CA1314813C - Porous solid phases having bioaffinity, a process for preparing porous solid phases having bioaffinity, and their use - Google Patents
Porous solid phases having bioaffinity, a process for preparing porous solid phases having bioaffinity, and their useInfo
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- CA1314813C CA1314813C CA000549922A CA549922A CA1314813C CA 1314813 C CA1314813 C CA 1314813C CA 000549922 A CA000549922 A CA 000549922A CA 549922 A CA549922 A CA 549922A CA 1314813 C CA1314813 C CA 1314813C
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- bioaffinity
- particles
- porous solid
- solid phases
- porous
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28054—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their surface properties or porosity
- B01J20/28095—Shape or type of pores, voids, channels, ducts
- B01J20/28097—Shape or type of pores, voids, channels, ducts being coated, filled or plugged with specific compounds
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
- B01J20/28026—Particles within, immobilised, dispersed, entrapped in or on a matrix, e.g. a resin
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
- B01J20/28028—Particles immobilised within fibres or filaments
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
- B01J20/28033—Membrane, sheet, cloth, pad, lamellar or mat
-
- 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
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
- G01N33/525—Multi-layer analytical elements
- G01N33/526—Multi-layer analytical elements the element being adapted for a specific analyte
-
- 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
-
- 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
- G01N33/5436—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand physically entrapped within the solid phase
-
- 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
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
Abstract
BEHRINGWERKE AKTIENGESELLSCHAFT 86/B 035 - Ma 555 Dr. Ha/9n Abstract of the disclosure Porous solid phases having bioaffinity, a process for preparing porous solid phases having bioaffinity, and their use A porous solid phase which has bioaffinity and which con-tains a binding partner having bioaffinity, and a process for preparing porous solid phases which contain a binding partner having bioaffinity are described, a dispersion of particles to which the binding partner having bioaffinity is bound being incorporated into a porous material, and the dispersion agent subsequently being removed. The por-ous solid phases mentioned can be used for removal of the counterpartner corresponding to the binding partner and for detection of both the counterpartner and the binding partner.
Description
~31~
BEHRINGWERKE AKTIENGESELLSCHAFT 8b/B 035 - Ma 555 Dr. Ha/8n Porous solid phases having bioaffin;ty, a process for preparing porous solid phases having bioaffinity, and their use The invention relates to a porous solid phase having bioaffinity and to a process for preparing a porous solid phase to which one or more binding partners of a binding ; system having bioaffini$y are bound. The invention fur-thermore relates to the use of porous solid phases prep-ared in such a fashion for binding to the counter partner corresponding to the binding partner.
A large number of porous solid phases are known, to which the binding partners of binding systems hav;ng bioa~finity are bound the binding partners being enzymes, lec~ins, antigens, antibodies or partners of other systems having bioaffinity.
Porous solid phases which have binding partners having bioaffinity and in which the binding partners are bound adsorptively or covalently to tne material o-f the porous solid phases are described, for example in EPA 0,063,810 and US Patent 4,366,241.
In addition, porous solid phases exist which contain binding partners having a bioaffinity and in which latex particles to ~hich the binding partners are coupled are incorporated in film coatings (EPA 97,952 and German Offenlegungsschrift 3,329,728).
Surprisingly, it has been found that it is possible to incorporate particles to which one or more different bind-ing partners having bioaffinity are coupled into preformed materials having an isotropically porous, i.e. foamed, or anisotropically porous, i.e. fibrous, structure in a 11 3 ~
fashion such that the particles are fi%ed in these materials.
The invention relates to a porous sol;d phase which con-tains a binding partner having a bioaffinity and which can be obtained by incorporating a dispersion or suspen-sion of particles to which one or more binding partners having bioaffin;ty are bound~ into a porous material, comprising fibers, and subsequently removing the disper-sion or suspension agent.
The invention also relates to a process for preparing porous solid phases which contain a binding partner having ; a bioaffinity, wherein a dispersion or suspension of par-ticles to which one or more binding partners having a bio-affinity are bound ;s incorporated into a porous material, and the dispersion or suspension agent is subsequentLy removed~
; The invention furthermore relates to the use of such a porous solid phase which conta;ns a binding partner having a bioaffinity, ;n apparatuses for separating off and det-ecting the corresponding counterpartners having a bioaffin-ity and for detecting the binding partner in dissolved form.
; 25 The porous material can be designed in various shapes.
Examples are spheres, cylinders and flat structures, such as fleeces, membranes and papers.
The porous structures can be produced by foaming or by precipitating using a precipitation bath of solutions or dispersions of synthetic, semisynthetic or natural poly-mers or by evaporating the solvent from solutions of mem-brane~forming polymers~ and by press-molding fibers which are prepared from the polymers mentioned below.
For the preparation of porous solid phases for diagnostic agents, fleeces, papers or sponges made from celluLose or cellulose derivatives, and also membranes made from ~3~8~ ~
cellulose derivatives or polyamides are preferred.
The particles may be latex particles wh;ch are composed entirely of a hard polymer or in the core of a hard poly-S mer. Examples of such polymers are polymers which can beprepared by suspension or emulsion polymerization, such as polystyrenes or polyacrylatesn The lattices xhich have a core/shell structure with a hydrophilic shell are preferred.
The binding partners hav;ng b;oaff;n;ty are bound adsorp-tively or preferably covalently to the surface of the lattices, it being possible ~or the binding partners to be receptors, lectins, protein A of Staphylococcus aureus, protein G from Streptococcus, antigens, haptens, anti-bod;es, enzymes or inhibitors thereof.
The particLes may also be composed of stabilized cells or cell fragments.
Such cells or fragments preferably carry components which are capable of specific binding, such as, for example, re-ceptors wl1ich bind the antibodies via the Fc portion, of these, for example, protein A of Staph. aureus or protein G
of Streptococcus of group C or G, lectins, antigens, haptens, antibodies and antibodies, enzymes or inhibitors thereof wh;ch are bound via protein A. Entire cells or membrane fragments of Staph. aureus, COWAN I stra;n, to wh;ch anti-bodies are bound as described by SuWO Kessler (J. Immunol.,1975, 115, 1617-1624~, are particularly preferred.
The process according to the invention can be carried out by apply;ng dispersions and suspensions of the particles onto the porous materials described above or incorporat-ing them by dipping and subsequently removing the liquid incorporated during this by evaporation. The pore and particle sizes, and the number of particles and the volume lS,~
of the porous matrix are matched so that the l;quid flow in the matrix ~hich is necessary for the respective test structure is not hindered.
In the case of membranes, pore sizes from 0.1-12 ~m and par-ticle sizes from ~.û1~1 ~m are preferred. The particle dis-persions or suspensions applied have a concentration of 1-100 g/l. The suspension can be appl;ed to the support in point or line form by dropping-on, pipett;ng~on or with the aid of pumps and cannulars. Drying can be effected by allow-ing to lie in air, or by blowing-over air at room temperature or elevated temperature ;n open or closed systems at atmos-pheric pressure or in vacuo. The drying condit;ons, ;n par-ticuLar the thermal load permitted, are determined by the stab;lity of the active component coupled to the part;cle.
The solid phases prepared accord;ng to the ;nvention can be used as biologically active solid phases, such as, for example, enzyme, antibody, antigen or hapten solid phases.
These are preferably employed in diagnostic test elements having zones which are alongside one another, above one another or mixed alongside and above one another.
In the following Examples, the preparation of latex and cellular solid phases and their use in diagnostic test elements having function zones lying alongside one another are shown. The solid phases here are both enzyme and antibody solid phases.
The Examples should in no way be regarded as limiting, but serve merely to further illustrate the subject-matter of the invention.
Example 1 . ~
Porous solid phase containing glucose oxidase 3 ml of a dispersion containing 50 g/l of latex having ~ 3 ~
acetal groups and prepared as described in Example 2b of EPA 0,080,614 were incubated for one hour at 20C with aqueous solutions of 300 ~l of 1 normal HCl and 300 ~l of 200 g/l (~ een 20. A pH of 6.5 was then produced by adding 250 ~l of 1 normal NaOH and a saturated solution of Na2HPO4. 1.5 ml of a solution of 1 g/l of glucose oxidase having an activity of 250 U/mg (~oehringer Mann-heim, degree of purity 1r Order No. 105 139) in phosphate-buffered physiological sodium chloride solution~ pH 7.Z
(PBS) and 1.5 ml of a solution of 5 g/l of sod;um cyano-borohydride in PBS were subsequently added, and the m;x-ture was kept at 4C for 15 hours. 1.2 ml of û.5 mol/l ethanolamine, adjusted to pH 8.5 using HCl, and 0.3 ml of a solution of 25 g/L of sodium borohydride ~ere then added, and the mixture was kept at 4C for a further hour.
After centrifugation, the sediment was resuspended in PBS
~ith 2 g/l (R)Tween 20, a voLume of 15 ml of the disper-sion of latex/glucose oxidase conjugate being obtained.
Filter paper strips from Macherey und Nagel, Duren, Federal Republic of Germany, Prod. No. 215, were soaked with 40~l/
cm of the dispersion of latex/glucose oxidase conjugate, and subsequently dried at 20-50C. In the same fashion, mem-branes of cellulose coesters from Millipore, USA, Prod. No.
SCWP, were soaked with 10 ~l/cm2 of dispersion and dried.
Example 2 Antibodies against porous soLid phase containing human myoglobin 2.1 A conjugate of latex and rabbit IgG with antibodies against human myoglobin was prepared by proceeding in accordance with Example 1, with the change that 1.5 ml of a solution of 2.0 mg/mL of rabbit Ig~ with antibodies against human myoglobin was used in place of the solution of glucose oxidase. Porous solid phases were prepared as described in Example 1.
11 3~481~
2~2 A conjugate of cells of Staphylococcus aureus (COWA~
I strain) and antibodies against human myoglobin was prepared as follows:
16 mL of a solution of 2 g/l of rabbit IgG containing antibodies against human myoglobin were added to 50 ml of 100 g/l suspension of Staphylococcus aureus cells in a 0.05 mol/l aqueous ssd;um phosphate buffer solution of pH 7.4 con$a;ning 9 g/l of sod;um chlor-ide. The suspension was stirred for one hour. The cells~ charged with antibodies, were subsequently separated from the supernatant by centrifuging and decant;ng. The cells were resuspended ;n 45 ml of the abovementioned buffer and, for tovalent binding of the ant;body to the ceLls, were treated with con-stant stirr;ng w;th 50 ml of a solut;on of 2 g/l of glutaraldehyde ;n the abovement;oned buffer and, after stirring for one hour, with 50 ml of a solution of 5û g/l of sodium sulfite in the abo~ementioned buffer.
ZO
After stirring for a further hour, the cells were separ-ated from the supernatant by centr;fug;ng and decanting.
The cells ~ere washed by resuspend;ng them ;n 200 ml of 0.05 mol/l aqueous sod;um phosphate buffer of pH 7.4 and Z5 separat;ng them from the wash;ng buffer by centrifug;ng and decant;ng.
The sol;d phases were prepared by resuspend;ng 20 g/l of the cells thus treated ;n PaS and applying them 3û onto filter paper as in Example 1 and drying.
Exam Test element for determination of myoglobin, containing a porous solid phase in accordance with Example 2.1 3.1 Myoglobin peroxidase conjugate Electrophoretically uniform human myoglobin and peroxidase from Boehringer Mannheim, Order No. 413 470, was used.
N-gamma-maleimidobutyryloxysuccinimide (GM~S) ~as obtained from Behring Diagnostics and reacted with human myoglobin as described by Tanimori et al.~ 1983, in J. Imm. Meth. 62, 123-131. 2-Iminothiolane hydro-chloricle (Sigma, Cat. No. I 6256) was reacted with peroxidase as described by King et al., 1978, in Bio-chemistry 17, 1499-1506~ A conjugate was prepared, as de~r~d by Tanimori et al., from the product of the reaction of GM~S with human myoglobin and the imino-thiolane peroxidase. The crude conjugate was purified by gel chromatography on Ultrogel ACA 44 (LKB). The fraction in which about 1-2 peroxidase molecules were coupled with each molecule of human myoglobir, was used for the test. The conjugate was diluted to 100 pyrogallol units/ml with Enzygnost (R) IgE incubation medium from Behringwerke, Order No. OSD.
3.2 Preparation of the components of a test element 3.2.1 Viscose fleece containing tetramethylben~i~ine and glucose Viscose fleece having a basis weight of 140-1~0 9/m2 from Kalle, ~iesbaden, Federal Republic of Germany, was soaked with an aqueous solution containing 375 mg/l of tetramethylbenzidene dihydrochloride and 50 g/l of D-glucose, in a fashion such that the fleece was not able to take up any further solution.
The fleece was dried at 20-50C and cut into pieces of size 10x5 mm.
3~2.2 Paper containing myoglobin peroxidase conjugate and glucose oxidase A number 1 filter paper fro~ Macherey und Nagel cut into 30x5 mm pieces was soaked in 2 separa~e areas 1 3 ~
once w;th 5 ~L of a solution of myoglobin perox;dase conjugate in accordance with Example 3.1 and once with 5 ~l of a solution of 200 ~/ml of glucose oxidase, the points of application being selected so that the wetted areas of the paper were 5-6 mm apart. The filter paper was dr;ed at 20-50C.
~ 3.2.3 Paper containing antibodies against myoglobin :~' Porous solid phase in accordance with Example 2.1 was cut into 10x5 mm pieces.
:`
3.2.4 Non-impregnated paper Schleicher und Schuell (Prod. No. 2668/8) paper was cut into 20x~ wm rectangular pieces.
BEHRINGWERKE AKTIENGESELLSCHAFT 8b/B 035 - Ma 555 Dr. Ha/8n Porous solid phases having bioaffin;ty, a process for preparing porous solid phases having bioaffinity, and their use The invention relates to a porous solid phase having bioaffinity and to a process for preparing a porous solid phase to which one or more binding partners of a binding ; system having bioaffini$y are bound. The invention fur-thermore relates to the use of porous solid phases prep-ared in such a fashion for binding to the counter partner corresponding to the binding partner.
A large number of porous solid phases are known, to which the binding partners of binding systems hav;ng bioa~finity are bound the binding partners being enzymes, lec~ins, antigens, antibodies or partners of other systems having bioaffinity.
Porous solid phases which have binding partners having bioaffinity and in which the binding partners are bound adsorptively or covalently to tne material o-f the porous solid phases are described, for example in EPA 0,063,810 and US Patent 4,366,241.
In addition, porous solid phases exist which contain binding partners having a bioaffinity and in which latex particles to ~hich the binding partners are coupled are incorporated in film coatings (EPA 97,952 and German Offenlegungsschrift 3,329,728).
Surprisingly, it has been found that it is possible to incorporate particles to which one or more different bind-ing partners having bioaffinity are coupled into preformed materials having an isotropically porous, i.e. foamed, or anisotropically porous, i.e. fibrous, structure in a 11 3 ~
fashion such that the particles are fi%ed in these materials.
The invention relates to a porous sol;d phase which con-tains a binding partner having a bioaffinity and which can be obtained by incorporating a dispersion or suspen-sion of particles to which one or more binding partners having bioaffin;ty are bound~ into a porous material, comprising fibers, and subsequently removing the disper-sion or suspension agent.
The invention also relates to a process for preparing porous solid phases which contain a binding partner having ; a bioaffinity, wherein a dispersion or suspension of par-ticles to which one or more binding partners having a bio-affinity are bound ;s incorporated into a porous material, and the dispersion or suspension agent is subsequentLy removed~
; The invention furthermore relates to the use of such a porous solid phase which conta;ns a binding partner having a bioaffinity, ;n apparatuses for separating off and det-ecting the corresponding counterpartners having a bioaffin-ity and for detecting the binding partner in dissolved form.
; 25 The porous material can be designed in various shapes.
Examples are spheres, cylinders and flat structures, such as fleeces, membranes and papers.
The porous structures can be produced by foaming or by precipitating using a precipitation bath of solutions or dispersions of synthetic, semisynthetic or natural poly-mers or by evaporating the solvent from solutions of mem-brane~forming polymers~ and by press-molding fibers which are prepared from the polymers mentioned below.
For the preparation of porous solid phases for diagnostic agents, fleeces, papers or sponges made from celluLose or cellulose derivatives, and also membranes made from ~3~8~ ~
cellulose derivatives or polyamides are preferred.
The particles may be latex particles wh;ch are composed entirely of a hard polymer or in the core of a hard poly-S mer. Examples of such polymers are polymers which can beprepared by suspension or emulsion polymerization, such as polystyrenes or polyacrylatesn The lattices xhich have a core/shell structure with a hydrophilic shell are preferred.
The binding partners hav;ng b;oaff;n;ty are bound adsorp-tively or preferably covalently to the surface of the lattices, it being possible ~or the binding partners to be receptors, lectins, protein A of Staphylococcus aureus, protein G from Streptococcus, antigens, haptens, anti-bod;es, enzymes or inhibitors thereof.
The particLes may also be composed of stabilized cells or cell fragments.
Such cells or fragments preferably carry components which are capable of specific binding, such as, for example, re-ceptors wl1ich bind the antibodies via the Fc portion, of these, for example, protein A of Staph. aureus or protein G
of Streptococcus of group C or G, lectins, antigens, haptens, antibodies and antibodies, enzymes or inhibitors thereof wh;ch are bound via protein A. Entire cells or membrane fragments of Staph. aureus, COWAN I stra;n, to wh;ch anti-bodies are bound as described by SuWO Kessler (J. Immunol.,1975, 115, 1617-1624~, are particularly preferred.
The process according to the invention can be carried out by apply;ng dispersions and suspensions of the particles onto the porous materials described above or incorporat-ing them by dipping and subsequently removing the liquid incorporated during this by evaporation. The pore and particle sizes, and the number of particles and the volume lS,~
of the porous matrix are matched so that the l;quid flow in the matrix ~hich is necessary for the respective test structure is not hindered.
In the case of membranes, pore sizes from 0.1-12 ~m and par-ticle sizes from ~.û1~1 ~m are preferred. The particle dis-persions or suspensions applied have a concentration of 1-100 g/l. The suspension can be appl;ed to the support in point or line form by dropping-on, pipett;ng~on or with the aid of pumps and cannulars. Drying can be effected by allow-ing to lie in air, or by blowing-over air at room temperature or elevated temperature ;n open or closed systems at atmos-pheric pressure or in vacuo. The drying condit;ons, ;n par-ticuLar the thermal load permitted, are determined by the stab;lity of the active component coupled to the part;cle.
The solid phases prepared accord;ng to the ;nvention can be used as biologically active solid phases, such as, for example, enzyme, antibody, antigen or hapten solid phases.
These are preferably employed in diagnostic test elements having zones which are alongside one another, above one another or mixed alongside and above one another.
In the following Examples, the preparation of latex and cellular solid phases and their use in diagnostic test elements having function zones lying alongside one another are shown. The solid phases here are both enzyme and antibody solid phases.
The Examples should in no way be regarded as limiting, but serve merely to further illustrate the subject-matter of the invention.
Example 1 . ~
Porous solid phase containing glucose oxidase 3 ml of a dispersion containing 50 g/l of latex having ~ 3 ~
acetal groups and prepared as described in Example 2b of EPA 0,080,614 were incubated for one hour at 20C with aqueous solutions of 300 ~l of 1 normal HCl and 300 ~l of 200 g/l (~ een 20. A pH of 6.5 was then produced by adding 250 ~l of 1 normal NaOH and a saturated solution of Na2HPO4. 1.5 ml of a solution of 1 g/l of glucose oxidase having an activity of 250 U/mg (~oehringer Mann-heim, degree of purity 1r Order No. 105 139) in phosphate-buffered physiological sodium chloride solution~ pH 7.Z
(PBS) and 1.5 ml of a solution of 5 g/l of sod;um cyano-borohydride in PBS were subsequently added, and the m;x-ture was kept at 4C for 15 hours. 1.2 ml of û.5 mol/l ethanolamine, adjusted to pH 8.5 using HCl, and 0.3 ml of a solution of 25 g/L of sodium borohydride ~ere then added, and the mixture was kept at 4C for a further hour.
After centrifugation, the sediment was resuspended in PBS
~ith 2 g/l (R)Tween 20, a voLume of 15 ml of the disper-sion of latex/glucose oxidase conjugate being obtained.
Filter paper strips from Macherey und Nagel, Duren, Federal Republic of Germany, Prod. No. 215, were soaked with 40~l/
cm of the dispersion of latex/glucose oxidase conjugate, and subsequently dried at 20-50C. In the same fashion, mem-branes of cellulose coesters from Millipore, USA, Prod. No.
SCWP, were soaked with 10 ~l/cm2 of dispersion and dried.
Example 2 Antibodies against porous soLid phase containing human myoglobin 2.1 A conjugate of latex and rabbit IgG with antibodies against human myoglobin was prepared by proceeding in accordance with Example 1, with the change that 1.5 ml of a solution of 2.0 mg/mL of rabbit Ig~ with antibodies against human myoglobin was used in place of the solution of glucose oxidase. Porous solid phases were prepared as described in Example 1.
11 3~481~
2~2 A conjugate of cells of Staphylococcus aureus (COWA~
I strain) and antibodies against human myoglobin was prepared as follows:
16 mL of a solution of 2 g/l of rabbit IgG containing antibodies against human myoglobin were added to 50 ml of 100 g/l suspension of Staphylococcus aureus cells in a 0.05 mol/l aqueous ssd;um phosphate buffer solution of pH 7.4 con$a;ning 9 g/l of sod;um chlor-ide. The suspension was stirred for one hour. The cells~ charged with antibodies, were subsequently separated from the supernatant by centrifuging and decant;ng. The cells were resuspended ;n 45 ml of the abovementioned buffer and, for tovalent binding of the ant;body to the ceLls, were treated with con-stant stirr;ng w;th 50 ml of a solut;on of 2 g/l of glutaraldehyde ;n the abovement;oned buffer and, after stirring for one hour, with 50 ml of a solution of 5û g/l of sodium sulfite in the abo~ementioned buffer.
ZO
After stirring for a further hour, the cells were separ-ated from the supernatant by centr;fug;ng and decanting.
The cells ~ere washed by resuspend;ng them ;n 200 ml of 0.05 mol/l aqueous sod;um phosphate buffer of pH 7.4 and Z5 separat;ng them from the wash;ng buffer by centrifug;ng and decant;ng.
The sol;d phases were prepared by resuspend;ng 20 g/l of the cells thus treated ;n PaS and applying them 3û onto filter paper as in Example 1 and drying.
Exam Test element for determination of myoglobin, containing a porous solid phase in accordance with Example 2.1 3.1 Myoglobin peroxidase conjugate Electrophoretically uniform human myoglobin and peroxidase from Boehringer Mannheim, Order No. 413 470, was used.
N-gamma-maleimidobutyryloxysuccinimide (GM~S) ~as obtained from Behring Diagnostics and reacted with human myoglobin as described by Tanimori et al.~ 1983, in J. Imm. Meth. 62, 123-131. 2-Iminothiolane hydro-chloricle (Sigma, Cat. No. I 6256) was reacted with peroxidase as described by King et al., 1978, in Bio-chemistry 17, 1499-1506~ A conjugate was prepared, as de~r~d by Tanimori et al., from the product of the reaction of GM~S with human myoglobin and the imino-thiolane peroxidase. The crude conjugate was purified by gel chromatography on Ultrogel ACA 44 (LKB). The fraction in which about 1-2 peroxidase molecules were coupled with each molecule of human myoglobir, was used for the test. The conjugate was diluted to 100 pyrogallol units/ml with Enzygnost (R) IgE incubation medium from Behringwerke, Order No. OSD.
3.2 Preparation of the components of a test element 3.2.1 Viscose fleece containing tetramethylben~i~ine and glucose Viscose fleece having a basis weight of 140-1~0 9/m2 from Kalle, ~iesbaden, Federal Republic of Germany, was soaked with an aqueous solution containing 375 mg/l of tetramethylbenzidene dihydrochloride and 50 g/l of D-glucose, in a fashion such that the fleece was not able to take up any further solution.
The fleece was dried at 20-50C and cut into pieces of size 10x5 mm.
3~2.2 Paper containing myoglobin peroxidase conjugate and glucose oxidase A number 1 filter paper fro~ Macherey und Nagel cut into 30x5 mm pieces was soaked in 2 separa~e areas 1 3 ~
once w;th 5 ~L of a solution of myoglobin perox;dase conjugate in accordance with Example 3.1 and once with 5 ~l of a solution of 200 ~/ml of glucose oxidase, the points of application being selected so that the wetted areas of the paper were 5-6 mm apart. The filter paper was dr;ed at 20-50C.
~ 3.2.3 Paper containing antibodies against myoglobin :~' Porous solid phase in accordance with Example 2.1 was cut into 10x5 mm pieces.
:`
3.2.4 Non-impregnated paper Schleicher und Schuell (Prod. No. 2668/8) paper was cut into 20x~ wm rectangular pieces.
4.3 Preparation of the test element Using a double-sided adhesive tape, in each case one of the rectangular pieces was stuck successively via its narrow sides in adsorptive contact to a polyester f;lm in the following sequence:
a) Viskose fleece, in accordance with Example 3.2.1, b) Paper, in accordance with Example 3.2.2, positioned such that the area containing the glucose oxidase was adjacent to the porous solid phase, c) Porous solid phase, in accordance with Example 3.2.3, and d) Paper in accordance with Example 3.2.4 4.4 Functional testing of the test element In each case 100 ~l of a solution con~aining 10~ 100, 1,000 and 10,000 mg/ml of human myoglobin in Enzygnost (R) IgE dilution buffer were applied to the viskose fleece of separate test elements, and the color inten-sities produced on the porous solid phase after 12 to ~3~48~3 _ 9 _ 13 minutes ~ere measured using a Quelle (R)Sanoquell reflection photometer calibrated for the content of glu-cose ;n blood, and the ~ollowing results were obtained:
Myoglobin Measured signal cal;brated for ~g/ml ~mg of glucose per dl of blood 1,000 70 10,000 0 It was possible to prepare a test element which can func-tion in the same fashion with latex-bound glucose oxidase in accordance with Example 1 had been fixed as the porous solid phase, as described in Example 3.2.2, and also when the antibodies had been bound to cells of Staphylococcus aureus and fixed in the porous solid phase according to Example 2.2.
a) Viskose fleece, in accordance with Example 3.2.1, b) Paper, in accordance with Example 3.2.2, positioned such that the area containing the glucose oxidase was adjacent to the porous solid phase, c) Porous solid phase, in accordance with Example 3.2.3, and d) Paper in accordance with Example 3.2.4 4.4 Functional testing of the test element In each case 100 ~l of a solution con~aining 10~ 100, 1,000 and 10,000 mg/ml of human myoglobin in Enzygnost (R) IgE dilution buffer were applied to the viskose fleece of separate test elements, and the color inten-sities produced on the porous solid phase after 12 to ~3~48~3 _ 9 _ 13 minutes ~ere measured using a Quelle (R)Sanoquell reflection photometer calibrated for the content of glu-cose ;n blood, and the ~ollowing results were obtained:
Myoglobin Measured signal cal;brated for ~g/ml ~mg of glucose per dl of blood 1,000 70 10,000 0 It was possible to prepare a test element which can func-tion in the same fashion with latex-bound glucose oxidase in accordance with Example 1 had been fixed as the porous solid phase, as described in Example 3.2.2, and also when the antibodies had been bound to cells of Staphylococcus aureus and fixed in the porous solid phase according to Example 2.2.
Claims (13)
1. A porous solid phase comprising particles immobilized in the pores of a solid support to which one or more binding partners having bioaffinity are bound, said particles being smaller than said pores of said solid support.
2. The porous solid phase as claimed in claim 1, wherein the particles have a size of from 0.01 to 1 µm.
3. The porous solid phase as claimed in claim 1 or 2, wherein the pores have a size of from 0.1 to 12 µm.
4. A process for preparing a porous solid phase device containing a binding partner having bioaffinity, wherein a dispersion or suspension of particles to which one or more binding partners having bioaffinity are bound is incorporated into a preformed porous material, and the dispersion or suspension agent is subsequently removed.
5. The process as claimed in claim 4, wherein the porous material is a paper.
6. The process as claimed in claim 4, wherein the porous material is a membrane.
7. The process as claimed in claim 4, wherein the porous material is a fleece.
8. The process as claimed in claim 4, wherein the cavities of the porous material have been produced by gas evolution.
9. The process as claimed in claim 4, wherein the particles are latex particle
10. The process as claimed in claim 4, wherein a binding partner having a bioaffinity has been bound covalently to the particles.
11. The process as claimed in claim 4, wherein the particles have been coated with protein A from Staphylococcus aureus or are cells of Staphylococcus aureus of COWAN I strain, and immunoglobin has been bound to the particles.
12. The process as claimed in claim 4, wherein the particles have been coated with protein G from group C or G
Streptococcus, and immunoglobin has been bound to the particles,
Streptococcus, and immunoglobin has been bound to the particles,
13. The use of a porous solid phase prepared as claimed in claim 4 in apparatuses for removal and detection of the corresponding counterpartner having bioaffinity and for detection of the binding partner in dissolved form.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19863636060 DE3636060C3 (en) | 1986-10-23 | 1986-10-23 | Bioaffine porous solid phase, process for their preparation and their use |
| DEP3636060.0 | 1986-10-23 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1314813C true CA1314813C (en) | 1993-03-23 |
Family
ID=6312304
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000549922A Expired - Fee Related CA1314813C (en) | 1986-10-23 | 1987-10-22 | Porous solid phases having bioaffinity, a process for preparing porous solid phases having bioaffinity, and their use |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP0264804B1 (en) |
| JP (1) | JPS63122700A (en) |
| AT (1) | ATE92525T1 (en) |
| AU (1) | AU8004587A (en) |
| CA (1) | CA1314813C (en) |
| DE (1) | DE3786875D1 (en) |
| ES (1) | ES2059339T3 (en) |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2033938T3 (en) * | 1987-01-21 | 1993-04-01 | Abbott Laboratories | A NEUTRALIZATION TEST TO CONFIRM THE PRESENCE OF A LEAGUE IN A BIOLOGICAL SAMPLE. |
| DE3814370A1 (en) * | 1988-04-28 | 1989-11-09 | Boehringer Mannheim Gmbh | TEST PROVIDER FOR THE ANALYSIS OF A SAMPLING FLUID, METHOD FOR CARRYING OUT SUCH ANALYSIS AND METHOD OF PRODUCTION |
| EP0474858A1 (en) * | 1990-03-28 | 1992-03-18 | Tenta Properties N.V. | A method for the collection of antibodies and the detection of a specific antibody species in a fluid sample and a device for carrying out said method as well as a kit comprising said device |
| US5993935A (en) * | 1991-10-11 | 1999-11-30 | 3M Innovative Properties Company | Covalently reactive particles incorporated in a continous porous matrix |
| DE4137261C2 (en) * | 1991-11-13 | 1995-06-29 | Meinhard Prof Dr Knoll | Miniaturized sensor element for determining substance concentrations in liquids and process for its production |
| US5344701A (en) * | 1992-06-09 | 1994-09-06 | Minnesota Mining And Manufacturing Company | Porous supports having azlactone-functional surfaces |
| US5965454A (en) * | 1993-02-03 | 1999-10-12 | Histaggen Incorporated | Automated histo-cytochemistry apparatus and encapsulation system for processing biological materials |
| ATE165917T1 (en) * | 1993-02-03 | 1998-05-15 | Histaggen Inc | AUTOMATED HISTO-CYTOCHEMICAL DEVICE AND ENCAPSULATION SYSTEM FOR TREATING BIOLOGICAL MATERIAL |
| US5476665A (en) * | 1994-04-13 | 1995-12-19 | Minnesota Mining And Manufacturing Company | Azlactone functional particles incorporated in a membrane formed by solvent phase inversion |
| US5510421A (en) * | 1994-05-26 | 1996-04-23 | Minnesota Mining And Manufacturing Company | Azlactone-functional membranes and methods of preparing and using same |
| US5942442A (en) * | 1997-04-02 | 1999-08-24 | The Perkin-Elmer Corporation | Detection of low level analytes in samples using agglutination reaction capillary slide test and apparatus therefor |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4258001A (en) * | 1978-12-27 | 1981-03-24 | Eastman Kodak Company | Element, structure and method for the analysis or transport of liquids |
| US4366241A (en) * | 1980-08-07 | 1982-12-28 | Syva Company | Concentrating zone method in heterogeneous immunoassays |
| DE3269567D1 (en) * | 1981-04-29 | 1986-04-10 | Ciba Geigy Ag | New devices and kits for immunological analysis |
| JPS5977356A (en) * | 1982-06-30 | 1984-05-02 | Fuji Photo Film Co Ltd | Multilayer analysis element for fluorescent assay and fluorescent assay using the same |
| JPS5934154A (en) * | 1982-08-19 | 1984-02-24 | Konishiroku Photo Ind Co Ltd | Determination by means of immunoanalytical element |
| CA1272127A (en) * | 1985-04-04 | 1990-07-31 | Hybritech Incorporated | Solid phase system for use in ligand-receptor assays |
-
1987
- 1987-10-14 DE DE8787114994T patent/DE3786875D1/en not_active Expired - Lifetime
- 1987-10-14 ES ES87114994T patent/ES2059339T3/en not_active Expired - Lifetime
- 1987-10-14 EP EP87114994A patent/EP0264804B1/en not_active Expired - Lifetime
- 1987-10-14 AT AT87114994T patent/ATE92525T1/en not_active IP Right Cessation
- 1987-10-22 CA CA000549922A patent/CA1314813C/en not_active Expired - Fee Related
- 1987-10-22 AU AU80045/87A patent/AU8004587A/en not_active Abandoned
- 1987-10-22 JP JP62265543A patent/JPS63122700A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| JPS63122700A (en) | 1988-05-26 |
| DE3786875D1 (en) | 1993-09-09 |
| AU8004587A (en) | 1988-04-28 |
| ES2059339T3 (en) | 1994-11-16 |
| EP0264804A1 (en) | 1988-04-27 |
| EP0264804B1 (en) | 1993-08-04 |
| ATE92525T1 (en) | 1993-08-15 |
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