved in 3 ml PBS1× and are added. The solution is end over end mixed overnight to ensure coupling.

The coupled beads are then centrifuged 5 min at 5000 g. Finally the bead pellet is resuspended in the conservation buffer (phosphate buffered saline supplemented with 0.5% bovine serum albumin and 0.25M saccharose) to get back the introduced bead solution weight.

3. Reaction Device Preparation and Detection Procedure

The device is a moulded plastic cassette in which a Porex HDPE hydrophobic membrane of 0.6 mm thickness and porosity between 9 to 12 μm, a draining pad (9610HY) and an absorbent pad (Cleanis) are assembled.

The porous membrane is hydrophilized with 0.5 μl of a surfactant solution (phosphate buffered saline supplemented with 2% Triton X-100 and 1% of green dye). 2 μl of p15, p17, p47T. Pallidumantigen coupled beads at 4%, are then spotted. The device is dried for 15 min at 37° C., 20% humidity before use.

The detection procedure is initiated by introducing in each well 10 μl of sample (Treponema pallidumantibody 20-TR89). Add 30 μl of AGL protein beads 1 μm at 0.1%. A first image before washes is acquired. The reaction zone is then washed with 120 μl of phosphate buffered saline supplemented with 0.2% Tween-20. An image is acquired after wash.

4. Results

FIG.17shows the resulting image on which is observed the presence of dark grey spots indicating an immune complex formation and therefore a positive reaction, while the presence of a colorless spot reflects a negative reaction and graphic with intensity of grey color.

Example 14: Detection of Antibodies Specific for Hepatitis B Virus Antigen (HbsAg) Using the Device of Invention with Beads 1 μm Red Detection Method

1. Antigen Coupling

On 1 ml of homogenized 4% beads, 100 μg of HBs Antigen (30-AH15) are dissolved in 3 ml PBS1× and are added. The solution is end over end mixed overnight to ensure coupling.

The coupled beads are then centrifuged 5 min at 2500 g. Finally the bead pellet is resuspended in the conservation buffer (phosphate buffered saline supplemented with 0.5% bovine serum albumin and 0.25M saccharose) to get back the introduced bead solution weight.

2. Antibodies Coupling For Detection

On 1 ml of homogenized 4% beads 1 μm, 200 μg of AGL protein are dissolved in 3 ml PBS1×and are added. The solution is end over end mixed overnight to ensure coupling.

The coupled beads are then centrifuged 5 min at 5000 g. Finally the bead pellet is resuspended in the conservation buffer (phosphate buffered saline supplemented with 0.5% bovine serum albumin and 0.25M saccharose) to get back the introduced bead solution weight.

3. Reaction Device Preparation and Detection Procedure

The device is a moulded plastic cassette in which a Porex HDPE hydrophobic membrane of 0.6 mm thickness and porosity between 9 to 12 μm, a draining pad (9610HY) and an absorbent pad (Cleanis) are assembled.

The porous membrane is hydrophilized with 0.5 μl of a surfactant solution (phosphate buffered saline supplemented with 2% Triton X-100 and 1% of green dye). 2 μl of HBs antigen coupled beads at 4%, are then spotted. The device is dried for 15 min at 37° C., 20% humidity before use.

The detection procedure is initiated by introducing in each well 10 μl of sample (HBsAg antibody 10-H05G). Add 30 μl of AGL protein beads 1 μm at 0.1%. A first image before washes is acquired. The reaction zone is then washed with 120 μl of phosphate buffered saline supplemented with 0.2% Tween-20. An image is acquired after wash.

4. Results

FIG.18shows the resulting image on which is observed the presence of dark grey spots indicating an immune complex formation and therefore a positive reaction, while the presence of a colorless spot reflects a negative reaction and graphic with intensity of grey color.

Example 15: Detection of the Hepatitis B Virus Antigen Using the Device of Invention

1. Antigen Coupling

On 1 ml of homogenized 4% beads, 100 μg of HBs Antibody (10-H05G) are dissolved in 3 ml PBS1× and are added. The solution is end over end mixed overnight to ensure coupling.

The coupled beads are then centrifuged 5 min at 2500 g. Finally the bead pellet is resuspended in the conservation buffer (phosphate buffered saline supplemented with 0.5% bovine serum albumin and 0.25M saccharose) to get back the introduced bead solution weight.

2. Antibodies Coupling For Detection

On 1 ml of homogenized 4% beads 1 μm, 200 μg of HBs Antibody (10-H05H) are dissolved in 3 ml PBS1× and are added. The solution is end over end mixed overnight to ensure coupling.

The coupled beads are then centrifuged 5 min at 5000 g. Finally the bead pellet is resuspended in the conservation buffer (phosphate buffered saline supplemented with 0.5% bovine serum albumin and 0.25M saccharose) to get back the introduced bead solution weight.

3. Reaction Device Preparation and Detection Procedure With HRP Method as Detection Method

The device is a moulded plastic cassette in which a Porex HDPE hydrophobic membrane of 0.6 mm thickness and porosity between 9 to 12 μm, a draining pad (9610HY) and an absorbent pad (Cleanis) are assembled.

The porous membrane is hydrophilized with 0.5 μl of a surfactant solution (phosphate buffered saline supplemented with 2% Triton X-100 and 1% of green dye). 2 μl of antigen HBs Antibody (10-H05G) coupled beads at 4%, are then spotted. The device is dried for 15 min at 37° C., 20% humidity before use.

The detection procedure is initiated by introducing in each well 50 μl of sample (kit 1701-12). Add 50 μl of antibody Anti-HBs antigen HRP conjugate (kit 1701-12). A first image before washes is acquired. The reaction zone is then washed with 100 μl of revelation buffer (TMB Substrate (kit 1701-12)). An image is acquired after 15 min.

4. Reaction Device Preparation and Detection Procedure With Beads Method

The device is a moulded plastic cassette in which a Porex HDPE hydrophobic membrane of 0.6 mm thickness and porosity between 9 to 12 μm, a draining pad (9610HY) and an absorbent pad (Cleanis) are assembled.

The porous membrane is hydrophilized with 0.5 μl of a surfactant solution (phosphate buffered saline supplemented with 2% Triton X-100 and 1% of green dye). 2 μl of HBs Antibody (10-H05G) coupled beads at 4%, are then spotted. The device is dried for 15 min at 37° C., 20% humidity before use.

The detection procedure is initiated by introducing in each well 10 μl of sample HBs antigen (kit 1701-12). Add 100 μl of HBs Antibody (10-H05H) beads 1 μm at 0.025%. A first image before washes is acquired. The reaction zone is then washed with 120 μl of phosphate buffered saline supplemented with 0.2% Tween-20. An image is acquired after wash.

5. Results

FIG.19ashows the resulting image on which is observed the presence of dark grey spots indicating an immune complex formation and therefore a positive reaction, while the presence of a colorless spot reflects a negative reaction and graphic with intensity of grey color.

FIG.19bshows the resulting image on which is observed the presence of dark grey spots indicating an immune complex formation and therefore a positive reaction, while the presence of a colorless spot reflects a negative reaction and graphic with intensity of grey color.