METHOD FOR DETECTING AN IMMUNE CELLULAR RESPONSE

Abstract

A method for detecting an immune cellular response including the steps of: (a) suctioning a defined amount of the sample by an automated suction/discharge device, (b) discharging this amount, using the device, into container R, containing, beforehand, at least one stimulant for secretion of the molecule by the at least one cell, or else not containing such a stimulant, (c) when the container does not contain the stimulant beforehand, introducing into container R the at least one secretion stimulant contained in container E, using the device, and (d) allowing the sample and stimulant to incubate, forming a mixture, by the at least one cell (i) in container R or (ii) in another container S after suctioning and discharge of the mixture using the device, and (e) detecting the immune cellular response, the detection of the immune cellular response indicates the presence of an immune cellular response specific for the stimulant.

Claims

1. A method for detecting an immune cellular response in a biological sample of animal origin, which may contain at least one cell which secretes at least one immune effector molecule, comprising the steps consisting in: (a) suctioning a defined amount of the biological sample as taken by means of an automated suction/discharge device, (b) discharging this defined amount, using the automated suction/discharge device, into a container R, the container R containing, beforehand, at least one stimulant for secretion of the molecule by the at least one cell, or else the container R not containing such an at least one stimulant, (c) when the container does not contain, beforehand, at least one secretion stimulant, introducing into the container R the at least one secretion stimulant contained in a container E, using the automated suction/discharge device, and (d) allowing the defined amount of sample and the at least one stimulant to incubate, forming a mixture, for the secretion of the molecule by the at least one cell (i) in the container R or (ii) in another container S after suctioning and discharge of the mixture using the automated suction/discharge device, and (e) detecting the immune cellular response, wherein the detection of the immune cellular response indicates the presence of an immune cellular response specific for the at least one secretion stimulant.

2. The method for detecting an immune cellular response as claimed in claim 1, wherein the secretion stimulant is a nonspecific stimulant or a stimulant specific for a pathogenic microorganism.

3. The method for detecting an immune cellular response as claimed in claim 1, wherein the at least one secretion stimulant is an antigen highly specific for Mycobacterium tuberculosis, which may be absent from the BCG (Bacillus Calmette-Gurin) vaccine.

4. The method for detecting an immune cellular response as claimed in claim 3, wherein the at least one secretion stimulant is chosen from the ESAT-6, CFP-10 and TB7.7 antigens.

5. The method for detecting an immune cellular response as claimed in claim 1, wherein the at least one cell is a cell capable of secreting at least one cytokine.

6. The method for detecting an immune cellular response as claimed in claim 5, wherein the at least one cytokine is chosen from interleukins, chemokines, tumor necrosis factor (TNF) and interferon gamma (IFN).

7. The method for detecting an immune cellular response as claimed in claim 1, wherein the cells are B cells capable of secreting at least one antibody.

8. The method for detecting an immune cellular response as claimed in claim 1, wherein the biological sample is a biological sample of human, bovine, canine, feline or equine origin.

9. The method for detecting an immune cellular response as claimed in claim 1, wherein the incubation step (d) is carried out under nonsterile conditions.

10. The method for detecting an immune cellular response as claimed in claim 1, wherein the container R is nonsterile after introduction of the defined amount of sample.

11. The method for detecting an immune cellular response as claimed in claim 1, wherein the duration of the incubation step is between 2 and 72 h.

12. The method for detecting an immune cellular response as claimed in claim 1, wherein step (e) uses a partner for binding to the immune effector molecule which is an immunoassay partner and in that the revealing of the binding or nonbinding of the molecule is carried out by means of a sandwich assay using another partner for binding to the molecule, which may or may not be different in nature, one of the two partners being labeled.

13. The method for detecting an immune cellular response as claimed in claim 1, wherein step (e) uses a partner for binding to the immune effector molecule which is an immunoassay partner and in that the revealing of the binding or nonbinding of the molecule is carried out by means of a competition assay using a labeled compound which competes with the molecule.

14. The method for detecting an immune cellular response as claimed in claim 1, wherein steps (a) to (e) are carried out with the same instrument.

15. The method for detecting an immune cellular response as claimed in claim 1, wherein steps (a) to (e) are carried out successively on the same day.

16. The method for detecting an immune cellular response as claimed in claim 1, wherein steps (a) to (d) are carried out on a first day or over the course of several days, and step (e) is then carried out on another day.

17. An automated method for stimulating at least one cell capable of secreting an immune effector molecule for the secretion of the molecule, the at least one cell being from a biological sample of animal origin, wherein it comprises or consists of the steps consisting in: (a) suctioning a defined (calibrated) amount of the biological sample as taken by means of an automated suction/discharge device, (b) discharging this defined amount, using the automated suction/discharge device, into a container R, the container R containing, beforehand, at least one stimulant for secretion of the molecule by the at least one cell, or else the container R not containing such an at least one stimulant, (c) when the container does not contain, beforehand, at least one secretion stimulant, introducing into the container R the at least one secretion stimulant contained in a container E, using the automated suction/discharge device, and (d) allowing the defined amount of sample and the at least one stimulant to incubate, forming a mixture, for the secretion of the molecule by the at least one cell (i) in the container R or (ii) in another container S after suctioning and discharge of the mixture using the automated suction/discharge device.

18. The automated stimulation method as claimed in claim 17, wherein the incubation step (d) is carried out under nonsterile conditions.

19. The automated stimulation method as claimed in claim 17, wherein the container R is nonsterile after introduction of the defined amount of sample.

20. The automated stimulation method as claimed in claim 17, wherein the secretion stimulant is a nonspecific stimulant or a stimulant specific for a pathogenic microorganism.

21. The automated stimulation method as claimed in claim 18, wherein the biological sample is a sample of human, bovine, canine, feline or equine origin.

22. The method for detecting an immune cellular response as claimed in claim 1, wherein the method is used for the diagnosis of a pathological condition, for a medicament or vaccine toxicity study or for determining the immune status of a patient.

Description

EXAMPLES

Example 1: Automation of the Stimulation Step, Taking of Total Blood Sample, Stimulation with a Nonspecific Stimulant, Assaying of Interferon Gamma

1.1. Carrying Out the Secretion Step

[0147] The blood samples used in this example were obtained from the Etablissements Franais du Sang (EFS) [French National Blood Bank] and collected from healthy donors. Five blood samples were taken sterily in Vacutainer tubes (Becton-Dickinson) containing lithium heparinate or equivalent reference from another supplier.

[0148] These samples were stimulated according to two conditions (1 and 2)

Condition 1: Stimulation in a Tube, According to the Prior ArtSterile Stimulation Conditions.

[0149] For this condition, sterile dry tubes (Cat. No. 367614, Becton-Dickinson) are used. Under sterile conditions, under a laminar flow hood, each blood sample is stimulated in three different ways:

Assay 1: PBS tube (negative control): 400 l of PBS buffer are mixed with 400 l of total blood.
Assay 2: Phorbol 12-myristate 13-acetate (PMA)-phytohemagglutinin (PHA) tube: 400 l of PMA (2.30 ng/ml) and PHA (0.46 g/ml) stimulants are mixed with 400 l of total blood.
Assay 3: Phorbol 12-myristate 13-acetate (PMA)-phytohemagglutinin (PHA) tube: 400 l of PMA (25 ng/ml) and PHA (5 g/ml) stimulants are mixed with 400 l of total blood.

[0150] The tubes were re-stoppered under a hood and then incubated vertically at 37 C. for 17 hours 30 minutes.

Condition 2: Automated Stimulation in a VIDAS CartridgeNonsterile Stimulation Conditions.

[0151] The strip of the VIDAS immunoanalysis automated device is described in FIG. 1. Said strip (11) is composed of 10 wells (1 to 10) partially covered with a labeled sealed aluminum foil (not shown). The last well (10) is an optical cuvette in which the fluorescence is measured, and contains the enzymatic substrate. The various reagents required for the stimulation and/or the analysis, such as the stimulant (in this case PBS wells or PMA-PHA wells), the washing solutions or the revealing antibodies, are contained in the various other wells (1 to 9).

[0152] The strips thus prepared were inserted into the VIDAS 3 automated device along with the tubes of total blood taken from the donors. Before loading the tubes in the automated device, their stopper is removed. From this moment, the total blood samples are no longer processed under sterile conditions. The VIDAS 3 instrument was programmed in order to automatically carry out the following steps: [0153] 1) Suctioning, by means of the suction/discharge device, a precise volume of total blood (400 l) from the primary sample tube, [0154] 2) Piercing the aluminum foil which covers the stimulation well using the suction/discharge device, [0155] 3) Discharging the total blood into the stimulation well, [0156] 4) Incubating the cartridge at 37 C. for 17 h 30 min.

[0157] Following this stimulation step, the VIDAS 3 can automatically link said step to the step of detecting the secreted IFN according to the procedure described hereinbelow in section 1.2.

[0158] The conditions for stimulation in a sterile tube (tube) or in the VIDAS strip (well) are summarized in Table 1 hereinafter.

TABLE-US-00001 TABLE 1 conditions for sterile-tube stimulation or automated-device-well stimulation Stimulation Conditions container Secretion stimulant PBS Tube PBS (negative control) PMA-PHA low Tube PMA 2.30 ng/ml + concentration PHA 0.46 g/ml in PBS PMA-PHA high Tube PMA 25 ng/ml + concentration PHA 5 g/ml in PBS PBS Well PBS (negative control) PMA-PHA low Well PMA 2.30 ng/ml + concentration PHA 0.46 g/ml in PBS PMA-PHA high Well PMA 25 ng/ml + concentration PHA 5 g/ml in PBS PMA: phorbol 12-myristate 13-acetate PHA: phytohemagglutinin

1.2. IFN Detection Step: Automated Immunoassay Procedure

Detection Principle

[0159] The detection of the IFN in the biological samples is carried out by one-step sandwich immunoassay using the VIDAS immunoanalysis automated device (bioMrieux). The single-use tip (represented as 12 in FIG. 1) serves both as solid phase for the reaction and as pipetting system. It is used in combination with the strip (11), as described in point 1.1 above. All the steps of the test are thus carried out automatically by the instrument. They consist of a succession of cycles of suction/discharge of the reaction medium.

Sensitization and Passivation of the Tips.

[0160] The tips were sensitized with 270 l of a solution of first anti-IFN monoclonal antibody (4G1E12, bioMrieux, France) at 5 g/ml in a carbonate buffer. After approximately 6-8 h of incubation at +18/25 C. with the sensitizing solution, the tips were emptied. Next, 300 l of phosphate buffer containing 10 g/l of bovine albumin are added for the passivation of the tips at +18/25 C. for approximately 18-24 h. The tips are then emptied, dried, then stored at +4 C. until use, in the dry.

Preparation of the Conjugated Antibody Solutions.

[0161] The solution of conjugate (detection partner) contains a second anti-IFN monoclonal antibody coupled to alkaline phosphatase (2A4B11, bioMrieux). The conjugated antibodies were diluted to approximately 500 ng/ml in a phosphate/BSA buffer.

Immunoassay.

[0162] As soon as the VIDAS tip is in contact with the sample, the immunological reaction begins since the capture antibodies are immobilized on this tip. The automated device mixes the sample to be tested (100 lin the present case the blood after stimulation) with 250 l of the solution of conjugate. The incubation lasts approximately 10 minutes at 37 C. and allows the specific binding of the IFN to, on the one hand, the antibody adsorbed onto the tip (capture antibody) and to, on the other hand, the conjugated antibody (detection antibody). The nonbound components are then removed by means of three washes with a Tris NaCl buffer. During the final revealing step, the 4-methylumbelliferyl phosphate substrate is suctioned then discharged in the tip; the enzyme of the conjugated antibody catalyzes the reaction for hydrolysis of this substrate to 4-methylumbelliferone, the emitted fluorescence of which is measured at 450 nm. The fluorescence signal value (RFV: relative fluorescence value) is proportional to the concentration of the antigen present in the sample.

[0163] The results of the assays are presented in Table 2:

TABLE-US-00002 TABLE 2 measurement of IFN by VIDAS IFN measurement by VIDAS Signal in RFV PMA-PHA high PMA-PHA low Sample Stimulation PMA/PHA high PMA/PHA low concentration/ concentration/ code container PBS concentration concentration PBS ratio PBS ratio 0312274 Tube 23 11447 229 498 10 Well 11 11516 72 1047 7 0312370 Tube 15 11319 111 755 7 Well 8 10928 48 1366 6 0312397 Tube 26 11216 767 431 30 Well 16 13588 543 43.6 34 0312303 Tube 16 11306 610 707 38 Well 7 8905 172 1272 25

[0164] A very strong secretion of IFN (signals >8000 RFV) is observed when the cells are stimulated using a high concentration of PHA in the PMA/PHA mixture, whereas the secretion is moderate (signals <800 RFV) if the PHA concentration is low in the PMA/PHA mixture. This phenomenon is observed both for the stimulation carried out in the tube and for that carried out in the well.

[0165] The nonspecific secretion in the absence of stimulant (PBS assay) is very low in the tube (signals <30 RFV) and virtually nonexistent in the well (signals <20 RFV).

[0166] The IFN secretion level obtained after stimulation is compared to that measured in the absence of stimulant by calculating a ratio between the two measurements. If the ratio is at least 3, a positive conclusion is drawn.

[0167] When the stimulation is carried out in the well, the ratios range from 6 to 1366, whereas, when the stimulation is carried out in the tube, the ratios range from 7 to 755. It can thus be concluded that the stimulation causing the IFN secretion is equivalent in the well and the tube or is better in the well than in the tube.

Example 2: Automation of the Stimulation Step, Taking of Total Blood Sample, Stimulation with a Specific Stimulant (Mycobacterium tuberculosis Antigens), Assaying of Interferon Gamma

[0168] The blood samples used in this example were obtained from Biotech Bank (Angers, France) and collected either from individuals having had tuberculosis or from their close entourage. Four blood samples were taken sterily in Vacutainer tubes (Becton-Dickinson) containing lithium heparinate or an equivalent reference from another supplier.

[0169] Each blood sample was stimulated according to three different protocols detailed in Table 3 hereinafter, using as stimulation agent the content of the Quantiferon TB tubes (Cat. No. T0590-0301, Qiagen). The Quantiferon TB tubes are particular vacuum blood sampling tubes which make it possible to collect the total blood under perfectly sterile conditions. There are three of them:

[0170] The AG tube, for antigen, contains peptides derived from the ESAT-6, CFP-10 and TB7.7 proteins of Mycobacterium tuberculosis; it is the tube for the specific stimulation. If there is IFN secretion in this tube, it is because the sample contains T cells which are capable of specifically reacting against the M. tuberculosis peptides.

[0171] The NIL tube corresponds to the negative control and the MIT tube, which contains a mitogen, corresponds to the positive control.

[0172] Each sample was stimulated by the content of the two Quantiferon AG and NIL tubes and according to two protocols, which corresponds to a total of four conditions per sample.

[0173] In routine use, according to the manufacturer's instructions, 0.8 to 1.2 ml of total blood are directly taken sterily on Quantiferon TB tubes and the stimulation phase is carried out sterily in the tubes containing beforehand the secretion stimulants. The samples thus stimulated are then centrifuged and the interferon gamma detection is carried out, in a microplate, in the plasma thus obtained. However, in order to be able to perform the comparison with the automated stimulation, this procedure was slightly modified, as indicated in Table 3:

TABLE-US-00003 TABLE 3 interferon gamma stimulation and detection conditions Reference condition 1. Under a laminar flow hood, under CENTRIFUGATION sterile conditions: add 400 l of PBS to each Quantiferon tube, then add 400 l of blood 2. Invert the tubes 10 times in order to mix 3. Incubate in the vertical position at 37 C. for 17 h 30 4. Centrifugation for 15 min at 1800 g 5. IFN detection in the plasma by VIDAS Automated condition, 1. Add 400 l of PBS to each Quantiferon tube according to the 2. Invert the tubes 10 times in order to mix invention 3. Recovery of 400 l of stimulant for transfer into the stimulation well of the VIDAS cartridge 4. Launching of the automated stimulation program (nonsterile) + incubation + assay on VIDAS: Automatic suction of 2 200 l of total blood from the primary tube Discharge of the 400 l in the stimulation well Incubation at 37 C. for 17 h 30 IFN detection in the sample thus stimulated by VIDAS

[0174] The detection of IFN in the blood samples after the stimulation was carried out according to the procedure detailed in section 1.2 of Example 1.

[0175] The results (RFV signal measured using VIDAS according to the experimental conditions) are given in Table 4 below:

TABLE-US-00004 TABLE 4 measurement of IFN by VIDAS IFN measurement by VIDAS Signal in RFV Patient Stimulation ANTIGEN AG/NIL Interpre- code protocol NIL (AG) Ratio tation BK-37 Reference 104 118 1.1 Neg Automated 121 204 1.7 Neg BK-38 Reference 14 15 1.1 Neg Automated 11 14 1.3 Neg BK-39 Reference 53 482 9.1 Pos Automated 31 1351 43.6 Pos BK-40 Reference 67 354 5.3 Pos Automated 64 268 4.2 Pos BK-33 Reference 42 118 2.8 Neg Automated 45 48 1.1 Neg BK-34 Reference 98 98 1.0 Neg Automated 113 142 1.3 Neg BK-20 Reference 179 1083 6.1 Pos Automated 114 1954 17.1 Pos

Results:

[0176] The automated protocol for stimulation and detection of the immune cellular response, without centrifugation and under nonsterile conditions, makes it possible to obtain signals (11-1954 RFV) equivalent to or greater than those obtained with the sterile-tube reference protocol (14-1083 RFV). The AG/NIL ratio ranges from 1.1 to 43.6 for the automated condition and from 1.1 to 17.1 for the reference condition, which reflects an equivalent or greater specific stimulation under the automated condition.

[0177] The samples BK-37, BK-38, BK-33 and BK-34 have AG/NIL ratios which range between 1.0 and 2.8 under the two conditions tested. The detected IFN secretion level is basal (ratio <3) and is not specific for the M. tuberculosis antigens. Consequently, the automated condition and the reference condition make it possible to conclude that these patients were not infected with M. tuberculosis: negative interpretation (Neg).

[0178] The sample BK-40 exhibits highly corresponding AG/NIL ratios between 4.2 and 5.3 depending on the stimulation protocol. In this sample, IFN secretion (ratio >3) specifically triggered by the M. tuberculosis antigens is observed. It can be concluded that the BK-40 patient has already been infected with M. tuberculosis: positive interpretation (Pos). The same interpretation is obtained for the samples BK-39 and BK-20 for which the highest specific IFN secretion levels are demonstrated. For these patients, the AG/NIL ratios are not equivalent between the two conditions: ratios of 43.6 and 17.1 are respectively observed for the automated condition compared with 9.1 and 6.1 for the reference condition, which shows an improved analytical sensitivity for the automated condition, without a centrifugation step and under nonsterile conditions with a calibrated amount of blood, compared with the reference condition (sterile conditions with amount of blood which varies).

Example 3: Automation of the Stimulation Step, Taking of Total Blood Sample, Nonspecific PMA+PHA Stimulation, TNF Alpha Assay

3.1. Implementation of the Secretion Step

[0179] The blood samples used in this example were obtained from the Etablissements Franais du Sang (EFS) [French National Blood Bank] and collected from healthy donors. Two blood samples were taken sterily into Vacutainer tubes (Becton-Dickinson) containing lithium heparinate or equivalent reference from another supplier, and stimulated either in a tube (reference sterile condition), or in a VIDAS well (according to the invention) according to the procedure described in section 1.1.

[0180] The conditions for sterile-tube stimulation (tube) or stimulation in the VIDAS strip (well) are summarized in Table 5 below:

TABLE-US-00005 TABLE 5 conditions for sterile-tube stimulation or automated-device-well stimulation Stimulation Conditions container Secretion stimulant PBS Tube PBS (negative control) PMA-PHA high Tube PMA 25 ng/ml + concentration PHA 5 g/ml in PBS PBS Well PBS (negative control) PMA-PHA high Well PMA 25 ng/ml + concentration PHA 5 g/ml in PBS

[0181] At the end of the stimulation step, the sample is taken in order to measure the TNF concentration with the Quantikine HS ELISA human TNF immunoassay kit from R&D Systems (Cat No. HSTA00D) by applying the procedure recommended by the producer (instruction sheet version 12/13).

3.2. Results of the TNF Alpha Assay

[0182] The experimental results obtained are presented in Table 6 below:

TABLE-US-00006 TABLE 6 TNF measurement by ELISA TNF measurement by Stimu- ELISA in pg/ml PMA-PHA high Sample lation PMA/PHA high concentration/ code container PBS concentration PBS ratio 59171003916 Tube 2.15 >32 >14.9 Well 2.95 >32 >10.8 5917100373- Tube 1.33 >32 >24.1 Well 0.97 >32 >33.0

[0183] As shown in Table 6, a very strong TNF secretion (concentration greater than 32 pg/ml) is observed when the cells are stimulated using a high concentration of PHA in the PMA/PHA mixture. This phenomenon is observed both for the stimulation carried out in the tube and for that carried out in the well.

[0184] The nonspecific secretion in the absence of stimulant (PBS assay) is very weak both in the tube and in the well (concentration <3 pg/ml).

[0185] When the stimulation is carried out in the well, the ratios are 10.8 and 33, comparable to those calculated when the stimulation is carried out in the tube, respectively 14.9 and 24.1. It can thus be concluded that the TNF secretion was stimulated by the PMA-PHA mixture in an equivalent manner in the tube and in the well.

Example 4: Automation of the Stimulation Step, Taking of Total Blood Sample, Nonspecific LPS Stimulation, TNF Alpha Assay

4.1. Implementation of the Secretion Step

[0186] The blood samples used in this example were obtained from the Etablissements Franais du Sang (EFS) [French National Blood Bank] and collected from healthy donors. Two blood samples were taken sterily into Vacutainer tubes (Becton-Dickinson) containing lithium heparinate or equivalent reference from another supplier.

[0187] The samples were stimulated either in a tube (REF sterile condition), or in a VIDAS well (according to the invention) according to the procedure described in section 1.1 of example 1, with the only difference being that the PMA+PHA mixture was replaced with a mixture in equal parts of E. coli lipopolysaccharides (LPS, Sigma, Cat. No. L3012, L2637 and L3137). The stimulation conditions are reproduced in Table 7:

TABLE-US-00007 TABLE 7 conditions for sterile-tube stimulation or automated-device-well stimulation Conditions Stimulation container Secretion stimulant PBS Tube PBS (negative control) LPS Tube LPS 50 ng/ml PBS Well PBS (negative control) LPS Well LPS 50 ng/ml

[0188] At the end of the stimulation step, the sample is taken in order to measure the TNF alpha concentration with the Quantikine HS ELISA human TNF alpha immunoassay kit from R&D systems (Cat. No. HSTA00D) by applying the procedure recommended by the producer (instruction sheet version 12/13).

4.2. Results of the TNF Alpha Assay

[0189] The experimental results obtained are presented in Table 8:

TABLE-US-00008 TABLE 8 TNF measurement by ELISA TNF measurement by Stimu- ELISA in pg/ml PMA-PHA high Sample lation PMA/PHA high concentration/ code container PBS concentration PBS ratio 59171003916 Tube 2.15 >32 >14.9 Well 2.95 >32 >10.8 5917100373- Tube 1.33 >32 >24.1 Well 0.97 >32 >33.0

[0190] As shown in Table 8, a very strong TNF secretion (concentration >32 pg/ml) is observed when the cells are stimulated using LPS. This phenomenon is observed both for the stimulation carried out in the tube and for that carried out in the well.

[0191] The nonspecific secretion in the absence of stimulant (PBS assay) is very weak both in the tube and in the well (concentration <3 pg/ml).

[0192] When the stimulation is carried out in the well, the ratios are 10.8 and 33, comparable with those calculated when the stimulation is carried out in the tube, respectively 14.9 and 24.1. It can thus be concluded that the TNF secretion was stimulated by LPS in an equivalent manner in the tube and in the well.

Example 5: Automation of the Stimulation Step, Taking of Total Blood Sample, Stimulation with a Specific Stimulant (Cytomegalovirus Antigens), Interferon Gamma Assay

[0193] The blood samples used in this example were obtained from the Etablissements Franais du Sang (EFS [French National Blood Bank] Rhne-Alpes, France). Thirteen blood samples were taken sterily into Vacutainer tubes (Becton-Dickinson) containing lithium heparinate or equivalent reference from another supplier.

[0194] Each blood sample was stimulated according to the Automated condition, according to the invention protocol described in Table 3 of example 2 using, as stimulating agent, the content of the Quantiferon CMV tubes (Cat. No. 0192-0301, Qiagen). The Quantiferon CMV tubes are particular vacuum blood sampling tubes, which make it possible to collect total blood under perfectly sterile conditions. There are three of them:

[0195] The AG, for antigen, tube contains Cytomegalovirus peptides; it is the tube for the specific stimulation. If there is INF secretion during this stimulation, this means that the blood sample contains T cells which are capable of reacting specifically against the Cytomegalovirus peptides.

[0196] The NIL tube corresponds to the negative control and the MIT tube, which contains a mitogen, corresponds to the positive control.

[0197] Each sample was stimulated by the content of the Quantiferon AG and NIL tubes.

[0198] The detection of IFN in the blood samples after stimulation was carried out according to the procedure detailed in section 1.2 of example 1.

[0199] The results (RFV signal measured by VIDAS according to the experimental conditions) are given in Table 9 below.

TABLE-US-00009 TABLE 9 IFN measurement by VIDAS IFN measurement by VIDAS RFV signal Sample ANTIGEN AG/NIL code NIL (AG) ratio Interpretation 59180446129 87 90 1.0 Neg 59180447383 24 26 1.1 Neg 59180447498 11 15 1.4 Neg 59180447500 47 41 0.9 Neg 59180404025 12 2505 209 Pos 59180404005 25 26 1.0 Neg 59180404324 3 2325 775 Pos 59180808113 20 27 1.4 Neg 59180404076 23 7463 324 Pos 59174894224 9 12053 1339 Pos 59174894208 14 54 3.9 Neg 59174890039 98 1686 17.2 Pos 59174890020 26 29 1.1 Neg

Results:

[0200] The automated protocol for stimulation and detection of the immune cellular response, without centrifugation and under nonsterile conditions, makes it possible to obtain signals from 3 to 12 053 RFV and AG/NIL ratios ranging from 0.9 to 1339.

[0201] Samples 59180446129, 59180447383, 59180447498, 59180447500, 5918040405-, 59180404113, 59174894208 and 59174890020 have AG/NIL ratios which range between 0.9 and 3.9. The IFN secretion level detected is basal (ratio <5) and is not induced by the Cytomegalovirus peptides. Consequently, the stimulation under automated conditions makes it possible to conclude that these patients have not been infected by the Cytomegalovirus: negative interpretation (Neg).

[0202] Samples 59180404025, 59180404324, 59180404076, 59174894224 and 59174890039 have AG/NIL ratios between 17.2 and 1339. In these samples, IFN secretion (ratio >5) specifically triggered by the Cytomegalovirus peptides is observed. Consequently, the stimulation under automated conditions makes it possible to conclude that these patients have been infected by Cytomegalovirus: positive interpretation (Pos).

Example 6: Automation of the Stimulation Step, Taking of Total Blood Sample, Stimulation with a Specific Stimulant (Epstein-Barr Virus Antigens)

[0203] The blood samples used in this example were obtained from the Etablissements Franais du Sang (EFS, Rhne-Alpes, France). Ten blood samples were taken sterily in Vacutainer tubes (Becton-Dickinson) containing lithium heparinate or equivalent reference from another supplier.

[0204] Each blood sample was stimulated according to the Automated condition, according to the invention protocol described in Table 3 of example 2 using, as stimulating agent, Epstein-Barr virus peptides (Pep Tivator). For the NIL condition, the sample was brought into contact solely with PBS.

[0205] The detection of IFN in the blood samples after stimulation was carried out according to the procedure detailed in section 1.2 of example 1.

[0206] The results (RFV signal measured by VIDAS according to the experimental conditions) are given in Table 10 below.

TABLE-US-00010 TABLE 10 IFN measurement by VIDAS IFN g measurement by VIDAS Siganl in RFV Sample ANTIGEN AG/NIL code NIL (AG) ratio Interpretation 59174878275 19 727 38.3 Pos 59174878101 49 1382 28.2 Pos 59174878136 37 4838 131 Pos 59174878208 47 21 0.4 Neg 59174878179 7 832 119 Pos 59180477726 15 268 17.9 Pos 59180477734 11 22 2.0 Neg 59180477718 32 1263 39.5 Pos 59180477697 83 79 1.0 Neg 5918047770 41 1163 28.4 Pos

Results:

[0207] The automated protocol for stimulation and detection of the immune cellular response, without centrifugation and under nonsterile conditions, makes it possible to obtain signals of 7 to 4838 RFV and AG/NIL ratios ranging from 0.4 to 131.

[0208] Samples 59174878208, 59180477734 and 59180477697 have AG/NIL ratios which vary between 0.4 and 2.0. These ratios are low (<4), which means that the IFN secretion is not linked to the EBV virus peptides. Consequently, the stimulation under the automated conditions makes it possible to conclude that these patients have not been infected with the Epstein-Barr virus: negative interpretation (Neg).

[0209] The samples 59174878275, 59174878101, 59714878136, 59174878179, 59180477726, 59180477718 and 5918047770have AG/NIL ratios between 17.9 and 131. These ratios are high (>4), which means that the IFN secretion is specifically linked to the Epstein-Barr virus peptides. Consequently, the stimulation under the automated conditions makes it possible to conclude that these patients have been infected with the Epstein-Barr virus: positive interpretation (Pos).

Example 7: Automation of the Stimulation Step, Taking of Total Blood Sample, Nonspecific LPS Stimulation, Assay by Molecular Biology

[0210] The blood samples used in this example were obtained from the Etablissements Franais du Sang (EFS, Rhne-Alpes, France). Five blood samples were taken sterily into Vacutainer tubes (Becton-Dickinson) containing lithium heparinate or equivalent reference from another supplier.

[0211] Each blood sample was stimulated according to the Automated condition, according to the invention protocol described in Table 3 of example 2, using LPS as stimulating agent. For the NIL condition, the sample was brought into contact only with PBS.

[0212] Detection of the immune cellular response by quantification of the mRNAs of the following genes: IFN gene, IL1 gene and ZAP70 gene (bioMrieux internal kits), in the blood samples after stimulation, was carried out as follows: the mRNAs were extracted after lysis of 100 l of the reaction mixture using an automated method (BOOM technology). The mRNAs were then amplified by two-step RT-nested PCR (1st PCR of 26 cycles, followed by a 2nd PCR of 35 cycles). The actual detection was carried out using an intercalating agent and the real-time measurements were acquired in order to construct an amplification curve and to obtain a point Cp (crossing point) for each target and for the three reference genes (DECR1, PPIB & FPGS). The signal, corresponding to the mRNA expression level, was standardized by subtracting, from the target Cp, a geometric mean of the three reference genes.

[0213] The results (standardized Cp valuesstandard deviation) are given in table 11 hereinafter. The lower the standardized Cp value, the higher the expression level.

TABLE-US-00011 TABLE 11 Standardized CP values SD NIL LPS P-value IFN-y 5.33 0.88 3.12 1.19 0.0159 IL1B 4.37 4.04 7.90 0.69 0.1508 ZAP70 1.75 0.61 2.14 0.86 0.5476

[0214] The results in Table 11 below show that it is possible to detect an immune cellular response by means of a molecular biology test after automated stimulation according to the invention.

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