CA2157796A1 - Liposomes incorporating density media - Google Patents
Liposomes incorporating density mediaInfo
- Publication number
- CA2157796A1 CA2157796A1 CA 2157796 CA2157796A CA2157796A1 CA 2157796 A1 CA2157796 A1 CA 2157796A1 CA 2157796 CA2157796 CA 2157796 CA 2157796 A CA2157796 A CA 2157796A CA 2157796 A1 CA2157796 A1 CA 2157796A1
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- liposomes
- density
- liposome
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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
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/49—Blood
- G01N33/491—Blood by separating the blood components
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/5432—Liposomes or microcapsules
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Abstract
Liposomes incorporating a density medium and having a defined density. The liposomes are useful as control reagents for monitoring density gradient centrifugation procedures, especially in centrifugal hematology and immunoassay procedures. The density of the liposomes can be adjusted by the practioner to correspond to the density of any component of a sample expected to band in a particular position in a density gradient centrifugation procedure.
Description
WO 94/26299 21~ 7 7 9 ~ PCT/US94/05188 C LIPOSOMES INCORP~ENSlTY MEDIA
.~
FIELD OF TH~ INVENIION
?
The present invention relates to liposomes and methods for their production. The10 invention further relates to uses of liposomes in analytical methods.
BACKGROUND OF THE INVENTION
Liposomes are vesicles or sacs having closed membranes of amphiphilic molecules in 15 equilibrium with an aqueous solution. Polar lipids such as phosphatidylcholines, phosphatidylethanol~mines~ ,go",yelins, cardiolipins, phosphatidic acids, cerebrosides and conlbillalions of fatty acids form such membranes when placed in an aqueous el~vi~o~ ent.
The bimolecular lipid sheets of the membrane are intercalated by aqueous spaces, and these structures can persist even in the ~,esence of excess water. Liposomes are the,effi,t; useful 20 vehicles for inco,~o,aling active agents for delivery to a desired therapeutic site and as model systems for cellular processes. Liposomes have also been used in the art for encapsulation of dyes and used as tracers in imm~nnassays. PERCOLL beads have also been ~nc~rs~ ted in liposomes for use as markers in spleen tissue for vi~ li7~tion by electron microscopy (EM) (Cudd and Mcolau. 1986. J. Microencapsulation 3(4):275-282). These authors did not 25 attempt to make liposomes of defined density, as the goal was only to produce an electron dense marker for EM studies which could be readily rli~tin~li~hed from subcellular organelles.
They also observed that PERCOLL seemed to render the liposomes less stable.
Materials having a specific, reproducible buoyant density have been used in the art as 30 density markers to calibrate or monitor density gradients in such applications as density gradient centrifugation. These materials in general have taken the ffirm of solid beads of SUBSTITUTE SHEET (RULE 26~
.~
FIELD OF TH~ INVENIION
?
The present invention relates to liposomes and methods for their production. The10 invention further relates to uses of liposomes in analytical methods.
BACKGROUND OF THE INVENTION
Liposomes are vesicles or sacs having closed membranes of amphiphilic molecules in 15 equilibrium with an aqueous solution. Polar lipids such as phosphatidylcholines, phosphatidylethanol~mines~ ,go",yelins, cardiolipins, phosphatidic acids, cerebrosides and conlbillalions of fatty acids form such membranes when placed in an aqueous el~vi~o~ ent.
The bimolecular lipid sheets of the membrane are intercalated by aqueous spaces, and these structures can persist even in the ~,esence of excess water. Liposomes are the,effi,t; useful 20 vehicles for inco,~o,aling active agents for delivery to a desired therapeutic site and as model systems for cellular processes. Liposomes have also been used in the art for encapsulation of dyes and used as tracers in imm~nnassays. PERCOLL beads have also been ~nc~rs~ ted in liposomes for use as markers in spleen tissue for vi~ li7~tion by electron microscopy (EM) (Cudd and Mcolau. 1986. J. Microencapsulation 3(4):275-282). These authors did not 25 attempt to make liposomes of defined density, as the goal was only to produce an electron dense marker for EM studies which could be readily rli~tin~li~hed from subcellular organelles.
They also observed that PERCOLL seemed to render the liposomes less stable.
Materials having a specific, reproducible buoyant density have been used in the art as 30 density markers to calibrate or monitor density gradients in such applications as density gradient centrifugation. These materials in general have taken the ffirm of solid beads of SUBSTITUTE SHEET (RULE 26~
2~5~ ~ 9 ~' PCT/US94/05188 defined buoyant density, such as the Density Marker Beads (cross-linked dextran) ava(~le from Pharmacia LKB Biotechnology, Uppsala, Sweden. POLYBEAD PMMA (polymethyl m.oth~crylate) Monodisperse Particles available from Polysciences, Inc. (Warrington, PA) have a defined buoyant density of 1.19 which allows more rapid separation of the particles when 5 used in immunoassays. However, these commercial- density markers and defined density t partides may not be m~n-lf~r,t~lred with thèi buoyant density required for a particular application and cannot easily be modified to obtain the buoyant density desired. For example, if a density marker for a particular blood cell type is required, density marker beads may not be commercially available with the correct buoyant density.
The complete blood cell count (CBC) is used extensively in hematological analysis and is frequently performed to obtain general information about the status of a patient. Recently, rapid methods and instn-mt?nt~tion have been developed which allow hematological analysis by centrifugation of a blood tube to provide hematocrit (HCT), platelet count (PLT), white blood 15 cell count ~WBC), and a partial differential cell count (Wardlaw and Levine. 1983. JAMA
249(5):617-620). A coll~.llelcially available ill~7llulll~ and method based on this technology is the QBC centrifugal hematology analyzer from Becton Dickinson Primary Care Diagnostics (Sparks, MD). The QBC method uses di~-~ Lial met~cl~lo~ lic fluorescence of acridine orange treated blood cells and density gradient cell l~e,ing within the buffy coat to measure 20 the sepal~led packed volumes of red blood cells, white blood cells and pl~t~let~ The QBC
instrument makes electro-optical measurements of the cell layers and computes the hematocrit, platelet count, WBC and subgroup counts of granulocytes and Iymphocytes/monocytes.
Hemoglobin concentration is derived from the hematocrit and measurements of red cell density.
To perform the QBC analysis, capillary or venous blood is placed in a glass capillary tube internally coated with acridine orange and potassium oxalate. The acridine orange stains white cells and platelets. The potassium oxalate osmotically removes water from the red cells SUBS~ITUT;E SHEET (RULE ~
WO 94/26299 . ~ l ~i 7 ~ ~ 6 PCT/US94/05188 to increase their density and improve separation from granulocytes. A float is fitted in the QBC capillary tube and settles within the buffy coat during centrifugation, thereby axially ~Yp~ntling the stained white cell and platelet layers appro~l,la~ely 10-fold.
The blood tube is centrifuged to separate the cell types into layers or bands within the tube. It is then ilhlmin~ted by blue-violet light in the QBC reader instrument to visualize the interfaces between packed and e~p~n-led red cell layers and between differentially fluorescing layers of granulocytes, lymphocytes/monocytes and platelets. Packed cell volumes and test values (numerical counts and percentages) are computed from the lengths of the five cell layers, as the length of the layer or band is a reflection of the number of cells present.
The pe,rollllance of the QBC reader and related methods such as that of Wardlaw, et al., supra, may be monitored by means of a standardized control reagent which upon centrifugation provides bands having positions in the tube and band lengths which would be expected upon centrifugation of normal and defined abnormal blood samples. Becton Dickinson Plhll~y Care Diagnostics sells such l.,age~lls under the name QBC Centrifugal ~em~tology Control. The normal and abnollllal QBC controls contain stabilized human erythrocytes, m~mm~ n leukocytes and sim~ ted platelets in a plasma-like fluid.
The present invention provides a means for gellelali,l~, particles of a desired density applupliale for a given application. The particles comprise liposomes incorporating a density medium such that the particles have the desired buoyant density. Such liposomes are useful as densit,v markers for monitoring or calibrating density gradients and centrifugal hematology instr~ ;on, as they can be prepared to have a buoyant density corresponding to various cell types, viruses, b~ct~-ri~ etc SUBSTITUTE SHEET (RULE 26 wo 94/26299 S~ PCTIUS94/05188 SU~vIMARY OF THE INVENTION ~
The present invention provides liposomes of defined density. These liposomes areproduced by incorporation of a density merii~lm at a concentration (w/v) which provides a 5 liposome of the desired density. As the density of the defined density liposomes can be easily adjusted by the practitioner, they may be used to calibrate or monitor density gradients for identification of the position of a desired band in the gradient or for marking a specific density point in a gradient. For example, defined density liposomes may be plepa~ed to correspond to the density of a particular cell type, virus, bacterium or molecule. In one embodiment, the 10 liposomes may be produced such that their buoyant density is app.o;~dmately equal to that of normal blood granulocytes, thus making the defined density liposome useful in control reagents for centrifugal hematology systems such as the QBC.
DETAILED DESCRIPTION OF TE~ INVENTION
The liposomes of the invention incorporate a density m~ lm which provides liposomes having the desired density distribution. The incorporated medium is a density medium which is coln~a~ible with the Jllelllblalle structure of the liposome, i.e., the me~lillm does not disrupt the liposome ~em~lane and there is no substantial loss of the m~ lm from 20 the liposome due to me",~ e lç~k~ge. The ~ led density media are particulate media, as salts and soluble compounds commonly used for density gradient centrifugation may be inco...pa~;hle with liposome membrane integrity and/or may leak from the liposome. The most plt;relled density me~ for incorporation comprises polyvinylpyllolidone (PVP) coated colloidal silica particles, for example PERCOLL (Pharmacia LKB Biotechnology, Uppsala, 25 Sweden). PERCOLL is cc~ nollly used to generate density gradients from 1.0-1.3 g/ml for use in purification and isolation of cells, viruses and subcellular particles. It can be made iso-osmotic and is therefore con,pa~ible with the membrane structure of the liposomes when incorporated. Without wishing to be limited by any particular structure of the defined density SUBSTITUTE SHEET (RULE 2~
WO 94/26299 ~ ~. 5 7 7 9 ~ PCT/US94/05188 llposomes, Applicant believes that the density m~ lm may be included within the lipid bilayer as well as encapsulated in the aqueous space. This belief is consistent with the fintling~ of Cudd and Nicolau, supra. The term "incorporated" and variations thereof are therefore used herein to include both encapsulated density merlillm and density me(lillm within the lipid 5 bilayer.
The liposomes of the invention may be prepared from a variety of lipids and lipid mixtures as are known in the art. Reviewed by Szoka and Papahadjopoulos (1980) Ann. Rev.
Biophys. Bioeng. 9: 467-508. Phospholipids are most often used in the pl-el)a,~lion of 10 liposomes and are ~r~felled in the present invention. l~nltil~mpll~r vesicles (MLV) are the simplest to prepare and may be produced by depositing lipids in a thin film from organic solvents by rotary evaporation under reduced pres~ e. An aqueous buffer is then added and the lipids are hydrated with agitation to induce incorporation. Vigorous agitation, brief sonication or extrusion through polycarbonate membranes may be used to produce a15 ple~ lion of MLV with a smaller and/or more UnirUllll size. Alternatively, MLV in dispersions can be reduced in size by extrusion through a small orifice under pies~u,e, such as in a French press. If small l-nil~m~ r vesicles (SUV) are desired, they may be prepared from a suspension of MLV by sonication under an inert atmosphere. SW may also be prepared by the solvent injection method. Non-incorporated material may be removed by dialysis, gel 20 filtration, c~ntrifi-~tion or a col,lbillaLion thereo Large ~lnil~m~ r vesicles (LUV) may be formed from water-in-oil emulsions of lipid and buffer in an excess organic phase, followed by removal of the organic phase under reduced pressure. This method is commonly referred to as the reverse phase evaporation technique. In 25 general, lipids are dissolved in organic solvents and the aqueous material to be incorporated is added to the lipid/solvent mixture. The p,~pa,~lion is then sonicated to form a homogeneous emulsion. The organic solvents are removed by rotary evaporation until a gel is formed.
Additional buffer is added to the gel and the evaporation vessel is vortexed to suspend the SUBSTITUTE SHEET (RULE 2~
WO 94/26299 215 ~ 7 ~ ~ PCT/US94/05188 liposomes. Rem~inin~ traces of solvent may be removed from the suspension by dialys~
column chlo,l,a~ography, a procedure which reduces the tendency of the vesicles to aggregate.
The extrusion method and the reverse phase evaporation method are prere,l~d for producing the defined density liposomes of the present invention.
After the liposomes have been produced, their size distribution may be analyzed. Many methods are known in the art for size analysis of liposomes, inclllrling gel permeation and electron microscopy. However, the simplest and prt;r~ d method for estim~tin~ the size distribution is analysis of the light scatter plope,Lies of the liposomes. Preferably, sizing is 10 done using a sub-micron particle analyzer such as the Coulter N4~ (Coulter Corporation, Hialeah, FL) which employs photon correlation spectroscopy to size particles by analyzing light intensity fl~ tions caused by the Bl ow"ian motion of the particles.
In a plert;lled embodiment of the invention, liposomes of a desired density are 15 produced by inco,~uo,~ion of a particulate density medil~m An aqueous p~ lion of the density ,llP.lill.ll, most plt;rt;lably PVP coated colloidal silica, is plepa~d such that, after addition of any other ingredients to the aqueous phase, the density of the incorporated mer1illm will be equal to or greater than the desired density of the liposome. In general, it has been found that the density of the incorporated medillm, the size of the liposomes and the 20 composition of the lipid bilayer all affect the final density. For eY~mpl~, it has been noted that increased amounts of di~lcaroyl phosphatidylglycerol (DSPG) incol~o,~ed in the lipid bilayer result in a lighter partide. The me~ m the liposome particle is in will also afect its appa, e"l buoyant density. These pa.~nelers are easily adjusted by one skilled in the art to obtain liposomes of the desired density using only routine ~ nt~tion to vary the pa~a"~elers.
Optionally, dyes may be in~l.lded in the density mellillm for incorporation to f~ilit~te detection of the liposomes. The dyes may be fluolescellL or colored dyes and are incl~lded in the density medi~lm at a fluorescent or visible conce,.l,~lion as approp,;ate. For example, SUBSTITUTE SHEET (RULE 26~
215779~
sulforhodamine G or sulforhodamine B may be included in the density mer~ m plel,a,~lion at a fluolesce~l concentration. Alternatively, lipophilic dyes may be inciuded in the lipid bilayer to f~ilit~te detection of the liposomes. Such lipophilic dyes are incorporated into the membrane bilayer upon formation of the liposome vesicle.
Liposomes incorporating the density medium may then be produced using any of theknown procedures described above as long as the procedure does not adversely affect the density m~di~lm In the pre~lled embodiment, a lipid film is swollen with an aqueous density medium prepal ~lion (with or without dye) and extruded through polycarbonate filters to obtain 10 the desired size and density distribution of liposomes. Liposome powder as described in Example 1 may be made in advance and dried for storage, allowing rapid lecon~ tion and liposome formation at a later time. The resultin~ liposome prep~lion is diluted with an aqueous buffer, centrifuged to pellet the liposomes, washed and resuspended in an aqueous buffer. If desired, the size distribution of the liposome plep~Lion may be ~stim~ted on a sub-15 micron particle analyzer, determining the size distribution of the liposomes in the plepal~lionby photon coll~,lalion spectroscopy. Electron microscopy is p.~;~lled for dete,l"il ing the siz e more accurately.
In addition to monitoring or calibrating analytical methods and instrl.",~"l~l;on based 20 on buoyant density analysis, the defined density liposomes of the invention may also be used in immllno~ ys. In this embodiment the defined density liposome is derivatized with a ligand applop,iate for the immlmoassay and is used to generate a detect~ble immllne binding reaction at a defined position or reaction area in a tube after cçntrifi-g~tion. As is known for imm~lnoaSsayS, the ligand may be a capture antibody, an antigen or a hapten noncovalently 25 associated with the liposome surface, covalently coupled to the liposome surface or intercalated into the lipid bilayer of the membrane. Exposing the derivatized defined density liposome to a fluid co,~f ~ g an analyte which is a receptor for the ligand (i.e., the SUBSTITUTE SHEET (RULE 26~
WO 94/26299 2 lS ~ ~ 9 ~ PCTIUS94/05188 corresponding antigen or antibody) allows the ligand and the analyte to bind and for( a complex associated with the defined density liposome.
In a sandwich assay format, the liposome is also exposed to a tracer conjugate 5 comprising an antibody or antigen associated with a detect~ble label and specific for the analyte. The detect~ble label may be a fluorescent compound or a colored absorbing dye. The tracer conjugate recognizes and binds to the analyte in the ligand/analyte complex on the liposome surface. Upon centrifugation, the defined density liposomes with associated analyte and tracer conjugate band at a defined position in the cçntrifi1~e tube. As unbound tracer 10 conjugate is lighter than the liposomes, detectable label above background levels ~vill be detected in the reaction area only when bound to analyte associated with the defined density liposomes. The amount of analyte may then be qu~ntit~ted by measuring fluorescence or absorbance from the detector conjugate in the reaction area. Alternatively, the immlln~assay may be performed in a competitive assay format. In this embodiment, the derivatized defined 15 density liposomes are exposed to analyte and a col"~eLing tracer conjugate. The competing tracer conjugate comprises an antigen or antibody which co",peles with the analyte for binding to the ligand-de~iv~lized liposome. Upon centrifugation, the defined density liposomes with an amount of associated tracer conjugate inversely proportional to the amount of analyte will band in the reaction area. A reduction in fluorescence or absorbance in the reaction area may 20 then be used to qu~..l;l~le the analyte. The ro,e~,oi"g centrifugal imm~lno~cs~ys using derivatized liposomes of defined density may also be pe,~""ed using other particles of defined density, for example POLYl~.EAD PMMA Monodisperse Particles, with applop,iate derivatization of the particles ~,vith antigen, antibody or hapten.
The following experimental examples are provided to illustrate certain embodiments of the invention but are not intended to limit the scope of the invention as defined by the appended claims. Variations and modifications of the invention disclosed herein will occur to those skilled in the art w`ithout departing from the spirit of the invention and without the SUBSTITUTE SHEET (RULE 26~
WO 94/26299 21~ 7 7 9 ~ PCT/US94/05188 exercise of inventive skill. These variations and modifications are intenrled to be incl~lded within the scope of the invention.
PREPARATION OF DEFINED DENSITY LIPOSOMES
Liposomes of defined density were prepared as follows: 1.88 g of lecithin, 0.206 g DSPG, 1.018 g cholesterol and 10 mg DiI C18(3) (Molecular Probes Inc., Eugene, OR) were added to a round bottom flask and dissolved in 150 ml of chloroform. The lipid film was prepared on a rotary evaporator using a 40C water bath. The film was rotated under 200 mbar of vacuum for 1 hr. The film was then swollen with 150 ml of dH2O. A tray was pre-cooled on the Iyophilizer shelf and the swollen film was poured into the tray and allowed to freeze before turning on the vacuum. The p~ l a~ion was Iyophilized over the weekend using a program in which the plep~Lion was held for 12 hr at -40C, following which the temperature was ramped up to 25 C over 8 hr. The shelf temperature was set at 15C.
Following Iyophilization, the dry powder was removed from the lyol)hili~, and scraped out of the tray. The powder was stored in two 50 ml Falcon tubes and referred to as "0.2% DiI
Powder. "
Saline iso-osmotic PERCOLL solutions were p.~)~ed at ~l~n~ities of 1.123 g/ml, 1.100 g/ml and 1.06 g/ml. The refractive index was measured using an ABBE Mark II refractometer (Reichert Scientific Instruments). The osmolality was measured using an Osmette A
instrument (Precision Systems Inc., Natick, MA). The results were as follows:
REFRACTrVE
SOLIJTION INDEX TEMP. mOsm 1.123 g/ml 1.3517 21.4C 313 1.100 g/ml 1.3481 23.0C 299 1.06g/ml 1.3425 23.0C 276 SUBSTITUTE SHEET (RUEE 2~
_ _ WO 94/26299 215 ~ ~ 9 PCT/US94/05188 .
Sixty six mg of 0.2% DiI Powder was weighed into each of three 50 ml round bottom flasks. Ten ml of the PERCOLL solutions was added to each fiask to hydrate the powder with each of the density media described above. The powders were swollen by rotating on a rotary 5 evaporator for 30 min. using a 60C water bath. The swollen mixtures were then extruded in a Lipex extruder at room temperature and passed three times through two stacked 8.0 ~m NUCLEOPORE PC membranes (Nucleopore Corp. Pleasanton CA) on a polyester drain disk. The same set of membranes was used repeatedly for each p~a~Lion of liposomes.
Three ml of the 8.0 um extruded mixture was removed and the r~?m~in~er was extruded 10 through two stacked 1.0 ~m NUCLEOPORE PC me,llb,~nes on a polyester drain disk passing through the same set of membranes three times. Three ml of the 1.0 ~m extruded mixture was removed. The r~m~in~er was extruded through two stacked 0.4 llm NUCLEOPORE PC
membranes on a polyester drain disk passing through the same set of membranes three times.
A solution co.. l~ ~.;.. g 1% PVP-10, 10 mM MOPSO pH 7.4, 1.05% NaCl (319 mOsm) was prepared ("1% PVP-MBS"). A 10X volume ofthis solution was added to each aliquot of extrusion mixture and the mixture centrifuged for 30 min. at 1500 xg. ~cer one 30 min.
centrifugation the 8.0 ~lm extruded and 1.0 llm extruded p,e~a,~Lions had pelleted. However, the 0.4 ~m extruded plepa,~ions had to be ce~trifil~ed a second time for 30 min. to pellet the 20 liposomes. The supe~,la~ s were removed and 5X the original volume of 1% PVP-MBS was added. The pellets were resuspended and centrifuged as before. A~er removing thesupt;llla~ s, the pellets were resuspended in 50 mM MOPSO pH 7.4, 20 mM EDTA 0.2%
NaN3, 1.25% glycerol. The 0.4 ~m extruded plepal~ion was resuspended in 0.5 ml and the other p~epa~Lions were resuspended in 1 ml. The liposome p,epa,~Lions were stored in the 25 refrigerator at 2-8C.
SUBYlTUTE SHEET (RULE 26 wO 94/26299 215 7 7 9 6 PCT/US94/05188 '~, This procedure provided liposome p~ tions of three different sizes incorporating O J
PERCOLL solutions of three di~lel-L den~ities i.e., nine di~erel,l cor .I)il~aLions of density and size.
ASSAYS USING DEFINED DENSITY LIPOSQMES
The nine pr~lions of liposomes described in Example 1 were mixed with aliquots of QBC control reagent without granulocytes (R&D Systems, Inc., Minneapolis, MN) and tested in venous and capillaly tubes for b~n~ing positions in the QBC instrument. Fifteen ~1 of liposomes were mixed with 450 ,ul of QBC control reagent. Each mixture was tested in duplicate in each tube type. The spun tubes were viewed under blue excitation using an Olympus BH2 microscope. I3 refers to the intçrf~ce between the granulocyte and red blood cell layers. I4 refers to the interface between the Iymphocyte and granulocyte layers. The results are ~11111111~1 l~ed in the following Table:
LIPOSOME
PREPARATION
(Densitv/Size) LIPOSOME BANDING POSlTION
1.123/8.0 Venous: Slightly below lymphocytes with sLle~l.ing into the RBC's at I3.
C9p:ll9~y: Same as above.
1.123/1.0 Venous: below Iymphocytes with a small "fragment line" between Iymphocytes and liposomes, good I3.
~sp~ ry: Bands as a granulocyte.
1.123/0.4 Venous: Slight mixing of liposomes and Iymphocytes at I4, good I3.
Capilla~y: Mixing at I4, good I3.
SUBSTIME SHEET (RUEE 26 WO 94/26299 215~ 9 ~ PCT/US94/05188 1.100/8.0 Venous: Small "fragment line" between liposomes ~nd lymphocytes. I3 slightly diffuse but not .7L~ rg.
Capillaly: Bands like a granulocyte.
1.100/1.0 Venous: Bands like a granulocyte. Good I3 and I4. Slight "fragment line" between Iymphocytes and liposomes.
Capillary: Bands like a granulocyte.
1.100/0.4 Venous: Liposomes mix with Iymphocytes at I4 and band just below.
CPri~ y: Liposomes band just below Iymphocytes and mix with them at I4.
1.060/8.0 Venous: Liposomes band below Iymphocytes and mix with them.
Capillary: Liposomes band below Iymphocytes and mix with them.
1.060/1.0 Venous: Liposomes mix with lymphocytes throughout the Iymphocyte band.
Capilla-y: Liposomes mix with Iymphocytes but are more concel.ll ~ted at the lower part of the Iymphocytes.
1.060/0.4 Venous: Liposomes mix with Iymphocytes throughout the Iymphocyte band.
Capillary: Liposomes mix with Iymphocytes but are more concelll~ ed in the upper part of othe lymphocytes, near the pl~tplp~tC
This e~y~"illlc~ll dçmon~ les the b~n~ patterns of liposomes hav~ng nine different combill~lions of incorporated rnç~ -m density and size. As the ~ ye~illlent illustrates, liposomes having a predçfined density and b~n~li~ pattern may be ylepaled for use as Ill~ke;l, in density gradients.
SUBSTITUTE SHEET (RULE 26
The complete blood cell count (CBC) is used extensively in hematological analysis and is frequently performed to obtain general information about the status of a patient. Recently, rapid methods and instn-mt?nt~tion have been developed which allow hematological analysis by centrifugation of a blood tube to provide hematocrit (HCT), platelet count (PLT), white blood 15 cell count ~WBC), and a partial differential cell count (Wardlaw and Levine. 1983. JAMA
249(5):617-620). A coll~.llelcially available ill~7llulll~ and method based on this technology is the QBC centrifugal hematology analyzer from Becton Dickinson Primary Care Diagnostics (Sparks, MD). The QBC method uses di~-~ Lial met~cl~lo~ lic fluorescence of acridine orange treated blood cells and density gradient cell l~e,ing within the buffy coat to measure 20 the sepal~led packed volumes of red blood cells, white blood cells and pl~t~let~ The QBC
instrument makes electro-optical measurements of the cell layers and computes the hematocrit, platelet count, WBC and subgroup counts of granulocytes and Iymphocytes/monocytes.
Hemoglobin concentration is derived from the hematocrit and measurements of red cell density.
To perform the QBC analysis, capillary or venous blood is placed in a glass capillary tube internally coated with acridine orange and potassium oxalate. The acridine orange stains white cells and platelets. The potassium oxalate osmotically removes water from the red cells SUBS~ITUT;E SHEET (RULE ~
WO 94/26299 . ~ l ~i 7 ~ ~ 6 PCT/US94/05188 to increase their density and improve separation from granulocytes. A float is fitted in the QBC capillary tube and settles within the buffy coat during centrifugation, thereby axially ~Yp~ntling the stained white cell and platelet layers appro~l,la~ely 10-fold.
The blood tube is centrifuged to separate the cell types into layers or bands within the tube. It is then ilhlmin~ted by blue-violet light in the QBC reader instrument to visualize the interfaces between packed and e~p~n-led red cell layers and between differentially fluorescing layers of granulocytes, lymphocytes/monocytes and platelets. Packed cell volumes and test values (numerical counts and percentages) are computed from the lengths of the five cell layers, as the length of the layer or band is a reflection of the number of cells present.
The pe,rollllance of the QBC reader and related methods such as that of Wardlaw, et al., supra, may be monitored by means of a standardized control reagent which upon centrifugation provides bands having positions in the tube and band lengths which would be expected upon centrifugation of normal and defined abnormal blood samples. Becton Dickinson Plhll~y Care Diagnostics sells such l.,age~lls under the name QBC Centrifugal ~em~tology Control. The normal and abnollllal QBC controls contain stabilized human erythrocytes, m~mm~ n leukocytes and sim~ ted platelets in a plasma-like fluid.
The present invention provides a means for gellelali,l~, particles of a desired density applupliale for a given application. The particles comprise liposomes incorporating a density medium such that the particles have the desired buoyant density. Such liposomes are useful as densit,v markers for monitoring or calibrating density gradients and centrifugal hematology instr~ ;on, as they can be prepared to have a buoyant density corresponding to various cell types, viruses, b~ct~-ri~ etc SUBSTITUTE SHEET (RULE 26 wo 94/26299 S~ PCTIUS94/05188 SU~vIMARY OF THE INVENTION ~
The present invention provides liposomes of defined density. These liposomes areproduced by incorporation of a density merii~lm at a concentration (w/v) which provides a 5 liposome of the desired density. As the density of the defined density liposomes can be easily adjusted by the practitioner, they may be used to calibrate or monitor density gradients for identification of the position of a desired band in the gradient or for marking a specific density point in a gradient. For example, defined density liposomes may be plepa~ed to correspond to the density of a particular cell type, virus, bacterium or molecule. In one embodiment, the 10 liposomes may be produced such that their buoyant density is app.o;~dmately equal to that of normal blood granulocytes, thus making the defined density liposome useful in control reagents for centrifugal hematology systems such as the QBC.
DETAILED DESCRIPTION OF TE~ INVENTION
The liposomes of the invention incorporate a density m~ lm which provides liposomes having the desired density distribution. The incorporated medium is a density medium which is coln~a~ible with the Jllelllblalle structure of the liposome, i.e., the me~lillm does not disrupt the liposome ~em~lane and there is no substantial loss of the m~ lm from 20 the liposome due to me",~ e lç~k~ge. The ~ led density media are particulate media, as salts and soluble compounds commonly used for density gradient centrifugation may be inco...pa~;hle with liposome membrane integrity and/or may leak from the liposome. The most plt;relled density me~ for incorporation comprises polyvinylpyllolidone (PVP) coated colloidal silica particles, for example PERCOLL (Pharmacia LKB Biotechnology, Uppsala, 25 Sweden). PERCOLL is cc~ nollly used to generate density gradients from 1.0-1.3 g/ml for use in purification and isolation of cells, viruses and subcellular particles. It can be made iso-osmotic and is therefore con,pa~ible with the membrane structure of the liposomes when incorporated. Without wishing to be limited by any particular structure of the defined density SUBSTITUTE SHEET (RULE 2~
WO 94/26299 ~ ~. 5 7 7 9 ~ PCT/US94/05188 llposomes, Applicant believes that the density m~ lm may be included within the lipid bilayer as well as encapsulated in the aqueous space. This belief is consistent with the fintling~ of Cudd and Nicolau, supra. The term "incorporated" and variations thereof are therefore used herein to include both encapsulated density merlillm and density me(lillm within the lipid 5 bilayer.
The liposomes of the invention may be prepared from a variety of lipids and lipid mixtures as are known in the art. Reviewed by Szoka and Papahadjopoulos (1980) Ann. Rev.
Biophys. Bioeng. 9: 467-508. Phospholipids are most often used in the pl-el)a,~lion of 10 liposomes and are ~r~felled in the present invention. l~nltil~mpll~r vesicles (MLV) are the simplest to prepare and may be produced by depositing lipids in a thin film from organic solvents by rotary evaporation under reduced pres~ e. An aqueous buffer is then added and the lipids are hydrated with agitation to induce incorporation. Vigorous agitation, brief sonication or extrusion through polycarbonate membranes may be used to produce a15 ple~ lion of MLV with a smaller and/or more UnirUllll size. Alternatively, MLV in dispersions can be reduced in size by extrusion through a small orifice under pies~u,e, such as in a French press. If small l-nil~m~ r vesicles (SUV) are desired, they may be prepared from a suspension of MLV by sonication under an inert atmosphere. SW may also be prepared by the solvent injection method. Non-incorporated material may be removed by dialysis, gel 20 filtration, c~ntrifi-~tion or a col,lbillaLion thereo Large ~lnil~m~ r vesicles (LUV) may be formed from water-in-oil emulsions of lipid and buffer in an excess organic phase, followed by removal of the organic phase under reduced pressure. This method is commonly referred to as the reverse phase evaporation technique. In 25 general, lipids are dissolved in organic solvents and the aqueous material to be incorporated is added to the lipid/solvent mixture. The p,~pa,~lion is then sonicated to form a homogeneous emulsion. The organic solvents are removed by rotary evaporation until a gel is formed.
Additional buffer is added to the gel and the evaporation vessel is vortexed to suspend the SUBSTITUTE SHEET (RULE 2~
WO 94/26299 215 ~ 7 ~ ~ PCT/US94/05188 liposomes. Rem~inin~ traces of solvent may be removed from the suspension by dialys~
column chlo,l,a~ography, a procedure which reduces the tendency of the vesicles to aggregate.
The extrusion method and the reverse phase evaporation method are prere,l~d for producing the defined density liposomes of the present invention.
After the liposomes have been produced, their size distribution may be analyzed. Many methods are known in the art for size analysis of liposomes, inclllrling gel permeation and electron microscopy. However, the simplest and prt;r~ d method for estim~tin~ the size distribution is analysis of the light scatter plope,Lies of the liposomes. Preferably, sizing is 10 done using a sub-micron particle analyzer such as the Coulter N4~ (Coulter Corporation, Hialeah, FL) which employs photon correlation spectroscopy to size particles by analyzing light intensity fl~ tions caused by the Bl ow"ian motion of the particles.
In a plert;lled embodiment of the invention, liposomes of a desired density are 15 produced by inco,~uo,~ion of a particulate density medil~m An aqueous p~ lion of the density ,llP.lill.ll, most plt;rt;lably PVP coated colloidal silica, is plepa~d such that, after addition of any other ingredients to the aqueous phase, the density of the incorporated mer1illm will be equal to or greater than the desired density of the liposome. In general, it has been found that the density of the incorporated medillm, the size of the liposomes and the 20 composition of the lipid bilayer all affect the final density. For eY~mpl~, it has been noted that increased amounts of di~lcaroyl phosphatidylglycerol (DSPG) incol~o,~ed in the lipid bilayer result in a lighter partide. The me~ m the liposome particle is in will also afect its appa, e"l buoyant density. These pa.~nelers are easily adjusted by one skilled in the art to obtain liposomes of the desired density using only routine ~ nt~tion to vary the pa~a"~elers.
Optionally, dyes may be in~l.lded in the density mellillm for incorporation to f~ilit~te detection of the liposomes. The dyes may be fluolescellL or colored dyes and are incl~lded in the density medi~lm at a fluorescent or visible conce,.l,~lion as approp,;ate. For example, SUBSTITUTE SHEET (RULE 26~
215779~
sulforhodamine G or sulforhodamine B may be included in the density mer~ m plel,a,~lion at a fluolesce~l concentration. Alternatively, lipophilic dyes may be inciuded in the lipid bilayer to f~ilit~te detection of the liposomes. Such lipophilic dyes are incorporated into the membrane bilayer upon formation of the liposome vesicle.
Liposomes incorporating the density medium may then be produced using any of theknown procedures described above as long as the procedure does not adversely affect the density m~di~lm In the pre~lled embodiment, a lipid film is swollen with an aqueous density medium prepal ~lion (with or without dye) and extruded through polycarbonate filters to obtain 10 the desired size and density distribution of liposomes. Liposome powder as described in Example 1 may be made in advance and dried for storage, allowing rapid lecon~ tion and liposome formation at a later time. The resultin~ liposome prep~lion is diluted with an aqueous buffer, centrifuged to pellet the liposomes, washed and resuspended in an aqueous buffer. If desired, the size distribution of the liposome plep~Lion may be ~stim~ted on a sub-15 micron particle analyzer, determining the size distribution of the liposomes in the plepal~lionby photon coll~,lalion spectroscopy. Electron microscopy is p.~;~lled for dete,l"il ing the siz e more accurately.
In addition to monitoring or calibrating analytical methods and instrl.",~"l~l;on based 20 on buoyant density analysis, the defined density liposomes of the invention may also be used in immllno~ ys. In this embodiment the defined density liposome is derivatized with a ligand applop,iate for the immlmoassay and is used to generate a detect~ble immllne binding reaction at a defined position or reaction area in a tube after cçntrifi-g~tion. As is known for imm~lnoaSsayS, the ligand may be a capture antibody, an antigen or a hapten noncovalently 25 associated with the liposome surface, covalently coupled to the liposome surface or intercalated into the lipid bilayer of the membrane. Exposing the derivatized defined density liposome to a fluid co,~f ~ g an analyte which is a receptor for the ligand (i.e., the SUBSTITUTE SHEET (RULE 26~
WO 94/26299 2 lS ~ ~ 9 ~ PCTIUS94/05188 corresponding antigen or antibody) allows the ligand and the analyte to bind and for( a complex associated with the defined density liposome.
In a sandwich assay format, the liposome is also exposed to a tracer conjugate 5 comprising an antibody or antigen associated with a detect~ble label and specific for the analyte. The detect~ble label may be a fluorescent compound or a colored absorbing dye. The tracer conjugate recognizes and binds to the analyte in the ligand/analyte complex on the liposome surface. Upon centrifugation, the defined density liposomes with associated analyte and tracer conjugate band at a defined position in the cçntrifi1~e tube. As unbound tracer 10 conjugate is lighter than the liposomes, detectable label above background levels ~vill be detected in the reaction area only when bound to analyte associated with the defined density liposomes. The amount of analyte may then be qu~ntit~ted by measuring fluorescence or absorbance from the detector conjugate in the reaction area. Alternatively, the immlln~assay may be performed in a competitive assay format. In this embodiment, the derivatized defined 15 density liposomes are exposed to analyte and a col"~eLing tracer conjugate. The competing tracer conjugate comprises an antigen or antibody which co",peles with the analyte for binding to the ligand-de~iv~lized liposome. Upon centrifugation, the defined density liposomes with an amount of associated tracer conjugate inversely proportional to the amount of analyte will band in the reaction area. A reduction in fluorescence or absorbance in the reaction area may 20 then be used to qu~..l;l~le the analyte. The ro,e~,oi"g centrifugal imm~lno~cs~ys using derivatized liposomes of defined density may also be pe,~""ed using other particles of defined density, for example POLYl~.EAD PMMA Monodisperse Particles, with applop,iate derivatization of the particles ~,vith antigen, antibody or hapten.
The following experimental examples are provided to illustrate certain embodiments of the invention but are not intended to limit the scope of the invention as defined by the appended claims. Variations and modifications of the invention disclosed herein will occur to those skilled in the art w`ithout departing from the spirit of the invention and without the SUBSTITUTE SHEET (RULE 26~
WO 94/26299 21~ 7 7 9 ~ PCT/US94/05188 exercise of inventive skill. These variations and modifications are intenrled to be incl~lded within the scope of the invention.
PREPARATION OF DEFINED DENSITY LIPOSOMES
Liposomes of defined density were prepared as follows: 1.88 g of lecithin, 0.206 g DSPG, 1.018 g cholesterol and 10 mg DiI C18(3) (Molecular Probes Inc., Eugene, OR) were added to a round bottom flask and dissolved in 150 ml of chloroform. The lipid film was prepared on a rotary evaporator using a 40C water bath. The film was rotated under 200 mbar of vacuum for 1 hr. The film was then swollen with 150 ml of dH2O. A tray was pre-cooled on the Iyophilizer shelf and the swollen film was poured into the tray and allowed to freeze before turning on the vacuum. The p~ l a~ion was Iyophilized over the weekend using a program in which the plep~Lion was held for 12 hr at -40C, following which the temperature was ramped up to 25 C over 8 hr. The shelf temperature was set at 15C.
Following Iyophilization, the dry powder was removed from the lyol)hili~, and scraped out of the tray. The powder was stored in two 50 ml Falcon tubes and referred to as "0.2% DiI
Powder. "
Saline iso-osmotic PERCOLL solutions were p.~)~ed at ~l~n~ities of 1.123 g/ml, 1.100 g/ml and 1.06 g/ml. The refractive index was measured using an ABBE Mark II refractometer (Reichert Scientific Instruments). The osmolality was measured using an Osmette A
instrument (Precision Systems Inc., Natick, MA). The results were as follows:
REFRACTrVE
SOLIJTION INDEX TEMP. mOsm 1.123 g/ml 1.3517 21.4C 313 1.100 g/ml 1.3481 23.0C 299 1.06g/ml 1.3425 23.0C 276 SUBSTITUTE SHEET (RUEE 2~
_ _ WO 94/26299 215 ~ ~ 9 PCT/US94/05188 .
Sixty six mg of 0.2% DiI Powder was weighed into each of three 50 ml round bottom flasks. Ten ml of the PERCOLL solutions was added to each fiask to hydrate the powder with each of the density media described above. The powders were swollen by rotating on a rotary 5 evaporator for 30 min. using a 60C water bath. The swollen mixtures were then extruded in a Lipex extruder at room temperature and passed three times through two stacked 8.0 ~m NUCLEOPORE PC membranes (Nucleopore Corp. Pleasanton CA) on a polyester drain disk. The same set of membranes was used repeatedly for each p~a~Lion of liposomes.
Three ml of the 8.0 um extruded mixture was removed and the r~?m~in~er was extruded 10 through two stacked 1.0 ~m NUCLEOPORE PC me,llb,~nes on a polyester drain disk passing through the same set of membranes three times. Three ml of the 1.0 ~m extruded mixture was removed. The r~m~in~er was extruded through two stacked 0.4 llm NUCLEOPORE PC
membranes on a polyester drain disk passing through the same set of membranes three times.
A solution co.. l~ ~.;.. g 1% PVP-10, 10 mM MOPSO pH 7.4, 1.05% NaCl (319 mOsm) was prepared ("1% PVP-MBS"). A 10X volume ofthis solution was added to each aliquot of extrusion mixture and the mixture centrifuged for 30 min. at 1500 xg. ~cer one 30 min.
centrifugation the 8.0 ~lm extruded and 1.0 llm extruded p,e~a,~Lions had pelleted. However, the 0.4 ~m extruded plepa,~ions had to be ce~trifil~ed a second time for 30 min. to pellet the 20 liposomes. The supe~,la~ s were removed and 5X the original volume of 1% PVP-MBS was added. The pellets were resuspended and centrifuged as before. A~er removing thesupt;llla~ s, the pellets were resuspended in 50 mM MOPSO pH 7.4, 20 mM EDTA 0.2%
NaN3, 1.25% glycerol. The 0.4 ~m extruded plepal~ion was resuspended in 0.5 ml and the other p~epa~Lions were resuspended in 1 ml. The liposome p,epa,~Lions were stored in the 25 refrigerator at 2-8C.
SUBYlTUTE SHEET (RULE 26 wO 94/26299 215 7 7 9 6 PCT/US94/05188 '~, This procedure provided liposome p~ tions of three different sizes incorporating O J
PERCOLL solutions of three di~lel-L den~ities i.e., nine di~erel,l cor .I)il~aLions of density and size.
ASSAYS USING DEFINED DENSITY LIPOSQMES
The nine pr~lions of liposomes described in Example 1 were mixed with aliquots of QBC control reagent without granulocytes (R&D Systems, Inc., Minneapolis, MN) and tested in venous and capillaly tubes for b~n~ing positions in the QBC instrument. Fifteen ~1 of liposomes were mixed with 450 ,ul of QBC control reagent. Each mixture was tested in duplicate in each tube type. The spun tubes were viewed under blue excitation using an Olympus BH2 microscope. I3 refers to the intçrf~ce between the granulocyte and red blood cell layers. I4 refers to the interface between the Iymphocyte and granulocyte layers. The results are ~11111111~1 l~ed in the following Table:
LIPOSOME
PREPARATION
(Densitv/Size) LIPOSOME BANDING POSlTION
1.123/8.0 Venous: Slightly below lymphocytes with sLle~l.ing into the RBC's at I3.
C9p:ll9~y: Same as above.
1.123/1.0 Venous: below Iymphocytes with a small "fragment line" between Iymphocytes and liposomes, good I3.
~sp~ ry: Bands as a granulocyte.
1.123/0.4 Venous: Slight mixing of liposomes and Iymphocytes at I4, good I3.
Capilla~y: Mixing at I4, good I3.
SUBSTIME SHEET (RUEE 26 WO 94/26299 215~ 9 ~ PCT/US94/05188 1.100/8.0 Venous: Small "fragment line" between liposomes ~nd lymphocytes. I3 slightly diffuse but not .7L~ rg.
Capillaly: Bands like a granulocyte.
1.100/1.0 Venous: Bands like a granulocyte. Good I3 and I4. Slight "fragment line" between Iymphocytes and liposomes.
Capillary: Bands like a granulocyte.
1.100/0.4 Venous: Liposomes mix with Iymphocytes at I4 and band just below.
CPri~ y: Liposomes band just below Iymphocytes and mix with them at I4.
1.060/8.0 Venous: Liposomes band below Iymphocytes and mix with them.
Capillary: Liposomes band below Iymphocytes and mix with them.
1.060/1.0 Venous: Liposomes mix with lymphocytes throughout the Iymphocyte band.
Capilla-y: Liposomes mix with Iymphocytes but are more concel.ll ~ted at the lower part of the Iymphocytes.
1.060/0.4 Venous: Liposomes mix with Iymphocytes throughout the Iymphocyte band.
Capillary: Liposomes mix with Iymphocytes but are more concelll~ ed in the upper part of othe lymphocytes, near the pl~tplp~tC
This e~y~"illlc~ll dçmon~ les the b~n~ patterns of liposomes hav~ng nine different combill~lions of incorporated rnç~ -m density and size. As the ~ ye~illlent illustrates, liposomes having a predçfined density and b~n~li~ pattern may be ylepaled for use as Ill~ke;l, in density gradients.
SUBSTITUTE SHEET (RULE 26
Claims (16)
- WHAT IS CLAIMED IS
A composition comprising liposomes of defined density, the liposomes incorporating a density medium compatible with liposome membrane structure at a concentration which provides liposomes having a selected buoyant density - 2. The composition of Claim 1 wherein the density medium is a particulate medium.
- 3 The composition of Claim 2 wherein the density medium is polyvinylpyrrolidone coated colloidal silica
- 4 The composition of Claims 1 or 3 wherein the liposomes have a buoyant density approximately equal to the buoyant density of a selected cell type
- 5. The composition of Claim 4 wherein the liposomes have a buoyant density approximately equal to the buoyant density of granulocytes
- 6 The composition of Claims 1 or 3 wherein the liposomes further comprise an incorporated dye
- 7. The composition of Claim 6 wherein the dye is a fluorescent dye
- 8. A method for monitoring the performance of density gradient centrifugation comprising:
a) centrifuging a reagent comprising liposomes of defined density such that the liposomes form a layer according to their buoyant density, the liposomes incorporating a density medium compatible with liposome membrane structure at a concentration which provides liposomes having a selected buoyant density;
b) determining the position of the liposome layer within the centrifuged controlreagent, and;
c) comparing the position of the liposome layer with a position expected for particles having similar buoyant density. - 9. The method of Claim 8 wherein liposomes incorporating a particulate density medium are centrifuged.
- 10. The method of Claim 9 wherein liposomes incorporating polyvinylpyrrolidone coated colloidal silica are centrifuged.
- 11. The method of Claim 10 wherein the liposomes form a layer having a buoyant density approximately equal to the buoyant density of a selected cell type.
- 12. The method of Claim 11 wherein the liposomes form a layer having a buoyant density approximately equal to the buoyant density of granulocytes.
- 13. The method of Claims 8 or 10 wherein the position of the liposomes in the centrifuged control reagent is determined by detecting a dye incorporated in the liposomes.
- 14. The method of Claim 13 wherein the dye is detected by detection of fluorescence.
- 15. An immmunoasay method for detecting an analyte comprising:
a) contacting the analyte with a composition comprising liposomes of defined density, the liposomes being derivatized with a ligand which is a receptor for the analyte, such that the analyte binds to the ligand to form a liposome/analyte complex;
b) centrifuging the complex such that it forms a layer according to its buoyant density, and;
c) detecting the layer as an indication of the presence of analyte by means of adetectable label associated with the complex. - 16. The method of Claim 15 wherein each one of two or more analytes forms a complex with liposomes which form separately detectable layers upon centrifugation, each liposome/analyte complex being associated with a separately detectable label.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US6164693A | 1993-05-13 | 1993-05-13 | |
| US08/061,646 | 1993-05-13 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2157796A1 true CA2157796A1 (en) | 1994-11-24 |
Family
ID=22037159
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2157796 Abandoned CA2157796A1 (en) | 1993-05-13 | 1994-05-10 | Liposomes incorporating density media |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0678032A4 (en) |
| AU (1) | AU6909594A (en) |
| CA (1) | CA2157796A1 (en) |
| WO (1) | WO1994026299A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10197480B2 (en) | 2012-11-07 | 2019-02-05 | Sandstone Diagnostics, Inc. | Methods and devices for processing samples and counting cells |
| CA2885845C (en) | 2012-11-07 | 2019-10-29 | Sandstone Diagnostics, Inc. | Methods and devices for processing samples and counting cells |
| EP2954302B1 (en) | 2013-02-07 | 2020-08-26 | Sandstone Diagnostics, Inc. | Automated sample processing, fluid distribution, and sedimentation assay |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IE50457B1 (en) * | 1979-01-18 | 1986-04-30 | Unilever Ltd | Method for detecting and determining proteinaceous specific binding agents and materials bindable thereto,test composition and test kit therefor |
| US4743560A (en) * | 1984-03-26 | 1988-05-10 | Becton Dickinson And Company | Solid phase assay |
| US4962022A (en) * | 1986-09-22 | 1990-10-09 | Becton Dickinson And Company | Storage and use of liposomes |
| US5188937A (en) * | 1989-04-06 | 1993-02-23 | Becton, Dickinson And Company | Layered sandwich assay method for chlamydia and materials therefor |
| US5369036A (en) * | 1992-07-02 | 1994-11-29 | Becton, Dickinson And Company | Enhancement of signal in immunoassays using microparticles which contain different detectable substances |
-
1994
- 1994-05-10 CA CA 2157796 patent/CA2157796A1/en not_active Abandoned
- 1994-05-10 AU AU69095/94A patent/AU6909594A/en not_active Abandoned
- 1994-05-10 EP EP94917343A patent/EP0678032A4/en not_active Withdrawn
- 1994-05-10 WO PCT/US1994/005188 patent/WO1994026299A1/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| WO1994026299A1 (en) | 1994-11-24 |
| EP0678032A4 (en) | 1997-08-20 |
| AU6909594A (en) | 1994-12-12 |
| EP0678032A1 (en) | 1995-10-25 |
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