Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
  • G01N33/6869—Interleukin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

• the present application relates to a new method for detecting or quantifying basophils and eosinophils in healthy or sick subjects, the methods for preparing the necessary reagents and their use, especially in the form of kits.
• Eosinophils have been shown to play a very important role in chronic allergic asthma. Eosinophils infiltrate the airways where they can be activated and degranulate. The release of granular contents, enzymes and basic proteins, causes damage to the bronchial epithelium. Some reports also mention dysfunctions of eosinophils.
• the bridging of specific IgE, present on basophils, by different allergens, such as pneumallergens, venoms, food proteins or drugs can lead to the release of mediators contained in granules or newly formed and capable of inducing anaphylactic shock.
• the methods for counting basophils and eosinophils are based on the coloring of the granules (manual counting) with, for example, ortho-toluidine blue for basophils and eosin for eosinophils, on their size and particle size. or on a differential immunophenotyping of their cell surface. These are cumbersome, time consuming, and unspecific methods. They have large uncertainties and significant statistical errors because of the small number of these cell types. On the other hand, most of these methods do not analyze the degree of activation of these cells. For all these reasons, it would be desirable to have available products and methods making it possible to detect and quantify more specifically eosinophils and basophils, activated or not.
• CDs Cluster Differentiation
• Leukocytes express on their surface proteins characterized in CDs (Cluster Differentiation) by groups of antibodies which recognize them. Expression of several CDs on the same cell can be used to characterize it. The same CD can be expressed on different cell types. This is why most of the flow cytometry methods use mixtures of antibodies recognizing several of these CDs as markers for basophils or eosinophils.
• the use of two mixtures of antibodies, anti-CD2, anti-CD14, anti-CD16 and anti-CD19 on the one hand and anti-CD32, anti-CD25, anti-lgG1 and anti-lgG4 from on the other hand can detect basophils using a flow cytofluorimeter.
• the anti-CD2 antibodies reveal the T cells
• the anti-CD19 antibodies make it possible to detect the B cells
• the anti-CD14 antibodies detect mainly the monocytes but also the granulocytes.
• CD16 a type III IgG receptor
• NK T cells
• CD32 a type II IgG receptor
• CD25 is an activation marker for T and B cells as well as an activation marker for macrophages.
• Basophils and eosinophils are minority populations of leukocytes. This is why most of the data on basophils and eosinophils were obtained from purified cells. For example, the basophils of subjects suffering from chronic myeloid leukemia could be studied after several stages of purification using monoclonal antibodies then lysis of the red blood cells by the complement. However, this process is impractical in routine because it is long and requires a large volume of blood.
• basophils use an anti-IgE antibody or a high affinity anti-receptor antibody for IgE.
• these methods do not allow a clear identification of basophils since small amounts of receptor with high affinity for IgE have been demonstrated on monocytes and eosinophils and B cells can carry IgE on their surface.
• the bridging of IgE or their receptors on the surface of basophils, by the antibodies used as probe, can lead to cellular activation modifying the properties of the membrane. Activation of basophils by an allergen has been shown to decrease the binding of an anti-IgE antibody.
• EP-A-0 811 691 offers antibodies of various types, monoclonal, humanized etc. directed against the ⁇ chain of the interleukin-5 receptor blocking the activity of this cytokine. It also proposes a method for detecting eosinophils. But the use of antibodies neutralizing the biological activity of IL-5 gives disappointing results, by flow cytometry analysis. The specific signal obtained on eosinophils, even when purified, does not differ from the signal obtained with a control antibody (background noise).
• the Applicant discovered that, surprisingly, basophils and eosinophils could, in the midst of other populations cell of human or animal blood, to be detected in isolation and jointly with the aid of certain antibodies specific for the alpha chain of the interleukin-5 receptor and which could also, if desired, count these same cells.
• the Applicant has shown by bioassay on TF1 cells, dependent on IL5 for their survival, that these selected antibodies do not inhibit the growth of these cells.
• the Applicant has also shown that the receptor linked to these antibodies immobilized on the solid phase is still capable of fixing the labeled IL5.
• the present application relates to a method for the detection or quantification of eosinophils and basophils, characterized in that it comprises bringing a sample possibly containing said eosinophils or basophils into contact with an anti-monoclonal antibody.
• -IL-5 receptor alpha chain which does not interfere with the binding of IL5 to its receptor and which does not inhibit the biological activity of IL-5 to detect and if desired carry out quantification of eosinophils and basophils.
• the invention therefore uses a monoclonal antibody anti-IL-5 receptor (alpha chain) and the expression of the alpha chain of the IL-5 receptor as a specific marker for eosinophils and basophils. It was observed that this labeling was specific for these blood cells and that none of the B, T, monocyte or neutrophil cells were labeled.
• the sample optionally containing said eosinophils or basophils may for example be a blood sample, in particular from a sick subject, preferably allergic or parasitic.
• the monoclonal antibody anti-IL-5 receptor is an antibody which does not interfere with the binding of IL5 to its receptor and which does not inhibit the biological activity of IL-5.
• the anti-IL-5 receptor monoclonal antibody is an antibody which does not interfere with IgE.
• IgE an antibody which does not prevent the binding of an allergen or of another anti-IgE antibody to these surface IgEs.
• the anti-IL-5 receptor monoclonal antibody is an antibody which does not interfere with the cellular activation of eosinophils or basophils.
• which does not interfere with the cellular activation of eosinophils or basophils is meant an antibody which by its binding to the cell surface does not induce or inhibit the appearance of a basophil or eosinophil surface activation marker.
• the detection and if desired the quantification of eosinophils and basophils uses a flow cytometer or optical scanning.
• Exclusion markers are therefore used to further improve the accuracy of the count by excluding erratic events.
• the subject of the present application is in particular a process above, characterized in that the other monoclonal antibodies are directed against the markers CD3, CD16 and CD19 which make it possible to exclude erratic events.
• the invention allows the specific detection and quantification of activated or non-activated basophils and eosinophils. It has recently been demonstrated that CD63, a lysosomal protein of the tetraspan family and initially described as a marker for activating platelets, is also present in the granules of basophils and neutrophils. The expression of CD63 on the cell surface is dependent on calcium. Upon activation of basophils, CD63 expressed on the surface can be recognized by a specific antibody. The intensity of the labeling is a function of the number of activated cells.
• the present application also relates to a process above, characterized in that the detection or quantification of the activated basophils is carried out by additionally bringing the sample into contact with one or more other directed monoclonal antibodies. against activation markers of basophils and in particular against the CD 63 antigen.
• the present application also relates to a process above characterized in that one proceeds to the detection or quantification of the activated eosinophils by further bringing the sample into contact with one or more other monoclonal antibodies directed against activation markers of eosinophils and in particular against the CD 69 antigen.
• the detection and counting of basophils and eosinophils separately is possible as soon as there is an additional means of discrimination such as
• the present application also relates to an anti-RIL-5 antibody characterized by:
• An advantageous embodiment of the invention consists in the use of a mixture of antibodies directed on the one hand against RIL-5, with or without "specific" markers of T and B lymphocytes, NK cells, monocytes and neutrophils and , if we are interested in activated eosinophils or basophils, on the other hand a marker for activation of basophils and eosinophils.
• the cell activation marker can be an antibody specific for a protein appearing on the surface of the cell membrane after activation or demonstration of an oxidative enzymatic activity. This allows the detection and, possibly, the quantification of cellular activation.
• kits for different applications said kits based on an anti-RIL-5 monoclonal antibody, preferably of mouse, rat or rabbit, or else genetically modified and firstly a kit for the detection or quantification of eosinophils or basophils containing - at least one anti-RIL-5 monoclonal antibody as defined above coupled to a first fluorochrome,
• the present application also relates to a kit above, characterized in that it also contains a specific substrate for the oxidative activity of eosinophils.
• the present application also relates to a kit for the detection or quantification of the oxidative activity of eosinophils or basophils containing
• the present application finally relates to a process, antibody or kit above characterized in that the monoclonal antibody anti-IL-5 receptor is an antibody of the lgG1 type whose corresponding hybridoma has been deposited in the Collection National Culture of Microorganisms (CNCM) under N ° I-
• the invention can be applied in the test, by clinical and pharmaceutical laboratories, of the response of basophils or eosinophils to different types of degranulating agents (allergens, chemical substances, parasites, etc.), in order to carry out a diagnosis or desensitization monitoring, or to highlight the ability of new molecules to modulate the degranulation of basophils or eosinophils and for the study of allergic, parasitic and leukemic pathologies.
• degranulating agents allergens, chemical substances, parasites, etc.
• Figure 1 is an illustration of a "dot plot" obtained with the anti-RIL-5 H17N monoclonal antibody (B) and an isotypic control (A) conjugated with phycoerythrin.
• the abscissas represent the intensity of fluorescence expressed on a logarithmic scale and the ordinates represent the lateral diffusion.
• the polygons marked R4 and R5 represent basophils and eosinophils respectively.
• Figures 2A and 2B illustrate a double labeling obtained with the anti-RIL-5 antibody conjugated to phycoerythrin for basophils and eosinophils, respectively.
• the abscissa represents the fluorescence emitted by fluorescein (FITC) and the ordinates the fluorescence emitted by phycoerythrin (PE).
• the left dot plot represents the double labeling between the anti-RIL-5-PE and an antibody specific for another marker coupled to FITC.
• the right dot plot represents the marking obtained with isotypic control and the anti-RIL-5-PE.
• the markers used are, from top to bottom, anti-CD16 (this marker is present on neutrophils, NK cells,
• FIG. 3 is an illustration of a double labeling obtained with an anti-RIL-5 antibody conjugated to phycoerythrin in combination with a mixture of antibodies CD3, CD19, CD16, all conjugated to phycoerythrin-Cyanine 5 (PECy ⁇ ) .
• the FL-1 channel corresponds to the fluorescence emitted by fluorescein
• the FL-2 channel corresponds to the fluorescence emitted by phycoerythrin
• the FL-3 channel corresponds to the fluorescence emitted by phycoerythrin Cyanine 5.
• Diagram A corresponds to the dispersion according to size and grain size. A distinction is made between the R1 region which corresponds to the lymphocytes, the R2 region which corresponds to the monocytes and the R3 region which corresponds to the granulocytes.
• Diagram B corresponds to the fluorescence of the anti-IL-5R-PE antibody as a function of lateral diffusion, a function of cell size.
• R4 corresponds to basophils and R5 corresponds to eosinophils.
• Diagram C represents the fluorescence of the mixture of anti-CD3, anti-CD16, anti-CD19 antibodies, as a function of the lateral diffusion.
• R6 corresponds to unlabeled eosinophils.
• Diagram D represents the fluorescence of the mixture of antiCD3, anti-CD16, anti-CD19 antibodies and the fluorescence of the anti-IL-5R-PE antibody. Eosinophils project into the R5 and R6 regions. Only the events present in R5 and R6 are taken into account in diagram D.
• FIG. 4 is an illustration of the correlation between the percentages given by a hematimeter and the method of the invention.
• the abscissas represent the percentage of eosinophils (A) and the percentage of basophils (B) obtained in flow cytometry and the ordinates represent the percentage of eosinophils (A) and the percentage of basophils (B) given by the hematimeter.
• the upper graph corresponds to the data obtained on eosinophils and the lower graph corresponds to the data obtained on basophils. Correlation coefficients are given inside each graph.
• FIG. 5 is a typical illustration of a result obtained after activation of basophils with an anti-IgE antibody.
• the “dot plot” 1 represents the size and the particle size of the blood cells
• the diagram 2 represents the labeling obtained with the anti-RIL-5-PE antibody (diagram similar to that presented in FIG. 1 )
• Diagram 3 represents the isolation of basophils and the cleansing of the population from other possible contaminants, such as lymphocytes and monocytes.
• Diagram 4 represents the basophils.
• FIG. 5B represents 4 “dot plots” of activated basophils. Each dot plot corresponds to stimulation of whole blood by decreasing concentrations of anti-IgE.
• the point cloud of each dot plot corresponds to the population, isolated according to the dot plot 5A-4.
• the abscissas represent the fluorescence emitted by fluorescein coupled with the anti-CD63 antibody, and the ordinates represent the fluorescence emitted by phycoerythrin coupled with the anti-RIL-5 antibody.
• Figure 6 is an illustration of the comparison between the percentages of CD63 + IL5R + cells and the percentage of histamine released.
• Each diagram represents a donor.
• the black symbols represent the% of CD63 + IL-5R + cells and the white symbols the percentage of histamine released.
• the abscissas represent the concentration of anti-IgE used by the stimulation of basophils expressed in ⁇ g / ml and the ordinates represent the percentage of basophils expressing CD63 or the percentage of histamine released.
• FIG. 7 is the illustration of the activation of eosinophils by a phorbol ester (PMA: phorbol myristate acetate).
• the abscissas represent the concentration of PMA added and the ordinates represent the percentage of eosinophils expressing the molecule CD69 or CD63.
• Each diagram represents a different donor.
• Anti-CD3, anti-CD16, anti-CD19, anti-CD49d, anti-CD63, anti-CD69 monoclonal antibodies conjugated to fluorescein N-isothiocyanate (FITC) or to phycoerythrin Cyanine 5 (PECy ⁇ ) and the anti-monoclonal antibody -lgE clone E124.2.8. are commercial products obtained from Immunotech, Marseille, France.
• the anti-RIL-5 antibody clone H17N, was obtained after immunization of mice with a recombinant soluble form of RIL-5, a protein produced from a RIL-5 - IL-2 fusion gene and expressed in cells. CHO, then fusion of the blastocytes with the X63 myeloma cells, according to the conventional technique described by Kôhler and Milstein (1975, Nature 256, 495). Antibody production was screened by ELISA using plates coated with either the RIL-5-IL-2 hybrid protein or with IL-2. Clones positive on plates coated with RIL-5-IL-2 and negative on plates coated with IL-2 were cloned by limiting dilution.
• the selection of the antibodies is carried out on TF-1 cells because of their ability to recognize RIL-5 in the presence and in the absence of IL-5, a criterion according to the present invention.
• the anti-RlL-5 monoclonal antibody called H17N
• H17N has a strong affinity for the antigen but does not inhibit the biological activity of IL-5. Consequently, despite the binding of IL-5 to its receptor, no inhibition is observed.
• the corresponding hybridoma was deposited in the National Collection of Culture of Microorganisms (CNCM) in Paris on September 3, 1998 under N ° I-2068.
• the anti-RIL-5 monoclonal antibody, called H17N is of the lgG1 type.
• the blood samples used for the correlation study were obtained from La Conception Hospital (Marseille, France).
• the blood formulation was carried out on tubes containing EDTA as an anticoagulant with an STKS-Coulter device, Miami, USA.
• the blood used for basophil activations was drawn from heparinized tubes. These samples were taken from people in the laboratory.
• RIL-5 The expression of RIL-5 in different cell populations, from whole blood, was analyzed using a double immunostaining, using on the one hand the anti-RIL-5 H17N antibody conjugated with phycoerythrin prepared in stage 3 of Example 1 and on the other hand specific markers of the cell populations studied.
• the protocol used is as follows: 100 ⁇ l of whole blood are incubated with 20 ⁇ l of anti-RIL-5 H17N-PE antibody and 20 ⁇ l of a second antibody specific for another marker. The sample is incubated for 15 min. at room temperature.
• the non-specific binding is controlled using an irrelevant antibody of the same isotype reference IM 0670 (Immunotech, Marseille). This allows the relevant device settings to be made. Cytometric analysis of cells labeled with different fluorochromes was performed on a FACScalibur device (Becton Dickinson, Mountain View, USA). For each sample, 100,000 events were analyzed.
• the anti-RIL-5 H17N antibody was conjugated to phycoerythrin (PE) in order to more easily characterize the cells which express the riL-5 receptor.
• PE phycoerythrin
• the immunostaining of whole blood with the antibody H17N-PE and a control antibody (irrelevant antibody of the same isotype reference IM 0670) is shown in FIG. 1 (A and B).
• FIG. 1A In the presence of the control antibody, an absence of labeling is observed (FIG. 1A).
• FIG. 1B In the presence of the anti-RIL-5-PE antibody, a shift in the fluorescence of certain cells is observed (FIG. 1B).
• the two clouds (windows R5 and R4) of cells labeled with the anti-RIL-5 antibody correspond to eosinophils and basophils.
• This observation was confirmed by several immunostaining using specific antibodies from different CD's (Clusters Differentiation).
• CD3 and CD16 CDs specific for lymphocytes
• CD14 specific for monocytes.
• the cells are positive for CD49d, a protein expressed by eosinophils and basophils, but also by lymphocytes, monocytes and thymocytes.
• EXAMPLE 3 Quantification of eosinophils and basophils.
• Basophils and eosinophils are the only cell types labeled with the anti-RIL-5 antibody according to the invention, but the cloud of basophils is very close to the cloud of cells corresponding to lymphocytes.
• Anti-RIL-5 antibody conjugated to PE was added to a mixture of anti-CD3, anti-CD16 and anti-CD19 antibodies conjugated to phycoerythrin Cyanine 5, which makes it possible to exclude erratic events. This combination makes it possible to obtain more exact values.
• the percentages of eosinophils and basophils obtained by the two methods were compared for 50 different samples. The results are shown in Figure 4.
• the release of histamine by the activated basophils was carried out on aliquots of 300 ⁇ l of blood, deposited in a tube and incubated in the presence of different concentrations of anti-IgE in order to establish a dose-response curve.
• the samples are gently mixed and incubated at 37 ° C for 15 minutes.
• Cellular activation is stopped by adding 300 ⁇ l of cold phosphate buffer containing 1 mM EDTA.
• the tubes are centrifuged at + 4 ° C for 3 minutes at 1200 revolutions per minute. The supernatant is collected to quantify the histamine.
• the cell pellet is resuspended in 300 ⁇ l of phosphate buffer, 1 mM EDTA and 100 ⁇ l of this cell suspension are analyzed after immunostaining.
• the quantification of the histamine released was carried out using a commercial radioimmunoassay (Reference 1659, Immunotech, Marseille, France), according to the manufacturer's instructions.
• Total histamine is determined after cell lysis by diluting 50 ⁇ l of blood in 950 ⁇ l of distilled water followed by two freeze-thaw cycles. The histamine released after cell activation is expressed as a percentage of total histamine.
• the expression of the activation marker CD69 was carried out on aliquots of 300 ⁇ l of blood, deposited in a tube and incubated in the presence of different concentrations of PMA (Phorbol Myristate Acetate) in order to establish a dose-response curve. The samples are mixed gently and incubated at 37 ° C for 6 hours. Cellular activation is stopped by centrifuging at + 4 ° C for 3 minutes at 1,200 rpm. The supernatant is collected to quantify the histamine. The cell pellet is resuspended in 300 ⁇ l of PBS, 1 mM EDTA and 100 ⁇ l of this cell suspension are analyzed after immunostaining.
• PMA Phorbol Myristate Acetate
• the quantification of the released histamine was carried out using a commercial radioimmunoassay (reference 1659, Immunotech, Marseille, France), following the manufacturer's instructions.
• Total histamine is determined after cell lysis by diluting 50 ⁇ l of blood in 950 ⁇ l of distilled water followed by two freeze-thaw cycles. The histamine released after cell activation is expressed as a percentage of total histamine.
• the analysis by flow cytometry was carried out after triple labeling on 100 ⁇ l of cell pellet taken up in PBS, with the anti-RIL-5 H17N-PE antibody, the anti-CD69-FITC antibody or the anti -CD63-FITC and the anti-CD3, anti-CD16, anti-CD19 antibodies, all three conjugated to PECy ⁇ .
• the procedure is identical to that described above.
• the analysis of 100,000 cells is acquired.
• the percentage of cells doubly labeled CD69 + and RIL-5 + on the one hand, CD63 + and IL-5R + on the other hand was determined after cellular activation.
• FIG. 7 A representative experiment is presented in FIG. 7. An increase in doubly labeled cells (CD69 + and RIL-5 + ) is observed as a function of the concentration of PMA. Interestingly, labeling with the anti-CD63 antibody on eosinophils did not give fluorescence demonstrating that CD63 is specific for basophils.
• EXAMPLE 6 Kit for detecting or quantifying eosinophils or basophils.
• EXAMPLE 8 Kit for detecting or quantifying eosinophils or basophils 0 A kit was prepared corresponding to the composition:

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• 1998
  • 1998-09-10 FR FR9811456A patent/FR2783326B1/fr not_active Expired - Fee Related
• 1999
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