EP0838034A1 - Immunoassay zur bestimmung der blutzellaktivierung - Google Patents

Immunoassay zur bestimmung der blutzellaktivierung

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Publication number
EP0838034A1
EP0838034A1 EP96918058A EP96918058A EP0838034A1 EP 0838034 A1 EP0838034 A1 EP 0838034A1 EP 96918058 A EP96918058 A EP 96918058A EP 96918058 A EP96918058 A EP 96918058A EP 0838034 A1 EP0838034 A1 EP 0838034A1
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European Patent Office
Prior art keywords
platelets
activation
platelet
enzyme
antibody
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EP96918058A
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English (en)
French (fr)
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EP0838034A4 (de
Inventor
David L. Amrani
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Milwaukee Heart Project
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Milwaukee Heart Project
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Publication of EP0838034A4 publication Critical patent/EP0838034A4/de
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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells

Definitions

  • TECHNICAL FIELD This invention relates to an immunoassay, particularly an enzyme-linked immunoabsorbent assay (ELISA) for monitoring activation of platelets and other types of blood cells which undergo activation.
  • ELISA enzyme-linked immunoabsorbent assay
  • P-selectin e.g., CD62
  • P-selectin is used as a means of detecting activated platelets in the systemic circulation by flow cytometry.
  • P-selectin also known as GMP-140 or PADGEM
  • GMP-140 or PADGEM is an a-granule protein which is expressed on the surface of endothelial cells and on platelets only after activation and release. P-selectin has been implicated in recognition of monocytes, macrophages and neutrophils during the inflammatory response, and in particular, as a first step in the regulation of leukocyte migration through endothelium.
  • a method for determining the activation level of blood cells by an immunoassay of the type wherein a primary antibody undergoes specific binding with the blood cells and a labelled secondary antibody undergoes specific binding with the primary antibody-blood cell complex includes the step of reacting a sample containing the blood cells in a liquid phase, e.g. an aqueous suspension, with an excess quantity of an activation-specific primary antibody prior to allowing the cells in the sample to bind to a solid surface.
  • a liquid phase e.g. an aqueous suspension
  • the activated platelet ELISA assay of the invention is a relatively simple procedure for quantitation of the amount of activated platelets in the bloodstream of patients who may be at risk for thrombosis or thrombosis- related conditions.
  • a method for controlling the activation level of platelets or other blood cells in a patient's bloodstream involves the steps of obtaining a sample of platelets or other blood cells which undergo activation, immunoassaying the sample with an antibody that selectively binds to activated platelets or other blood cells while the sample preferably remains in liquid suspension, correlating the results with a standard to determine the degree of activation of the platelets or cells prior to the assay, and administering a drug to the patient in an amount effective to maintain activation at a predetermined level.
  • the physician then administers an anticoagulant or antiplatelet agent or increase the anticoagulant or antiplatelet agent dosage. If the opposite is true, the anticoagulant or antiplatelet agent dosage can be decreased.
  • the assay includes the steps of:
  • step (C) further comprises adding a labelled secondary antibody to the suspension which reacts with and specifically binds to the primary antibody.
  • the label is one of a pair of specific binding substances, such as biotin, which binds specifically to the other substance, such as streptavidin.
  • the platelets are separated from unbound labelled secondary antibody, and an enzyme labelled with the other of the pair of specific binding substances is added to the platelets so that the enzyme becomes bound to the secondary antibody.
  • the platelets are then separated from unbound enzyme.
  • the secondary antibody may be linked to the enzyme in advance, and the resulting complex reacted directly with the platelet having primary antibody bound thereto.
  • the invention further contemplates a kit for carrying out the assay of the invention.
  • the kit includes necessary reagents, including standards containing fixed platelets activated at different, predetermined levels for generating a standard curve, and the necessary primary and optionally secondary antibody preparations, as well as written directions giving a protocol for carrying out the assay.
  • the principle of measuring P-selectin is used as a specific marker for platelet activation to develop a whole platelet ELISA for detecting activated platelets in platelet-rich plasma (PRP) .
  • the accuracy and specificity of the assay is verified via fluorescence-activated flow cytometric analysis.
  • the quantitative assay is based on the use of a standard curve generated from activation of platelets in pooled PRP with phorbol yristate acetate (PMA) or thrombin receptor peptide.
  • PMA phorbol yristate acetate
  • the assay is applied to assess platelet activation in patients diagnosed with unstable angina compared to normal controls.
  • Figures 1 and 2 are plots of optical density (OD) against collagen concentration (CC) in ⁇ g/ml for two examples of assays according to the invention.
  • Figures 3 and 4 are plots of optical density (OD) against thrombin concentration (TC) in ⁇ g/ml for two examples of assays according to the invention.
  • Figures 5A and 5B are agonist-induced dose response curves for washed platelets. Washed platelets were treated with increasing concentrations of either thrombin (5A) or collagen (5B) and subsequently measured by enzyme-linked indirect antibody detection.
  • Figs. 6A and 6B show a flow cytometric analysis of resting and thrombin-activated platelets.
  • the degree of platelet activation as indicated by CD-62 (6A) or CD-63 (6B) antigen expression was carried out to assess relative changes in platelet activation during preparation of homologous whole blood (WB) , PRP, and washed platelets (PLTS) .
  • the symbols for the respective lines are indicated within the legends.
  • the exception is the resting PRP in Fig. 6A, which is superimposed on the whole blood sample.
  • WB 0.75%, PRP, 0.84%, resting PLTS, 59.9%, and thrombin-activated PLTS, 97.2%
  • Fig. 6B - WB 0.39%, PRP, 0.46%, resting PLTS, 8.6%, and thrombin-activated PLTS, 90%.
  • Figures 7A-7C are agonist-induced dose response curves of PRP.
  • the dose-response curves corresponding to collagen (7A) , thrombin receptor peptide (7B) , and PMA (7C) activation of PRP were measured by flow cytometry.
  • Figures 8A and 8B are standard curves for PMA- activated platelet ELISA.
  • Figs. 7A and 7B demonstrate curves that have correlation coefficients of 0.976 and 0.993, respectively.
  • Figures 9A-9D show the results of flow cytometric analysis of samples from a representative control subject (9A) and a patient with unstable angina (9B) together with whole blood resting (9C) and activated (9D) controls.
  • Samples were fixed in 1.8% paraformaldehyde for 30 min and after washing, were stained with CD-61- FITC (FLl) and CD-62-PE (FL2; % of platelets expressing P-selectin) and evaluated for the degree of activated platelets.
  • the resting and activated controls were prepared from whole blood obtained from a normal volunteer where the resting control was immediately fixed and a sample of the blood was activated with 20 micromoles APP for 15 min, and then fixed and stained.
  • Figure 10 is a scattergram of flow cytometric and ELISA data on the percent P-selectin expression in fixed PRP samples from control subjects and patients with unstable angina.
  • the assay of the present invention can eliminate the need for any equipment other than that commonly found in an ordinary clinical/or research laboratory.
  • the following description deals with a platelet assay, but the procedure is also applicable to samples of other types of cells which undergo activation, for example, blood cells other than red blood cells, such as monocytes, lymphocytes, and granulocytes.
  • the invention is based in part on the discovery that affixing platelets or the associated primary antibody that binds thereto to a plastic surface as part of the ELISA assay causes surface activation of the platelets, rendering the assay inaccurate. This effect is most pronounced for plastics which have a substantial electrical surface charge, such as polyethylene and polystyrene.
  • plastics which have a substantial electrical surface charge, such as polyethylene and polystyrene.
  • ELISA assay for platelet activation can be carried out if binding of the primary antibody occurs in aqueous suspension. In practice, this means taking care to avoid binding the platelets to a plastic surface, such as the bottom of a microtiter well, until after the platelets have reacted with the primary antibody. Accordingly, according to assay of the invention, the platelet suspension is placed in a plastic microtiter well or test tube but not centrifuged until after the reaction between the primary antibody and the platelets has taken place. In accordance with one embodiment of the assay method, platelets are obtained from whole blood by standard centrifugation procedures, for example, using a standard laboratory centrifuge at room temperature and spinning for 15 minutes at 350 times gravity (xG) .
  • xG times gravity
  • the assay can be performed with either platelet-rich plasma, or washed platelets.
  • the procedure for washing the platelets may be the one described in Amrani et al., Blood. Vol. 72, No. 3, 1988, pp. 919-924, the contents of which are incorporated by reference herein.
  • washed platelets are preferred to provide better uniformity in the results.
  • the procedure may be simplified by using platelet-rich plasma. After washing, the platelets are then resuspended in a buffer solution at a pH of about 7.3 to 7.4.
  • buffer solutions according to the invention are formulated to maintain the platelets in a stable condition by maintaining salt concentrations and pH at normal plasma levels.
  • a calcium inhibitor e.g., a chelating agent such as ethylenediaminetetraamine (EDTA) or citrate, is preferably added to the platelet suspension prior to activation.
  • the calcium inhibitor is added in an amount effective to chelate substantially all calcium ions in the aqueous suspension so that calcium does not bind to FC receptors on the platelet surface, leading to unwanted binding of S12 antibody containing FC regions of the antibody at these sites.
  • the amount of calcium inhibitor added is generally in the range of 2 to 10 mM in the suspension.
  • the platelets are then activated at room temperature with one of a number of known agonists such as thrombin (for washed platelets only) in an amount ranging from 0 units/ml to 1.0 units/ml, or collagen (for either platelet-rich plasma or washed platelets) in an amount ranging from 0 ⁇ g/ml to 400 ⁇ g/ml.
  • thrombin for washed platelets only
  • collagen for either platelet-rich plasma or washed platelets
  • a specific primary antibody which is directed at platelet-activatiorr specific antigens (receptors) is also added, and the mixture is allowed to react for a time sufficient to allow the binding reaction to go to substantial completion, generally 30-60 minutes.
  • Suitable primary antibodies include S12 or CD63, and others which are commercially available.
  • CD63 available from AMAC Inc., Westbrook, ME binds to glycoprotein (GP) 53 lysosomal protein.
  • Antibody S12 described in McEver and Martin, J. Biol. Chem. 259, 9799 (1984), is directed against granule membrane protein 140.
  • one or more inhibitors of platelet activation are then optionally added to minimize further activation and antibody binding.
  • Such inhibitors include prostaglandin El (PGE-L) , preferably used in an amount of 5 ng to 100 ng per ml of carrier liquid, preferably ethanol, typically 10 ng/ l ethanol, and hirudin, a specific thrombin inhibitor used at the same concentration as thrombin.
  • PGE-L prostaglandin El
  • a secondary antibody is added to link to the platelet-attached primary antibody (i.e., the activation- specific antibody) .
  • the secondary antibody is generally either coupled in advance with an enzyme, as in Example 1 below, or else is labelled with a substance such as biotin.
  • a biotin-labeled anti- mouse immunoglobulin preferably goat or rabbit anti- mouse Ig depending on the antibody used, is added to link to the platelet-attached primary antibody. The reaction is allowed to proceed in the mixture for a sufficient time, typically 30-60 minutes.
  • the platelet-antibody mixture is then separated by centrifugation from a supernatant material containing any antibody remaining unbound either directly, or through a dense medium such as sucrose or silicone oil. Centrifugation time may vary from about 2 seconds to 10 minutes depending on the medium used. For example, a 6 minute spin at 10,000 xG is preferred for a 5:1 silicone- oil mixture for washed platelets, and 8 minute spins at 10,000 xG are used for 8:1 silicone-oil mixture.
  • the platelet pellet is then washed 1 to 6 times with buffer, and an enzyme bound to streptavidin, such as streptavidin peroxidase, is added and allowed to react for 15-60 minutes.
  • streptavidin is a molecule that binds specifically and with high affinity to biotin.
  • Preferred enzymes include peroxidase and alkaline phosphatase. If the enzyme is pre-linked to the secondary antibody as in Example 1 below, then the use of labels (specific binding substances) such as biotin and streptavidin may be omitted, along with the associated separation steps.
  • the label for the secondary antibody could also be a
  • the pellet is again washed, e.g. 2-6 times, and a substrate such as 2,2- azino-di(3-ethylbenzylthiazoline) sulfonic acid (ABTS) is added to the pellet to develop the color.
  • ABTS 2,2- azino-di(3-ethylbenzylthiazoline) sulfonic acid
  • the foregoing procedure is used to construct a standard curve by activating platelets to predetermined levels based on the amount of activating agent used.
  • the procedure can then be repeated without prior in vitro activation to determine activation levels for unknown samples.
  • Whole blood is centrifuged at a relatively low speed, e.g. 350 xG, to obtain platelet-rich plasma.
  • the steps of adding the activator and inhibitor are likewise omitted. Otherwise, the procedure remains substantially the same.
  • the activation level of the unknown sample is then read from the linear portion of the standard curve.
  • each successive step is preferably carried out with the platelets remaining in the same tube or microtiter well. Since polypropylene has less surface charge and less tendency to cause the platelets to bind thereto spontaneously, use of a polypropylene microtiter plate, particularly a Nunc pre- siliconized polypropylene microtiter plate, is most preferred.
  • the standard according to the invention may be a predetermined standard curve that correlates activation level with the observed color change, fluorescence or similar indication.
  • the invention provides a method for identifying patients who may be at risk for thrombosis or thrombosis-related conditions, such as artificial heart recipients. Anticoagulant or antiplatelet drugs are commonly administered to such patients.
  • the assay of the invention is carried out on a patient, such as a person on anticoagulant or antiplatelet drug therapy, and the activation level is compared to a normal activation level.
  • a normal activation level is generally around 5-6%, i.e., in a normal person only 5 to 6 percent of platelets will be activated at any given time.
  • the activation level may become as high as 25-50%; 5 to 10 times the normal level. This can be readily determined by, for example, carrying out the assay of the invention on several normal individuals and comparing the values obtained on the standard curve with that of the patient.
  • the dosage of the anticoagulant drug can be increased, a stronger anticoagulant can be used, or the like.
  • the dosage of the anticoagulant drug can be decreased, or a weaker anticoagulant can be used.
  • the immunoassay used in the method for controlling platelet or other blood cell activation according to the invention need not be the primary and secondary antibody- based assay described above.
  • Any suitable assay system could be used, for example, standard radioimmunoassay or immunofluorescence procedures, so long as the assay is conducted in the fluid phase so that the result of the assay accurately reflects the activation level of the cells in the patient's bloodstream.
  • the primary antibody could be directly labelled with an enzyme, fluorescent label, radioactive isotope or similar label, and the secondary antibody could be omitted.
  • the platelet ELISA assay of the invention provides a sensitive, accurate, and economical means of detecting circulating activated platelets. Fixed platelet samples can be prepared and analyzed fresh or after storage. Either procedure is useful or detecting systemic platelet activation by whole platelet ELISA as well as by flow cytometry.
  • platelets kept in suspension with a 6-rpm elliptical rotator caused a loss of one-third of the platelets in the form of clumps, a 50% decrease in GPIb in remaining single platelets, and an increase in P-selectin expression.
  • the forgoing data suggest that physical manipulation can lead to selective release of platelet vesicles.
  • Anticoagulants and inhibitors can also effect the expression of platelet receptors.
  • the possibility that hemolysis and release of APP from red blood cells could induce P-selectin expression during physical manipulation can be minimized by the use of apyrase in either the whole blood samples prior to centrifugation or in PRP during washing procedures.
  • Washed platelets prior to agonist-induced activation, demonstrate an essentially unactivated fibrinogen receptor, and together with the low levels of GP53 expression in the presence of 50-60% expression of P-selectin (Fig. 6) , suggest that P-selectin can be selectively expressed on the surface of platelets and that platelet vesicle release is a more highly regulated process than previously believed.
  • the following examples illustrate the invention:
  • EXAMPLE 1 The following solutions were prepared. In each case, sufficient water was added to the listed ingredients to bring the volume of the solution to the level indicated:
  • Buffer pH was adjusted to 6.5 by titration of the solution with 1 N NaOH.
  • PGE-L and apyrase were added as needed to inhibit activation.
  • 10 ml wash buffer 21.2 ⁇ l PGE- solution, 3.5 ⁇ g/ml and 25 ⁇ l, 1.25 units/ml apyrase were used; for 20 ml wash buffer, the amounts were 42.3 ⁇ l PGE- ⁇ and 50 ⁇ l apyrase.
  • HEPES is an amine commercially available from Sigma. Buffer pH was adjusted to 7.3-7.4 by titration of the solution with 1 N NaOH. PGE ] ⁇ is added as needed as needed to inhibit activation, here in an amount that provided a final concentration of 5 ng/ml.
  • Pre-siliconized polypropylene Eppendorf tubes were filled with 0.5 ml of silicone oil.
  • the silicone oil was a 5:1 mixture of Hiphenyl DC-550 to methyl DC-200 silicone oil.
  • Fresh blood was collected into 15 ml Vacutainer tubes containing 2.14 ml ACD anticoagulant in a ratio of 9 parts by volume blood to 1 part ACD. Washed platelets were then prepared from the blood samples. Blood was drawn into the prepared Vacutainer tubes and centrifuged at 800 rpm (350 xG) for 15 minutes in an IEC-Centra-7 tabletop centrifuge. The platelet- rich plasma was carefully removed from the red cell layer and placed into a clean 13 ml polypropylene tube.
  • PGE-L solution used in this example contained 3.545 ⁇ g of prostaglandin E- ⁇ per ml of 100% ethanol.
  • the platelets were then pelleted by centrifugation at 700 xG for 5 minutes.
  • the platelet-poor plasma supernatant may be saved as a source of fibrinogen for a platelet aggregation assay, or may be discarded.
  • the platelet pellet usually contains red cells.
  • the platelets were carefully resuspended in wash buffer containing inhibitors, as described above, at one half the original volume of the platelet-rich plasma. Platelets were removed only from around the red cells at the center, and the red cells were discarded.
  • the platelets were centrifuged at 1500 rpm (500 xG) for 5 minutes to pellet the platelets. The supernatant was poured off, and the platelets were resuspended in the wash buffer and inhibitors. The platelets were then centrifuged again at 500 xG for 5 minutes. The supernatant from the second wash was discarded, and the platelets were resuspended in the wash buffer containing inhibitors. A 0.5 ml sample of the washed platelet suspension was removed to a 4 ml polypropylene tube for counting. Platelet count is platelets/ml times total ml. The platelets were then centrifuged again at 500 xG for 5 minutes. The supernatant from the third wash was discarded, and the platelets were resuspended in platelet resuspension buffer containing no inhibitors.
  • the platelet count was adjusted to 175,000 platelets per microliter to provide a final concentration of 100,000 platelets/microliter.
  • EDTA was added to the final suspension of platelets to provide a final concentration of 1 mM (micromole) .
  • a 200 microliter sample of the washed platelets was pipetted into a number of reaction tubes.
  • Collagen from BioData was reconstituted with distilled water to yield 1.9 mg collagen/ml.
  • 63.2 microliters of the reconstituted collagen suspension were pipetted into each reaction tube to provide final collagen concentrations of 0, 25, 75, 100, 150, 200, 250, 300, 350, and 400 micrograms/ml.
  • 36.8 microliters of S12 antibody solution were then gently pipetted into each tube in amounts to provide a final concentration of 2.0 micrograms of the antibody per ml.
  • the S12 antibody was centrifuged at the top speed of the microcentrifuge (10,000 xG) about for 15 minutes to remove microaggregates.
  • the reaction tubes were allowed to incubate at room temperature for 30 minutes.
  • GAM IgG(H & L)-HRP horseradish peroxidase
  • the GAM IgG(H & L)-HRP contained 0.9 mg/ml IgG and 0.6 mg/ml peroxidase.
  • the PGE-L solution contained PGE ! in an amount calculated to provide a final concentration of 10 ng/ml in the reaction tubes, and the GAM IgG(H & L)-HRP solution was used at a final dilution of 1:300.
  • the GAM IgG(H & L)-HRP was centrifuged before use for 15 minutes to remove microaggregates in the same manner as for the S12 antibody. The reaction tubes were then incubated for 60 minutes as room temperature.
  • Figure 4 shows much less uncertainty and a more linear plot than Figure 3.
  • the four graphs together show that greater convergence occurs as the number of individuals contributing to the platelet pool increases.
  • the standards for use in the present invention are preferably prepared using platelets pooled from several (4 or more) individuals. It should also be possible according to the invention to generate a standard curve from a large number of individuals that can be used as a general standard, potentially eliminating the need to generate a new standard curve each time the assay is performed.
  • EXAMPLE 2 The following illustrates preparation of fixed platelets for use in a kit according to the invention.
  • Platelets were prepared in accordance with Example 1, except as follows. After activation, the platelets were treated with 1% para-formaldehyde in phosphate-buffered saline (100 millimolar sodium phosphate, 150 millimolar NaCl, pH 7.4) for 5 minutes at room temperature. The platelets were spun down by centrifugation, washed 3 times, then counted. After the third spin, the platelets were resuspended in platelet resuspension buffer containing 5% dimethyl sulfoxide (DMSO) at a concentration of 10 9 platelets per ml. The platelets were then stored for 1 day at -70°C. Samples were then thawed out using a water bath at 37°C.
  • DMSO dimethyl sulfoxide
  • Example 1 The procedure of Example 1 was then followed as described above using the thawed platelets. The results were substantially the same as shown in Figs. 1-4, except that the background level, reflected as the baseline of the graph from the X-axis, was somewhat higher.
  • This example indicates that platelets for use as standards in a kit according to the invention can be prepared in advance and stored frozen until the time of use.
  • EXAMPLE 3 The following reagents were used needed: bovine serum albumin (RIA grade, fraction V) (BSA) ; prostaglandin El (PGE i) ; potato apyrase (grade III) ; ethylene diamine tetraacetic acid (EDTA) ; adenosine-5'- diphosphate (grade X) from equine muscle (ADP) ; acetate (PMA)3 hirudin, dimethylsulfoxide (DMSO), and leupeptin were purchased from Sigma Chemical Co., St. Louis, MO. Tris(hydroxymethyl)aminomethane (Tris) was purchased from Aldrich Chemical Co. Inc., Milwaukee, WI.
  • Calf skin collagen (soluble form) was purchased from BioData Corporation, Hatboro, PA.
  • N-hydroxy ethylpiperazine N'-2- ethane sulfonic acid (HEPES) was purchased from Boehringer Mannheim Biochemicals, Indianapolis, IN. Trasylol was purchased from Mobay Chemical Corp. , New York, NY.
  • Purified S-12 an antibody specific for P-selectin (6) was provided by Dr. Rodger McEver, Oklahoma Medical Research Foundation, Oklahoma City, OK. Other antibodies against receptor glycoproteins, p3, P-selectin, and GP53 corresponding to CD61-FITC, CD62-PE, and CD63-FITC, respectively, were purchased from Becton-Dickinson (Torrance, CA) . Thrombin receptor peptide was purchased from Bachem. Inc. , Torrance, CA. Goat anti-mouse (GAM) IgG alkaline phosphatase and para-nitrophyenlyphosphate (PNPP) were purchased from Zymed, Inc. Purified S-12 was radioactively labeled with 125 I using the iodine monochloride technique as previously described for fibrinogen. 125 I-labeled S-12 had a specific radioactivity of approximately 7.5 ⁇ Ci/ ⁇ g protein.
  • Venous blood was drawn into acid-citrate dextrose anticoagulant from normal donors who had given informed consent.
  • Platelet-rich plasma (PRP) and washed platelets were subsequently prepared as previously described.
  • Radiolabeled ligands were tested for binding to thrombin- or collagen-activated washed platelets as previously described for fibrinogen. Washed platelets at a final concentration of 2 x 108/ml were preincubated at room temperature with 125 IS12 for 1 hr.
  • non-radioactively labeled ligands were also added to reaction mixtures to test for non-specific binding. Bound radioactive counts were determined using a Packard Multi-Prias 4 gamma counter (Packard Instrument Co. , Downers Grove, IL) . Background values were obtained in the absence of activation and in the presence of hirudin (1 unit/ml) and PGE 1(5 ng/ml) .
  • Platelet rich plasma and washed platelets were obtained as indicated above for platelet binding experiments. Platelet aliquots (1.75 x 108/ml, final concentration) were activated with increasing concentrations of thrombin followed by the addition of hirudin (1 unit/ml, final concentration) . Most studies with PRP were carried out by activation with increasing concentrations of PMA; two studies were carried with the thrombin receptor peptide.
  • PMA-activated platelets (1.8 x 108/ml, final concentration) were fixed with paraformaldehyde, washed in 50 mM sodium phosphate buffer, pH 7.4, containing saline (PBS) and resuspended at a final concentration of l X l ⁇ 9 /ml in platelet storage buffer [platelet resuspension buffer (see below) containing 5% dimethyl-sulfoxide, 5 mM EDTA, 400 ⁇ M leupeptin] .
  • Platelet storage buffer [platelet resuspension buffer (see below) containing 5% dimethyl-sulfoxide, 5 mM EDTA, 400 ⁇ M leupeptin] .
  • Fixed platelets can be used immediately or stored at -70°C for up to 10 months.
  • a standard curve was prepared from PMA- or thrombin receptor peptide-activated, paraformaldehyde-fixed (1%) platelets, which had been diluted with platelet resuspension buffer (137 mM NaCl, 2mM KC1, 0.3 mM Ma phosphate, 2 mM CaC12, 1 mM MgCl2, 5.5 mM glucose, 5 mM Hepes, 12 mM NaHC03, 0.35% bovine serum albumin, pH 7.4) to a stock concentration of 1.75 X 108/ml.
  • platelet resuspension buffer 137 mM NaCl, 2mM KC1, 0.3 mM Ma phosphate, 2 mM CaC12, 1 mM MgCl2, 5.5 mM glucose, 5 mM Hepes, 12 mM NaHC03, 0.35% bovine serum albumin, pH 7.4
  • platelet resuspension buffer 137 mM NaCl, 2m
  • the primary antibody, S-12 or CD-62 was added to a further diluted suspension of fixed platelets (4 x 10 7 /ml) , and incubated at room temperature for 30 min. The platelets were centrifuged at 10,000 X g for 3 min, the supernatant solution decanted, and platelets then washed once with TBS-Tween 20. A secondary antibody-GAM IgG (H+L) Fab'2- alkaline phosphatase was added for an additional incubation period of 60 min, followed by centrifugation at 10,000 X g for 3 min. The substrate, PNPP (1 mg/ml), was then added and color developed over 60 min at 37°C. The reaction was stopped by addition of 1 N NaOH, the solution transferred to a microELISA plate, and the absorbance read at 405 nm.
  • EDTA-anticoagulated whole blood was obtained by venipuncture or from an arterial line from patients with unstable angina or from individuals who demonstrated no apparent acute disease following catherization as well as from normal individuals who had given written informed consent. The first 2 ml of the blood draw was discarded, and blood then collected by syringe into an anticoagulant solution containing EDTA (2 mM) , Trasylol (100 units/ml) , hirudin (1 unit/ml) and PGE1 (5 ng/ml) , final concentrations, respectively.
  • resting control platelets were prepared by direct fixation of whole blood with paraformaldehyde (final concentration, 1.8%) for 30 minutes at 4°C.
  • Platelets for the positive control were activated with either ADP or soluble collagen at final concentrations of 20 kM and 190 micrograms/ml, respectively.
  • Whole blood samples from patients or control subjects were incubated at room temperature for 15 minutes and then fixed with paraformaldehyde as above. Fixed samples were then washed two times with PBS containing 2% fetal bovine serum, incubated 20 minutes with CD-61-FITC (B 3 integrin) at approximately 3 microgram/ml and/or CD-62-PE at 0.4 micrograms/ml in the dark at room temperature. The samples were again washed and resuspended in PBS for flow cytometric analysis. The CD-61-FITC was used solely to gate on the platelet population and CD-62-PE to detect only those platelets that were activated.
  • Collagen which does not induce coagulation directly, could cause significant (>90%) P-selectin expression in washed platelets.
  • the result is shown in Table 1, a comparison of P-selectin expression by washed platelets after thrombin or collagen addition.
  • Flow cytometric analysis of thrombin- and collagen-activated washed platelets revealed, however, that P-selectin expression (Fig.
  • PRP 4 X 10-platelets/ml
  • PRP 4 X 10-platelets/ml
  • Two representative standard curves are presented as Figs. 8A, 8B.
  • a linear standard range of activation was obtained between 0 and 100 nM PMA.
  • a representative flow cytometric pattern demonstrates the comparison of control subject platelets (Fig. 9C) versus platelets from an angina patient (Fig. 9D) . Resting control (Fig. 9A) and ADP-activated control (Fig. 9B) platelet samples are also shown. Similar samples were prepared during each flow cytometric run in order to control for the blood drawing process and to provide a positive control for maximal expression of platelet P-selectin. Control data from flow cytometric and ELISA analysis showed a mean of approximately 0.5% for control samples evaluated within the first 3 hrs (Table 2) . In contrast, control values after 10 months storage demonstrated 8.0% and 9.6% P-selectin expression by flow and ELISA analyses, respectively.

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EP96918058A 1995-06-07 1996-06-05 Immunoassay zur bestimmung der blutzellaktivierung Withdrawn EP0838034A4 (de)

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US47352095A 1995-06-07 1995-06-07
PCT/US1996/008776 WO1996041176A1 (en) 1995-06-07 1996-06-05 Immunoassay for determining blood cell activation
US473520 1999-12-28

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