JP2012505639A - Apparatus, kit and method for pulsing a biological sample with an agent and stabilizing the pulsed sample - Google Patents

Abstract

The present invention relates to a device or kit for pulsing a biological sample with a pulsing agent and then stabilizing such pulsed biological sample, wherein a control reaction is provided. The present invention is applied in the field of medical diagnosis, particularly with respect to immunity.
[Selection] Figure 19

Description

  The present invention relates to devices, methods and kits for use in diagnostic assays and is applied in the field of immunity.

Introduction Monitoring of nucleic acid levels, eg, mRNA levels, is important in directly confirming the effect of an agent on a biological system. For example, when an agent is introduced into a biological system for a predetermined length of time, the biological system's response to the agent can be determined by measuring the level of mRNA. This can be useful in monitoring immunity (eg, the agent is an antigen and the monitored mRNA is a cytokine, eg, an interleukin mRNA).

  Introducing variability delay time between blood out of circulation and stimulation by agent by testing the effect of the agent by collecting blood from the individual and later adding the agent Is done. During the delay, the blood can undergo a slow or rapid chemical modification, for example depending on the holding temperature. Furthermore, the variation in lag time means that there is no basis for comparative studies between consecutively collected samples.

  When testing nucleic acids, the main challenge stems from in vitro RNA instability, especially when low level RNA or unstable RNA detection is required. Even a fractional degradation of RNA can change the interpretation of RNA levels. Some transcripts are known to be present in cells in low copies, and some transcripts have a “high AU (AU-rich)” sequence at their 3 ′ ends and are endogenous RNases. Some promote the rapid degradation of RNA by. Studies have shown that RNA degrades rapidly and rapidly within hours after sample collection. Furthermore, when a sample is collected, certain types of RNA increase due to the gene induction process. Both RNA degradation and in vitro gene induction can result in underestimation or overestimation of the number of gene transcripts in vivo.

  Therefore, when measuring the effect of an agent on collected blood, the challenge in the art is to manage the mRNA degradation process that occurs immediately after blood collection and resumes after the introduction of antigen. Since the “before” decomposition can affect the “after” decomposition, the error is linked to two processes, which increases the possibility of the error and makes it more difficult to elaborate on the error.

  Another challenge in the art is that several instruments are required to analyze the nucleic acid after exposing a biological sample to the agent. Usually, quantitative measurement requires at least a reagent bottle, an accurate pipetter, and refrigeration means. In the absence of suitable research facilities, such as taking a sample in a private home or a clinic with basic equipment, the home or clinic is not suitable or inconvenient for accurate substrate addition. Furthermore, there are cases where refrigeration facilities cannot be used.

  When a sample is exposed to an agent, there are many methods for isolating and measuring nucleic acids, such as mRNA, in the sample. In some methods, it is even possible to determine low level transcripts from a pool of transcripts. However, none of these methods can determine the level (s) of transcript (s) present in a biological sample at the time of sampling. Even under refrigerated conditions, storage of biological samples results in inaccurate mRNA levels. In practice (Indeed, in practice), since the sampling place and the RNA analysis place are provided separately, the analysis of a fresh sample cannot be executed.

  In recent years, PreAnalytiX (a joint project between Becton Dickinson and Qiagen) has created the PAXgene ™ Blood RNA System. The PAXgene ™ Blood RNA System (also referred to as the Qiagen method) is an integrated standardization system for the collection and stabilization of whole blood specimens and the isolation of cellular RNA. According to PreAnalytiX, in the PAXgene ™ Blood RNA System, blood is collected directly into the PAXgene ™ Blood RNA Tube, and then RNA is isolated using the PAXgene ™ Blood RNA Kit. Using this system, intact cellular RNA can be removed from whole blood.

  PAXgene ™ Blood RNA Tube is a plastic vacuum tube for whole blood collection and stabilization of cellular RNA profiles. These tubes stabilize cellular RNA, eliminate ex vivo induction of gene transcription, and suppress the dramatic changes in cellular RNA expression profiles that normally occur in vitro (proprietary blend reagents) ). The RNA is then isolated using silica gel membrane technology as given in the PAXgene ™ Blood RNA Kit. According to PreAnalyticiX, the resulting RNA accurately represents the expression profile in vivo and is suitable for use in a wide range of downstream applications. According to the supplier, this system can be used to accurately quantify gene transcripts. The main drawback of this PAXgene (TM) Blood RNA System is that each PAXgene (TM) Blood RNA Tube must be combined with the PAXgene (TM) Blood RNA Kit (PAXgene (TM) Blood RNA Tube instruction manual Please refer to the manual). However, the need for this combination has limited further improvements in the system.

  More recently, Tempus (TM) Blood RNA Tube (Applied Biosystems), a plastic vacuum tube for collection of whole blood and stabilization of cellular RNA profiles, has also been developed. Each tube contains additives (a proprietary blend of reagents) that stabilize cellular RNA, eliminate ex vivo introduction of gene transcription, and suppress the dramatic changes in cellular RNA expression profiles that normally occur in vitro. ing.

  One of the objects of the present invention is to provide an apparatus, kit and method for exposing a biological sample to an agent and stabilizing nucleic acids in the sample so exposed.

  One of the objects of the present invention is to provide an apparatus, kit and method for exposing a biological sample to an agent and stabilizing nucleic acids in the exposed sample, which also provides a control reaction. It is to be.

  Another object of the present invention is to provide an apparatus, kit and method for reducing, constant, or nearly constant the time between obtaining a biological sample and exposing the sample to the agent. is there.

  Another object of the present invention is to provide an apparatus, kit and method that exposes a sample to an agent and reduces, makes constant or nearly constant the time to stabilize nucleic acids in the sample.

  Another object of the present invention is to provide an apparatus, kit and method for exposing an agent to a sample without the need to measure the amount of the sample and / or agent.

  Another object of the present invention is to simply expose the agent and the control substance to the sample, and expose the sample and the control substance to the agent so that the time for stabilizing the nucleic acid in the sample is constant or nearly constant. One device is to provide.

  Another object of the present invention is to provide an apparatus, kit and method for exposing an agent to a sample without the need to measure the amount of stabilizer.

  Another object of the present invention is to provide an apparatus, kit and method for exposing a biological sample to an agent, stabilizing the nucleic acid in the exposed sample, and extracting the nucleic acid from the biological sample for further analysis. That is.

  Another object of the present invention is to provide a device for exposing a biological sample to an agent and stabilizing nucleic acids in the sample so exposed, which also provides a control reaction and facilitates robotic automation. It is to be.

  Another object of the present invention is to provide apparatus, kits and methods that address one or more combinations of the above objects.

One embodiment of the present invention is a kit for examining a liquid biological sample,
A container suitable for receiving a liquid biological sample and exposing the sample to a first substance and then to a nucleic acid stabilizer, the container comprising: a) a first substance present in the container;
b) a container in which the stabilizer is present;
c) a connection between the interior of the container and the interior of the container;
d) a physical barrier that temporarily blocks the connection;
A container containing,
A control container suitable for receiving a liquid biological sample and exposing the sample to a control substance and then to a nucleic acid stabilizer, the control container being a) a control substance present in the control container;
b) a control container in which the stabilizer is present;
c) a connection between the inside of the control container and the inside of the control container;
d) a physical barrier that temporarily blocks the connection;
A container containing,
It is a kit containing.

  Another embodiment of the present invention is the kit as described above, wherein the first substance is fixed to a part or all of the inner surface of the container.

  Another embodiment of the present invention is a kit as described above, wherein the first substance is immobilized on a solid support.

  Another embodiment of the present invention is a kit as described above, wherein the first substance is a liquid.

  Another embodiment of the present invention is a kit as described above, wherein the first substance is a solid.

  Another embodiment of the invention is a kit as described above, wherein the container and / or control container comprises one or more regions suitable for puncture with a syringe needle.

  Another embodiment of the present invention is a kit as described above, wherein the region is a resealable septum.

  Another embodiment of the invention as described above, wherein the container and / or control container includes a fitting suitable for receiving a syringe and delivering the contents therein to the interior of the container or the control container. It is a kit.

  Another embodiment of the invention is a kit as described above, wherein the container and / or control container includes a suitable attachment for receiving a syringe needle.

  Another embodiment of the present invention is as described above, wherein the container and / or control container includes a valve that minimizes gas / liquid flow from the container and allows liquid biological sample to flow to the container. It is a kit.

  Another embodiment of the present invention is a kit as described above, wherein the container and / or the control container comprise means by which the exhaust gas can be pushed out.

  Another embodiment of the present invention is a kit as described above, wherein the container and / or the control container are under is held under pressure.

  Another embodiment of the present invention is a kit as described above, wherein the physical barrier of item d) is opened by application of physical force to the container or the control container.

  Another embodiment of the invention is a kit as described above, wherein the force sends an opening means to the physical barrier.

  Another embodiment of the invention is a kit as described above, wherein the force irreversibly opens the physical barrier.

  Another embodiment of the present invention is a kit as described above, wherein said container and / or said control container includes an indication for dispensing a known volume of stabilizer therein.

  Another embodiment of the present invention is a kit as described above, wherein said first substance comprises one or more immune system antigens.

  Another embodiment of the present invention is a kit as described above, wherein the immune system antigen is a vaccine component.

  Another embodiment of the present invention is a kit as described above, wherein the immune system antigen is an antigen that induces a hyperallergic reaction.

  In another embodiment of the present invention, the immune system antigen is a histocompatibility antigen, bacterial LPS, tetanous toxoid, cancer immunotherapy antigen, MAGE-3, cat allergen, Feld1, antigen-presenting cell from an organ donor , A kit as described above, which is one or more selected from autoantigen, GAD65.

  Another embodiment of the present invention is a kit as described above, wherein the stabilizing agent is an inhibitor of cellular RNA degradation and / or gene induction.

  Another embodiment of the present invention is a kit as described above, wherein said inhibitor of cellular RNA degradation and / or gene induction is that found in PAXgene ™ or Tempus ™ Blood RNA Tube .

  Another embodiment of the present invention is a kit as described above, wherein the exterior of the container and the control container are connected to form a single body.

Another embodiment of the invention is an assay device for a liquid biological sample that facilitates separate exposure of the sample to a first material and a control material and then to a nucleic acid stabilizer, the device comprising: ,
A first compartment in which the first substance is present;
A second compartment in which the control substance is present;
A third compartment in which the stabilizer is present;
A support for a biological sample tube;
Is an assay device.

  Another embodiment of the invention is an assay device as described above, wherein one or more of the compartments are sealed and contain one or more regions suitable for puncturing with a hollow needle.

  Another embodiment of the invention is an assay device as described above, wherein the region is a resealable septum.

  Another embodiment of the present invention further includes a support for removably connecting to the transfer tube, wherein the transfer tube is capable of liquid between any two compartments or between the compartment and the biological sample tube. An assay device as described above, comprising a flexible or rigid hollow tube with hollow needles at both ends, suitable for transfer.

  Another embodiment of the present invention is an assay device as described above further comprising the transfer tube as described above.

  Another embodiment of the present invention includes a flexible hollow tube with one needle of a transfer tube connected in parallel with the hollow needle of a pressure tube, the pressure tube having the needle at one end, and a compartment or An assay device as described above, which is suitable for applying a vacuum or pressure to a biological sample tube and forcing liquid through the transfer tube.

  Another embodiment of the present invention is the assay device as described above, wherein the first substance is fixed to a part or all of the inner surface of the container.

  Another embodiment of the invention is an assay device as described above, wherein one or more of the compartments includes a vent.

  Another embodiment of the invention is an assay device as described above, wherein the first compartment and / or the second compartment is under negative pressure.

  Another embodiment of the invention is an assay device as described above, wherein the first compartment and / or the second compartment includes an indication for dispensing a known volume of stabilizer therein.

  Another embodiment of the present invention is an assay device as described above, wherein said first substance is as defined above.

  Another embodiment of the present invention is an assay device as described above, wherein said stabilizing agent is as defined above.

  One embodiment of the present invention relates to a container suitable for holding a biological sample, the container holding a predetermined amount of a first substance (which may be a pulsing agent).

  As used herein, a “pulse agent” includes any substance that can expose a biological sample. Examples of substances include peptides, nucleic acids, and antigens. In addition to the substance, the pulsatile agent may contain other components such as a stabilizer, an indicator, a linker, and a matrix.

  The term “biological sample” refers to clinical samples (eg, cell fraction, whole blood, plasma, serum, urine, tissue, cells, etc.), agricultural samples, environmental samples (eg, soil, mud, ore, water, air), food samples By (optional food), we mean a sample containing a nucleic acid / biological agent such as a forensic sample or other possible sample. “Whole blood” means blood that is collected by venous sampling, ie, contains white blood cells and red blood cells, platelets, plasma, and thus infectious agents. Infectious agents can be viral, bacterial or parasitic. Clinical samples can be from humans or animals. The analytical sample can be both solid or liquid in nature. If solid materials are used, it is apparent that they are first dissolved in a suitable solution (which may be an RNAlater reagent sold by Qiagen). According to the present invention, this solution need not necessarily be an actual “buffer” having at least two well balanced components. This solution may be a strong hypotonic solution such as NaCl alone or an extraction solution with alcohol or the like.

  The vessel (also known herein as a reaction vessel) may hold the pulsing agent in several ways. According to one aspect of the present invention, the pulsing agent may be immobilized on the inner wall of the container. The inner wall of the container may be lined with a suitable coating to which the pulsing agent can adhere. Alternatively, the pulsing agent may be adhered directly to part or all of the inner wall of the container. Suitable coatings, methods and container materials for such adhesion are known in the art. According to another aspect of the invention, the pulsing agent is present as a solid. The solid can be a powder, lyophilized pellet, gel, cream. Suitable solid compositions and methods for their preparation are known in the art. According to another aspect of the invention, the pulsing agent is immobilized on a solid support. A solid carrier may be adhered to the inside of the container. Alternatively, the solid carrier may not be inside the container. Examples of solid carriers include, but are not limited to, chromatography matrices and magnetic beads. According to another aspect of the invention, the pulsing agent is present as a liquid. Suitable liquid compositions and methods for their preparation are known in the art.

  Performing analytical pulse experiments outside laboratory conditions requires a calibrated measuring instrument such as a pipettor. Errors due to uncalibrated measuring devices can lead to inherent errors, and artificial errors in dispensing can lead to errors between different samples, which makes the comparative analysis unfounded. By preparing a container to supply a predetermined amount of pulsing agent, the need for additional equipment is eliminated and human measurement errors are eliminated. Furthermore, by preparing a control container separately, it is possible to normalize the results and facilitate error detection. Use of controls is important because RNA is unstable and degrades rapidly when taken from the circulation. Inconsistent delays when pulsing whole blood make it impossible to compare results. During incubation with pulsing agents, different (biological) pathways are involved depending on the pulsing agent. The present invention allows the measurement of such biological processes by quantifying biomarkers such as cytokines, chemokines, transcription factors and other gene products or messenger RNA of surrogate markers typical of a particular process. Without a reference value, quantification of these biomarkers in the assay has little meaning, and the use of integrated controls allows for quick control reactions. Comparing assays and controls is meaningful for the amount of biomarker and the effect of pulsing agents on biological systems. For example, it is generally accepted that the Mx1 gene is under the control of type I IFN. We used this knowledge to use this gene product as a surrogate marker to measure the therapeutic effect of type I IFN. After incubating whole blood with type I IFN, the Mx1 gene product is quantified. The amount of Mx1 gene product can be expressed as a normalized gene product, ie, mRNA copy number relative to the mRNA copy number of a gene whose transcriptional activity is not affected by the action of IFN. However, this quantification becomes meaningless under unstimulated control conditions, ie without a control reaction, which is the quantification of surrogate markers under the same treatment without type I IFN. The same reasoning is possible for any immune response. In order to be a strict control, it is very important that the control sample is processed in the same way, ie the same collection, the same incubation time and temperature, and parallel processing. The combination of the assay on the single device and the control compartment makes it possible to do this.

  The type of container according to the present invention may be any suitable for storage of biological samples. According to one embodiment of the present invention, the container containing the pulse agent is sealed. According to one aspect of the invention, the container containing the pulsing agent has a resealing means such as a screw cap, a push-on cap, a flip cap. See, for example, FIG. According to one aspect of the present invention, a biological sample or other fluid can be introduced into a container by puncturing the wall of the container using a syringe needle. The container wall can be resealable after puncture, or the container wall cannot be resealed after puncture, or the container wall has a resealable region such as a septum Also good. For example, see FIG.

  According to one aspect of the present invention, a biological sample or other liquid is introduced by a transfer tube that includes a hollow tube with needles (eg, tubular needles) at both ends. One needle is configured to puncture a sample tube holding a sample or a partition wall of another container, and the other needle is configured to puncture a partition wall of a container. The biological sample can be transferred from the sample tube to the container using a needle. The tube may be flexible or rigid. The shape of the transfer tube may be U-shaped. See, for example, FIG.

  According to one aspect of the invention, a biological sample can be introduced into a container by one or more attachments or other containers attached to the container to receive a syringe. For example, a luer type attachment that can receive a needleless syringe may be attached to the container. See, for example, FIG. In another example, the container may be fitted with a non-luer attachment that can be combined with a container having a non-luer coupling design that is reciprocating.

  According to one embodiment of the present invention, a biological sample can be introduced into the container by a tubular needle or a hypodermic needle that is suitable for collecting a biological sample directly from an individual. See, for example, FIG.

  According to one embodiment of the present invention, the biological sample can be introduced into the container by opening the resealing means. See, for example, FIG.

  As is known to those skilled in the art, introduction of a sample into a sealed container causes the same volume of air or gas to be exhausted therefrom, or the pressure therein to increase. The container may therefore be provided with suitable means for releasing the exhausted gas from the container or for adjusting the pressure increase. Such means are known in the art and include the use of valves, non-drip holes, vents, clothed-vents, expandable container walls, and negative pressure within the container. For example, see arrow 31 in FIG.

  In one embodiment of the present invention, the pressure in the sealed container is a negative pressure. By utilizing negative pressure, an increase in pressure when the biological sample is introduced into the sealed container can be mitigated. Alternatively or additionally, the negative pressure may be at a predetermined level and can be utilized to allow the introduction of a fixed volume of biological sample.

  According to another aspect of the invention, the container has a first needle (tubular) through which a liquid biological sample can be placed into the container and a second needle through which positive or negative pressure is applied to the container. And a sealable partition wall as described above, which is suitable for receiving the inside. When negative (vacuum) pressure is applied through the second needle, the biological sample can be withdrawn through the first needle into the container. Preferably, the first needle is part of the transfer tube previously described. The second needle can be part of a pressure tube as described below connected to an air (discharge or pressure) pump. The first needle and the second needle may be connected in parallel. See, for example, FIG.

  A container already supplied with a predetermined amount of pulsing agent can be used to perform a diagnostic test on an individual without the need for an instrument for measuring the antigen. In addition, if diagnostic tests are performed outside of laboratory conditions, incorrect results may be obtained in quantitative assays due to problems with contamination and dispensing accuracy. Containers as described herein overcome these problems.

  Another aspect of the invention is a control container, which can be a container as described above, except that it contains a control pulsing agent (control substance) instead of containing a biological pulsing agent. As mentioned elsewhere for this description, containers such as those described herein are used to hold pulsing agents and are sometimes referred to as “reaction vessels”, but are described herein. A control container such as this is used to hold a control pulsing agent. The container and the control container operate independently, receive the sample and the stabilizer independently, and the interior is not connected. However, the container and the control container can exist together as part of the kit. Alternatively, the outer surfaces of the container and the control container can be mechanically connected to form a single body that includes both the container and the control container. According to one aspect of the invention, the control container containing the control pulsing agent is sealed. The control container may have resealing means such as a screw cap, push-in lid, flip cap. The control container may have a breakable seal such as a peel-back adhesive seal, a snap-off seal. According to one aspect of the invention, the wall of the control container may be provided with a resealable region such as a septum through which the substance can be introduced into the control container by puncturing with a syringe needle. it can. The septum of the control container may be resealable after puncture. According to one aspect of the present invention, a biological sample or other liquid is introduced by the transfer tube described above with needles (eg, tubular needles) at both ends. One needle is configured to puncture the septum of the tube or other container that holds the sample tube, and the other needle is configured to puncture the septum of the control container. The liquid biological sample can be transferred from the sample tube to the control container using a needle. The tube may be flexible or rigid. The shape of the transfer tube may be U-shaped.

  In one aspect of the invention, the control container may have means for withdrawing the liquid biological sample from the biological sample tube when in fluid connection with the biological sample tube. Examples include, but are not limited to, syringe-type plungers or any means of applying negative pressure.

  It should be understood that the control container receives a portion of the same biological sample introduced into the reaction container. It should be understood that the biological sample can be divided into two, one for the control container and one for the reaction container. Alternatively, one aliquot of the biological sample can be introduced into the reaction vessel and a second equal volume aliquot can be introduced into the control vessel.

  According to another aspect of the present invention, the control container comprises a first needle (tubular) through which a liquid biological sample can be placed into the control container and a second needle (through which positive or negative pressure is applied). Which can be applied to the inside of the control container). When negative (vacuum) pressure is applied through the second needle, the biological sample can be withdrawn through the first needle into the control container. Preferably the first needle is part of a transfer tube as described above. The second needle may be part of a pressure tube as described below connected to an air (discharge or pressure) pump. The first needle and the second needle may be connected in a parallel arrangement. See, for example, FIG.

  As is well understood by those skilled in the art, the control pulsing agent depends on the pulsing agent and the parameters under study. For example, if the pulsing agent is a peptide, a set of peptides or a pool of peptides, the control pulsing agent may contain the same length peptide (s), but the sequence of the peptide (s) may be randomized. And / or are not identical to existing protein or peptide segments. This control pulsing agent is incubated with the same whole blood as the pulsing agent, with the same amount of agent, the same volume of blood, for the same period and at the same temperature. In another example, the pulsing agent can be a protein therapeutic, eg, IFN-β dissolved in an excipient such as water, phosphate buffered saline, or saline to the desired concentration. Thus, the control pulsing agent can be an excipient. In a further example, the pulsing agent can be an antigen, eg Feld1, dissolved in an excipient such as water, phosphate buffered saline or saline to the desired concentration. Thus, the control pulsing agent can be an excipient of the antigen. Alternatively, the control container may lack a control agent or is empty, for example when comparing inducible immune response or inducible gene expression to an untreated sample.

  Another aspect of the invention is a container as described herein, further comprising a container in which a stabilizer is present, wherein the stabilizer contacts the pulse agent or a biological sample exposed to the pulse agent. It is related with the container which prevents temporarily.

  In one embodiment of the invention, the stabilizer comprises a nucleic acid stabilizer and / or a cellular RNA degradation inhibitor and / or a gene induction inhibitor, and / or the stabilizer is a PAXgene ™ Blood RNA Tube. Same as found and / or the stabilizer is the same as found in Tempus ™ Blood RNA Tube. Agents and combinations thereof are known in the art or can be inferred by one skilled in the art.

  PAXgene ™ and Tempus ™ Blood RNA Tubes can be supplied with a solution containing an additive that stabilizes cellular RNA to eliminate ex vivo induction of gene transcription. Detailed information explaining the nature of this additive is not given. The catalog attached to the PAXgene ™ tube for this purpose refers to US Pat. No. 5,906,744. Nevertheless, the tubes described in this patent document allow one skilled in the art to prepare nucleic acids from plasma rather than whole blood as is done in the present invention. In particular, the device of US Pat. No. 5,906,744 preferably includes a plastic or glass tube, means for inhibiting blood clotting, and means for separating plasma from whole blood (US Pat. No. 5,906,744). 2nd column, I. 42-43). Thus, according to the present invention, the content described in US Pat. No. 5,906,744 is not related to the actual content of the PAXgene ™ Blood RNA Tube, as it relates to another application.

  According to the present invention, the solution retained in the PAXgene ™ Blood RNA Tube can contain a quaternary amine surfactant. Therefore, according to the present invention, a quaternary amine surfactant may be used as a stabilizer. The use of quaternary amine surfactants to stabilize nucleic acids in biological samples has been previously described in US Pat. No. 5,010,183. This patent document provides a method for purifying DNA or RNA from a mixture of biomaterials. The above method lyses the cells and solubilizes any contaminating proteins and lipids in the mixture, so that RNA or DNA is present in an amount sufficient to form an insoluble hydrophobic complex between the nucleic acid and the detergent. Adding a cationic detergent to the containing mixture. In this way, the complex containing RNA or DNA together with the detergent is separated from solubilized contaminants. In recent patent literature, the same inventors have used inactive surfactants such as those described in US Pat. No. 5,010,183, and other commercially available surfactants, and inefficient precipitation of RNA and blood. It states that incomplete lysis of cells occurs. Because of this, it was necessary to improve the cationic surfactant, the inventors of US Pat. No. 5,010,183 decided to use an aqueous cationic surfactant solution containing a selected quaternary amine. A new method for isolating RNA from biological samples (including blood) was sought (US Pat. No. 5,985,572). New aqueous quaternary amine surfactants capable of stabilizing RNA from biological samples are also described in WO 94/18156 and WO 02/00599. The synthesis of various potential surfactants that can be used in any of the methods of the invention can be performed according to the instructions mentioned above or published in the relevant patent literature. An example of a quaternary amine that can be used in the process of the present invention is tetradecyltrimethyl-ammonium oxalate (US Pat. No. 5,985,572). Alternatively, the cationic detergent can be Catrimox-14 ™ as shown in Example 1 of the present invention (US Pat. No. 5,010,183). In addition to stabilizing the biological sample, the application describes the isolation of nucleic acids using conventional separation techniques such as column chromatography. Since the PAXgene ™ Blood RNA Tube must be combined with the PAXgene ™ Blood RNA kit (which also applies to column chromatography), only the compounds present in the PAXgene ™ Blood RNA Tube are supplied by the supplier. An impression is given that it cannot be adapted to the above chromatographic method.

  In one embodiment of the present invention, the stabilizer is contained in the container until such time as the biological sample is mixed with the pulsing agent and / or until the user needs to introduce the stabilizing agent. .

  The container may be incorporated into the container, which means that the container may be mechanically coupled with the container to form a one-piece unit. According to one aspect of the present invention, there is a physical barrier that connects the interior of the container and the interior of the container and blocks the connection. Application of force in a timely manner opens the physical barrier and allows the stabilizer to be mixed with the biological sample so pulsed. According to one aspect of the invention, the physical barrier opens and closes reversibly in response to the applied physical force. The applied force can be sent to the physical barrier itself or via a stabilizer to the physical barrier. The physical barrier can be any mechanical barrier. Examples of such physical barriers include rotary valves, open valves, slit valves, partition valves, ball valves, and flap valves. According to another aspect of the invention, the physical barrier may be irreversibly opened by the application of force. The physical force can be any mechanical force. The applied force can be transmitted to the physical barrier itself (see, eg, FIG. 7) or via a stabilizer to the physical barrier (see, eg, FIG. 8). Other examples of such physical barriers include plugs that are threaded out of place (see, for example, FIG. 1), and barriers that break upon application of force (see, for example, FIG. 7).

  According to another aspect of the present invention, the inside of the container and the inside of the container are connected, and the stabilizer flows from the container to the container due to the surface tension of the stabilizer together with the opening size of the connection. prevent. According to this aspect of the invention, the stabilizer flows from container to container by the application of the force transmitted to the stabilizer in a timely manner. For example, the force can be applied by pressing, continuous inversion and stirring.

  Another aspect of the invention is a control container as described herein further comprising a control container in which a stabilizer is present, wherein the stabilizer is exposed to the control pulse or the control pulse. It relates to a control container that temporarily prevents contact with the sample. A container with an accumulation container as described herein may be present together as part of a kit along with a control container with an accumulation control container. Alternatively, the outside of the control container with the accumulation control container, together with the outside of the container with the accumulation container described herein, may be linked or connected using a bridging member so that it forms a single device ( 19 and 20).

  A control container may be incorporated into the control container, which means that the control container may be mechanically coupled with the control container to form a one-piece unit. According to another aspect of the present invention, there is a physical barrier that connects the interior of the control container and the interior of the control container and blocks the connection. Application of force in a timely manner opens the physical barrier and allows the stabilizer to be mixed with the biological sample so pulsed. According to one aspect of the invention, the physical barrier opens and closes reversibly in response to the applied physical force. The applied force can be transmitted to the physical barrier itself or via a stabilizer to the physical barrier. Examples of such physical barriers include rotary valves, open valves, slit valves, partition valves, ball valves, and flap valves. According to another aspect of the invention, the physical barrier may be irreversibly opened by the application of force. The applied force can be transmitted to the physical barrier itself (see, eg, FIG. 17) or to the physical barrier via a stabilizer (see, eg, FIG. 18). Other examples of such physical barriers include plugs that have been removed from place (see, eg, FIG. 12), and barriers that break upon application of force (see, eg, FIG. 17).

  According to another aspect of the present invention, the stabilizer is connected from the control container to the control container by the surface tension of the stabilizer connecting the inside of the control container and the inside of the control container and adjusting to the opening size of the connection. To prevent the flow. According to this aspect of the invention, the stabilizer flows from the control container to the control container by applying a force that is transmitted to the stabilizer in a timely manner. For example, the force can be applied by pressing, continuous inversion and stirring. Containers or control containers as described herein, each containing a container or control container for dispensing stabilizers, allows technicians who have not received special training to use pulsing agents or controls to dispense blood. It is possible to stabilize the pulsed and pulsed blood for later analysis by those skilled in the art. Thus, if a large number of samples need to be collected, a container such as disclosed herein or a control container can be used to employ operators who have not received special training to pulse and stabilize blood. Can be reduced. In addition, a known amount of pulsing agent and stabilizer may be pre-fed into the container by the container or control container, thus providing reproducibility with minimal errors associated with pipetting. Furthermore, because the sample can be withdrawn directly into the tube or via, for example, a syringe, the time between taking a biological sample and exposing the sample to a pulsed antigen or control is greatly reduced. The In addition, the introduction of the stabilizer into the sample is easily achieved by applying force, thereby eliminating the delay caused by pipetting the stabilizer, so that the biological sample is pulsed and the biological sample is stabilized. The time between can be set accurately.

  Another embodiment of the present invention is a kit suitable for pulsing a biological sample with a pulsing agent and then introducing a stabilizing agent into the pulsing agent to test RNA components in such a pulsed biological sample. A kit comprising one or more containers as disclosed above and one or more containers in which the stabilizer is present. The kit may further comprise a control container in which a biological control pulsing agent is present.

  In one embodiment of the kit, the container in which the stabilizer is present is not incorporated in the container in which the pulse agent is present, and / or the control container in which the stabilizer is present is in the control container in which the control substance is present. Is not incorporated.

  This allows the container or control container to be separated and can be any container in the art suitable for holding the stabilizer in the kit. According to one aspect of the invention, the container containing the stabilizer or the control container is sealed. The container or control container may have resealing means such as screw caps, push-in lids, flip caps. The container or control container may have a breakable seal such as a peel-off adhesive seal, a snap-off seal. According to one aspect of the present invention, the wall of the container or control container may comprise a resealable region such as a septum, and the stabilizer is removed from the container or control container by puncturing with a syringe needle. Can be pulled out. The septum of the container or control container may be resealable after puncture. According to one particular aspect of the present invention, a container or control container is used in conjunction with a transfer tube with needles (eg, tubular needles) at both ends. One needle is configured to puncture the septum of the container or control container, and the other needle is configured to puncture the respective septum of the container or control container (described below). The stabilizer can be transferred from the container or control container to the container or control container, respectively, using a transfer tube. The tube may be flexible or rigid. The shape of the transfer tube may be U-shaped. The container or control container may comprise one or more attachments suitable for the connection of the containers to which reciprocal coupling means are attached and for the transfer of stabilizers to the container or the control container. For example, the container or control container receives a reciprocal luer attachment attached to the container as described above (see, eg, FIGS. 9, 10 and 11) or attached to the control container. The lure type attachment part which can be attached may be attached. In another example, the container or control container may be fitted with a non-luer attachment that can be associated with a container or control container that has an interrelated non-luer coupling design. By opening the resealing means of the container, the stabilizer can be transferred to the container or control container. The stabilizer can be discharged into the container or control container through any of the attachments or openings described above. The container or control container may optionally have means to allow air to flow in while discharging the stabilizer. In one aspect of the present invention, the container or control container may have means for extruding the stabilizer from the container or control container, such as a syringe-type plunger, a compressed wall of the container or control container, Alternatively, any means for applying a positive pressure can be mentioned, but the present invention is not limited thereto. The container or control container optionally has a measuring means such as a scale that establishes the volume of stabilizer dispensed. In one aspect of the invention, the container or control container holds a sufficient volume of stabilizer for a single use. In another aspect of the invention, the container or control container holds a sufficient volume of stabilizer for multiple pulse experiments.

  According to another aspect of the present invention, the container or control container comprises a first needle (tubular) through which the stabilizer can be discharged from the container and a second needle (through which positive pressure or negative). A sealable septum as described above suitable for receiving pressure (which can be applied to the inside of the container). When positive pressure is applied through the second needle, the stabilizer can be pushed through the first needle into the container or control container. Preferably, the first needle is part of the transfer tube previously described. The second needle can be part of a pressure tube as described below connected to an air (discharge or pressure) pump. The first needle and the second needle may be connected in parallel.

  In another embodiment of the kit, the container in which the stabilizer is present is connected to the container in which the pulsing agent is present, and the embodiment of the container is shown above. In another embodiment of the kit of the present invention, a control container in which the stabilizer is present is connected to a control container in which a control substance is present, and an embodiment of the control container is shown above.

  Optionally, the kit of the present invention may include instructions including a description of a method for pulsing a biological sample.

  Another aspect of the invention relates to a container as disclosed herein and a kit comprising the container, wherein the pulsing agent comprises an antigen. According to one aspect of the invention, the antigen is bacterial LPS. According to another aspect of the invention, the antigen is an immune response recall antigen. According to another aspect of the invention, the antigen is a tetanus toxoid. According to another aspect of the invention, the antigen is a cancer immunotherapy antigen. According to another aspect of the invention, the antigen is MAGE-3. According to another aspect of the invention, the antigen is a cat allergen. According to another aspect of the invention, the antigen is Feld1. According to another aspect of the present invention, the antigen is an antigen-presenting cell derived from an organ donor. According to another aspect of the invention, the antigen is a self antigen. According to another aspect of the invention, the antigen is GAD65.

  As is well understood by those skilled in the art, the control pulsing agent depends on the pulsing agent and the parameters under study. For example, if the pulsing agent is a peptide, a set of peptides or a pool of peptides, the control pulsing agent may contain the same length peptide (s), but the sequence of the peptide (s) may be randomized. And / or are not identical to existing protein or peptide segments. A control pulsing agent is incubated with the same whole blood as the pulsing agent, with the same amount of agent, the same volume of blood, for the same period and at the same temperature. In another example, the pulsing agent can be a protein therapeutic, eg, IFN-β dissolved in an excipient such as water, phosphate buffered saline, or saline to the desired concentration. The control pulsing agent can thus be an excipient. In a further example, the pulsing agent can be an antigen, eg Feld1, dissolved in an excipient such as water, phosphate buffered saline or saline to the desired concentration. Thus, the control pulsing agent can be an excipient of the antigen. Alternatively, the control container may lack a control agent or is empty, for example when comparing inducible immune response or inducible gene expression to an untreated sample.

  Another aspect of the invention relates to a method of testing RNA components in a biological sample so pulsed with a biological sample pulsed with an antigen, after which the nucleic acid is stabilized therein. The method includes the use of containers, control containers and / or kits as disclosed herein for optionally collecting, pulsing and stabilizing the sample.

One embodiment of the present invention is an apparatus for receiving a liquid biological sample that facilitates separate exposure of the sample to a first substance and a control substance and then to a nucleic acid stabilizer, the apparatus comprising:
A first compartment in which a first substance is present;
A second compartment in which a control substance is present;
A third compartment in which a stabilizer is present;
A support for a biological sample tube;
Including the device.

  An example of the device is shown in FIGS. 21a and 21b. According to one aspect of the invention, at least one (eg one, two, three, all), preferably all compartments are sealed. One or more (eg, one, two, three, all) compartments may have resealing means such as screw caps, push lids, flip caps, for example. One or more (eg one, two, three, all) compartments may have a breakable seal such as a peel-off adhesive seal, a snap-off seal. According to one aspect of the invention, one or more (eg, one, two, three, all) compartments are provided with a resealable region, such as a septum, through which a puncture is made using a syringe needle. Material can be introduced into the compartment. The compartment septum can be resealed after puncture.

  According to certain embodiments, one or more (e.g. one, two, three, all) compartments and biological sample tubes are connected to a transfer tube, e.g. For example, it is used in combination with a hollow tube equipped with a tubular hypodermic needle. One needle is configured to puncture the septum of one compartment (eg, the first compartment) and the other needle is configured to puncture the septum of the other compartment (eg, the third compartment). ing. Alternatively, one needle is configured to pierce the septum of one compartment (eg, the first compartment) and the other needle draws liquid from the biological sample tube (held by the support). It is configured as follows. In this way, two compartments or one compartment and a biological sample tube can be temporarily fluidly connected by a tube. The fluid connection allows the stabilizer to be transferred from the third compartment to the first compartment and / or the second compartment, for example using a transfer tube. The tube may be flexible or rigid. The transfer tube may be U-shaped.

  The biological sample tube according to the present invention may be any suitable for storage of biological samples. According to one aspect of the invention, the biological sample tube is sealed. According to one aspect of the present invention, the biological sample tube has resealing means such as a screw cap, a push lid, and a flip cap. According to one embodiment of the present invention, a biological sample can be introduced into a tube by puncturing the wall of the tube using a syringe needle. The wall of the tube may be resealable after puncture, or the wall of the tube may not be resealable after puncture, or the wall of the sample tube comprises a resealable region such as a septum It may be. According to one aspect of the present invention, as described elsewhere herein, a biological sample is withdrawn from a tube introduced by a transfer tube that includes a hollow tube with needles (eg, tubular needles) at both ends. Can do. One needle is configured to puncture the septum of the sample tube holding the sample or other liquid, and the other needle is configured to puncture the compartment septum. A tube can be used to transfer a liquid biological sample from a sample tube to one of the compartments. The tube may be flexible or rigid. The transfer tube may be U-shaped. According to one aspect of the invention, a biological sample can be introduced into a sample tube by one or more attachments or other containers attached to a sample tube adapted for a syringe. For example, a luer type attachment that can receive a needleless syringe may be attached to the tube. In another example, the tube may be fitted with a non-luer attachment that can be brought together with a container having an interrelated non-luer coupling design. According to one embodiment of the present invention, a biological sample can be introduced into the tube by a tubular needle or a hypodermic needle attached to the tube, which is suitable for directly collecting the biological sample from an individual. According to one embodiment of the present invention, the biological sample can be introduced into the sample tube by opening the resealing means.

  In one aspect of the invention, the first compartment may have means for withdrawing the liquid biological sample from the biological sample tube to the first compartment when in fluid connection with the biological sample tube. Similarly, the second compartment may have means for withdrawing the liquid biological sample from the biological sample tube to the second compartment, for example when in fluid connection with the biological sample tube using a transfer tube. Examples of such means include, but are not limited to, the use of a syringe-type plunger or any means of applying a negative pressure to the receiving compartment or a positive pressure to the supply compartment. According to a preferred embodiment of the present invention, the pressure in the compartment is controlled using the insertable pressure tube described below. The first compartment and / or the second compartment may be partially under vacuum.

  In one aspect of the invention, the first compartment may have means for withdrawing the stabilizer from the third compartment to the first compartment when in fluid connection with the third compartment. The second compartment may also have means for withdrawing the stabilizer from the third compartment to the second compartment, for example when in fluid connection with the third compartment using a transfer tube, for example. Examples of such means include, but are not limited to, the use of a syringe-type plunger or any means of applying a negative pressure to the receiving compartment or a positive pressure to the supply compartment. According to a preferred embodiment of the present invention, the pressure in the compartment is controlled using the insertable pressure tube described below. The first compartment and / or the second compartment may be partially under vacuum.

  In another aspect of the invention, the third compartment is fluidly connected to the first compartment and / or the second compartment using, for example, a transfer tube, from the third compartment to the first compartment and / or Or it may have means to push the stabilizer into the second compartment. Examples of such means include, but are not limited to, any means for applying a positive pressure to a syringe-type plunger or a third compartment. According to a preferred embodiment of the present invention, the pressure in the compartment is controlled using the insertable pressure tube described below. The third compartment can be under positive pressure.

  According to one aspect of the invention, the first compartment is sterilized. According to another aspect of the invention, the second compartment is sterilized. According to another aspect of the invention, the third compartment is sterilized. According to another aspect of the invention, all the compartments are sterilized.

  According to one embodiment of the invention, each compartment applies a first needle (through which liquid can pass) and a second needle (through which positive or negative pressure is applied to the interior of the compartment) A separable septum as described above suitable for receiving. When positive pressure is applied through the second needle, liquid can be pushed out of the compartment through the first needle. Alternatively, if negative pressure is applied through the second needle, liquid can be withdrawn through the first needle into the compartment. Preferably the first needle is part of a transfer tube as previously described. The second needle is part of the pressure tube. The first needle and the second needle may be connected in parallel. See, for example, FIG.

  If desired, one or more (eg, one, two, three, all) compartments may optionally include vents that balance the pressure in the compartment with the ambient pressure. The vent may include a one-way valve (eg rotary valve, opening valve, slit valve, septum valve, ball valve, flap valve) configured to give a gas flow in only one direction, for example, allowing the release of extruded gas Can be.

  According to a further aspect of the invention, the apparatus further comprises a support for a biological sample tube, also known herein as a sample tube support. The sample tube support can be any suitable physical or other structure that holds the sample tube in an upright position. In a preferred embodiment, the sample tube support is a walled opening having a shape that is interrelated with at least the base region of the sample tube.

  According to a further aspect of the invention, the apparatus further comprises a support for a biotransport tube, also known herein as a tube support. The tube support may be any suitable physical or other structure suitable for a removable connection between the transfer tube and the device. The tube support portion enables the transfer tube to be stored during transfer and when not in use. The tube support provides a clean environment at the tip of the transfer tube needle. In a preferred embodiment, the transfer tube support includes two slots each having a shape that is interrelated with each end of the transfer tube.

  The device may take any form that provides requirements for compartments and support elements. It should be understood that the device is preferably optimal with respect to size, weight, supportability, durability and recyclability. In a preferred embodiment of the invention, the compartments are mechanically connected to each other. Preferably, the sample tube support is also mechanically connected to the compartment. Preferably, the tube support is also mechanically connected to the compartment. When connected in this way, the device is a single body with the convenience of being a self-supporting unit. The mechanical connection may be made using any suitable arrangement known in the art, such as biology such as polycarbonate, polypropylene, cyclic olefin copolymer (COC) or glass mechanically connected to a support of the same or different material. This can be achieved using a compartment formed from a single block of electrically inert material.

  A preferred embodiment of the present invention is shown in a three-dimensional view in FIG. 21a and a cross-section in FIG. 21b, which represents a device 30 consisting of a solid block 44 of a material such as a cyclic olefin copolymer (COC), the first substance 2 A first compartment 32 in which is present, a second compartment 34 in which the control substance 35 is present, and a third compartment 36 in which the stabilizer 5 is present. The compartments 32, 34, 36 are cylindrical openings provided in the solid block 44 and are each sealed with lids 38, 48, 50 which may be at least partially made from COC. Alternatively, the compartments 32, 34, 36 each consist of a cylindrical vial made of a suitable material (eg glass, polypropylene, polycarbonate) secured with a cylindrical opening provided in the solid block 44. obtain. Each vial is sealed with a lid 38, 48, 50, respectively. The lids 38, 48, 50 each include a resealable septum 46, 40, 42 that provides access to the interior of the compartment using a needle. A sample tube support 52 is provided on the apparatus. The sample tube support 52 is a cylindrical opening configured to support the base region of the sample tube. The apparatus further comprises tube supports 54, 56 each including two slots having shapes that are interrelated with each end of the transfer tube. A U-shaped transfer tube can be used to transfer the contents of the sample tube to each of the first (pulse agent) compartment 32 and the second (control) compartment 34. The compartment is under negative pressure. The same needle may be used to transfer the stabilizer from the third compartment 36 to each of the first compartment 32 and the second compartment 34.

  As described elsewhere, transfer tubes, including hollow tubes with needles (eg, tubular needles) at both ends, are used to transfer liquid from one compartment or container to another. The liquid is pushed into the supply compartment (or container or control container) by the application of positive pressure or withdrawn by application of negative pressure in the receiving compartment (or container or control container).

  The transfer tube needle is suitable for piercing a resealable (eg rubber or silicone) septum so that fluid or air can only enter the compartment (container or control container) through the needle . The tip of the needle is preferably beveled at both sides, and the core need not be removed. The needle may be 22G, 23G, 24G or 25G, or a value in the range between any two of the above values, preferably 23G. Each needle can be protected with a removable waterproof cover to prevent entry of microorganisms before use. Each needle cover remains on the tube support and can be adhered to the tube support so that when the U-shaped needle is lifted from the device, the cover will disengage from the respective needle and the cover will remain in the tube support.

  The length (L) of the needle of the transfer tube in the transfer tube, which represents the length of the straight portion of the needle, may be the same or different. Its length depends on the depth of the compartment and the volume of liquid transferred. As a general guideline, the length (L1) of one transfer tube needle is 25 mm, 26 mm, 28 mm, 30 mm, 32 mm, 33 mm, 34 mm, 35 mm, 36 mm, 38 mm, 40 mm, 42 mm, 44 mm, 46 mm, 48 mm, 50 mm, It can be 55 mm or a value in the range between any two of the above values, preferably 25 mm to 35 mm, more preferably 30 mm to 35 mm. The length (L2) of the needle of the other transfer tube is 25 mm, 26 mm, 28 mm, 30 mm, 32 mm, 33 mm, 34 mm, 35 mm, 36 mm, 38 mm, 40 mm, 42 mm, 44 mm, 46 mm, 48 mm, 50 mm, 55 mm, or above. Can be in the range between any two of the values, preferably 40 mm to 50 mm, more preferably 42 mm to 46 mm. The tube height (L3) varies depending on the available gap space. As a general guideline, the length of one transfer tube is 5mm, 7mm, 8mm, 9mm, 10mm, 11mm, 12mm, 13mm, 14mm, 15mm, 16mm or 17mm, or in the range between any two of the above values It may be a certain value, preferably 10 mm to 15 mm, more preferably 12 mm to 14 mm. The hollow tube may be rigid or flexible. Preferably the hollow tube is rigid. According to a preferred embodiment of the invention, the transfer tube consists of a long needle bent in a U-shape, and the non-pointed tip so that the second needle forms the transfer tube of the invention having a rigid hollow tube. Adhered to (non-pointed end). When a rigid tube is used, the distance (D) between the two needles depends on the spacing between the compartments. According to a preferred embodiment of the present invention, the distance (D) between two needles is essentially the same as the distance between two adjacent compartments, which is generally square with 2 × 2 compartments and sample support. This is achieved when arranged in an array. As a general guide, the distance between needles is 20 mm, 22 mm, 24 mm, 26 mm, 28 mm, 30 mm or 32 mm, or a value in the range between any two of the above values, preferably 24 mm to 28 mm, more preferably 26 mm. It can be.

  The transfer tube is suitable for connection to a robotic system (eg, a robotic arm or a robotic platform) and can facilitate automatic sample processing. When utilizing a rigid hollow tube, the robot system is connected to the transfer tube via a rigid hollow tube. Through the use of a robotic system, automated assays can be performed using several devices of the present invention even in sterile environments that are potentially devoid of other stimulating factors. Furthermore, the device can facilitate the transfer of liquid reagents due to the availability of a single transfer tube.

  Provide the pressure on the compartment, container or container with a needle at one end for insertion into the compartment, container or container bulkhead and the other end to provide the required control pressure on the compartment, container or container Can be provided by a hollow pressure tube (FIG. 23) including a flexible tube suitable for connection to an air pump. The pressure tube needle can be mechanically bonded to one of the transfer tube needles by a joint such as a welded joint, adhesive joint or clamp (see FIG. 24). The pressure tube needle has a hole in the resealable (eg rubber or silicone) septum so that air or gas can only enter the compartment, container or container or exit the compartment, container or container through the needle. Suitable for opening. The tip of the needle is preferably beveled at both sides, and the core need not be removed. The needle may be 22G, 23G, 24G or 25G, or a value in the range between any two of the above values, preferably 23G.

  It is also within the scope of the present invention that the device comprises a further sealing compartment. The compartments may be arranged along a straight line or in an array (eg 2 × 2, 2 × 4, etc.). Preferably, the compartments and the sample support are arranged in a 2 × 2 square array, and the distance between the adjacent compartment and the sample support is equal.

In one embodiment of the present invention, a method for pulsing a biological sample comprises:
i) introducing a biological sample into the container;
ii) optionally stirring the container;
iii) introducing a stabilizer into the container after a predetermined period; and iv) testing the nucleic acid level in the biological sample;
including.

Another embodiment of the present invention is a method for testing an individual's immune response to an antigen that has been pre-immunized to an individual, comprising the use of a container as disclosed herein (where the pulsing agent is a study). A) introducing a blood sample collected from the individual into the container,
b) optionally stirring the container;
c) introducing the nucleic acid stabilizer into the container after a predetermined period; and d) testing cytokine mRNA levels.
Including a method.

  According to one aspect of the invention, the cytokine is one or more of IL-2, IL-4, IL-13, IFN-γ.

Another embodiment of the invention is a method of testing an individual for hyperallergenicity against an antigen, the use of a container as disclosed herein (where the pulsing agent is the antigen under study). And e) introducing a blood sample collected from the individual into the container,
f) optionally stirring the container;
g) introducing the nucleic acid stabilizer into the container after a predetermined period of time; and h) testing the mRNA level of IL-4.
Including a method.

Another embodiment of the invention is a method of testing an individual for rejection of an organ transplant, wherein the use of a container as disclosed herein (where the pulsing agent is a tissue compatible antigen of the organ donor). And i) introducing a blood sample collected from the individual into the container,
j) optionally stirring the container;
k) introducing the nucleic acid stabilizer into the container after a predetermined period of time; and l) testing IL-2 mRNA levels.
Including a method.

The above method may be suitable for use with the apparatus of the present invention. For example, one embodiment of the present invention is a method of pulsing a liquid biological sample present in a biological sample tube using the apparatus of the present invention, comprising:
i) using a U-shaped needle to transfer the liquid biological sample from the biological sample tube to the second compartment where the control agent is present;
ii) using a U-shaped needle to transfer the liquid biological sample from the biological sample tube to the first compartment where the pulsing agent is present;
iii) transferring the stabilizer from the third compartment to the biological sample tube using a U-shaped needle;
iv) using a U-shaped needle to transfer the stabilizer from the third compartment to the second compartment in which the control agent and liquid biological sample are present; and v) using the U-shaped needle to Transferring the stabilizer from the compartment of the first to the first compartment in which the pulsing agent and the liquid biological sample are present, thereby pulsing the liquid biological sample with the pulse control agent and stabilizing the pulsed sample. ,
Including a method.

  Other methods described above, such as testing an individual's immune response to an antigen previously immunized to the individual, testing an individual for hyperallergenicity to the antigen, and testing an individual for organ transplant rejection It may be equally suitable for use with the device of the present invention.

Another aspect of the present invention is a method of pulsing a biological sample with an antigen, then stabilizing the nucleic acid in the biological sample, and testing the RNA component in the pulsed stabilized biological sample comprising:
A) Using the kit, apparatus and / or method as described above, pulsing the biological sample with a pulsing agent, and adding a compound that inhibits RNA degradation and / or gene induction to the biological sample;
B) forming a precipitate containing nucleic acid;
C) separating the precipitate from step (B) from the supernatant;
D) dissolving the precipitate in step (C) using a buffer solution to form a suspension, dissolving step;
E) isolating nucleic acid from the suspension in step (D) using an automated device;
F) Dispensing / dispensing the reagent mixture for RT-PCR using an automatic device,
G) Dispensing / distributing the nucleic acid isolated in step (E) within the dispensing reagent mixture of step (F) using an automated device, and H) RT-PCR of nucleic acid / (G) in an automated configuration Determining an in vivo level of the transcript using the reagent mixture;
Including a method.

  The above methods may optionally utilize the controls described herein to which a biological sample is exposed. Controls contain no reagent or contain a control pulsing agent. The method is carried out in the same manner as that utilizing a container, essentially simultaneously with exposure to the pulsing agent. The control container results can be used to normalize the results obtained in the container.

  Inhibition of RNA degradation and / or gene induction at the time of biological sampling is critical for removing a pool of RNA that can be used to determine in vivo transcript levels. Cellular RNA can be purified using the PAXgene (TM) Blood RNA System in its entirety, but it has been found that the present invention cannot be used to measure actual in vivo levels using this system "by itself". (See Example 2).

  In vivo levels of nucleic acid transcripts only when starting from a pool of RNA prepared from a stabilized biological sample using compounds that inhibit extracellular and / or intracellular RNA degradation and / or gene induction according to the present invention. It can be measured / determined / quantified. In this way, the nucleic acid is isolated using an automatic device, whereby the reagent mixture and the isolated nucleic acid used for the RT-PCR reaction are dispensed using the automatic device, thereby determining the transcript level in an automated configuration. Do. According to the present invention, only this approach allows reproducible in vivo RNA quantification. In order to avoid errors, the number of steps performed in the above method is reduced to a minimum. An “error” can be an error due to pipetting, manipulation, procedure and / or calculation, or any error that can occur by one skilled in the art. In this regard, the present invention proposes performing RT and PCR reactions in one step. The method of the present invention becomes more accurate when more intermediate steps are combined. For example, in the method of the present invention, step (A) and step (B) can be combined.

  In another embodiment of the present invention, after dispensing the reagent mixture necessary for RT-PCR (step (F)), the nucleic acid is dispensed (step (G)) before or simultaneously with the dispensing. be able to.

  According to the method of the present invention, it is not necessary to perform OD measurement, and errors occurring in the calculation of the nucleic acid concentration are eliminated. On the other hand, when the complete PAXgene ™ Blood RNA kit is used, it is necessary to perform OD measurement. This also shows that the method according to the present invention is more reliable and accurate than the latter system. This better accuracy of the present invention is shown by the reproducibility study represented in Table 1.

  In another aspect of the present invention, the resulting suspension is used in combination with fully automated RNA extraction and analysis methods when dissolving the precipitate formed in step (D) of the method according to the present invention. can do. This combination alone allows accurate optimization and reproducibility of the method performed, and allows accurate and reproducible determination of RNA levels after the pulse. The PAXgene (TM) Blood RNA System catalog states that the corresponding tube cannot be used in combination with other isolation methods, and detailed information describing the various configurations of the kit is available. Since it is not possible, it is not clear that one skilled in the art will use a portion of this PAXgene ™ Blood RNA System and develop a new method therefrom.

  There are only a few commercial systems that allow fully automatic RNA isolation. Examples of such automated nucleic acid extraction devices include MagNA Pure LC Instrument (Roche Diagnostics), AutoGenprep 960 (Autogen), ABI Prism ™ 6700 Automated Nucleic Acid Workstation (Applied Biosystems), optionally WAVE (Registered Trademark) FC200 With WAVE® Nucleic Acid Analysis System and BioRobot 8000 (Qiagen).

  The present invention is most important to start with materials that are as fresh or stabilized as possible in all of these systems (minimum RNA degradation) to allow determination of transcript levels after pulse It points out that. The problem with all of these systems is that the biological sample can be collected in a tube containing no or only one conventional additive and transported to the laboratory so that the mRNA can still degrade rapidly. It is. As a result, there is no doubt that mRNA quantification using these methods will result in quantification of transcripts present in the tube, but this quantification is not present in transcripts present in cells / biological agents at the time of sampling. It does not represent a level. This experimental evidence is given in FIG. 32.2 of Example 1 of the present invention.

  The term “quantification” means the accurate and reproducible determination of RNA copy number, but qualitative or semi-quantitative studies can also be performed using RNA isolated by methods as described in the present invention. It is obvious to those skilled in the art that this can be done.

  The definition of “transcript” is not limited to messenger RNA (mRNA) but also to other types of RNA molecules known to exist by those skilled in the art. According to the method of the present invention, mRNA and total RNA can be extracted. This makes it possible to obtain an accurate estimate of nuclear RNA in vivo and provides a powerful tool for assessing gene transcription.

  The term “nucleic acid” refers to a single-stranded or double-stranded nucleic acid sequence, which nucleic acid may consist of deoxyribonucleotides (DNA) or ribonucleotides (RNA), RNA / DNA hybrids, or amplified cDNA or amplified genomic DNA Or a combination thereof. The nucleic acid sequence according to the present invention may also comprise any modified nucleotide as defined in the art.

  According to the present invention, the nucleic acid can be present extracellularly or intracellularly in the biological sample.

  The “separation” of the precipitate from the supernatant in step (C) of the method of the invention can be performed by centrifugation, filtration, absorption or other means known to those skilled in the art. The precipitate may comprise cells, cells / debris, nucleic acids or combinations thereof. The basis for this concept is to stop nucleic acid containing agents (ie biological agents) from coming into contact with external sources / pulses / signals. This can be done by immobilizing, dissolving and / or disintegrating the nucleic acid-containing agent or by other means known to those skilled in the art.

  The buffer solution used in step (D) of the method of the present invention may be a buffer solution that dissolves the precipitate obtained in step (C) of the above method. This buffer may have additional effects such as lysis or further lysis of the nucleic acid containing agent.

  The “automatic device” used may be an automatic pipetting device or another automatic device known to those skilled in the art suitable for performing the specified action.

  By “reagent mixture for RT-PCR” is meant all reagents required for simultaneous RT and PCR reactions (except for oligonucleotides where explicitly mentioned). According to the present invention, an “oligonucleotide” can include a short stretch of nucleic acid, such as found in primers or probes. According to the present invention, this method can be used in combination with a microarray or RNase protection assay.

  As pointed out earlier, storage of biological samples such as blood results in inaccurate determination of mRNA levels. Actually, since a sampling place and an RNA analysis place are provided separately, analysis of a fresh sample cannot be executed. The method according to the invention makes it possible to transport a biological sample from a remote location to a suitable laboratory without affecting the in vivo transcript content. The biological sample can be transferred after step (A) or step (B) in the method of the present invention.

  When using a normal blood sample, red blood cells are selectively excluded before isolating the nucleic acid. Red blood cells are abundant in hemoglobin, and their presence results in a viscous lysate. Therefore, the nucleic acid can be further improved by removing these red blood cells. However, the method of the present invention eliminates this step because insoluble precipitates containing nucleic acids are immediately formed and these nucleic acids are separated from all other components of the biological sample. This indicates, in addition to other advantages, that the method of the present invention is superior to most prior art methods.

  According to the present invention, the buffer used in step (D) of the method of the present invention may be a guanidine-thiocyanate-containing buffer.

  In an example of the present invention, the precipitate formed with PAXgene ™ Blood RNA Tube is dissolved in a lysis buffer as provided by MagNA Pure LC mRNA Isolation Kit I (Roche Diagnostics, Molecular Biochemicals). Therefore, in the present invention, one of the buffers that can be used in the method of the present invention is a guanidine-thiocyanate-containing lysis buffer as provided by MagNA Pure LC mRNA Isolation Kit I (Roche Diagnostics, Molecular Biochemicals). Proposed.

  MagNA Pure LC mRNA Isolation Kit I (Roche Diagnostics, Molecular Biochemicals) for use with MagNA Pure LC Instrument to ensure the isolation of high quality undegraded RNA from whole blood, leukocytes and peripheral blood lymphocytes Designed specially. According to its product description, the resulting RNA is sensitive and quantitative LightCycler RT-PCR reaction and standard block cycler RT-PCR reaction, Northern blotting and other standard RNA applications It is suitable for. Nonetheless, according to the present invention, it has been found that the use of this method “by itself” cannot result in an accurate transcript level determination. The present invention shows that RNA needs to be stabilized prior to RNA isolation (see Example 1). The present invention describes a particular combination of compounds that stabilize RNA and the use of automated isolation / analysis procedures.

  According to the present invention, if the precipitate of step (D) is dissolved in a lysis buffer such as a buffer provided by MagNA Pure LC mRNA Isolation Kit I, the method of the present invention relates to MagNA Pure LC mRNA Isolation Kit I. Procedures as described can be followed. After the sample is lysed by the presence of chaotropic salt in the lysis buffer, streptavidin-coated magnetic particles are added along with biotin-labeled oligo-dT, and the mRNA binds to the surface of the particles. This is followed by a DNase digestion process. The mRNA is then isolated from unbound material using a magnet and several washing steps. Finally, purified mRNA is eluted. This isolation kit allows for the automatic isolation of pure mRNA as a “walk away” system. This isolation kit allows for the isolation of high quality and integrity mRNA suitable for all major subsequent applications for gene expression analysis. Different protocols are given depending on the sample material used. Samples can be constructed directly at the MagNA pure LC Instrument stage. When whole blood is used, cells present in the sample are selectively lysed manually. Therefore, mRNA isolation can be postponed later or further processed directly on this instrument.

  According to the present invention, by use of MagNA Pure LC Instrument (Roche Diagnostics, Molecular Biochemicals) as an automated device in step (E), step (F) and / or step (G) of the method according to the invention in an embodiment of the invention. It has been found that exposure to pulsing agents results in a pool of RNA that can be used to determine the exact level of transcript. In order to separate mRNA from cell debris, RNA capture beads, such as magnetic beads, coated with oligo-dT by a streptavidin-biotin system or equivalent system can be applied to the method of the invention.

  Alternatively, according to the present invention, other automated devices can be used, such as ABI Prism ™ 6700 Automated Nucleic Acid Workstation (Applied Biosystems) or any other automated device that can be used for this purpose.

  The catalog of MagNA pure LC mRNA Isolation Kit I (catalog number 3 004 015) does not mention in detail the composition of the buffer used in this kit. Therefore, it is not self-evident that one skilled in the art would expect to be able to dissolve the pellet obtained by the PAXgene ™ Blood RNA Tube method with the buffer provided by this kit. Furthermore, those skilled in the art combine both methods based on information provided by the PAXgene ™ Blood RNA Tube catalog stating that these tubes can only be combined with the corresponding PAXgene ™ Blood RNA Kit. (See page 3, limitations of the system; see page 6, ordering information).

  As pointed out above, when using a blood sample, red blood cells are selectively lysed after step (A) of the method of the invention. The design of MagNA Pure LC mRNA Isolation Kit I (Roche Diagnostics, Molecular Biochemicals) may lyse and eliminate red blood cells before isolating mRNA from white blood cells. Nevertheless, because of this step, the sample cannot be processed quickly enough to avoid mRNA degradation. The inventors have decided to use the stabilizer contained in PAXgene ™ Blood RNA Tube in conjunction with MagNA Pure mRNA Isolation Kit from MagNA Pure Instrument. Use of PAXgene ™ Blood RNA Tube provides a precipitate of nucleic acid that does not dissolve in the lysis buffer of MagNA Pure mRNA Isolation Kit. Despite this, the present inventors have found that dissolution is actually possible. In accordance with this view, we combined the use of stabilizers in the PAXgene ™ Blood RNA Tube with the use of an automated RNA isolation system. It is surprising that the inventors have found that this combination is possible and that this combination provides a powerful technique for accurate mRNA quantification from biological samples.

  RNA isolated using the method according to the present invention is for example quantitative RT-PCR including RT-PCR and nucleic acid amplification techniques such as NASBA®, expression arrays and chip analysis, TaqMan® technology, It is ready for use in a wide range of subsequent applications, including cDNA synthesis, RNase and S1 nuclease protection, Northern blot analysis, dot blot analysis and slot blot analysis, and primer extension.

  The inventors have found that in vivo levels of transcripts can be determined by the use of compounds that inhibit RNA degradation and / or gene induction in conjunction with automated RNA isolation and analysis methods such as real-time PCR. It showed in Example 1 and Example 2 of this invention. However, according to the present invention, analysis methods other than real-time PCR may be applied as long as they are given in an automated configuration.

  The main advantage of the method according to the invention is that a small amount of sample can be analyzed by using this method. This is most important when only a small amount is available, for example when analyzing neonatal blood samples or when blood loss is large. According to the present invention, RNA quantification can be performed using a biological sample as small as 100 μl. The Qiagen kit (PAXgen ™ Blood RNA System), which requires a larger amount of blood (2.5 ml according to the kit handbook), cannot analyze RNA from a sample as small as 100 μl.

As described above, one embodiment of the present invention is a kit suitable for pulsing a biological sample with a pulsing agent and then stabilizing nucleic acid derived from the biological sample so pulsed. In another aspect of the invention, the kit includes additional components to isolate quantifiable RNA from the stabilized pulsed biological sample. According to one aspect of the invention, the kit comprises:
Reagents for automated RNA isolation,
Reagent mixtures for simultaneous RT and real-time PCR reactions that allow automatic dispensing of the mixture, or separate compounds thereof,
Optionally, a specific oligonucleotide for performing the RT-PCT reaction, and optionally a method of automatically isolating RNA, automatically dispensing reagent mixtures and separating the isolated nucleic acids for RT-real-time PCR. Instruction manual explaining how to pour and automatically analyze RNA,
And other additional components such as

  In an example of the present invention, we applied the “Lightcycler mRNA hybridization probe kit” (Catalog No. 3 018 954) from Roche Diagnostics, Molecular Biochemicals to perform RT-PCR reactions in one step. Yes. All necessary reagents are included in this kit except for oligonucleotides (synthesized by Biosource). Nevertheless, real-time PCR as described in the present invention can also be performed with other equipment such as Applied Biosystems equipment. The kit may further comprise a buffer such as a guanidine-thiocyanate-containing buffer that can be used in step (b) of the method according to the present invention.

  The method according to the invention can also be used for quantification / detection of DNA (double-stranded or single-stranded) in a biological sample. Therefore, the present invention is a method for quantifying DNA from a biological sample, which is used for quantification of RNA according to the present invention, the RT reaction is omitted, and the compound of step (a) is DNA It also relates to a method that prevents the decomposition of the material. Since these nucleic acids are more stable than RNA, their stabilization is less important than that of RNA.

  Furthermore, the present invention relates to a kit for isolating quantifiable DNA from a biological sample according to the present invention, which lacks a reagent mixture / compound for performing the RT reaction. The need to determine an accurate DNA level in a biological sample is when determining the “presence” of infection (s) / contamination (s) in the biological sample by an unexpected gene, pathogen or parasite, and / or Or it may be when determining the “level” of infection / contamination. For example, the method can be used to determine the proportion of transgenic material in a cereal batch.

  The present invention provides an apparatus, kit, and apparatus for monitoring / detecting in vivo biological marker nucleic acid changes in a biological sample after pulsing with an agent to diagnose a particular disease Also relates to the use of the method.

  The present invention monitors / changes in vivo biological marker nucleic acid changes in a biological sample after pulsing with an agent to screen for a compound (the compound is used in the manufacture of a drug that cures the disease). It also relates to the use of the device, kit and method according to the invention for detection.

  The invention therefore also relates to compounds identifiable by the method according to the invention.

  The devices, kits and methods disclosed herein can be used to treat and / or diagnose a disease. An example of a disease to be cured or diagnosed is an immune related disease. According to the present invention, examples of immune related diseases are autoimmunity, rheumatoid arthritis, multiple sclerosis, type 1 diabetes mellitus, cancer (eg in cancer immunotherapy), immune deficiency (eg in AIDS), allergy, transplant rejection or It can be graft versus host disease (GVHD) (eg in transplantation). Examples included in this application describe the application in detail. Thus, an immunomodulatory compound or immunomodulator can affect one of the diseases, and changes in immune-related transcripts or epitope-specific CTL-related or T-helper lymphocyte-related transcripts The presence and / or condition of one of them and an immunological condition that can indicate one of the above conditions.

  Nucleic acids that can be quantified using the devices, kits and methods of the present invention to study the immune-related diseases include, for example, chemokines, cytokines, growth factors, cytotoxic markers, transcription factors, TNF-related cytokine receptor super It may be a nucleic acid encoding a member of the family and their ligands, apoptosis markers, immunoglobulins, T cell receptors and any markers associated with activation or inhibition of the known or discovered immune system.

  According to the present invention, the nucleic acid is IL-1ra, IL-1β, IL-2, IL-4, IL-5, IL-9, IL-10, IL-12p35, IL-12p40, IL-13, TNF. -Can encode markers such as α, IFN-γ, IFN-α, TGF-β and any interleukin or cytokine involved or not involved in the immune response. Housekeeping genes such as β-actin or GAPDH (glyceraldehyde phosphate dehydrogenase) can be used as internal markers.

  According to the present invention, the epitope-specific CTL-related or T helper lymphocyte-related transcripts are cytokines, cytokine receptors, cytotoxins, inflammatory or anti-inflammatory mediators, members of the TNF-related cytokine receptor superfamily And their ligands, G protein-coupled receptors and their ligands, tyrosine kinase receptors and their ligands, transcription factors, and nucleic acids encoding proteins involved in intracellular signaling pathways (be).

  According to the present invention, the nucleic acid comprises granzyme, perforin, prostaglandin, leukotriene, immunoglobulin and immunoglobulin superfamily receptor, Fas and Fas ligand, T cell receptor, chemokine and chemokine receptor, protein tyrosine kinase C , Protein tyrosine kinase A, Signal Transducer and Activator of Transcription (STAT), NF-kB, T-bet, GATA-3, Oct-2.

  The invention relates to the use of a device, method or kit according to the invention for the detection / monitoring / screening of a compound, wherein said compound is a eukaryote, prokaryote, virus, phage, parasite, drug (natural extract) Also described are uses, which are immunomodulatory compounds that can be selected from the group consisting of: organic molecules, peptides, proteins, nucleic acids), medical treatments, vaccines and transplants. The use of such methods is not limited to single compound detection / monitoring / screening. You can also study the synergistic effects of substance groups.

  The invention also relates to the use of any of the devices, kits and methods as described above for the detection / monitoring of epitope-specific CTLs or immune-related transcripts.

  The devices, kits and methods according to the invention can also be applied to monitoring in vivo immune responses after treatment of patients with drugs / treatments / vaccines that are likely to alter the patient's immune status. According to the present invention, cytokine mRNA (chemokine, chemokine, by the method described in whole blood of patients undergoing treatment or enrolled in clinical trials with immunomodulatory drugs or immunomodulatory therapy or with vaccines (therapeutic or prophylactic) Detection of growth factors, cytotoxicity markers, apoptosis markers, or any marker of known or discovered immune system activation) can detect the effectiveness, safety and / or ultimate side effects of the therapy Can be used to evaluate.

  The present invention also relates to a device, kit and method for detecting an immune state in vivo for diagnosis / prognosis of diseases (cancer, autoimmune diseases, allergies, transplant rejection, GVHD, etc.) affecting the immune system.

  In accordance with the present invention, cytokine mRNA (chemokines, growth factors, cytotoxic markers, apoptosis markers, or by the methods described in whole blood of patients suffering from diseases that directly or indirectly affect the immune system, The detection of a known or discovered marker that may extend to any marker associated with activation of the immune system is intended to be diagnostic or prognostic.

The present invention is a method for identifying an agent capable of altering a subject's immune status by analysis of epitope-specific CTL comprising:
(A) applying an immunomodulatory agent (s) to the subject;
(B) sampling whole blood from the subject,
(C) Using the apparatus as described above, pulsing blood cells present in the whole blood sample of step (b) with the same / similar and / or different immunomodulating agent as applied in step (a) ,
(D) recovering the pulsed blood cells of step (c) or non-pulsed blood cells of step (b) into a tube containing a compound that inhibits RNA degradation and / or gene induction, or pulsed with the compound / Adding to non-pulsed cells,
(E) forming a precipitate containing nucleic acid;
(F) separating the precipitate from step (e) from the supernatant;
(G) The step of dissolving the precipitate in step (f) using a buffer solution to prepare a suspension,
(H) isolating nucleic acids from the suspension of step (g) using an automated device;
(I) Dispensing / dispensing the reagent mixture for RT-PCR using an automatic device;
(J) Dispensing / distributing the nucleic acid isolated in step (h) in the dispensing reagent mixture of step (i) using an automatic device;
(K) detecting / monitoring / analyzing in vivo levels of epitope-specific CTL-related transcripts in the dispensing solution of step (j) in an automated configuration; and (l) altering the immune status of the subject. Identifying possible agents,
A method is also described wherein step (a) is omitted if the agent of step (a) already exists in the subject.

  The invention also relates to a kit comprising at least the components that make it possible to carry out step (c) above. The kit may contain additional reagents and instructions to allow one or more of the other steps to be performed. The disclosure made herein indicates to those skilled in the art the components required to construct the desired kit.

  According to the present invention, the immunomodulator (s) may be present in the subject in the case of disease or in the presence of an implant. In the present invention, an “epitope-specific CTL-related transcript” is a cytokine, cytokine receptor, cytotoxin (granzyme, perforin, etc.), a member of the TNF-related cytokine receptor superfamily and their ligands (eg, Fas and Fas ligand) or It can be a transcript encoding another cell receptor.

The present invention is a method for identifying an agent capable of altering a subject's immune status, comprising:
(A) applying an immunomodulatory agent (s) to the subject;
(B) sampling whole blood from the subject,
(C) Using the apparatus or kit as described above, pulsing blood cells present in the whole blood sample of step (b) with the same / similar and / or different immunomodulating agent as applied in step (a) The process of
(D) recovering the pulsed blood cells of step (c) or non-pulsed blood cells of step (b) into a tube containing a compound that inhibits RNA degradation and / or gene induction, or pulsed with the compound / Adding to non-pulsed cells,
(E) forming a precipitate containing nucleic acid;
(F) separating the precipitate from step (e) from the supernatant;
(G) The step of dissolving the precipitate in step (f) using a buffer solution to prepare a suspension,
(H) isolating nucleic acids from the suspension of step (g) using an automated device;
(I) Dispensing / dispensing the reagent mixture for RT-PCR using an automatic device;
(J) Dispensing / distributing the nucleic acid isolated in step (h) in the dispensing reagent mixture of step (i) using an automatic device;
(K) a step of detecting / monitoring / analyzing in vivo levels of immune-related transcripts in the dispensing solution of step (j) in an automated configuration; and (l) an agent capable of altering the immune status of the subject. Identifying the steps,
A method is also described wherein step (a) is omitted if the agent of step (a) already exists in the subject.

  The invention also relates to a kit comprising at least the components that make it possible to carry out step (c) above. The kit may contain additional reagents and instructions to allow one or more of the other steps to be performed. The disclosure made herein indicates to those skilled in the art the components required to construct the desired kit.

  In the present invention, “immune-related transcript” is, for example, cytokine (s), chemokine (s), growth factors, cytotoxic markers, transcription factors, members of the TNF-related cytokine receptor superfamily and their ligands, or known It can be a transcript that encodes any marker associated with or discovered activation of the immune system. According to the present invention, the immunomodulator (s) may be present in the subject in the case of disease or when a graft is present. A subject according to the invention can be of both human or animal origin.

The present invention is a method for diagnosing / prognosing / monitoring a clinical condition affecting the immune system in a subject, comprising:
(A) sampling whole blood from the subject,
(B) using the apparatus or kit as described above, pulsing blood cells present in the whole blood sample of step (a) with the same / similar and / or different immunomodulating agent as present in the subject;
(C) collecting the pulsed blood cells of step (b) into a tube containing a compound that inhibits RNA degradation and / or gene induction, or adding the compound to the pulsed cells;
(D) forming a precipitate containing nucleic acid;
(E) separating the precipitate from step (d) from the supernatant;
(F) dissolving the precipitate in step (e) using a buffer solution to prepare a suspension;
(G) isolating nucleic acid from the suspension of step (f) using an automatic device;
(H) Dispensing / dispensing the reagent mixture for RT-PCR using an automatic device;
(I) Dispensing / distributing the nucleic acid isolated in step (g) within the dispensing reagent mixture of step (h) using an automatic device;
(J) detecting / monitoring / analyzing in vivo levels of immune-related transcripts in the dispensing solution of step (i) in an automated configuration;
(K) detecting / monitoring changes in in vivo levels of immune-related transcripts, and (l) diagnosing / prognosing / monitoring diseases affecting the immune system,
A method is also provided comprising:

  In the present invention, a “clinical condition” is any change in a subject's physical condition, such as the presence of various diseases or transplants.

  The invention also relates to a kit comprising at least the components that make it possible to carry out step (c) above. The kit may contain additional reagents and instructions to allow one or more of the other steps to be performed. The disclosure made herein indicates to those skilled in the art the components required to construct the desired kit.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs (those skilled in the art). Have Illustrative methods and materials are described below, but methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications and other references mentioned herein are incorporated by reference in their entirety. In the case of conflicts, conflicts are adjusted according to this specification, including definitions. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the invention will be apparent from the following drawings, detailed description, and appended claims.

FIG. 2 shows an example of a container and method according to the present invention. FIG. 1 a shows a container 1 in which antigen particles 2 are present. A resealing means 3 for entering the syringe needle is attached to the container. Container 4 is also part of container 1. The stabilizer 5 is present in the container, and the connection between the interior of the container 1 and the interior of the container 4 is temporarily interrupted by a physical barrier, in this case a plug 25. Means for transmitting physical forces and removing the plug are provided in the form of a plunger 28. In this example, the plunger is covered with a cap 23. In FIG. 1 b, the biological sample 24 is introduced by the syringe needle 6 through the resealing means 3 for entering and into the container, and the biological sample 24 is exposed to the antigen 2. In FIG. 1c, the plunger cap 23 is removed (26). In FIG. 1 d, the shaft 7 is introduced and pressure is applied thereto (27), causing the plunger 28 to push the plug 25 away from the container 4. When the plug 25 is released, the stabilizer 5 is released into the container 1 and mixed with the biological sample 24 and the antigen 2. FIG. 2 shows an example of a container 1 according to the invention in which an antigen 2 is present. As shown here, the container is attached with a lid open (7), closures, or means for introducing a sample or stabilizer (examples shown in FIGS. 3-6) It may be done. The body of the container 1 may include a container in which a stabilizer is present as shown in FIGS. FIG. 3 is a diagram showing an example of an attachment portion suitable for the type of container shown in FIG. On the upper part 7 of the container, a luer type attaching part 8 capable of receiving a mutual luer type attaching part with a syringe 11 is attached. The syringe may contain a biological sample or a stabilizer according to an embodiment of the present invention. FIG. 4 is a diagram showing an example of an attachment portion suitable for the type of container shown in FIG. A resealable septum 9 capable of receiving a syringe needle is attached to the upper part 7 of the container. The syringe may contain a biological sample or a stabilizer according to an embodiment of the present invention. FIG. 5 is a diagram showing an example of an attachment portion suitable for the type of container shown in FIG. A means for receiving a screw cap 10 is attached to the upper part 7 of the container. FIG. 6 is a diagram showing an example of an attachment portion suitable for the type of container shown in FIG. A hypodermic syringe needle 19 is attached to the upper part 7 of the container. Containers can be used to collect samples directly from an individual. FIG. 7 is a diagram illustrating an example of the main body of the container 1 that can be used in combination with the container and the attachment unit illustrated in FIGS. 2 to 6, for example. The container 1 in which the antigen 2 is present includes a container 12 in which the stabilizer 5 is present. The wall of the container consists entirely or in part 15 of a material that pulverizes when a certain force is applied. The container is equipped with means for transmitting a force from a user that pulverizes part or all of the container, including a descending region 13 associated with the pointed tip 14. When the region 13 is lowered, the pointed tip 14 comes into contact with the grindable material 15 and material crushing occurs, thus removing the physical barrier between the container and the stabilizer at a time determined by the user. Can flow into the container 1. FIG. 8 is a diagram illustrating an example of the main body of the container 1 that can be used in combination with the container and the attachment unit illustrated in FIGS. 2 to 6, for example. The container 1 in which the antigen 2 is present includes a container 16 in which the stabilizer 5 is present. The connection 17 between the interior of the container and the container is physically interrupted by a septum 18 that can be breached by the application of force. When pressure is applied to the wall of the container 16, the pressure is transmitted to the partition wall, the partition wall is breeched, and thus the stabilizer 5 enters the container 1. FIG. 9 is a diagram showing an example of the container 20 of the present invention that is not connected to a container. The stabilizer 5 is present in the container 20, and the container 20 is provided with a luer type attachment portion 22 suitable for connection with a container having an attachment portion (for example, as shown in FIGS. 3 and 11). ). Pressure can be applied to the wall of the container 20 and when a force is applied to the container, the stabilizer is released. FIG. 10 is a diagram showing an example of the container 29 of the present invention that is not connected to a container. The stabilizer 5 is present in the container 29, and the container 29 is provided with a luer type attachment portion 22 for connection with a container having an interconnection portion (for example, as shown in FIGS. 3 and 11). ). A plunger 30 is further attached to the container, and the stabilizer 5 is released by applying a force thereto. FIG. 11 is a view showing an example of the kit according to the present invention including the container 1 to which the luer type attaching portion 8 and the valve 31 for exhausting air from the container are attached. The kit also includes a container 29 in which the stabilizer 5 is present, similar to that shown in FIG. The attachment portion 8 of the container can be connected to the attachment portion of the container 22. Figures 12a to 12d show an example of a control container and method according to the present invention. FIG. 12a shows a control container 37 in which a control substance 35 is present. The control container is fitted with an ingress resealing means 3 'for a syringe needle. The control container 4 ′ is also part of the control container 37, the stabilizer 5 ′ is present in the control container, and the connection between the inside of the control container 37 and the inside of the control container 4 ′ is a physical barrier, In this case, it is temporarily blocked by the plug 25 '. Means for transmitting physical force and removing the plug are provided in the form of a plunger 28 '. In this example, the plunger is covered with a cap 23 '. In FIG. 12b, the biological sample 24 is introduced by the syringe needle 6 'through the ingress resealing means 3' into the control container and the biological sample 24 is exposed to the control substance 35. In FIG. 12c, the plunger cap 23 'is removed (26'). In FIG. 12d, the shaft 7 'is introduced and pressure is applied thereto (27'), causing the plunger 28 'to push the plug 25' away from the container 4 '. When the plug 25 ′ is released, the stabilizer 5 ′ is released into the control container 37 and mixed with the biological sample 24 and the control substance 35. FIG. 13 shows an example of a control container 37 according to the present invention in which a control substance 35 is present. As shown here, the control container 37 is open (7 ') or attached with a closure or means for introducing a sample or stabilizer (examples shown in FIGS. 14-16). It may be done. The body of the control container 37 may include control containers 12 ', 16' in which a stabilizer is present as shown in Figs. FIG. 14 is a diagram showing an example of an attachment portion suitable for the control container of the type shown in FIG. On the upper part 7 ′ of the control container 35, a luer type attaching part 8 ′ which can receive a mutual luer type attaching part with a syringe 11 ′ is attached. The syringe may contain a biological sample or a stabilizer according to an embodiment of the present invention. FIG. 15 is a diagram showing an example of an attachment portion suitable for the control container of the type shown in FIG. The top 7 'of the control container 37 is fitted with a resealable septum 9' that can receive a syringe needle. The syringe may contain a biological sample or a stabilizer according to an embodiment of the present invention. FIG. 16 is a diagram showing an example of an attachment suitable for the control container 37 of the type shown in FIG. A means for receiving the screw cap 10 ′ is attached to the upper part 7 ′ of the control container 37. FIG. 17 is a view showing an example of the main body of the control container 37 that can be used in combination with the control container and the attachment part shown in FIGS. 14 to 16, for example. The control container 37 in which the control substance 35 is present contains the control container 12 'in which the stabilizer 5' is present. The wall of the control container is made entirely or partly of material 15 'that grinds when a certain force is applied. The control container is fitted with means for transmitting a force from the user to crush some or all of the control container, including a descending region 13 'associated with a pointed tip 14'. As the region 13 'descends, the pointed tip 14' comes into contact with the grindable material 15 'and material crushing occurs, thus removing the physical barrier between the control container and the control container, as determined by the user It will be possible to allow the stabilizer to flow into the control container 37 over time. FIG. 18 is a view showing an example of the main body of the control container 37 that can be used in combination with the control container and the attachment part shown in FIGS. 14 to 16, for example. A control container 37 in which the control substance 35 is present contains a control container 16 ′ in which the stabilizer 5 is present. The connection between the interior of the control container and the control container 37 is physically interrupted by a septum 18 'that is broken by the application of force. When pressure is applied to the wall of the control container 16 ′, the pressure is transmitted to the partition wall, the partition wall is destroyed, and thus the stabilizer 5 enters the control container 37. FIG. 19 shows an example of a device according to the invention comprising a control container 37 according to FIG. 17 connected externally to the container 1 according to FIG. This device allows the biological sample to be delivered separately to the first substance 2 and the control substance 35 before the stabilizer 5. Since the reaction sample and the control sample are kept side by side, errors that can be caused by mixing the control sample and the reaction sample are avoided. FIG. 20 shows an example of the device of the present invention comprising a control container 37 according to FIG. 18 mechanically connected to the container 1 according to FIG. 8 by a bridging member 70. This device allows the biological sample to be delivered separately to the first sample 2 and the control substance 35 before the stabilizer 5. Since the reaction sample and the control sample are kept side by side, errors that can be caused by mixing the control sample and the reaction sample are avoided. FIG. 21a is a schematic diagram of an example of a device 30 of the present invention for receiving a liquid biological sample. The device facilitates separate exposure of the sample to the first and control substances and then to the nucleic acid stabilizer. The device comprises a first compartment 32 in which the first substance 2 is present, a second compartment 34 in which the control substance 35 is present, a third compartment 36 in which the stabilizer 5 is present, and a cylindrical opening. And a support portion 52 for a biological sample tube. Each compartment is formed as an opening in the solid material block 44. Each compartment is sealed by a lid 46, 48, 50 with a sealable septum 38, 40, 42 that can be broken using a hollow needle, such as a hypodermic tubular needle. The device further comprises a support for the transfer tube, which is in the form of two slots 54, 56 adapted to receive the needle tip of the transfer tube. FIG. 21b shows a cross section of the device of the invention along the A-A 'line. This details the contents of the second (control) compartment 34 and the third (stabilizer) compartment 36. FIG. 22 shows a cross-sectional view of a transfer tube 60 of the present invention that includes a hollow elongate tube 64. Here, each end of the tube is equipped with a hollow (eg tubular or hypodermic) needle 62,66. The transfer tube 60 is configured to allow liquid to flow from one needle 62 through the tube 64 to the other needle 66. In the embodiment shown, the lengths of the needles 62, 66 are different. FIG. 23 shows a cross-sectional view of a pressure tube 88 of the present invention that includes a hollow flexible elongated tube 82. Here, one end of the tube is connected to a hollow (eg tubular or hypodermic) needle 80 and the other end 85 is an outlet connection of a (pressure or exhaust) gas medium (eg air) pump 86. It is suitable for connection with the part 87. The pressure tube 88 is configured to puncture the septum of the container or compartment and create different pressures therein by discharging or introducing a gaseous medium (eg, air). FIG. 24 shows a cross-sectional view of a composite set including the pressure tube 88 and transfer tube 60 of the present invention. One or more joints 84 are used to connect one needle 62 of the transfer tube 60 to the needle 80 of the pressure tube 88 so that both needles 62, 80 are in a parallel arrangement. For this reason, both the needles 62 and 80 simultaneously make a hole in the partition wall. FIG. 25 shows a cross-sectional view of the transfer tube 60 of the present invention, showing dimensions. D represents the minimum distance between the two needles 62, 66. L1 is the length of one needle, particularly the straight portion, L2 is the length of the other needle, particularly the straight portion, and L3 is a first length extending from the straight portion of the needles 62, 66 toward the tube 64. The distance L3 is the distance of the virtual straight line 65, and the distance L3 is the end point 67 of the straight part of the needle and the second virtual straight line 63 (the second virtual straight line 63 extends from the upper part of the tube 64 (intersects)). Is defined by a point 69 that intersects the first virtual straight line 65. FIG. 26 shows an example of the device 30 shown in FIG. 21a in use. A biological sample tube 58 containing a liquid biological sample (for example, blood, urine) is inserted into the tube support portion 52. One end of the transfer tube 60 is inserted through the septum 61 into the biological sample tube 58, and the other end is inserted through the septum 40 into the second (control) compartment 34. A liquid biological sample flows from the biological sample tube 58 along the transfer tube 60 in the direction of the arrow to the second compartment 34 where the liquid biological sample contacts the control substance 35. By using a pressure tube 88 (not shown), a liquid biological sample can be pushed out and a hole in the septum 38 of the second compartment 34 can be pierced with a pressure tube needle to create a vacuum or the biological sample tube 58 A hole can be made in the partition wall 61 to increase the pressure therein. FIG. 27 shows an example of the device 30 shown in FIG. 21a in use. The transfer tube is removed and the end of the transfer tube 60 that was in the second compartment 34 is reinserted through the septum 38 to be located in the first compartment 32, but the transfer tube 60 that was in the biological sample tube 58 is Leave the edges as they are. The liquid biological sample is flowed from the biological sample tube 58 through the transfer tube 60 to the first compartment 32 in the direction of the arrow, where the liquid biological sample comes into contact with the first substance 2 (pulse agent). The use of a pressure tube 88 (not shown) allows the liquid biological sample to be pushed out, and the pressure tube needle is used to puncture the septum 40 of the first compartment 32 to create a vacuum or the biological sample tube 58. A hole can be made in the partition wall 61 to increase the pressure therein. FIG. 28 shows an example of the device 30 shown in FIG. 28 and shown in FIG. 21a in use. The end of the transfer tube 60 that has been in contact with the biological sample tube 58 is inserted into the third (stabilizer) compartment 36 through the partition wall 42, and the other end of the transfer tube 60 is inserted through the partition wall 61 into the biological sample tube 58. FIG. 5 shows washing of the transfer tube 60 after transfer of the liquid biological sample by removing and reinserting the transfer tube 60 as shown in FIG. The liquid stabilizer 5 is allowed to flow from the third compartment 36 through the transfer tube 60 in the direction of the arrow into the biological sample tube 58 where the liquid stabilizer is stored for disposal or for further processing. The use of a pressure tube 88 (not shown) can push out the liquid stabilizer 5 and puncture the septum 42 of the third compartment 36 with a pressure tube needle to increase the pressure therein, or A hole can be made in the partition wall 61 of the biological sample tube 58 to create a vacuum. Note that samples are incubated after washing. Incubation period and temperature will vary depending on several factors including volume and concentration of pulsing agent, sample, and reagent. As a general guide, incubations can be carried out at 37 ° C. for 30 minutes to 16 hours, most commonly 2 hours to 4 hours. FIG. 29 shows an example of the device 30 shown in FIG. 21a in use. Remove the transfer tube 60 so that one end is inserted through the septum 42 into the third (stabilizer) compartment 36 and the other end is inserted through the septum 40 into the second (control) compartment 34; Reinsert. The liquid stabilizer 5 is allowed to flow from the third compartment 36 through the transfer tube 60 to the second compartment 34 in the direction of the arrow, where the liquid stabilizer contacts the mixture of the biological sample and the control substance 2. The use of a pressure tube 88 (not shown) can push out the liquid stabilizer 5 and puncture the septum 42 of the third compartment 36 with a pressure tube needle to increase the pressure therein, or A hole can be drilled in the septum 40 of the second compartment 34 to create a vacuum. FIG. 30 shows the transfer such that one end is inserted through the septum 42 into the third (stabilizer) compartment 36 and the other end is inserted through the septum 38 into the first (pulse reaction) compartment 32. Remove tube 60 and reinsert. A liquid stabilizer is flowed from the third compartment 36 through the transfer tube 60 to the first compartment 32 in the direction of the arrow, where the liquid stabilizer contacts the mixture of the biological sample and the first substance 35. The use of a pressure tube 88 (not shown) can push out the liquid stabilizer 5 and puncture the septum 42 of the third compartment 36 with a pressure tube needle to increase the pressure therein, or A hole can be drilled in the septum 38 of the first compartment 32 to create a vacuum. At the end of the sequence, both the first compartment and the second compartment have received the biological sample and the stabilizing agent. The transfer tube 60 and, if used, the pressure tube 88 can be actuated by a robot, for example by the use of a robot arm that moves to some extent or by an XY robot table. When robotic actuation is used, the transfer tube typically includes a rigid tube 64 that can grasp and accurately place the transfer tube 60. FIG. 31 illustrates the strategy followed in a given example. Figure 31.1 shows ex vivo monitoring of immune response to tetanus toxoid. FIG. 31.2 is a diagram showing a strategy according to the third embodiment. FIG. 31.3 is a diagram showing a strategy according to the fourth embodiment. FIG. 31.4 is a diagram showing a strategy according to the fifth embodiment. FIG. 31 illustrates the strategy followed in a given example. Figure 31.1 shows ex vivo monitoring of immune response to tetanus toxoid. FIG. 31.2 is a diagram showing a strategy according to the third embodiment. FIG. 31.3 is a diagram showing a strategy according to the fourth embodiment. FIG. 31.4 is a diagram showing a strategy according to the fifth embodiment. FIG. 31 illustrates the strategy followed in a given example. Figure 31.1 shows ex vivo monitoring of immune response to tetanus toxoid. FIG. 31.2 is a diagram showing a strategy according to the third embodiment. FIG. 31.3 is a diagram showing a strategy according to the fourth embodiment. FIG. 31.4 is a diagram showing a strategy according to the fifth embodiment. FIG. 31 illustrates the strategy followed in a given example. Figure 31.1 shows ex vivo monitoring of immune response to tetanus toxoid. FIG. 31.2 is a diagram showing a strategy according to the third embodiment. FIG. 31.3 is a diagram showing a strategy according to the fourth embodiment. FIG. 31.4 is a diagram showing a strategy according to the fifth embodiment. FIG. 32.1 is a diagram showing RT-PCR results concerning spontaneous production of IFN-γ and IL-10 mRNA in peripheral blood. Total RNA was extracted from whole blood and PBMC from 6 different healthy volunteers (columns 1 to 6) as described. Whole blood: Immediately after sample collection, 0.6 ml whole blood was mixed with 6 ml Catrimox-14 ™. The sample was then centrifuged at 12000 g for 5 minutes. The resulting nucleic acid pellet was carefully washed with water and dissolved in 1 ml of Tripure ™. RNA extraction was then performed according to the manufacturer's instructions for Tripure ™. PBMC: Cells were prepared from 15 ml heparinized venous blood according to standard procedures and lysed in 1 ml Tripure ™ for RNA extraction. As described (Stordeur et al., (1995), Prader et al., (1996)) RT-PCR of IFN-γ, IL-10 and the housekeeping gene HPRT in all samples from 1 μg total RNA. went. FIG. 32.2 shows real-time PCR results regarding IFN-γ and IL-10 mRNA stability in whole blood. A sample of citrate venous blood was collected from a healthy donor. Immediately after blood collection and every hour up to 5 hours, 100 μl aliquots from this sample were mixed with 900 μl Catrimox-14 ™. Blood samples were simply maintained at room temperature during each aliquot collection. The resulting nucleic acid pellet (see legend to FIG. 13.1) was dissolved in 300 μl of lysis buffer from “MagNA Pure LC mRNA Isolation Kit I” (Roche Diagnostics, Molecular Biochemicals). MRNA was extracted using MagNA Pure LC Instrument (Roche Diagnostics, Molecular Biochemicals) according to the manufacturer's instructions (final elution volume: 100 μl). Starting with 5 μl of mRNA preparation, reverse transcription and real-time PCR were performed in one step according to standard procedures described in “Lightcycler-RNA Master Hybridization Probes Kit” (Roche Diagnostics, Molecular Biochemicals). Primer and probe sequences and PCR conditions are described in Stordeur et al, J Immunol Methods, 259 (1-2): 55-64, 2002). The vertical axis represents the number of mRNA copies per 1 × 10 7 copies of 1β-actin, and the horizontal axis represents the time after blood collection. FIG. 34 shows a schematic comparison of RNA extraction methods from whole blood suggested by PreAnalyticiX compared to the method proposed by the present invention. Blood sampling in PAXgene Tube = 2.5 ml blood + 6.9 ml stabilizer, from which we have 1) 4.7 ml (contains 1.25 ml blood) or 2) for Qiagen extraction 0.4 ml (containing 0.11 ml of blood) was taken for MagNA Pure extraction. After centrifugation, the nucleic acid pellets were as follows 1) and 2): 1) PAXgene + Qiagen kit: Washed with water and dissolved in buffer BRI of PAXgene blood RNA Kit (Qiagen). Total RNA extraction was performed as described in the PAXgene blood RNA Kit handbook. ↓ Total RNA concentration measured by optical density. 500 ng (various volumes depending on concentration) was used for reverse transcription. This reverse transcription and real-time PCR was performed as described (Stordeur et al., J Immunol Methods, 259 (1-2): 55-64, 2002). Results: Recommended procedure, see Table 3.1 2) PAXgene + MagNA Pure ... dissolved in lysis buffer contained in MagNA Pure mRNA isolation kit. Extraction was performed with MagNA Pure instrument as recommended by Roche. ↓ It is not necessary to measure mRNA concentration. 5 μl was used for reverse transcription and real-time PCR performed in one step using the LC RNA Master hybridization kit (Roche). Real-time PCR conditions were as described in (Stordeur et al., J Immunol Methods, 259 (1-2): 55-64, 2002): Proposed procedure, see Table 3.2 FIG. 34 shows ex vivo induction of cytokine blood mRNA by tetanus toxoid. Tetanus toxoid (10 μg / ml, Aventis) was added to 500 μl of whole blood collected from healthy volunteers vaccinated against tetanus 7 years ago. After various periods at 37 ° C. in a 5% CO ₂ atmosphere, 1.4 ml of the reagent contained in the PAXgene tube was added. 300 μl of the resulting lysate was used to isolate total mRNA with MagNA Pure instrument and RT-PCR was performed as described in the present invention. In the figure, the vertical axis represents the number of mRNA copies per million actin mRNA copies, and the horizontal axis represents time. FIG. 36 is a diagram showing the mRNA dynamics of IL-1β and IL-1RA after stimulation of whole blood with LPS. 200 μl of heparinized blood was incubated with 10 ng / ml LPS for 0 hours (at the start of culture), 0.5 hours, 1 hour, 2 hours and 6 hours. At the end of the culture, 500 μl of PAXgene ™ tube reagent was added for whole cell lysis and nucleic acid precipitation. Then RT and real-time PCR of IL-1β, IL-1RA and β-actin mRNA was performed in one step as described in the present invention. The results are expressed as the number of mRNA copies per million of β-actin mRNA copies. The mean of 5 independent experiments and the standard error of the mean are shown. In the figure, the horizontal axis indicates the time after addition of LPS. FIG. 36 shows linear regression: mRNA copy number at the starting blood volume. Various whole blood volumes (X axis, in the range of 20 μl to 200 μl) were cultured for 6 hours in the presence of 10 ng / ml LPS. At the end of the culture, RT and real-time PCR of IL-1β and β-actin mRNA was performed as described in the present invention. The Y axis represents the raw copy number. The line is due to linear regression. One of six representative experiments is shown. In the figure, the horizontal axis indicates blood volume. FIG. 37 shows mRNA cytokine dynamics after whole blood stimulation with tetanus toxoid. Heparinized blood was collected from 5 healthy volunteers who had been vaccinated against tetanus at least 5 years ago. In each donor, 200 μl whole blood aliquots were incubated with 10 μg / ml tetanus toxoid for 0 hours (at the start of culture), 4 hours, 8 hours, 16 hours, 24 hours and 48 hours. At the end of the culture, 500 μl of reagent contained in PAXgene ™ tube was added and various transcripts were quantified using the method of the present invention. The results are expressed as the number of mRNA copies per million of β-actin mRNA copies. The mean of 5 independent experiments and the standard error of the mean are shown. FIG. 39 shows in vivo regulation of blood cytokine mRNA after intravenous injection of LPS. Five healthy volunteers were injected with a single dose of 4 ng / kg LPS. Ten minutes before LPS injection, 0.5 hours, 1 hour, 1.5 hours, 2 hours, 3 hours and 6 hours, 2.5 ml blood samples were collected in PAXgene ™ tubes. . Cytokine mRNA was quantified according to the method of the present invention. The results are expressed as the number of mRNA copies per million of β-actin mRNA copies. The mean at each time point and the standard error of the mean are shown. In the figure, the horizontal axis indicates the time after LPS injection. FIG. 40 shows FIG. 39 and shows follow-up of anti-tetanus vaccine response. Six healthy volunteers were selected for anti-tetanus recall. IL-2 mRNA levels were quantified from whole blood cultured for 20 hours with (full circles) or without (open circles) with 10 μg / ml tetanus toxoid and at the time of recall (0 Day), 14 days before, 3 days, 7 days, 14 days, 21 days and 90 days (X-axis) before recall. The results are expressed as the number of mRNA copies per 1 million mRNA copies of β-actin (Y axis). Each of the 6 panels (numbered 1-6) represents individual data from 6 different donors (one donor per panel). In the figure, the horizontal axis indicates the number of days before / after vaccine administration. FIG. 41 shows the outline of the procedure according to Example 7, Example 8, Example 9, Example 10 and Example 11 for analyzing blood cytokine mRNA expression shown in FIG. Incubation of whole blood samples in the presence of potential immune modulators to be tested (antigens, allergens, cells, xenobiotics ...) Blood cell lysis and mRNA stability using reagents contained in PAXgene tubes -Automated mRNA extraction for one-step RT-PCR using Magna Pure instrument (Roche Diagnostics) and preparation of reaction mixture-Cytokine mRNA levels by real-time PCR using Lightcycler instrument (Roche Diagnostics) Quantification FIG. 42 is a diagram showing a direct correlation between the amount of starting biomaterial and the actual copy number, showing FIG. 41 and automatic mRNA extraction and reagent mixture preparation with MagNA Pure. The Y axis represents the raw copy number. The line is due to linear regression. In the figure, the vertical axis represents the mRNA copy number, and the horizontal axis represents the PBMC number. FIG. 43 shows automatic mRNA extraction and reagent mixture preparation with MagNA Pure: direct correlation between the amount of starting biomaterial and the actual copy number. The Y axis represents the raw copy number. The line is due to linear regression. It is a figure which shows FIG. 43 and shows the summary of the case report of the patient registered into cancer immunotherapy. Diagnosed as melanoma in July 1999. In August 2001, multiple metastases were revealed and immediately after the orchydectomy in April 2002, patients were enrolled to receive a cancer vaccine. Vaccination consisted of several injections of MAGE-3 purified protein (antigen specifically expressed by melanoma cells) in combination with adjuvant. * July 1999: Melanoma of the right scapula. Resection * August 2001: SC M + Arm, back, abdomen, right testis * April 2002: Right testicle removal * April 2002: Pre-vaccination check * May 2002: Vaccine program started FIG. 44 shows a schematic diagram of vaccination protocol and monitoring of immune response by real-time PCR. Whole blood was stimulated in vitro and the immune response to MAGE-3 was evaluated. The patient received 3 vaccinations and a blood sample was taken once a week for 9 weeks. An aliquot of 200 μl of each patient's whole blood sample was incubated in the presence of 10 μg / ml MAGE-3 protein or 10 μg / ml TRAP (Plasmodium falciparum antigen) as a negative control. At the end of the culture, reagents contained in the PAXgene tube were added and IL-2 mRNA was quantified as described in Example 6. The results are shown in FIG. * Patient number 3 received 3 injections of anti-MAGE-3 vaccine (MAGE-3 + adjuvant) ↓ * Heparinized blood once a week for 9 weeks ↓ * 200 μl of MAGE-3 or TRAP antigen Overnight incubation of whole blood ↓ * mRNA extraction and RT-PCR using Magna Pure and Lightcycler FIG. 45 shows IL-2 mRNA in whole blood after MAGE-3 vaccination in patient number 3. Following boost of MAGE-3 vaccine, higher IL-2 mRNA levels are seen in MAGE-3-stimulated whole blood. The Y-axis represents the number of IL-2 mRNA copies per million β-actin mRNA copies, and the X-axis represents the week of blood sample collection. Vaccine injection was administered at 0, 2 and 6 weeks. The black solid columns are for whole blood incubated in the presence of MAGE-3, and the hatched columns are for whole blood incubated in the presence of TRAP. FIG. 46 shows in vitro stimulation of whole blood: evaluation of immune response to allergen. Experiments were performed on IL-4 mRNA quantification after whole blood incubation with allergens. Blood samples were collected from a cat allergic subject and two healthy subjects. The whole blood is then incubated at various incubation times in the absence or presence of cat allergen (ie Feld1), at the end of which the reagents contained in the PAXgene tube are added and IL as described in Example 6 -4 mRNA was quantified. The results are shown in FIG. * Cat allergic or non-allergic control ↓ * Heparinized blood ↓ * + Feline allergen (Feld1) ↓ * After various culture periods, + PAXgene tube reagents ↓ * mRNA extraction using MagNA Pure and Lightcycler RT-PCR FIG. 47 shows in vitro stimulation of whole blood: evaluation of immune response to Feld1. The Feld1 allergen significantly induces higher IL-4 mRNA levels in whole blood from subjects allergic to cats compared to non-allergic subjects. The Y-axis represents the number of IL-4 mRNA copies per million β-actin mRNA copies, and the X-axis represents various incubation times. The green bar graph represents IL-4 mRNA levels found in normal whole blood incubated with allergens, the red bar graph (blood incubated in the presence of Feld1) and the yellow bar graph (blood incubated without Feld1) ) Represents IL-4 mRNA levels found in whole blood of allergic subjects. Non-allergenic + allergen (1), non-allergenic + allergen (2), allergenic + allergen, allergenic, non-allergen. In vitro stimulation of whole blood: dose response to Feld1 FIG. 48 shows that the immune response to Feld1 in this whole blood system is specific and dose dependent. Whole blood from allergic subjects for 2 hours, 1) in the presence of increasing concentrations of Feld1 (shaded bar), 2) in the presence of 10 μg / ml of another allergen, β-lactoglobulin (BLG) (Horizontal broken line bar graph) 3) Incubated in the presence of cross-linked IgE (dotted line bar graph). The Y-axis represents the number of IL-4 mRNA copies per million copies of β-actin mRNA. IL-4 mRNA levels after whole blood stimulation with Feld1 are higher in allergic patients compared to healthy controls FIG. 49 shows a control with healthy IL-4 mRNA levels after whole blood stimulation with Feld1 It is a figure which shows that it is higher in the patient who is allergic to a cat compared with. The experiments described in slides 9 to 11 were repeated with blood samples from 10 healthy subjects (CTR bar graph) and patients allergic to 10 cats (ALL bar graph). Whole blood samples were incubated for 2 hours in the presence of 10 μg Feld1 or in the presence of cross-linked IgE as a positive control. Mean and standard error of the mean are shown. In the figure, the vertical axis shows the relative mRNA copy number of IL-4. Healthy controls and cat allergic patients are shown. FIG. 50 shows in vitro stimulation of whole blood: evaluation of T cell response to GAD65. FIG. 2 is a schematic of an experiment performed for IL-2 mRNA quantification after whole blood incubation with purified GAD65 protein. Blood samples were taken from 6 type 1 diabetic patients and from 5 healthy subjects. The whole blood was then incubated for 18 hours with or without 10 μg / ml GAD65, and then the culture was stopped by adding the reagents contained in PAXgene tube. IL-2 mRNA levels were then quantified as described in Example 6. The results are shown in FIG. * Type 1 diabetic or healthy control ↓ * Heparinized blood ↓ * 18 hours, + GAD65 ↓ * + PAXgene tube reagent ↓ * mRNA extraction using MagNA Pure and Lightcycler and RT-PCR for IL-2 mRNA FIG. 51 is a diagram showing in vitro stimulation of whole blood: evaluation of T cell response to GAD65. Whole blood from type 1 diabetic patients shows higher IL-2 mRNA levels after GAD65 stimulation compared to healthy subjects. The results are expressed as IL-2 mRNA copy numbers calculated relative to the copy numbers found in whole blood cultured without GAD65 after correction for β-actin. Use a log scale. Mean and standard error of the mean are shown. Healthy donor: CTR bar graph, autoimmune diabetic patient: PAT bar graph. In the figure, the vertical axis represents the relative copy number (log scale) of IL-2 mRNA. FIG. 52 shows alloreactive immune response monitoring: quantification of IL-2 mRNA in whole blood + dendritic cell line. Experiments were performed for mRNA quantification of IL-2 after whole blood incubation with unrelated dendritic cells (DCs) to assess alloreactive T cell responses. Dendritic cells from two unrelated healthy volunteers (MT and MA) were generated in vitro in the presence of IL-4 and GM-CSF. Whole blood samples from each donor were cultured in the presence of other donor dendritic cell populations (1) or in the presence of donor's own dendritic cells (2). Whole blood samples (3) from both donors and both dendritic cell populations (4) were mixed. After 12 hours of incubation, the culture was stopped by adding the reagents contained in the PAXgene tube. IL-2 mRNA levels were then quantified as described in Example 6. The results are shown in FIG. English indicates heparinized blood, dendritic cells, healthy volunteers, 12 hour incubation, and the like. FIG. 53 shows alloreactive immune response monitoring: quantification of IL-2 mRNA in whole blood + dendritic cell line. Evaluation of alloreactive T cell response by quantification of IL-2 mRNA in whole blood. The number of IL-2 mRNA copies per million of β-actin mRNA copies is shown. Conditions from left to right: whole blood from donor MA alone, whole blood from donor MA + DC from donor MA, whole blood from donor MA + DC from donor MT, from donor MT Whole blood alone, donor MT-derived whole blood + donor MT-derived DC, donor MT-derived whole blood + donor MA-derived DC, donor MT-derived whole blood + donor MA-derived whole blood, donor MT-derived DC + donor DC from MA. In the figure, the vertical axis represents the number of IL-2 mRNA copies per million of β-actin mRNA copies.

Example 1 Analysis of Natural Production of Cytokine mRNA in Peripheral Blood Quantification of cytokine mRNA synthesized by peripheral blood cells makes it possible to estimate “peripheral statute”. However, accurate quantification can only be performed from fresh whole blood samples where the mRNA is protected against nuclease digestion and gene transcription is inhibited. As described in this note, the use of a surfactant reagent such as tetradecyltrimethylammonium oxalate enables accurate quantification. RT-PCR was performed for quantification of IL-10 and IFN-γ mRNAs naturally produced in peripheral blood. The results showed significantly higher IFN-γ transcript levels in whole blood compared to peripheral blood mononuclear cells (PBMC) from the same individual, but no significant differences were observed in IL-10 mRNA. . The higher amount of IFN-γ mRNA observed in blood may be due to at least mRNA degradation. Using real-time PCR techniques, it can be demonstrated that indeed blood IFN-γ mRNA is rapidly degraded in vitro, with t1 / 2 corresponding to approximately 1 hour at room temperature.

  Recently, Hartelet al. Analyzed the effect of cell purification procedures on the spontaneous production of cytokine mRNA in peripheral blood (Hartel etal., 2001). Hartel et al. Found that freshly isolated peripheral blood mononuclear cells (PBMC) had higher levels of IL-2, IL-4 and TNF-α mRNA than whole blood freshly recovered from the same individual. However, no difference was observed at the mRNA level of IFN-γ. A comparison of IFN-γ in 6 different individuals was performed and different results were found. Strong expression of IFN-γ mRNA was observed in whole blood from all donors, but this expression is clearly reduced in PBMC (FIG. 32.1). Despite the fact that Hartel et al.'S experiment used quantitative real-time PCR, this difference between the results obtained and the results of the Hartelet al. May relate to the procedure used. Hartelet al. Used heparinized blood that had been hemolyzed within 2 hours by isotonic ammonium chloride treatment. The method of the present invention avoids the use of anticoagulants and is a cationic surfactant tetradecyltrimethyl oxalate called Catrimox-14 ™ (Qiagen, Westburg, Leusden, The Netherlands) that mixes directly with blood. Ammonium was used (Dahle and Macfarlane, (1993), Schmidt et al., (1995)). In addition, this reagent induces nucleic acid precipitation and nuclease inhibition immediately after sample collection. This gives a total RNA preparation that is considered to be closest to the mRNA state in vivo. This is particularly important for cytokine mRNAs that are sensitive to endogenous nucleases by high AU sequences located in the 3 'untranslated region. Using real-time PCR, it has actually been observed that IFN-γ mRNA in actual peripheral blood is naturally and rapidly degraded, and that the level is already reduced by approximately 50% after 1 hour of blood collection. However, this phenomenon may not be true for all cytokines since IL-10 mRNA levels were found to be stable for at least 5 hours after blood sampling (FIG. 32.2). Furthermore, no significant difference was found in IL-10 mRNA levels in whole blood compared to IL-10 mRNA levels in PBMC (FIG. 32.1).

  Nucleic acid pellets obtained after lysis of Catrimox-14 ™ (see legend to Fig. 32.1) were prepared from the guanidinium / thiocyanate solution described in Chomczynski and Sacchi (1987), and commercial versions thereof such as Tripure ™ ) (Roche Diagnostics, Molecular Biochemicals, Brussels, Belgium), which makes the use of this surfactant particularly easy. This means that the RNA isolation procedure is the same for whole blood and cells except for the first step with Catrimox-14 ™. Alternatively, PAXgene ™ Blood RNA Tube (Qiagen, Westburg, Leusden, The Netherlands) can be used in place of Catrimox-14 ™. In this case, the resulting pellet can be dissolved in the lysis buffer of “MagNA Pure LC mRNA Isolation Kit I” as described for Catrimox-14 ™ in the legend to FIG. 32.2. Characterization of spontaneous production of IL-10 mRNA by human mononuclear blood cells (Stordeur et al., (1995)) and monitoring of tissue factor mRNA induction in vivo by OKT3 monoclonal antibody (Pradier et al., ( 1996)) are two examples of successful use of Catrimox-14. Strong IL-2 mRNA induction was also observed after addition of ionophore A23187 + phorbol myristate acetate to whole blood (data not shown), and it was proposed to use it in in vitro studies on whole blood.

  The observations made in this example highlight the importance of performing RT-PCR from whole blood that has been lysed as quickly as possible to accurately quantify peripheral blood cytokine mRNA. To this end, the use of reagents such as additives contained in Catrimox-14 or PAXgene ™ Blood RNA Tube along with real-time RT-PCR appears to represent the best procedure to date. This allows for the study of the natural state of peripheral blood cells without the use of strong in vitro stimuli such as ionomycin or phytohemagglutinin.

Example 2: Comparison of PAXgene (TM) Blood RNA System with the proposed method according to the present invention "PAXgene (TM) Blood RNA System" means "PAXgene (TM) Blood RNA Tube" and "PAXgene (TM) Blood" It means a combination with “RNA Kit”. “Qiagen method” means “PAXgene ™ Blood RNA Kit”.

  Based on the experimental evidence described in Stordeur et al, J Immunol Methods, 259 (1-2): 55-64, 2002, the present invention proposes a new procedure for isolating mRNA from whole blood. In vivo transcript levels can be determined using an easy and reproducible method. The PAXgene ™ blood RNA System and the method according to the invention are schematically compared in FIG.

Materials and methods:
All experiments were performed with peripheral venous blood collected directly on the PAXgene ™ Blood RNA Tube as recommended by the PAXgene ™ Blood RNA System (Qiagen) (ie, 6.9 ml of 2.5 ml of blood). Collected in vacuum in a tube containing unknown reagent). After lysis was complete, the contents of the tube were transferred to two other tubes: 4.7 ml was used for the PAXgene blood RNA kit and 0.4 ml was used for MagNA Pure extraction. The remainder of the lysate was discarded. These two tubes were centrifuged at 2000 g for 10 minutes and the supernatant was discarded. Then the nucleic acid pellet
a) (PAXgene ™ Blood RNA Tube + PAXgene ™ Blood RNA Kit) Washed with water prior to dissolution in BR1 buffer for total RNA extraction as recommended in the corresponding instruction manual. The procedure for the PAXgene ™ Blood RNA System is as follows: A blood sample (2.5 ml) may be collected in a PAXgene Blood RNA Tube and stored or moved at room temperature as desired. RNA isolation began with a centrifugation step and the nucleic acids were pelleted in a PAXgene Blood RNA Tube. Protein digestion occurs by washing the pellet and adding proteinase K. Binding conditions were adjusted by adding alcohol, and the sample was applied to a spin column as provided by PAXgene ™ Blood RNA Kit. During a brief centrifugation, the RNA selectively binds to the silica gel membrane as provided by the PAXgene ™ Blood RNA Kit and contaminants pass through. After the washing step, the RNA is eluted in an optimized buffer. Reverse transcription and real-time PCR of IFN-γ and β-actin mRNA were performed as described by Stordeur et al. ("CytokinemRNA Quantification by Real Time PCR") J Immunol Methods, 259 (1-2) : 55-64, 2002).
b) (PAXaene ™ Blood RNA Tube ++++ MagNA Pure LC mRNA Isolation Kit I) MagNA Pure mRNA Isolation Kit was dissolved in 300 μl of lysis buffer. Then, extraction and purification of mRNA with a final elution volume of 100 μl were performed with MagNA Pure LC Instrument according to the instruction manual of Roche Diagnostics, Molecular Biochemicals. Reverse transcription and real-time PCR were performed in one step, starting with a 5 μl mRNA preparation, following the standard procedure described in “Lightcycler-RNA Master Hybridization Probes Kit” (Roche Diagnostics, Molecular Biochemicals).

result:
Extraction method recommended by Qiagen in combination with PAXgene ™ Blood RNA Tube (PAXgene ™ Blood RNA System), as well as comparison of MagNA Pure LC Instrument extraction method in combination with PAXgene ™ Blood RNA Tube went. In both methods, the use of PAXgene ™ blood RNA Tube can stabilize RNA from blood cells. The results are listed in Table 1.1 and Table 1.2. The results of this experiment show better reproducibility with MagNA Pure LC Technique (variation coefficient of IFN-γ mRNA copy number corrected for β-actin is 16% for MagNA Pure LC vs. Qiagen, respectively). 26%).

  Interestingly, note that MagNA Pure extraction was performed from a lower starting blood volume than that used in the Qiagen method (0.11 ml for MagNA Pure versus 1.25 ml for Qiagen). When the Qiagen method is performed with such a small volume, it becomes impossible to measure the RNA concentration and to perform reverse transcription. This highlights another advantage of the technique described in the present invention: mRNA can be quantified with a very small volume of blood (about 100 μl).

Conclusion:
Example 2 can use PAXgene ™ Blood RNA Tube in combination with MagNA Pure LC mRNA Isolation Kit I, or more precisely, PAXgene ™ Blood RNA Tube in the lysis buffer contained in this kit. This lysis buffer needs to be used with the other components of the kit.

  In this example, in contrast to other combinations, it has been demonstrated that only combinations as described in the present invention provide accurate / true in vivo transcript quantification.

Example 3: Ex vivo monitoring of immune response to tetanus toxoid In Example 3, exogenous blood is ex- Stimulate in vivo. RT-PCR is performed according to the method (Figure 31.1). Cytokine mRNA is measured as a read out of the ability to react to antigens of Borandia's immune system. Although IL-2, IL-4, IL-13 and IFN-γ mRNA are selectively analyzed, quantification of their corresponding mRNAs allows all potential reactive proteins to be analyzed. The results of Example 3 are shown in FIG. In general, the strategy followed in this example can be schematically represented as shown in FIG. 31.2.

Potential application: cancer immunotherapy For several years, basic strategies for cancer immunotherapy have been developed by vaccination. Indeed, advances in genetics and immunology have made it possible to identify many growing tumor antigens that are expressed on the surface of tumor cells. These antigens are presented on the surface of tumor cells in the form of peptides related to the major histocompatibility complex (HLA). Examples of antigens that can be considered tumor antigens are described in Fong and Engleman (Annu. Rev. Immunol. 2000.18: 245-273). The principle of anti-cancer vaccination consists of presenting these antigens to the patient's immune system according to the most immunogenic way immunogenic. This shifts from the injection of the antigen or corresponding peptide in the presence of additives to the presentation of the peptide on autoantigen presenting cells (eg dendritic cells). The ultimate goal of vaccination anti-cancer vaccination is tumor regression, but especially in patients with advanced disease who can only benefit from limited window of treatment The effectiveness of anti-cancer vaccination remains difficult in some cases. This is why anti-cancer vaccination can be particularly interesting as an adjuvant therapy or in a prevention framework. Therefore, experimental anti-cancer vaccines to identify how to administer these vaccines and to discover implied biological mechanisms that help to better define future treatment protocols It is very important to develop a sensitive and accurate monitoring technique to assess the immunological effects of inoculation. The difficulty in measuring the immunological efficacy of these vaccines is inherently in the absence of assays that are sensitive enough to detect cellular immune responses in vivo. Techniques that have been used to date have included thorough in vitro culture of patient's PBMC in the presence of antigen for long periods of time, and costimulation that tends to induce changes in the original functional properties of lymphocytes. This gives reversibility of their functional state after extensive in vitro incubation in the presence of antigen, so analysis of the anergy state or resistance state of lymphocyte precursors directed against tumor antigens is very Have difficulty. On the other hand, tetramer-based techniques of MHC-peptide complexes used to detect low frequency epitope-specific CTL precursors usually lack sensitivity to detection of tumor-specific lymphocytes. Furthermore, these techniques do not give any information regarding the functional reactivity of these lymphocytes.

  Only techniques that are sensitive enough to detect the original functional reactivity of lymphocytes to a given antigen after a very short period of stimulation in vitro with the antigen, for example, are anti-cancer vaccination protocols Allows for an actual assessment of the effectiveness of

  Recently (Kammula, US, Marincola, FM, and Rosenberg, SA (2000) “Real-time quantitative polymerase chain reaction assessment of immune reactivity in melanoma patients after tumor peptide vaccination. J. Natl. Cancer Inst. 92: 1336-44), detection of cytokine mRNA associated with short-term in vitro stimulation of PBMC (2 hours) using a tumor antigen. It has been shown that it was possible to detect specifiq CTL in the PBMCs of patients receiving vaccination. However, according to the present invention, this short ex vivo pulse is not essential.

Example 4: Detection of Recipient Immune System Activation by Donor Histocompatibility Antigen In Example 4, donor-derived organs (eg, liver, kidney, bone marrow, etc.) are transplanted into the recipient. Recipient whole blood is collected in a tube containing a compound that inhibits RNA degradation and / or gene induction according to the present invention. RT-PCR is performed according to the method. Cytokine mRNA is measured as a readout of activation of the recipient's immune system by the donor histocompatibility antigen (Figure 31.3).

Example 5 Detection of Recipient's Immune System Reactivity to Donor's Histocompatibility Antigen In Example 5, donor-derived organs (eg, liver, kidney, bone marrow, etc.) are transplanted into the recipient. Recipient whole blood is collected into tubes and incubated ex vivo with donor histocompatibility antigens. A compound that inhibits RNA degradation and / or gene induction according to the present invention is added to the blood. RT-PCR is performed according to this method. Cytokine mRNA is measured as a readout of the recipient's immune system response by the donor histocompatibility antigen (Figure 31.4).

Applications: Monitoring rejection after organ transplantation Monitoring transplant rejection is essentially in the patient's urine or blood (in the case of kidney transplants, blood urea nitrogen (BIN) or creatinine), or the life of the transplanted organ This is based on the detection of markers measured during the analysis of the test. However, these indicators are only detected if the rejection mechanism is already well advanced. Indeed, transplant rejection is the result of an immunological mechanism that precedes the deterioration of the transplanted organ. By detecting these immunological mechanisms before the transplanted organ is damaged, it is possible to greatly reduce the loss of the transplanted organ by adapting the immunosuppressive treatment earlier. On the other hand, it has also been recognized that asymptomatic episodes of rejection (no clinical signs are elicited) themselves frequently occur after transplantation. These episodes sub-slinical rejection episodes can cause chronic rejection. Some authors have detected early immunologiques markers of organ rejection, especially alloreactive T lymphocytes directed against donor alloantigens in the recipient's circulation. Researching sphere detection. The method essentially involves a relationship between the mixed culture and the continuous measurement of the proliferation of the receiver lymphocytes by various methods (ELISA, ELISPOT, flow cytometry, etc.) or the measurement of cytokine production. In recent years, other researchers have tried to characterize lymphocyte activation marker patterns that are likely to show early induction of rejection mechanisms. Detection of mRNA of genes expressed by activated cytotoxic T lymphocytes that have been T-activated (granzyme B, perforin, various cytokines) by a highly sensitive quantitative PCR method is an excellent tool for measuring the induction of rejection. I know that there is. To this end, according to the present invention, messengers encoding various types of cytokines can be studied, selective targets being IL-2, IFN-γ, IL-4, IL-5, granzyme, perforin And FasFas-ligand.

Example 6: Immune monitoring in whole blood using real-time PCR In Example 6, a method in whole blood is described that allows measurement of induction of cytokine synthesis at the mRNA level. The originality of this method is the PAXgene ™ tube containing an mRNA stabilizer for blood collection and MagNA Pure ™ instrument as an automated system for mRNA extraction and RT-PCR reagent mixture preparation. , In combination with the Lightcycler ™ real-time PCR method for accurate and reproducible quantification of transcript levels. This example first measures the induction of IL (interleukin) -1β and IL-1 receptor antagonist (IL-1RA) mRNA when this method adds bacterial lipopolysaccharide (LPS) to whole blood. It is shown that it is appropriate. This example further shows that this approach is also suitable for detecting the production of mRNA encoding cytokines derived from T cells in whole blood incubated with tetanus toxoid as a model for in vitro immune response to recall antigens. . Finally, this example allows detection of IL-1β and IL-1RA induction after LPS injection in healthy bolandia and IL-2 induction upon recall immunization with tetanus vaccine. It has been shown that this method can be successfully used to assess inflammation and T cell responses in vivo.

Materials and Methods Blood collection for in vivo studies For accurate quantification of peripheral blood mRNA levels, 2.5 ml blood samples were collected in PAXgene ™ tubes for immediate cell lysis and nucleic acid precipitation. The mRNA was stable in this blood lysate for up to 5 days and the tubes were kept at room temperature until mRNA extraction.

In Vitro Whole Blood Culture In vitro whole blood LPS stimulation or tetanus toxoid rechallenge was performed with 200 μl heparinized whole blood and started at the latest 4 hours after blood collection. The culture was stopped by adding 500 μl of PAXgene ™ tube reagent, which induces total cell lysis and mRNA stabilization. This allowed the same mRNA extraction protocol to be used for both in vitro and in vivo studies.

mRNA extraction Blood lysate obtained in the PAXgene ™ tube or at the end of whole blood culture is mixed briefly and then 300 μl for centrifugation at maximum speed (12000 g to 16000 g depending on the instrument) for 5 minutes. An aliquot was transferred to a 1.5 ml Eppendorf tube. The supernatant was discarded and the nucleic acid pellet was completely lysed by vortexing in 300 μl lysis buffer contained in the MagNA Pure ™ mRNA extraction kit (Roche Applied Science). The mRNA was then extracted from 300 μl of this solution using this kit in MagNA Pure ™ instrument (Roche Applied Science) according to the manufacturer's instructions (“mRNA I cell” Roche protocol, final elution volume 100 μl). . Northern blot analysis has so far documented the quality of extracted mRNA (Roche Applied Science, unpublished data).

Real-time PCR and reagent mixture preparation Reverse transcription and real-time PCR were performed in one step according to standard procedures described in “Lightcycler ™ -RNA Master Hybridization Probes” Kit (Roche Applied Science). More precisely, 1) up to 20 μl of H ₂ O; 2) 7.5 μl of RNA Master Hybridization Probe 2.7 fold (RNA Master Hybridization Probe Kit (Roche Applied Science)); 3) 1.3 μl of 50 mM Mn ( OAc) ₂ ; 4) 1 μl, 2 μl or 3 μl 6 pmol / μl forward primer and reverse primer (final concentration 300 nM, 600 nM or 900 nM depending on the mRNA target; conditions specific for each mRNA target are listed in Table 2 Except for IL-2 and IL-4, which are fully described in Stordeur et al, J Immunol Methods, 259 (1-2): 55-64, 2002); 5) 1 μl of 4 pmol / μl TaqMan Probe (final concentration 200 nM); 6) 5 μl of purified mRNA or standard dilution RT-PCR was performed reactions in a final volume of 20μl containing. After a 20 minute incubation period at 61 ° C. to reverse transcribe the mRNA, followed by an initial denaturation step at 95 ° C. for 30 seconds, a temperature cycle was initiated. Each cycle consisted of 95 ° C., 0 seconds and 60 ° C., 20 seconds, and fluorescence was read at the end of this second step (F1 / F2 channel, no color correction). A total of 45 cycles were performed. All primers were selected to span intron sequences, so genomic DNA amplification was not possible.

  An RT-PCR reaction mixture containing all of the reagents, oligonucleotides and sample was prepared completely directly into the capillary used in the Lightcycler ™ by MagNA Pure ™ instrument. The top caps of these capillaries were closed and centrifuged before introduction into the Lightcycler ™ for one-step RT-PCR. In this way, sampling of all RT-PCR components is fully automated and manual sampling errors are avoided.

  The results were expressed as copy number normalized to β-actin mRNA (copy number of cytokine mRNA per million copies of β-actin mRNA). For each sample, the mRNA copy number was calculated by instrument software using the Ct value (“Arimetric Fit point analysis”) from the calibration curve. This standard curve was prepared by PCR performed from purified DNA serially diluted as described in Stordeur et al, J Immunol Methods, 259 (1-2): 55-64, 2002.

Experimental Endotoxemia Five healthy male volunteers (21-28 years old) who had not taken any drug for at least 10 days prior to the experiment were LPS (from E. coli, lot G; United States Pharmacopeial Convention, Rockville, MD; 4 ng / kg body weight) was received intravenously. 2.5 ml blood sample collected in PAXgene ™ tube 10 minutes before LPS injection and 0.5 hours, 1 hour, 1.5 hours, 2 hours, 3 hours and 6 hours did. For in vitro studies, 200 μl of heparinized whole blood collected from healthy individuals was collected in a 5% CO ₂ atmosphere at 37 ° C. for 0 hour (at the start of culture), 0.5 hour, 1 hour, 2 hours. And 6 hours of incubation with 10 ng / ml LPS (serotype 0128: derived from E. coli B12, Sigma-Aldrich, Bornem, Belgium).

Anti-tetanus recall vaccination Healthy bolandia (2 men, 4 women, 27-53 years old) who had been vaccinated at least 5 years ago with a previous tetanus toxoid vaccination was called intramuscular vaccine recall (Tevax, Smith Kline Beecham Biologicals, Rixensart, Belgium). Heparinized blood tubes were collected on the day of administration, 14 days before administration, 3 days, 7 days, 14 days, 21 days and 90 days after administration. Incubate 200 μl of blood with or without 10 μg / ml tetanus toxoid (donated by Dr. E. Trannoy, Aventis Pasteur, Lyon, France) at 37 ° C. for 20 hours in a 5% CO ₂ atmosphere. did.

Results Measurement of IL-1β and IL-1RA mRNA upon addition of bacterial LPS to whole blood As shown in FIG. 35, addition of LPS (10 ng / ml) to whole blood resulted in IL-1β and IL-1 Rapid induction of -1RA mRNA resulted. As is apparent even 30 to 60 minutes after addition of LPS, this induction resulted in a 47-fold and 22-fold increase in mRNA levels in IL-1β and IL-1RA, respectively, 6 hours after the addition. The curve pattern suggests a rapid and sustained increase in both cytokine mRNA levels. To assess the accuracy of the system for mRNA quantification, mRNA was quantified for β-actin and IL-1β from whole blood stimulated with various volumes of LPS ranging from 20 μl to 200 μl. As shown in FIG. 36, the actual mRNA copy number of both β-actin and IL-1β was directly correlated with the starting volume of blood.

In Vitro Response to Tetanus Toxoid Tetanus is a well-established recall antigen when all individuals are vaccinated in childhood to determine whether this method may be suitable for analysis of T cell responses Cytokine mRNA levels in whole blood cultures after addition of toxoid were quantified. Rapid and transient induction of IFN-γ, IL-2, IL-4 and IL-13 mRNA following incubation of whole blood with this antigen was found (FIG. 37). When comparing the amplitude of response to each cytokine, the induction of IL-2 mRNA appeared to be most prominent. Indeed, the overall increase in IL-2 mRNA copy number after 16 hours of incubation in the presence of toxoid was approximately 220-fold in the five independent experiments shown in FIG. 37, compared to the same experiment. The greatest increase in IL-4 and IFN-γ mRNA did not exceed 5-fold. Therefore, quantification of IL-2 mRNA appears to be the most sensitive parameter in this whole blood system for assessing T cell responses. The data given in Table 3 shows that the amplitude of response to tetanus toxoid in this study is probably highly variable depending on the last vaccine recall. In fact, induction of IL-2 mRNA was not observed after adding tetanus toxoid to neonatal cord blood, and only previously primed and non-naive T cells were used in this assay. It has been shown that it can respond (Table 3).

Induction of IL-1RA and IL-1β mRNA in whole blood after intravenous injection of LPS As a first application of the in vivo cytokine induction detection method, low dose (4 ng / kg) bacterial lipopolysaccharide was injected A series of blood samples from healthy bolandia were analyzed. A clear induction of both IL-1RA and IL-1β mRNA was observed (FIG. 38). The induction of IL-1β mRNA is rapid because IL-1β mRNA has already been detected 30 to 60 minutes after endotoxin administration, and the IL-1β mRNA level is 6 hours before injection. It was temporary because it returned to the value of. Although the reaction rate was delayed as compared with IL-1β mRNA, IL-1RA mRNA was also induced.

Detection of anti-tetanus toxoid immune response after recall vaccination In vitro experiments suggested that IL-2 mRNA was the most sensitive parameter for monitoring anti-tetanus toxoid response. Selected to analyze changes in T cell response to tetanus toxoid in whole blood upon recall vaccination in vivo. For this purpose, whole blood incubations were performed in the presence or absence of tetanus toxoid before and at several times after vaccine administration. As shown in FIG. 39, IL-2 mRNA production in whole blood exposed to antigen was significantly increased in all vaccinated individuals. IL-2 mRNA induction was already apparent 7 days after vaccination and reached a maximum level on day 14 or 21. Inter-individual variability appears to be related to differences in the basic state of anti-tetanus immunity (see also Table 3). The IL-2 response measured in whole blood after vaccination was specific for the immunized antigen, as IL-2 mRNA levels measured without in vitro restimulation did not change significantly (Table 3).

DISCUSSION Real-time PCR is so called because it can directly monitor the accumulation of amplicons during the PCR process using fluorescent molecules that bind to the PCR product. As a result, a fluorescence curve is created for each sample, and (c) DNA copy number of the sample can be determined by comparing the calibration standard with the obtained fluorescence curve. In order to increase specificity, the fluorescent molecule can be an oligonucleotide complementary to the sequence of the PCR product, located between the two primers. The novel methods as described in this application provide a method for sensitive and accurate quantification of nucleic acids in biological samples that was not possible using prior art methods. This application describes this method by quantifying cytokine mRNA from purified cells or tissues that are representative of the in vivo situation.

  One of the problems faced by using whole blood for RT-PCR analysis is cell lysis prior to RNA extraction. Due to the high amount of protein present in plasma and red blood cells, the majority of methods for isolating RNA from whole blood involve the purification of potential cellular sources of mRNA to be analyzed prior to RNA extraction, or the elimination of red blood cells. These intermediate steps may be related to mRNA degradation and / or gene induction and thus to changes in mRNA levels. In addition, a simple fact of collecting blood can cause some mRNA degradation. This is especially true for cytokine mRNAs that are sensitive to endogenous nucleases due to high AU sequences located in the 3 'untranslated region. In fact, it has been shown so far that the mRNA level of peripheral blood IFN-γ has already been reduced by about 50% after 1 hour of blood collection (Stordeur et al., (2002) J. Immunol Meth. 261: 195). . Quaternary amine interfaces such as tetradecyltrimethylammonium oxalate, a cationic surfactant called Catrimox-14 ™ (Qiagen, Westburg, Leusden, The Netherlands), which induces whole cell lysis and simultaneously nucleic acid precipitation This can be avoided by using an activator. According to this example, it is surprisingly observed that the nucleic acid precipitate obtained with PAXgene ™ tube can be dissolved in a guanidinium / thiocyanate solution. An example of such a solution is the lysis buffer provided by the mRNA isolation MagNA Pure ™ LC kit (Roche Applied Science). This takes advantage of the high reproducibility and accuracy of MagNA Pure ™ instrument with automated preparation of all the components of the PCR reaction mixture, allowing us to use PAXgene ™ tube and MagNA Pure ™ instrument. Urged to use together.

  Interestingly, the present method has been successfully applied to detect cytokine gene induction in whole blood upon endotoxin challenge in vivo, thereby using the present method to monitor systemic inflammatory responses. It has been demonstrated that it can be done. The temporal traits of the IL-1 response following in vivo challenge are in contrast to the sustained increase in IL-1 mRNA following in vitro addition of LPS to the blood. This may be related not only to the rapid clearance of LPS in vivo, but also to the redistribution of cytokine-producing cells in vivo, which is associated with the upregulation of adhesion molecules and chemokine receptors. Another possible application of this method in whole blood is vaccination, as suggested by the apparent induction of IL-2 mRNA observed after re-inoculation in vitro in tetanus toxoid vaccinated individuals. Monitoring of the time T cell response. This is particularly interesting for organizing large-scale vaccination studies where cell isolation can be difficult, especially in developing countries where several new vaccines are under evaluation. obtain. To further study the applicability of this method in vaccine testing, this method will soon be tested as a readout of the T cell response upon the first vaccination against hepatitis B.

  A direct correlation between the starting volume of blood and the mRNA copy number (FIG. 36) suggests that it is not absolutely necessary to measure mRNA concentration for the resulting expression using this method. ing. However, since a quantitative error may be caused by a small variation in the sample volume, it is preferable to correct the copy number measured by simultaneous measurement of a housekeeping gene such as β-actin. This is still not optimal as housekeeping gene expression may change under certain conditions of stimulation. Therefore, it is possible to add an external standard to the sample before mRNA extraction. If the cell source of the cytokine is well established, such as in the case of T cells for IL-2, the copy number of the cytokine gene encodes a gene that is specifically expressed in the corresponding cell type, for example, CD3 in the latter example It may be appropriate to correct by the number of copies. Similarly, international standardization of calibrators for cytokine mRNA quantification by real-time PCR should be developed to facilitate comparison of data generated at various laboratories. Cytokine mRNA measurement in whole blood is useful for monitoring the innate adaptive immune response required for evaluation of new vaccines and immunotherapy.

Example 7 Automated mRNA Extraction and Reagent Mixture Preparation with MagNA Pure: Direct Correlation Between Amount of Starting Biomaterial and Actual Copy Number The procedure followed in this example is summarized in FIG. To show the accuracy of the system, a linear regression of the mRNA copy number at the starting cell number was calculated (FIG. 41). mRNA was extracted from various peripheral blood mononuclear cell (PBMC) numbers (cells ranging from 100,000 to 600,000, X axis), and one-step RT-real-time PCR was performed on β-actin mRNA. As described in the section “Materials and Methods”. This experiment was performed with PBMC for β-actin and TNF-α mRNA (FIG. 42, Panel B and Panel D), and with whole blood for β-actin mRNA (FIG. 42, Panel A) and with CD4 ⁺ purified T cells ( FIG. 42, panel C) was repeated.

Example 8: Cancer immunotherapy The procedure followed in this example is summarized in FIG. The method was applied to monitoring immune responses induced by cancer vaccines. 43, 40 and 41 show the results obtained in this field using melanoma patients.

Example 9 Allergy The procedure followed in this example is summarized in FIG. The method was then applied to allergies. Responses induced by in vitro incubation of whole blood from allergic subjects with related allergens were analyzed by mRNA quantification of IL-4 using real-time PCR. 46, 47, 48 and 49 show the results obtained in this field.

Example 10: Autoimmunity The procedure followed in this example is summarized in FIG. The method was then applied to autoimmunity. IL-2 mRNA quantification using this whole blood system was applied to assess T cell responses to glutamate decarboxylase 65 (GAD65), where autoantigens are expressed in autoreactive T cells in type 1 autoimmune diabetes. It was a target. 40 and 41 show the results obtained in this field.

Example 11: Transplantation The procedure followed in this example is summarized in FIG. The method was then applied to transplantation. IL-2 mRNA quantification by real-time PCR after whole blood incubation with alloreactive non-T cells provides an alternative to conventional mixed lymphocyte reaction (MLR) monitoring of alloreactive T cell responses Given. 52 and 53 show the results obtained in this field.

  While the invention has been described in conjunction with the detailed description thereof, the above description is illustrative and is not intended to limit the scope of the invention as defined by the appended claims. I want you to understand. Other aspects, advantages, and modifications are within the scope of the following claims.

Table 1. Comparison of Qiagen method and MagNA Pure LC extraction method 1.1. Qiagen mRNA extraction method. Blood mRNA derived from the same blood sample was extracted nine times.

1.2. MagNA Pure LC (kit + instrument) mRNA extraction method. Blood mRNA derived from the same blood sample was extracted nine times.

Table 2. Oligonucleotides for (real-time) PCR1

1. For a complete description, see Stordeur et al, J Immunol Methods, 259 (1-2): 55-64, 2002.
2. F, R, and P represent a forward primer, a reverse primer, and a probe, respectively. Numbers indicate sequence positions from Genebank accession number X01586 for IL-2 and NM_000589 for IL-4.
3. Final concentration of forward primer (F) and reverse primer (R).
4). A standard curve was generated from serially diluted PCR products prepared by “conventional” PCR, with specific conditions as follows: denaturation at 95 ° C. for 20 seconds, annealing at 58 ° C. for 20 seconds and 72 Elongation at 45 ° C for 45 seconds, total 35 cycles. The final concentration of MgCl2 was 1.5 mM.

Table 3.

Claims (39)

An assay device (30) for a liquid biological sample, facilitating separate exposure of said sample to a first substance (2) and a control substance (35) and then to a nucleic acid stabilizer (5); The device is
A first compartment (32) in which the first substance (2) is present;
A second compartment (34) in which the control substance (35) is present;
A third compartment (36) in which the stabilizer (5) is present;
A support (52) for the biological sample tube;
An assay device.   The device according to claim 1, wherein one or more of the compartments (32, 34, 36) are sealed and comprise one or more regions suitable for puncturing with a hollow needle.   The device according to claim 2, wherein the region is a resealable septum (40, 42, 46).   Further comprising a support (54, 56) for removably connecting with the transfer tube (60), the transfer tube transferring liquid between any two compartments or between the compartment and the biological sample tube 4. The device according to any one of claims 1 to 3, comprising a flexible or rigid hollow tube (64) with hollow needles (62, 66) at both ends, suitable for.   The apparatus of claim 4, further comprising the transfer tube (60) as defined in claim 4.   One needle (62) of the transfer tube is connected in parallel with the hollow needle (80) of the pressure tube (88), and the pressure tube is a flexible hollow tube (82 with the needle (80) at one end. 6) and is suitable for applying a vacuum or pressure to the compartment or biological sample tube and forcing the liquid through the transfer tube.   The device according to any one of the preceding claims, wherein the first substance (2) is fixed to part or all of the inner surface of the first compartment (32).   8. Device according to any one of the preceding claims, wherein one or more of the compartments (32, 34, 36) comprises a vent.   Device according to any one of the preceding claims, wherein the first compartment (32) and / or the second compartment (34) is under negative pressure.   10. A first compartment (32) and / or a second compartment (34) comprising an indication for dispensing a known volume of stabilizer therein. Equipment.   The device according to any one of the preceding claims, wherein the first substance (5) comprises one or more immune system antigens.   The device of claim 11, wherein the immune system antigen is a vaccine component.   12. The device of claim 11, wherein the immune system antigen is an antigen that induces a hyperallergic reaction.   The immune system antigen is one selected from histocompatibility antigen, bacterial LPS, tetanus toxoid, cancer immunotherapy antigen, MAGE-3, cat allergen, Feld1, antigen-presenting cell from organ donor, autoantigen, GAD65 or 12. The apparatus of claim 11, wherein there are a plurality.   The device according to any one of claims 1 to 14, wherein the stabilizer (35) is an inhibitor of cellular RNA degradation and / or gene induction.   16. The device of claim 15, wherein the inhibitor of cellular RNA degradation and / or gene induction is that found in PAXgene ™ or Tempus ™ Blood RNA Tube. A kit for testing a liquid biological sample,
A container (1) suitable for receiving a liquid biological sample and exposing the sample to a first substance (2) and then to a nucleic acid stabilizer, the container being a) in the container (2) The first substance (1) present,
b) Container (4, 12, 16) in which said stabilizer (35) is present,
c) a connection between the interior of the container (2) and the interior of the container (4, 12, 16);
d) a physical barrier (25, 15, 18) that temporarily interrupts the connection;
A container containing,
A control container (37) suitable for receiving a liquid biological sample and exposing the sample to a control substance and then to a nucleic acid stabilizer, the control container being a) in the control container (37) Control substance (35),
b) Control containers (4 ′, 12 ′, 16 ′) in which the stabilizer (35 ′) is present,
c) a connection between the inside of the control container (37) and the inside of the control container (4 ′, 12 ′, 16 ′);
d) physical barriers (15 ′, 18 ′, 25 ′) that temporarily interrupt the connection;
A container containing,
Including kit.   18. Kit according to claim 17, wherein the first substance (2) is fixed to part or all of the inner surface of the container (1).   18. Kit according to claim 17, wherein the first substance (2) is immobilized on a solid support.   The kit according to any one of claims 17 to 19, wherein the first substance (1) is a liquid.   The kit according to any one of claims 17 to 19, wherein the first substance (1) is a solid.   The kit according to any one of claims 17 to 21, wherein the container (1) and / or the control container (37) comprises one or more regions suitable for puncture with a syringe needle.   The kit according to claim 22, wherein the region is a resealable partition.   The container (1) and / or the control container (37) includes a fitting suitable for receiving a syringe and delivering the contents therein to the interior of the container (1) or the control container (37). Item 24. The kit according to any one of Items 17 to 23.   25. Kit according to any one of claims 17 to 24, wherein the container (1) and / or the control container (37) comprises a fitting suitable for receiving a syringe needle.   26. The container of claim 17-25, wherein the container (1) and / or the control container (37) comprises a valve that minimizes gas / liquid flow from the container and allows liquid biological sample to flow to the container. The kit according to any one of the above.   27. Kit according to any one of claims 17 to 26, wherein the container (1) and / or the control container (37) comprises means capable of pushing out the exhaust gas.   28. Kit according to any one of claims 17 to 27, wherein the container (1) and / or the control container (37) are under negative pressure.   The physical barrier (15, 18, 25, 15 ', 18', 25 ') of item d) is opened by application of a physical force to the container or the control container. A kit according to claim 1.   30. Kit according to claim 29, wherein the force sends an opening means to the physical barrier (15, 18, 25, 15 ', 18', 25 ').   30. Kit according to claim 29, wherein the force irreversibly opens the physical barrier (15, 18, 25, 15 ', 18', 25 ').   32. Kit according to any one of claims 17 to 31, wherein the container (1) and / or the control container (37) comprises an indication for dispensing a known volume of stabilizer therein.   The kit according to any one of claims 17 to 32, wherein the first substance (2) comprises one or more immune system antigens.   The kit of claim 32, wherein the immune system antigen is a vaccine component.   33. The kit of claim 32, wherein the immune system antigen is an antigen that induces a hyperallergic reaction.   The immune system antigen is one selected from histocompatibility antigen, bacterial LPS, tetanus toxoid, cancer immunotherapy antigen, MAGE-3, cat allergen, Feld1, antigen-presenting cell from organ donor, autoantigen, GAD65 or 33. The kit of claim 32, wherein the kit is plural.   The kit according to any one of claims 17 to 36, wherein the stabilizer is an inhibitor of cellular RNA degradation and / or gene induction.   38. A kit according to any one of claims 17 to 37, wherein the inhibitor of cellular RNA degradation and / or gene induction is that found in PAXgene ™ or Tempus ™ Blood RNA Tube.   39. Kit according to any one of claims 17 to 38, wherein the exterior of the container (1) and the control container (37) are connected to form a single body.