EP2321646A1 - Procédé de caractérisation, en particulier de quantification, de marqueurs moléculaires absorbés de manière intracellulaire à partir des tissus par des macrophages du sang provenant des tissus et remis en circulation dans le flux sanguin - Google Patents

Procédé de caractérisation, en particulier de quantification, de marqueurs moléculaires absorbés de manière intracellulaire à partir des tissus par des macrophages du sang provenant des tissus et remis en circulation dans le flux sanguin

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Publication number
EP2321646A1
EP2321646A1 EP09781481A EP09781481A EP2321646A1 EP 2321646 A1 EP2321646 A1 EP 2321646A1 EP 09781481 A EP09781481 A EP 09781481A EP 09781481 A EP09781481 A EP 09781481A EP 2321646 A1 EP2321646 A1 EP 2321646A1
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EP
European Patent Office
Prior art keywords
clone
polyclonal
blood
cells
beads
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP09781481A
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German (de)
English (en)
Inventor
Wolfang Brozek
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Synmed Research GmbH
SynMed Res GmbH
Original Assignee
Synmed Research GmbH
SynMed Res GmbH
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Publication date
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Publication of EP2321646A1 publication Critical patent/EP2321646A1/fr
Withdrawn legal-status Critical Current

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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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/5055—Cells of the immune system involving macrophages

Definitions

  • the invention relates to a method for the characterization, in particular quantification of phagocytosed, intracellular molecular markers of blood mononuclear leukocytes, in particular blood macrophages according to the preamble of patent claim 1 and an analysis arrangement for carrying out the method according to the preamble of patent claim 13.
  • CD14 is a cell surface protein that is typically expressed by monocytes and macrophages.
  • the cell-surface protein CD16 allows cells to bind to the constant (Fc) region of IgG.
  • Antibodies it is present in two isoforms: as CD16a (FcyRIIIA) on the surface of NK cells, macrophages and so-called activated monocytes, and as CD16b (FcyRIIIB) on neutrophil granulocytes.
  • CD16a FcyRIIIA
  • CD16b FcyRIIIB
  • activate monocytes of the blood, which express both CD14 and CD16.
  • Previous opinion has simply considered CD14 / CD16 positive (CD14 + CD16 + ) cells of the blood as “activated monocytes” that have not (yet) migrated into the tissue, for example at the site of inflammation, infection, or tumor (Ziegler-Heitbrock, L., 2007.
  • the CD14 + CD16 + blood monocytes their role in infection and inflammation, J. Leukocyte Biol., 81, 584-592).
  • activated monocytes are macrophages recirculated from the tissue into the bloodstream and have tissue components in their intracellular phagosomes, e.g. Epithelial proteins derived, for example, from the site of inflammation or a tumor (Herwig, R., Horninger, W., Rehder, P. et al., 2005.
  • CD14 + CD16 + blood monocytes their role in infection and inflammation., J. Leukocyte Biol. 584-592
  • a total loss of CD14 Bazil and Strominger, 1991: Shedding as a mechanism of down modulation of CD14 on stimulated human monocytes, J. Immunol., 147, 1567-1574).
  • PSA prostate-specific antigen
  • imPSA Intracellular Macrophage PSA
  • the basis of the flow cytometric characterization of cells is the labeling of the target molecules to be detected (the "antigens") with fluorescent dye-labeled antibodies.
  • the cells are passed in suspension by hydrodynamic focusing on a bundled laser beam of suitable wavelength. This excites the fluorescent dye to emit energy in the form of photons registered by a detector.
  • a disadvantage of this method lies in the lack of quantification of the amount of bound antibodies and thus in a lack of information about the amount of detected antigen.
  • Another disadvantage is the considerable apparatus and financial expense that causes the operation of a flow cytometer.
  • the object of the invention is to provide an improved, simplified and cost-effective method and an analysis arrangement, with their help a characterization of mononuclear leukocytes of the blood, especially blood macrophages, including their phagocytosed molecular marker from tissue quantitatively, in particular by a representation as Mass unit per cell number is possible.
  • the object is achieved by a method for the characterization, in particular quantification, of molecular markers (intracellularly taken up from tissue) by blood macrophages recirculated from the tissue into the bloodstream, wherein the following steps are carried out:
  • An inventive principle consideration is molecular markers, z.
  • epithelial proteins in phagocytic mononuclear cells of the bloodstream, namely in blood macrophages detect, and it is possible by means of the inventive method in an advantageous manner to lead a direct and specific and quantitative detection.
  • Marker fragments as defined above for imPSA, macrophages taken in the tissue and at least partially phagocytosed molecules that are detected intracellularly in blood macrophages.
  • the selection and / or enrichment and / or separation of the blood macrophages is carried out by means of a positive selection using antibodies directed against the surface marker CD16.
  • a consideration of all CD16-expressing cells is thus carried out in order to ensure detection of all activated monocytes / macrophages.
  • a positive selection is carried out using antibodies directed against the surface marker CD14.
  • CD14 + selection offers the advantage according to the invention that the CD14 + population, which is large compared to the CD16 + population, has undifferentiated monocytes which have no CD16 surface markers and are not the subject of the method according to the invention for characterizing, in particular quantifying, tissue recirculated into the bloodstream, blood macrophages intracellularly taken up by tissue molecular marker (s) are already excluded in a first step.
  • NK cells NK Cells expressed surface proteins CD56 or CD57 or CD161 can be connected.
  • a negative selection for the exclusion of cells with the surface markers CD56 and / or CD57 and / or CD161 using antibodies which are directed against the surface markers CD56 and / or CD57 and / or CD161.
  • Such a negative selection can be carried out as a separate selection step or, according to another advantageous Embodiment also be made simultaneously with a positive selection of CD16 + cells, as exemplified below.
  • the invention provides that a positive or negative selection is carried out using, preferably reversibly, coupled magnetic "beads" to separate the respective selected cells in a very simple manner by means of a suitable magnet.
  • a quantitative determination of a mononuclear leukocyte populations in an Elisa plate preferably by measuring the lactate dehydrogenase activity by Elisa plate reader, more preferably in the same Elisa plate, in which a qualitative and quantitative Determination of molecular markers, in particular non-blood macrophage antigens, is provided.
  • a quantitative determination of the amount of non-blood macrophage tissue-derived molecular markers present is carried out by means of Elisa plate reader and / or chemiluminescence measuring device, according to the invention as molecular markers, especially tissue markers and / or serological markers abnormal DNA methylation, AFP ( ⁇ -1-fetoprotein), AHCY (S-adenosyl-homocysteine hydrolase), AMY2 (pancreatic amylase), CA 15-3 (synonyms: MUCl, EMA, CD227), CA 19-9, CA 50, CA 72-4 (synonym: TAG72), CA 125 (synonym: MUC16), calcitonin, calprotectin, CCSA-2 (colon cancer-specific antigen-2), CCSP-2
  • whole blood is first of all mixed with an anti-caking and / or coagulating agent, for example heparin or another suitable anti-coagulant, eg. As citrate solution, added.
  • an anti-caking and / or coagulating agent for example heparin or another suitable anti-coagulant, eg. As citrate solution, added.
  • Perforation or lysis of the macrophages or macrophage-containing leukocyte populations is also carried out according to the invention, for example by means of a saponin treatment or a treatment with Triton solution.
  • non-blood macrophage antigens optionally conjugated with biotin, HRP (horseraddish peroxidase), AP (alkaline phosphatase), or luminescent, antibodies, in particular the immunoglobulin class G (IgG) and Fab or F (ab) 2 fragments, and / or aptamers, in particular selected from: anti-mouse IgG (polyclonal), anti-rabbit IgG (polyclonal), anti-goat IgG (polyclonal), anti-rat IgG (polyclonal), anti -Selective IgG (polyclonal), anti-AFP (clone AFP-Ol), anti-AFP (clone AFP-II), anti-AFP (clone 4A3), anti-AFP (clone 5H7), anti-AFP (clone M803209) , anti-AFP (clone M0151611), anti-AFP (clone M803209) , anti
  • anti-cystatin B (clone B-02), anti-cystatin B (clone RJMW-2E7), anti-cystatin B (polyclonal), anti-DDH (clone TlOl), anti-DDH (polyclonal), anti-cystatin DKK-1 (clone 141135), anti-DKK-1 (polyclonal), anti-GP73 / GOLPH2 (clone YA-14), anti-GP73 / GOLPH2 (clone 5B10), anti-GP73 / GOLPH2 (polyclonal), anti- HE4 (clone C-12), anti-HE4 (polyclonal), anti-HER2 / neu (clone 10C7), anti-HER2 / neu (clone 191924), anti-HER2 / neu (clone N3 / D10), anti-HER2 / neu (polyclonal), anti-HSP-27 (clone G3.1), anti
  • a positive selection against CD14 + cells or negative selection this is followed by another separation step characterized by positive selection of the cells using an antibody directed against the cell surface protein CD16, which is either coupled to magnetic beads or is coated on an Elisa plate.
  • a further positive selection of the cells against CD14 + cells is followed by an antibody which is either coupled to magnetic beads or coated on an Elisa plate, or it follows a negative Selection against CD56 + or CD57 + or CD161 + NK cells using a magnetic antibody directed against the cell surface protein CD56 or CD57 or CD161.
  • positive selection against CD16 + cells and negative selection against CD56 + or CD57 + or CD161 + NK cells can be done simultaneously in one step.
  • further selection steps may alternatively be omitted.
  • the now separated and enriched mononuclear leukocytes are subsequently destroyed by treatment with a perforation solution and / or a cell lysis reagent.
  • a perforation solution and / or a cell lysis reagent can be previously, either immediately before the lysis of the cells or immediately after
  • the quantification of the molecular marker contained in the cells is possible.
  • the activity of the enzyme lactate dehydrogenase (LDH) in the lysate is determined in an Elisa plate for the purpose of determining the number of cells used, wherein the amount of mononuclear leucocytes present can be calculated by means of a standard.
  • LDH lactate dehydrogenase
  • antibodies are specifically directed against the respective molecular marker (s) and / or against the respective molecular antibodies directed antibody (s), preferably in the same Elisa plate, in which the cell quantification was carried out by measuring the LDH activity, an enzymatic color reaction or a chemiluminescent signal is triggered in proportion to the amount of marker studied.
  • Elisa plate reader or chemiluminescence detector the respective molecular marker can be quantified by a standard.
  • the object according to the invention is further achieved by an analysis arrangement for carrying out the method according to the invention.
  • the method of analysis according to the invention comprises a device for adding an agent which inhibits the clumping and / or coagulation of whole blood (eg heparin, citrate solution, or another suitable anti-coagulant); a device for performing a preselection or selection and enrichment of all mononuclear leucocytes present in the blood sample by density gradient centrifugation;
  • an agent which inhibits the clumping and / or coagulation of whole blood eg heparin, citrate solution, or another suitable anti-coagulant
  • a device for performing a preselection or selection and enrichment of all mononuclear leucocytes present in the blood sample by density gradient centrifugation e.g heparin, citrate solution, or another suitable anti-coagulant
  • CD56 or CD57 or CD161 a device for perforating and / or lysing the mononuclear leukocyte populations; a device for the quantitative determination of mononuclear leukocyte populations in an Elisa plate (eg, measurement of lactate dehydrogenase activity by Elisa plate reader), in the same Elisa plate, in which the qualitative and quantitative determination of molecular weight Marker takes place;
  • a device for the qualitative and quantitative determination of intracellular molecular markers phagocytosed by blood macrophages eg using Elisa plate reader, chemiluminescence meter, et c.
  • intracellular molecular markers phagocytosed by blood macrophages eg using Elisa plate reader, chemiluminescence meter, et c.
  • a device for fixation and permeabilization of mononuclear leucocytes, prior to lysis thereof, preferably before or optionally after the selection and / or enrichment and / or separation of the blood macrophages or blood macrophage-containing leukocyte populations can be provided in order to advantageously produce a To perform intracellular binding of antibodies directed against intracellular phagocytosed molecular marker to be quantified before the cell lysis.
  • the starting point is 6 ml of whole blood taken in a tube containing a polyester gel and a 0.1 M sodium citrate solution.
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • 2 mM EDTA ethylene-diamine-tetra-acetate
  • the isolated mononuclear leukocytes are suspended in 0.5 ml of PBS solution (pH 7.4) containing 0.1% BSA and 2 mM EDTA but no Ca 2+ or Mg 2+ ions, an aliquot can be removed and stored at 2-8 ° C be kept.
  • the suspension is spiked with an adequate amount of magnetic "beads" carrying directly on its surface an antibody directed against the cell surface antigen CD14, for example 10 8 "beads" in 250 ⁇ l.
  • the suspension may be incubated with 10 ⁇ g of a DSB-X biotin-conjugated antibody to the cell-surface antigen CD14 at 2-8 ° C for 10 minutes with shaking or panning, after which the suspension is centrifuged (350 ⁇ g, 8 minutes), the cells are resuspended after discarding the supernatant in 0.5 ml of PBS solution (pH 7.4) containing 0.1% BSA and 2 mM EDTA but no Ca 2+ or Mg 2+ ions.
  • the beads in a cell-release buffer may contain the modified biotin contains, be resuspended.
  • the suspension is shaken for 10 minutes at room temperature, followed by magnetic separation of beads and supernatant.
  • the supernatant is removed, centrifuged (350 xg, 8 minutes), and dissolved in 0.5 ml of PBS solution (pH 7.4) containing 0.1% BSA and 2 mM EDTA but no Ca 2+ or Mg 2+ ions.
  • a defined volume of the homogenized suspension (maximum 100 ⁇ l) is pipetted per well into a 96-well Maxi-Sorp plate containing both a mouse antibody directed against the cell surface antigen CD16, for example 5 ⁇ g / ml of the clone 3G8, as well as with a mouse antibody directed against PSA, for example 1 ⁇ g / ml of the clone ER-PR8, and has been blocked (for example with a 5% FCS (fetal calf serum) solution). Serial dilutions of the suspension can be made. An aliquot of the suspension should be kept at 2-8 ° C. The plate is sealed (for example with cling film or aluminum foil) and incubated for one hour at room temperature or overnight in the refrigerator at 2-8 ° C.
  • the plate is washed five times with PBS solution (pH 7.4) containing 0.1% BSA and 2 mM EDTA but no Ca 2+ or Mg 2+ ions. Subsequently, 50 ⁇ l of cell lysis solution containing 1% Triton and ImM PMSF (phenyl methyl sulfonyl fluoride) are pipetted into each well.
  • aliquots of the retained suspensions of all mononuclear leukocytes or of the "beads" with all cells carrying the antigen CD14 on their surface are pipetted into any number, for example two, wells and with the volume of 50 l of missing cell lysis solution containing 1% Triton and ImM PMSF (phenyl methyl sulfonyl fluoride).
  • a serial dilution of recombinant PSA for example, between 50 pg / ml and 2 ng / ml, prepared, of which 50 .mu.l of each concentration in at least two free wells are pipetted.
  • the plate is then placed for 5 minutes at 2-8 ° C and then treated for 5 minutes in an ultrasonic bath (only the plate bottom immersed in the water, a just below the water surface mounted basket serves as a support).
  • HRP horseradish peroxidase
  • results are displayed, for example, as pg imPSA / cell number (CD14 + CD16 + cells, or also CD14 + cells, or else mononuclear leukocytes).
  • the starting point is 6 ml of whole blood taken in a tube containing a polyester gel and a 0.1 M sodium citrate solution.
  • centrifugation of the blood for example 1800 x g, 20 minutes, all mononuclear leukocytes are isolated together with the platelets above the gel.
  • the isolated cells are washed twice with PBS (phosphate buffered saline) solution (pH 7.4), which may additionally contain 0.1% BSA (bovine serum albumin) and 2 mM EDTA (ethylene-diamine-tetra-acetate), to the platelets to remove.
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • EDTA ethylene-diamine-tetra-acetate
  • the suspension is spiked with an adequate amount of magnetic "beads" carrying directly on its surface an antibody directed against the cell-surface antigen CD16, for example 10 8 "beads" in 250 ⁇ l.
  • the suspension may be incubated with 10 ⁇ g of a DSB-X biotin-conjugated antibody to the cell surface antigen CD16 at 2-8 ° C for 10 minutes with shaking or panning, after which the suspension is centrifuged (350 xg, 8 minutes), the cells are resuspended after discarding the supernatant in 0.5 ml of PBS solution (pH 7.4) containing 0.1% BSA and 2 mM EDTA but no Ca 2+ or Mg 2+ ions.
  • the "beads" in a cell-release buffer may contain the modified biotin contains, be resuspended.
  • the suspension is shaken for 10 minutes at room temperature, followed by magnetic separation of beads and supernatant.
  • the supernatant is removed, centrifuged (350 xg, 8 minutes), and suspended in 0.5 ml of PBS solution (pH 7.4) containing 0.1% BSA and 2 mM EDTA but no Ca 2+ or Mg 2+ ions.
  • a defined volume of the homogenized suspension (maximum 100 ⁇ l) is pipetted per well into a 96-well Maxi-Sorp plate which has both a mouse antibody directed against the cell-surface antigen CD14, for example 5 ⁇ g / ml of the clone M ⁇ P9, as well as coated with a mouse antibody directed against PSA, for example 1 ⁇ g / ml of the clone ER-PR8, and blocked (for example with a 5% FCS (fetal calf serum) solution). Serial dilutions of the suspension can be made. An aliquot of the suspension should be kept at 2-8 ° C. The plate is sealed (for example with cling film or aluminum foil) and incubated for one hour at room temperature or overnight in the refrigerator at 2-8 ° C.
  • the plate is washed five times with PBS solution (pH 7.4) containing 0.1% BSA and 2 mM EDTA but no Ca 2+ or Mg 2+ ions. Subsequently, 50 ⁇ l of cell lysis solution containing 1% Triton and ImM PMSF (phenyl methyl sulfonyl fluoride) are pipetted into each well.
  • aliquots of the retained suspensions of all mononuclear leucocytes or of the "beads" with all cells carrying the CD16 antigen on their surface are pipetted into any number, for example two, wells and with the volume of lysis solution missing to 50 ⁇ l containing 1% Triton and ImM PMSF (phenyl methyl sulfonyl fluoride).
  • a serial dilution of recombinant PSA for example, between 50 pg / ml and 2 ng / ml, prepared, of which 50 ul of each concentration in at least two free wells are pipetted.
  • the plate is then placed for 5 minutes at 2-8 ° C and then treated for 5 minutes in an ultrasonic bath (only the plate bottom immersed in the water, a just below the water surface mounted basket serves as a support).
  • the presentation of the results is carried out, for example, as pg imPSA / cell number
  • CD14 + CD16 + cells or also CD16 + cells, or even mononuclear leukocytes.
  • the starting point is 6 ml of whole blood taken in a tube containing a polyester gel and a 0.1 M sodium citrate solution.
  • centrifugation of the blood for example 1800 x g, 20 minutes, all mononuclear leukocytes are isolated together with the platelets above the gel.
  • the isolated cells are washed twice with PBS (phosphate buffered saline) solution (pH 7.4), which additionally contains 0.1% BSA (bovine serum albumin) and 2 mM EDTA (ethylene buffered saline).
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • 2 mM EDTA ethylene buffered saline
  • the isolated mononuclear leukocytes are suspended in 0.5 ml PBS solution (pH 7.4) containing 0.1% BSA and 2 mM EDTA but no Ca 2+ or Mg 2+ ions, an aliquot can be taken, in a defined volume of cell Lysis solution, which is 1%
  • Triton and ImM contains PMSF (phenyl methyl sulfonyl fluoride), dissolved, and stored at 2-8 ° C.
  • the suspension is incubated with 10 ⁇ g of a DSB-X biotin-conjugated antibody to the cell surface antigen CD14 at 2-8 ° C for 10 minutes with shaking or panning.
  • the cells are centrifuged (350 xg, 8 minutes) and, after discarding the supernatant, resuspended in 0.5 ml of PBS solution (pH 7.4) containing 0.1% BSA and 2 mM EDTA but no Ca 2+ or Mg 2+ ions.
  • PBS solution pH 7.4
  • the supernatant is removed, centrifuged (350 xg, 8 minutes), and suspended in 0.5 ml of PBS solution (pH 7.4) containing 0.1% BSA and 2 mM EDTA but no Ca 2+ or Mg 2+ ions. An aliquot of the suspension is removed and centrifuged, the pellet is dissolved in a defined volume of cell lysis solution containing 1% Triton and ImM PMSF (phenyl methyl sulfonyl fluoride) and stored at 2-8 ° C.
  • PBS solution pH 7.4
  • PMSF phenyl methyl sulfonyl fluoride
  • the cell suspension is now “beads” in 100 uL, added with an adequate amount of magnetic "beads”, which bear on their surface an antibody directed against the cell surface antigen CD16 antibody, for example, 4 x 10 7 and 20 minutes at 2-8 ° C shaken or swiveled. This is followed by the separation of the cells bound to the magnetic beads by means of a suitable magnet, the supernatant being discarded.
  • the "beads" with the cells are washed several times with a PBS solution (pH 7.4) containing 0.1% BSA and 2 mM EDTA but no Ca 2+ or Mg 2+ ions, before a defined volume of cell lysis Solution containing 1% Triton and ImM PMSF (phenyl methyl sulfonyl fluoride).
  • the lysate is placed at 2-8 ° C for at least 5 minutes.
  • All cell lysates are now sonicated for 5 minutes, a suitable magnet used to remove the beads from the appropriate lysates, and still centrifuged once (14000 xg, 10 minutes).
  • a defined volume of the supernatants (maximum 100 ⁇ l) is pipetted per well into a 96-well Maxi-Sorp plate which is coated with a mouse antibody directed against PSA, for example 1 ⁇ g / ml of the clone ER-PR8 and blocked was (for example with a 5% FCS (fetal calf serum) solution).
  • a serial dilution of recombinant PSA for example between 50 pg / ml and 2 ng / ml, is prepared, of which a defined volume of each concentration is pipetted into at least two free wells.
  • a cell number standard a dilution series of the calibrated LDH positive control in cell lysis solution, for example starting from a 1/2500 dilution, prepared, of which 50 .mu.l of each concentration in at least two free wells are pipetted.
  • the plate is sealed (for example, with cling film or aluminum foil) and incubated for one hour at room temperature.
  • HRP horseradish peroxidase
  • the presentation of the results is carried out, for example, as pg imPSA / cell number
  • CD14 + CD16 + cells or even CD14 + cells, or even mononuclear leukocytes.
  • the starting point is 6 ml of whole blood taken in a tube containing a polyester gel and a 0.1 M sodium citrate solution.
  • centrifugation of the blood for example 1800 x g, 20 minutes, all mononuclear leukocytes are isolated together with the platelets above the gel.
  • the isolated cells are washed twice with PBS (phosphate buffered saline) solution (pH 7.4), which additionally contains 0.1% BSA (bovine serum albumin) and 2 mM EDTA (ethylene buffered saline).
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • 2 mM EDTA ethylene buffered saline
  • the isolated mononuclear leukocytes are suspended in 0.5 ml PBS solution (pH 7.4) containing 0.1% BSA and 2 mM EDTA but no Ca 2+ or Mg 2+ ions, an aliquot can be taken, in a defined volume of cell Lysis solution, which is 1%
  • Triton and ImM contains PMSF (phenyl methyl sulfonyl fluoride), dissolved, and stored at 2-8 ° C.
  • the suspension is incubated with 5 ⁇ g of a DSB-X biotin-conjugated antibody against the cell surface antigen CD16 at 2-8 ° C for 10 minutes with shaking or panning.
  • the cells are centrifuged (350 ⁇ g, 8 minutes), and after discarding the supernatant at 0.5 ml of PBS solution (pH 7.4) but no Ca 2+ or Mg 0.1% BSA and 2 mM EDTA 2+ ions resuspended.
  • An adequate amount of magnetic "beads" carrying streptavidin on their surface, for example 2 x 10 7 "beads" in 50 ⁇ l, are added.
  • a defined volume of the suspension is removed therefrom and centrifuged, the pellet is dissolved in a defined volume of cell lysis solution containing 1% Triton and ImM PMSF (phenyl methyl sulfonyl fluoride) and at 2-8 ° C keep on.
  • Triton and ImM PMSF phenyl methyl sulfonyl fluoride
  • the cell suspension is then treated with an adequate amount of magnetic "beads” carrying on its surface a directed against the cell surface antigen CD14 antibody, for example, 2 x 10 7 "beads” in 50 ul, and 20 minutes at 2-8 ° C shaken or swiveled. This is followed by the separation of the cells bound to the magnetic beads by means of a suitable magnet, the supernatant being discarded.
  • an adequate amount of magnetic "beads” carrying on its surface a directed against the cell surface antigen CD14 antibody, for example, 2 x 10 7 "beads” in 50 ul, and 20 minutes at 2-8 ° C shaken or swiveled.
  • the "beads" with the cells are washed several times with a PBS solution (pH 7.4) containing 0.1% BSA and 2 mM EDTA but no Ca 2+ or Mg 2+ ions, before a defined volume of cell lysis Solution containing 1% Triton and ImM PMSF (phenyl methyl sulfonyl fluoride) is added.
  • the lysate is placed at 2-8 ° C for at least 5 minutes.
  • All cell lysates are now sonicated for 5 minutes, a suitable magnet used to remove the beads from the appropriate lysates, and centrifuged again (14,000 x g, 10 minutes).
  • a defined volume of the supernatants (maximum 100 ⁇ l) is pipetted per well into a 96-well Maxi-Sorp plate which is coated with a mouse antibody directed against PSA, for example 1 ⁇ g / ml of the clone ER-PR8 and blocked was (for example with a 5% FCS (fetal calf serum) solution).
  • a serial dilution of recombinant PSA for example between 50 pg / ml and 2 ng / ml, is prepared, of which a defined volume of each concentration is pipetted into at least two free wells.
  • a cell number standard a dilution series of the calibrated LDH positive control in cell lysis solution, for example starting from a 1/2500 dilution, prepared, of which 50 .mu.l of each concentration in at least two free wells are pipetted.
  • the plate is sealed (for example, with cling film or aluminum foil) and incubated for one hour at room temperature.
  • results are displayed, for example, as pg imPSA / cell number (CD14 + CD16 + cells, or also CD16 + cells, or else mononuclear leukocytes).
  • the starting point is 6 ml of whole blood taken in a tube containing a polyester gel and a 0.1 M sodium citrate solution.
  • centrifugation of the blood for example 1800 x g, 20 minutes, all mononuclear leukocytes are isolated together with the platelets above the gel.
  • the isolated cells are washed twice with PBS (phosphate buffered saline) solution (pH 7.4), which may additionally contain 0.1% BSA (bovine serum albumin) and 2 mM EDTA (ethylene-diamine-tetra-acetate), to the platelets to remove.
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • EDTA ethylene-diamine-tetra-acetate
  • the isolated mononuclear leukocytes are suspended in 0.5 ml PBS solution (pH 7.4) containing 0.1% BSA and 2 mM EDTA but no Ca 2+ or Mg 2+ ions, an aliquot can be taken, in a defined volume of cell Lysis solution, which is 1% Triton and ImM contains PMSF (phenyl methyl sulfonyl fluoride), dissolved, and stored at 2-8 ° C.
  • the suspension is spiked with an adequate amount of magnetic beads carrying on its surface an antibody directed against the cell surface antigen CD16, for example 2 x 10 7 "beads" in 50 ⁇ l.
  • DSB-X biotin-conjugated antibody to the cell surface antigen CD16 can be incubated at 2-8 ° C for 10 minutes with shaking, followed by the addition of an adequate amount of magnetic beads. which carry streptavidin on their surface, for example 2 ⁇ 10 7 "beads" in 50 ⁇ l, followed.
  • the suspension of mononuclear leucocytes and magnetic beads is shaken or swirled at 2-8 ° C for 20 minutes.
  • the "beads” coupled to antibodies or the “beads” labeled with streptavidin bind to cells which carry the CD16 antigen on their surface or to DSB-X biotin-conjugated antibody bound to cells with the surface antigen CD16 are.
  • All cell lysates are then sonicated for 5 minutes, a suitable magnet used to remove the beads from the appropriate lysates, and centrifuged again (14,000 xg, 10 minutes).
  • a defined volume of the supernatants (maximum 100 ⁇ l) is pipetted per well into a 96-well Maxi-Sorp plate which is coated with a mouse antibody directed against PSA, for example 1 ⁇ g / ml of the clone ER-PR8 and blocked was (for example with a 5% FCS (fetal calf serum) solution).
  • a serial dilution of recombinant PSA for example between 50 pg / ml and 2 ng / ml, is prepared, of which a defined volume of each concentration is pipetted into at least two free wells.
  • a cell number standard a dilution series of the calibrated LDH positive control in cell lysis solution, for example starting from a 1/2500 dilution, of which 50 ⁇ l of each concentration are pipetted into at least two free wells.
  • the plate is sealed (for example, with cling film or aluminum foil) and incubated for one hour at room temperature.
  • the starting point is 6 ml of whole blood taken in a tube containing a polyester gel and a 0.1 M sodium citrate solution.
  • centrifugation of the blood for example 1800 x g, 20 minutes, all mononuclear leukocytes are isolated together with the platelets above the gel.
  • the isolated cells are washed twice with PBS (phosphate buffered saline) solution (pH 7.4), which may additionally contain 0.1% BSA (bovine serum albumin) and 2 mM EDTA (ethylene-diamine-tetra-acetate), to the platelets to remove.
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • EDTA ethylene-diamine-tetra-acetate
  • the isolated mononuclear leukocytes are suspended in 0.5 ml PBS solution (pH 7.4) containing 0.1% BSA and 2 mM EDTA but no Ca 2+ or Mg 2+ ions, an aliquot can be taken, in a defined volume of cell Lysis solution containing 1% Triton and ImM PMSF (phenyl methyl sulfonyl fluoride) dissolved and stored at 2-8 ° C.
  • the suspension is spiked with an adequate amount of magnetic beads carrying on its surface an antibody directed against the cell surface antigen CD16, for example 2 x 10 7 "beads" in 50 ⁇ l.
  • DSB-X biotin-conjugated antibody to the cell surface antigen CD16 can be incubated at 2-8 ° C for 10 minutes with shaking, followed by the addition of an adequate amount of magnetic beads. which carry streptavidin on their surface, for example 2 ⁇ 10 7 "beads" in 50 ⁇ l, followed.
  • the suspension of mononuclear leucocytes and magnetic beads is shaken or swirled at 2-8 ° C for 20 minutes. In doing so, they bind to antibodies coupled "beads" or streptavidin-labeled "beads” on cells carrying on their Oberflumble the antigen CD16 or on DSB-X biotin-conjugated antibody bound to cells with the surface antigen CD16.
  • a defined volume of the suspension is removed therefrom and centrifuged, the pellet is dissolved in a defined volume of cell lysis solution containing 1% Triton and ImM PMSF (phenyl methyl sulfonyl fluoride) and at 2-8 ° C keep on.
  • Triton and ImM PMSF phenyl methyl sulfonyl fluoride
  • the cell suspension is then mixed with an adequate amount of magnetic "beads" carrying on its surface a directed against the cell surface antigen CD56 or CD57 or CD161 antibody, for example, 2 x 10 7 "beads” in 50 ul.
  • an adequate amount of magnetic "beads” carrying on its surface a directed against the cell surface antigen CD56 or CD57 or CD161 antibody for example, 2 x 10 7 "beads” in 50 ul.
  • 5 ⁇ g of a DSB-X biotin-conjugated antibody to the cell surface antigen CD56 or CD57 or CD161 can be incubated at 2-8 ° C for 10 minutes with shaking, followed by the addition of an adequate amount of magnetic "beads” carrying streptavidin on their surface, for example 2 ⁇ 10 7 "beads” in 50 ⁇ l, followed.
  • All cell lysates are then sonicated for 5 minutes and centrifuged again (14,000 x g, 10 minutes).
  • a defined volume of the supernatants (maximum 100 ⁇ l) is pipetted per well into a 96-well Maxi-Sorp plate which is coated with a mouse antibody directed against PSA, for example 1 ⁇ g / ml of the clone ER-PR8 and blocked was (for example with a 5% FCS (fetal calf serum) solution).
  • a serial dilution of recombinant PSA for example between 50 pg / ml and 2 ng / ml, is prepared, of which a defined volume of each concentration is pipetted into at least two free wells.
  • a cell number standard a dilution series of the calibrated LDH positive control in cell lysis solution, for example starting from a 1/2500 dilution, prepared, of which 50 .mu.l of each concentration in at least two free wells are pipetted.
  • the plate is sealed (for example, with cling film or aluminum foil) and incubated for one hour at room temperature.
  • results are displayed, for example, as pg imPSA / cell count (CD16 + CD56 " or CD16 + CD57 " or CD16 + CD161 " cells, or else CD16 + cells, or else mononuclear leukocytes).
  • the starting point is 6 ml of whole blood taken in a tube containing a polyester gel and a 0.1 M sodium citrate solution.
  • centrifugation of the blood for example 1800 x g, 20 minutes, all mononuclear leukocytes are isolated together with the platelets above the gel.
  • the isolated cells are washed twice with PBS (phosphate buffered saline) solution (pH 7.4), which additionally contains 0.1% BSA (bovine serum albumin) and 2 mM EDTA (ethylene buffered saline).
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • 2 mM EDTA ethylene buffered saline
  • the isolated mononuclear leukocytes are suspended in 0.5 ml of PBS solution (pH 7.4) containing 0.1% BSA and 2 mM EDTA but no Ca 2+ or Mg 2+ ions Aliquot can be removed, dissolved in a defined volume of cell lysis solution containing 1% Triton and ImM PMSF (phenyl methyl sulfonyl fluoride), and stored at 2-8 ° C.
  • the suspension is spiked with an adequate amount of magnetic beads carrying on its surface an antibody directed against the cell surface antigen CD56 or CD57 or CD161, for example 2 x 10 7 "beads" in 50 ⁇ l.
  • DSB-X biotin-conjugated antibody to the cell surface antigen CD56 or CD57 or CD161 can be incubated at 2-8 ° C for 10 minutes with shaking, followed by the addition of an adequate amount of magnetic "beads" carrying streptavidin on their surface, for example 2 ⁇ 10 7 "beads" in 50 ⁇ l, followed.
  • the suspension of mononuclear leucocytes and magnetic beads is shaken or swirled at 2-8 ° C for 20 minutes.
  • the "beads” coupled to antibodies or the “beads” labeled with streptavidin bind to cells which carry on their surface the antigen CD56 or CD57 or CD161 or to DSB-X biotin-conjugated antibody which binds to cells with the surface -Antigen CD56 or CD57 or CD161 are bound.
  • a defined volume of the suspension is removed therefrom and centrifuged, the pellet is dissolved in a defined volume of cell lysis solution containing 1% Triton and ImM PMSF (phenyl methyl sulfonyl fluoride) and stored at 2-8 ° C keep on.
  • Triton and ImM PMSF phenyl methyl sulfonyl fluoride
  • the cell suspension is then mixed with an adequate amount of magnetic "beads" carrying on its surface a directed against the cell surface antigen CD16 antibody, for example 2 x 10 7 "beads” in 50 ul.
  • an adequate amount of magnetic "beads” carrying on its surface a directed against the cell surface antigen CD16 antibody for example 2 x 10 7 "beads” in 50 ul.
  • 5 ⁇ g of a modified DSB-X biotin-conjugated antibody to the Cell surface antigen CD16 are incubated at 2-8 ° C for 10 minutes with shaking or panning, followed by the addition of an adequate amount of magnetic beads carrying streptavidin on their surface, for example 2 x 10 7 "beads" 50 ⁇ l, followed.
  • a serial dilution of recombinant PSA for example between 50 pg / ml and 2 ng / ml, is prepared, of which a defined volume of each concentration is pipetted into at least two free wells.
  • a dilution series of the calibrated LDH positive control in cell lysis solution for example starting from a 1/2500 dilution, prepared, of which 50 .mu.l of each concentration in at least two free wells are pipetted.
  • the plate is sealed (for example, with cling film or aluminum foil) and incubated for one hour at room temperature.
  • HRP horseradish peroxidase
  • results are displayed, for example, as pg imPSA / cell count (CD16 + CD56 “ or CD16 + CD57 “ or CD16 + CD161 " cells, or else CD56 " or CD57 “ or CD161 " cells, or else mononuclear leukocytes).
  • the starting point is 6 ml of whole blood taken in a tube containing a polyester gel and a 0.1 M sodium citrate solution.
  • centrifugation of the blood for example 1800 xg, 20 minutes, all mononuclear leukocytes are isolated together with the platelets above the gel.
  • the isolated cells are washed twice with PBS (phosphate buffered saline) solution (pH 7.4), which may additionally contain 0.1% BSA (bovine serum albumin) and 2 mM EDTA (ethylene-diamine-tetra-acetate), to the platelets to remove.
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • EDTA ethylene-diamine-tetra-acetate
  • the isolated mononuclear leukocytes are suspended in 0.5 ml PBS solution (pH 7.4) containing 0.1% BSA and 2 mM EDTA but no Ca 2+ or Mg 2+ ions, an aliquot can be taken, in a defined volume of cell Lysis solution containing 1% Triton and ImM PMSF (phenyl methyl sulfonyl fluoride) dissolved and stored at 2-8 ° C.
  • the suspension is spiked with an adequate amount of magnetic beads carrying on its surface an antibody directed against the cell surface antigen CD56 or CD57 or CD161, for example 2 x 10 7 "beads" in 50 ⁇ l.
  • DSB-X biotin-conjugated antibody to the cell surface antigen CD56 or CD57 or CD161 can be incubated at 2-8 ° C for 10 minutes with shaking, followed by the addition of an adequate amount of magnetic "beads" carrying streptavidin on their surface, for example 2 ⁇ 10 7 "beads" in 50 ⁇ l, followed.
  • the suspension of mononuclear leucocytes and magnetic beads is shaken or swirled at 2-8 ° C for 20 minutes.
  • the "beads” coupled to antibodies or the “beads” labeled with streptavidin bind to cells which carry on their surface the antigen CD56 or CD57 or CD161 or to DSB-X biotin-conjugated antibody which binds to cells with the surface -Antigen CD56 or CD57 or CD161 are bound.
  • a defined volume of the cell suspension (maximum 100 .mu.l) is pipetted per well into a 96-well Maxi-Sorp plate, both with a directed against the cell surface antigen CD16 mouse antibody, for example 5 ug / ml of Clone 3G8, as well as with a mouse antibody directed against PSA, for example 1 ⁇ g / ml of the clone ER-PR8, coated and blocked (for example with a 5% FCS (fetal calf serum) solution). Serial dilutions of the suspension can be made. A residual volume of the suspension should be retained, but not frozen for the time being. The plate is sealed (for example with cling film or aluminum foil) and incubated for one hour at room temperature or overnight in the refrigerator at 2-8 ° C.
  • the plate is washed five times with PBS solution (pH 7.4) containing 0.1% BSA and 2 mM EDTA but no Ca 2+ or Mg 2+ ions. Subsequently, 50 ⁇ l of cell lysis solution containing 1% Triton and ImM PMSF (phenyl methyl sulfonyl fluoride) are pipetted into each well. In addition, 25 .mu.l of the retained suspension from all cells which do not carry the antigen CD56 or CD57 or CD161 on their surface are pipetted into any number of wells, for example two, and with the same volume of cell lysis solution containing 1% Triton and ImM PMSF (phenyl methyl sulfonyl fluoride), added.
  • a serial dilution of recombinant PSA for example, between 50 pg / ml and 2 ng / ml, prepared, of which 50 .mu.l of each concentration in at least two free wells are pipetted.
  • the plate is then placed for 5 minutes at 2-8 ° C and then treated for 5 minutes in an ultrasonic bath (only the plate bottom immersed in the water, a just below the water surface mounted basket serves as a support).
  • results are displayed, for example, as pg imPSA / cell count (CD16 + CD56 “ or CD16 + CD57 “ or CD16 + CD161 " cells, or else CD56 " or CD57 “ or CD161 " cells, or else mononuclear leukocytes).
  • the starting point is 6 ml of whole blood taken in a tube containing a polyester gel and a 0.1 M sodium citrate solution.
  • centrifugation of the blood for example 1800 x g, 20 minutes, all mononuclear leukocytes are isolated together with the platelets above the gel.
  • the isolated cells are washed twice with PBS (phosphate buffered saline) solution (pH 7.4), which may additionally contain 0.1% BSA (bovine serum albumin) and 2 mM EDTA (ethylene-diamine-tetra-acetate), to the platelets to remove.
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • EDTA ethylene-diamine-tetra-acetate
  • the isolated mononuclear leukocytes are suspended in 0.5 ml PBS solution (pH 7.4) containing 0.1% BSA and 2 mM EDTA but no Ca 2+ or Mg 2+ ions, an aliquot can be taken, in a defined volume of cell Lysis solution containing 1% Triton and ImM PMSF (phenyl methyl sulfonyl fluoride) dissolved and stored at 2-8 ° C.
  • PBS solution pH 7.4
  • BSA bovine serum
  • ImM PMSF phenyl methyl sulfonyl fluoride
  • the suspension is spiked with 5 ⁇ g of a DSB-X biotin-conjugated antibody to the cell surface antigen CD16 and the same amount (5 ⁇ g) of a biotin-conjugated antibody to the cell surface antigen CD56 or CD57 or CD161 ,
  • the suspension is incubated at 2-8 ° C for 10 minutes with shaking or panning, followed by the addition of an adequate amount of magnetic beads carrying streptavidin on its surface, for example 2 ⁇ 10 7 beads in 50 ⁇ l ,
  • the suspension of mononuclear leucocytes and magnetic beads is shaken or swirled at 2-8 ° C for 20 minutes.
  • the labeled with streptavidin "beads” bind to biotin as well as with DSB-X biotin conjugated antibodies that are bound to cells with the surface antigen CD56 or CD57 or CD161 or with the surface antigen CD16. This is followed by the separation of the cells bound to the magnetic "beads" using a suitable magnet, the supernatant being discarded.
  • the "beads” are washed several times with a PBS solution (pH 7.4) containing 0.1% BSA and 2 mM EDTA but no Ca 2+ or Mg 2+ ions, the washing solution is discarded, the "beads” are subsequently in a cell release buffer containing modified biotin resuspended. The suspension is shaken for 10 minutes at room temperature, followed by magnetic separation of beads and supernatant.
  • the supernatant containing only CD16 + CD56 " or CD16 + CD57 " or CD16 + CD161 " cells is removed, centrifuged (350xg, 8 minutes), the pellet is placed in a defined volume cell lysis solution containing 1% Triton and ImM contains PMSF (phenyl methyl sulfonyl fluoride), dissolved and placed at 2-8 ° C for at least 5 minutes.
  • PMSF phenyl methyl sulfonyl fluoride
  • a serial dilution of recombinant PSA for example between 50 pg / ml and 2 ng / ml, is prepared, of which a defined volume of each concentration is pipetted into at least two free wells.
  • a dilution series of the calibrated LDH positive control in cell lysis solution for example starting from a 1/2500 dilution, prepared, of which 50 .mu.l of each concentration in at least two free wells are pipetted.
  • the plate is sealed (for example, with cling film or aluminum foil) and incubated for one hour at room temperature.
  • HRP horseradish peroxidase
  • results are displayed, for example, as pg imPSA / cell count (CD16 + CD56 " or CD16 + CD57 " or CD16 + CD161 " cells, or else mononuclear leukocytes).
  • the starting point is 6 ml of whole blood, which in one is a polyester gel and a 0.1 M
  • the isolated cells are washed twice with PBS (phosphate buffered saline) solution (pH 7.4), which may additionally contain 0.1% BSA (bovine serum albumin) and 2 mM EDTA (ethylene-diamine-tetra-acetate), to the platelets to remove.
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • EDTA ethylene-diamine-tetra-acetate
  • the isolated mononuclear leukocytes are suspended in 0.5 ml of PBS solution (pH 7.4) containing 0.1% BSA and 2 mM EDTA but no Ca 2+ or Mg 2+ ions and conjugated with 5 ⁇ g of a modified DSB-X biotin Antibody against the cell surface antigen CD16 and with the same amount (5 ug) of a biotin-conjugated antibody to the cell surface antigen CD56 or CD57 or CD161 added.
  • the suspension is incubated at 2-8 ° C for 10 minutes with shaking or panning.
  • the cells After a washing step with PBS solution (pH 7.4), the cells are suspended in 0.5 ml fixation reagent (for example 1% formalin solution), washed after 10 min with PBS solution (pH 7.4), then taken up in 100 ⁇ l permeabilization medium and incubated for approx For 20 minutes, incubate with an anti-PSA enzyme conjugated with the HRP (horseraddish peroxidase) or biotin-conjugated mouse antibody, for example 1 ⁇ g / ml of the clone ER-PR8.
  • fixation reagent for example 1% formalin solution
  • HRP horseradish peroxidase
  • biotin-conjugated mouse antibody for example 1 ⁇ g / ml of the clone ER-PR8.
  • the cells are washed with a PBS solution (pH 7.4) and suspended in 0.5 ml of PBS solution (pH 7.4) containing 0.1% BSA and 2 mM EDTA but no Ca 2+ or Mg 2+ ions; an aliquot can be taken, dissolved in a defined volume of zeolysis solution containing 1% Triton and ImM PMSF (phenyl methyl sulfonyl fluoride), and stored at 2-8 ° C. This is followed by the addition of an adequate amount of magnetic "beads" carrying streptavidin on their surface, for example 2 ⁇ 10 7 "beads" in 50 ⁇ l.
  • PBS solution pH 7.4
  • BSA bovine serum
  • ImM PMSF phenyl methyl sulfonyl fluoride
  • treatment of the cells with fixative reagent and permeabilization medium may be omitted at this point in the protocol, and after removal of an aliquot, take place in a defined manner Volume cell lysis solution containing 1% Triton and ImM PMSF (phenyl methyl sulfonyl fluoride) dissolved and stored at 2-8 ° C, the cells immediately with an adequate amount of magnetic beads, the carry on their surface streptavidin, for example, 2 x 10 7 "beads" in 50 ul, incubated.
  • Triton and ImM PMSF phenyl methyl sulfonyl fluoride
  • the pellet is discarded after discarding the supernatant, even if an addition of the antibody directed against imPSA to permeabilization of the cells, in a defined volume cell lysis solution containing 1% Triton and ImM PMSF (phenyl methyl sulfonyl fluoride) , dissolved and placed at 2-8 ° C for at least 5 minutes. Otherwise, the pellet is discarded after discarding the supernatant in 0.5 ml
  • Fixing reagent for example 1% formalin solution
  • PBS solution pH 7.4
  • HRP horseradish peroxidase
  • biotin conjugated, mouse antibody for example, 1 ⁇ g / ml of the clone ER-PR8, incubated.
  • HRP horseradish peroxidase
  • biotin conjugated, mouse antibody for example, 1 ⁇ g / ml of the clone ER-PR8, incubated.
  • the cells are washed with a PBS solution (pH 7.4) and dissolved in a defined volume of cell lysis solution containing 1% Triton and ImM PMSF (phenyl-methyl-sulfonyl-fluoride) for at least 5 minutes -8 ° C.
  • Triton and ImM PMSF phenyl-methyl-sulfonyl-fluoride
  • All cell lysates are then sonicated for 5 minutes and centrifuged again (14,000 xg, 10 minutes).
  • a defined volume of the supernatants (maximum 100 ⁇ l) is pipetted per well into a 96-well Maxi-Sorp plate coated with a polyclonal antibody directed against mouse immunoglobulin (using a HRP-conjugated antibody directed against PSA), e.g. 2 ⁇ g / ml, or coated with avidin or streptavidin (using a biotin-conjugated anti-PSA antibody) and blocked (for example with a 5% FCS (fetal calf serum) solution).
  • a polyclonal antibody directed against mouse immunoglobulin using a HRP-conjugated antibody directed against PSA
  • avidin or streptavidin using a biotin-conjugated anti-PSA antibody
  • FCS fetal calf serum
  • a dilution series of the HRP (horseraddish peroxidase) or biotin-conjugated mouse antibody clone ER-PR8 is prepared, of which a defined volume of each concentration is pipetted into at least two free wells. Of this, the amount of bound imPSA can be recalculated.
  • a dilution series of the calibrated LDH positive control in cell lysis solution for example starting from a 1/2500 dilution, is prepared as the cell number standard, of which 50 ⁇ l of each concentration are pipetted into at least two free wells. The plate is sealed (for example, with cling film or aluminum foil) and incubated for one hour at room temperature.
  • the incubation period with TMB-containing substrate solution is 15 minutes at room temperature, then 50 ⁇ l of a stop solution (for example IM H 2 SO 4 ) are pipetted into each well. Finally, the light absorption of the wells is measured in an Elisa plate reader at a wavelength of 450 nm, the absorption at 570 nm is subtracted. The intensity of the measured absorbance is proportional to the amount of imPSA which can be determined by the standard.
  • a stop solution for example IM H 2 SO 4
  • results are displayed, for example, as pg imPSA / cell count (CD16 + CD56 " or CD16 + CD57 " or CD16 + CD161 " cells, or else mononuclear leukocytes).

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Abstract

L'invention concerne un procédé de caractérisation, en particulier de quantification, de marqueurs moléculaires absorbés de manière intracellulaire à partir des tissus par des macrophages du sang provenant des tissus et remis en circulation dans le flux sanguin. Ce procédé comprend les étapes suivantes : application au sang total d'un moyen inhibant le caillement et/ou la coagulation du sang total ; à partir du sang total, réalisation d'une sélection et/ou d'un enrichissement et/ou d'une séparation de macrophages sanguins ou de populations de leucocytes contenant des macrophages sanguins ; réalisation d'une perforation et/ou d'une lyse des macrophages sanguins ou des populations de leucocytes contenant des macrophages sanguins, éventuellement après une perméabilisation préalable ; exécution d'une détermination qualitative et quantitative de marqueurs n'appartenant pas aux macrophages sanguins, c'est-à-dire des marqueurs moléculaires provenant des tissus, après perforation et/ou lyse préalables des macrophages sanguins ou des populations de leucocytes contenant des macrophages sanguins. L'invention concerne en outre un agencement pour la mise en oevre du procédé.
EP09781481A 2008-08-04 2009-08-04 Procédé de caractérisation, en particulier de quantification, de marqueurs moléculaires absorbés de manière intracellulaire à partir des tissus par des macrophages du sang provenant des tissus et remis en circulation dans le flux sanguin Withdrawn EP2321646A1 (fr)

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CN107164555A (zh) * 2017-07-19 2017-09-15 深圳市南山区慢性病防治院 一种腺苷高半胱氨酸酶基因启动子区甲基化程度检测试剂盒及测定方法
CN109576272A (zh) * 2018-07-02 2019-04-05 广西医科大学 一种dkk-1核酸适配体及其应用
CN111551717B (zh) * 2020-04-10 2023-04-07 深圳大学 一种基于有机光电化学晶体管的胃泌素释放肽前体传感器及其制备方法与应用
CN112129952B (zh) * 2020-09-21 2023-06-06 普健生物（武汉）科技有限公司 一种检测人可溶性cd14的化学发光试剂盒
CN114507644B (zh) * 2022-03-18 2023-11-14 江苏南农高科技股份有限公司 一种副猪嗜血杆菌MnSOD蛋白单克隆抗体及其阻断ELISA试剂盒
CN114965392B (zh) * 2022-04-26 2024-05-03 桂林电子科技大学 一种基于NGQDs-MoS2荧光共振能量转移结合适配体检测GP73的方法
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