SE459095B - Fixed biologically active materials - Google Patents
Fixed biologically active materialsInfo
- Publication number
- SE459095B SE459095B SE8104876A SE8104876A SE459095B SE 459095 B SE459095 B SE 459095B SE 8104876 A SE8104876 A SE 8104876A SE 8104876 A SE8104876 A SE 8104876A SE 459095 B SE459095 B SE 459095B
- Authority
- SE
- Sweden
- Prior art keywords
- protein
- biologically active
- silica
- receptors
- gel
- Prior art date
Links
- 239000011149 active material Substances 0.000 title abstract 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 30
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 24
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 24
- 239000002245 particle Substances 0.000 claims abstract description 9
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 8
- 239000011148 porous material Substances 0.000 claims abstract description 6
- 101710120037 Toxin CcdB Proteins 0.000 claims abstract description 3
- OGIDPMRJRNCKJF-UHFFFAOYSA-N titanium oxide Inorganic materials [Ti]=O OGIDPMRJRNCKJF-UHFFFAOYSA-N 0.000 claims description 7
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000003446 ligand Substances 0.000 claims description 5
- 108010087819 Fc receptors Proteins 0.000 claims description 3
- 102000009109 Fc receptors Human genes 0.000 claims description 3
- 108010018927 conglutinin Proteins 0.000 claims description 3
- QCUFYOBGGZSFHY-UHFFFAOYSA-N depsidomycin Chemical compound O=C1C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(NC=O)C(C)CC)C(C)OC(=O)C2CCCNN2C(=O)C(CC(C)C)NC(=O)C2CCCNN21 QCUFYOBGGZSFHY-UHFFFAOYSA-N 0.000 claims description 3
- 102000015612 Complement 3b Receptors Human genes 0.000 claims description 2
- 108010024114 Complement 3b Receptors Proteins 0.000 claims description 2
- 239000000945 filler Substances 0.000 claims description 2
- 238000001179 sorption measurement Methods 0.000 claims description 2
- 239000003547 immunosorbent Substances 0.000 claims 2
- 108010042653 IgA receptor Proteins 0.000 claims 1
- 102100034014 Prolyl 3-hydroxylase 3 Human genes 0.000 claims 1
- 108020003175 receptors Proteins 0.000 claims 1
- 102000005962 receptors Human genes 0.000 claims 1
- 108060003951 Immunoglobulin Proteins 0.000 abstract description 10
- 102000018358 immunoglobulin Human genes 0.000 abstract description 10
- 239000011521 glass Substances 0.000 abstract description 8
- 229940072221 immunoglobulins Drugs 0.000 abstract description 8
- 229940088623 biologically active substance Drugs 0.000 abstract description 3
- 239000000463 material Substances 0.000 abstract description 3
- 238000012856 packing Methods 0.000 abstract description 3
- 239000000427 antigen Substances 0.000 abstract description 2
- 108091007433 antigens Proteins 0.000 abstract description 2
- 102000036639 antigens Human genes 0.000 abstract description 2
- 230000001900 immune effect Effects 0.000 abstract description 2
- 238000012544 monitoring process Methods 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract description 2
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 abstract 1
- 239000013543 active substance Substances 0.000 abstract 1
- AZDRQVAHHNSJOQ-UHFFFAOYSA-N alumane Chemical compound [AlH3] AZDRQVAHHNSJOQ-UHFFFAOYSA-N 0.000 abstract 1
- 238000003745 diagnosis Methods 0.000 abstract 1
- 238000002955 isolation Methods 0.000 abstract 1
- 229910052710 silicon Inorganic materials 0.000 abstract 1
- 239000010703 silicon Substances 0.000 abstract 1
- 239000000741 silica gel Substances 0.000 description 13
- 229910002027 silica gel Inorganic materials 0.000 description 13
- 239000000499 gel Substances 0.000 description 10
- 150000002009 diols Chemical class 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 125000003277 amino group Chemical group 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000004593 Epoxy Substances 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- -1 C1q Proteins 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 125000003700 epoxy group Chemical group 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 3
- 239000010452 phosphate Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 125000006850 spacer group Chemical group 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- MLIWQXBKMZNZNF-KUHOPJCQSA-N (2e)-2,6-bis[(4-azidophenyl)methylidene]-4-methylcyclohexan-1-one Chemical compound O=C1\C(=C\C=2C=CC(=CC=2)N=[N+]=[N-])CC(C)CC1=CC1=CC=C(N=[N+]=[N-])C=C1 MLIWQXBKMZNZNF-KUHOPJCQSA-N 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 108010058683 Immobilized Proteins Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- PFKFTWBEEFSNDU-UHFFFAOYSA-N carbonyldiimidazole Chemical compound C1=CN=CN1C(=O)N1C=CN=C1 PFKFTWBEEFSNDU-UHFFFAOYSA-N 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- MGNCLNQXLYJVJD-UHFFFAOYSA-N cyanuric chloride Chemical compound ClC1=NC(Cl)=NC(Cl)=N1 MGNCLNQXLYJVJD-UHFFFAOYSA-N 0.000 description 1
- 230000002542 deteriorative effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- SOQBVABWOPYFQZ-UHFFFAOYSA-N oxygen(2-);titanium(4+) Chemical class [O-2].[O-2].[Ti+4] SOQBVABWOPYFQZ-UHFFFAOYSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 1
- 235000019345 sodium thiosulphate Nutrition 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 125000000446 sulfanediyl group Chemical group *S* 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 125000002088 tosyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1C([H])([H])[H])S(*)(=O)=O 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
-
- 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
- B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
- B01J20/10—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate
- B01J20/103—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate comprising silica
-
- 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
- B01J20/28002—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 physical properties
- B01J20/28004—Sorbent size or size distribution, e.g. particle size
Landscapes
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Inorganic Chemistry (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
459 095 2 Ändamålen uppnås enligt uppfinningen med den substans respektive den kolonn och det sätt som definieras i efterföljande patent- krav och som kommer att beskrivas närmare i det följande. The objects are achieved according to the invention with the substance and the column and the method, respectively, which are defined in the following claims and which will be described in more detail in the following.
Beskrivning av uppfinningen Enligt uppfinningen åstadkommas en immunoadsorbent för HPLAC bestå- ende av en oorganisk, porös bärare till vilken kovalent bundits adsorptionsspecifika ligander, kännetecknad av att bäraren består av silika med en partikelstorlek av 2-200 Pm och en porstorlek av 1000-4000 Å eller titanoxid med en partikelstorlek av 2-200 pm och en porstorlek av 500-1000 Å, och liganderna utgöres av Protein A, Protein G, Conglutinin, Clq, Fc receptorer och C3b receptorer.Description of the invention According to the invention there is provided an immunoadsorbent for HPLAC consisting of an inorganic, porous support to which covalently attached adsorption-specific ligands, characterized in that the support consists of silica with a particle size of 2-200 μm and a pore size of 1000-4000 Å or titanium oxide with a particle size of 2-200 μm and a pore size of 500-1000 Å, and the ligands are Protein A, Protein G, Conglutinin, C1q, Fc receptors and C3b receptors.
Föredragna exempel på silikamaterial enli gt uppfinningen är partik- lar av kiselgel eller glas, företrädesvis med storlekar av 5-10fnn.Preferred examples of silica materials according to the invention are particles of silica gel or glass, preferably with sizes of 5-10 microns.
Titanoxiden har företrädesvis storlekar av 20-50 pm.The titanium oxide preferably has sizes of 20-50 μm.
Uppfinningen avser även användning som fyllmedel i en HPLAC- kolonn av en immunoadsorbent enligt krav 1.The invention also relates to the use as a filler in an HPLAC column of an immunoadsorbent according to claim 1.
Uppfinningen kan användas i ett förfarande för separation av im- monoglobuliner i en vätska, i synnerhet en blodvätska, varvid vätskan ledes genom en kolonn med en immunoadsorbent enligt krav 1 med affinitet för nämnda immunoglobuliner.The invention can be used in a process for the separation of immunoglobulins in a liquid, in particular a blood liquid, wherein the liquid is passed through a column with an immunoadsorbent according to claim 1 with affinity for said immunoglobulins.
I det följande kommer uppfinningen att belysas med hjälp av kon- kreta utföringsexempel med användning av protein A (biologiskt aktiv substans) immobiliserat på föredragna exempel på bärare en- ligt uppfinningen för separation av immunoglobuliner.In the following, the invention will be elucidated by means of concrete exemplary embodiments using protein A (biologically active substance) immobilized on preferred examples of carriers according to the invention for separation of immunoglobulins.
Framställning av epoxi-kiselgel: 11,2 g kiselgel (Li Chrospher SI 4000, 10_pm, E. Merck, Darmstadt, Västtyskland) torkad vid 160°C, suspenderades i 375 ml Na-torkad toluen och 275 pl trietylamin. 8 ml V -glycidoxipropyltrimetoxi- silan tillsattes och blandningen återloppskøkades under N i b h.Preparation of epoxy silica gel: 11.2 g of silica gel (Li Chrospher SI 4000, 10 pm, E. Merck, Darmstadt, West Germany) dried at 160 ° C, suspended in 375 ml of Na-dried toluene and 275 μl of triethylamine. 8 ml of V -glycidoxypropyltrimethoxysilane were added and the mixture was refluxed under N for b h.
Den epoxisilaniserade silikan filtrerades på glasfilter och tvätta- des med aceton och eter (500 ml av vardera). Kiselgelen torkades 3 ~ 459 095 över natt vid 110°C.The epoxysilanized silica was filtered through a glass filter and washed with acetone and ether (500 ml each). The silica gel was dried 3 ~ 459 095 overnight at 110 ° C.
Koncentrationen av epoxigrupper bestämdes på känt sätt enligt den metod, som beskrivs av R. Axén et al, Acta Chem. Scand., B 29, 471-474 (1975), där 200 mg kiselgelprov suspenderades i 2 ml 3M natriumtiosulfat och 4 ml H20. De bildade hydroxijonerna titrera- des med 1 mM HCl. Mängden epoxigrupper uppskattades därvid till 13 pmol/g kiselgel (torr).The concentration of epoxy groups was determined in a known manner according to the method described by R. Axén et al, Acta Chem. Scand., B 29, 471-474 (1975), in which 200 mg of silica gel sample was suspended in 2 ml of 3M sodium thiosulfate and 4 ml of H 2 O. The hydroxy ions formed were titrated with 1 mM HCl. The amount of epoxy groups was then estimated to be 13 pmol / g silica gel (dry).
Framställning av aldehyd-kiselgel: 8,5 g epoxi-kiselgel hydrolyserades med HCl (pH 2,0, 90-9500, 2 h, 75 ml), varvid dihydroxigrupper (diol) bildades. Gelen filtrerades av på glasfilter och tvättades med H20. Diol-kiselgelen suspende- rades i 20 ml perjodatreagens (2,8 g kaliumperjodat i 100 ml H20 och 400 ml isättika). Oxidationen fick fortgå i 1 h vid 22°C.Preparation of aldehyde silica gel: 8.5 g of epoxy silica gel were hydrolyzed with HCl (pH 2.0, 90-9500, 2 hours, 75 ml) to give dihydroxy groups (diol). The gel was filtered off on a glass filter and washed with H 2 O. The diol silica gel was suspended in 20 ml of periodate reagent (2.8 g of potassium periodate in 100 ml of H 2 O and 400 ml of glacial acetic acid). The oxidation was allowed to proceed for 1 hour at 22 ° C.
Därefter tvättades aldehydgelen på glasfilter med H20 (250 ml) och torkades över natt vid QOOC.Then the aldehyde gel was washed on glass filters with H 2 O (250 mL) and dried overnight at QOOC.
Framställning av protein A kovalent immobiliserad på deaktiverad kiselgel: ~ 4 g torr aldehydkiselgel suspenderades i 9,0 ml 0,1M NaHC03-buf- fert (pH 8,3) innehållande 15,0 mg protein A. 5 mg Na(CN)BH3 till- sattes och blandningen omskakades på vippbord i 48 h (22°C).Preparation of protein A covalently immobilized on deactivated silica gel: 44 g of dry aldehyde silica gel was suspended in 9.0 ml of 0.1M NaHCO 3 buffer (pH 8.3) containing 15.0 mg of protein A. 5 mg of Na (CN) BH 3 was added and the mixture was shaken on a tilting table for 48 hours (22 ° C).
Reaktionen avslutades genom tillsats av 2 x 3 mg NaBH4 (20 min. mel- lanrum). Protein A-gelen filtrerades av och tvättades med 3 x 1 port. 1mM HCl, 0,1M NaHCO3, pH = 8,3, Och 0,5M NaCl, H20.The reaction was terminated by the addition of 2 x 3 mg NaBH 4 (20 min. Intervals). The protein A gel was filtered off and washed with 3 x 1 port. 1mM HCl, 0.1M NaHCO 3, pH = 8.3, and 0.5M NaCl, H 2 O.
Mängden immobiliserad protein A på kiselgel bestämdes genom sur hydrolys av det uppbundna proteinet med efterföljande kolorimet- risk analys med ninhydrin av hydrolysatets aminosyrainnehåll. 50 mg torr protein A-kiselgel suspenderades i 1 ml 6M HCI. Bland- ningen fick reagera vid 90-9500 i 4 h. Efter avslutad hydrolys neutraliserades blandningen till pH 5 med 6M Na0H. Till superna- tanten tillsattes 0,5 ml 0,2M citrat, pH 5,0 och 0,5 ml 2% (w/v) ninhydrin (Et0H). Lösningen inkuberades vid 90-95°C i 1 h. Den uppkomna färgutvecklingen mättes spektrofotometriskt vid 570 nm.The amount of immobilized protein A on silica gel was determined by acid hydrolysis of the bound protein with subsequent colorimetric analysis with ninhydrin of the amino acid content of the hydrolyzate. 50 mg of dry protein A silica gel was suspended in 1 ml of 6M HCl. The mixture was allowed to react at 90-9500 for 4 hours. After completion of hydrolysis, the mixture was neutralized to pH 5 with 6M NaOH. To the supernatant were added 0.5 ml of 0.2M citrate, pH 5.0 and 0.5 ml of 2% (w / v) ninhydrin (EtOH). The solution was incubated at 90-95 ° C for 1 hour. The resulting color development was measured spectrophotometrically at 570 nm.
Med denna metod bestämdes protein A-innehållet i gelen till 2,9- 3,0 mg protein A/g kiselgel (torr). 459 095 4 En referenskolonn utan protein A framställdes på analogt sätt en- ligt ovan.Using this method, the protein A content of the gel was determined to be 2.9-3.0 mg protein A / g silica gel (dry). A reference column without protein A was prepared in an analogous manner as above.
Kromatografi med protein Arsilika: Ca 2,3 g protein A-silika (i det följande benämnt pA-Si) "slurry“- packades i en kolonn ('316' rostfritt stål, 200 x 5 mm) vid 100- 200 bar tryck. Som packningsmedium användes 50 mM Na-fosfat, pH 7,5 (250 ml). Kromatograferingsutrustningen bestod av pump (Water Associates, 6000A), injektor (Waters, UGK) och detektor (UV, Waters 440A). All kromatografi utfördes vid rumstemperatur (20-2200) med ett normalt tryck av 600 p.s.i. (1 ml/min.) och injektionsvolymer 03; 0,01 -1ml.Chromatography with protein Arsilica: About 2.3 g of protein A-silica (hereinafter referred to as pA-Si) "slurry" - packed in a column ('316' stainless steel, 200 x 5 mm) at 100-200 bar pressure. The packing medium used was 50 mM Na-phosphate, pH 7.5 (250 ml) The chromatography equipment consisted of a pump (Water Associates, 6000A), an injector (Waters, UGK) and a detector (UV, Waters 440A). 20-2200) with a normal pressure of 600 psi (1 ml / min.) And injection volumes 03; 0.01 -1ml.
För att utröna funktionen hos pA- av hund-IgG på pA-Si. fat, Si gjordes ett inbindningstest En lösning av hund-IgG (0,5 mg/ml, 0,1M fos- pH 7,5) applicerades kontinuerligt vid 1,0 ml/min. Den mängd IgG som därvid adsorberades utgör ett bra mått citeten under operativa förhållande. nen av hund- på inbindningskapa- Den icke-specifika adsorptio- IgG på en referenskolonn utan pA är försumbar (< 1%) under motsvarande betingelser. Med den här frontanalystekniken upp- skattades den operativa kapaciteten hos pA-Si (2,4 g, till 29 mg IgG/g Si (torr). Återvinningen (" eluering med citrat (0,1M, 200 x 5 mm) recovery") vid step- pH 2,5) uppgick till > 95%.To ascertain the function of pA- of dog IgG on pA-Si. A solution of canine IgG (0.5 mg / ml, 0.1M phosphorus pH 7.5) was applied continuously at 1.0 ml / min. The amount of IgG adsorbed is a good measure of the operating conditions. The non-specific adsorption- IgG on a reference column without pA is negligible (<1%) under corresponding conditions. Using this frontal analysis technique, the operative capacity of pA-Si (2.4 g, was estimated to be 29 mg IgG / g Si (dry). at step pH 2.5) amounted to> 95%.
Separation av immunoglobuliner: Ett normalplasma (0,5 ml) applicerades på pA- Si-kolonnen med 0,1M fosfat, pH 7,5 (1 ml/min.) som rörlig fas.Separation of immunoglobulins: A normal plasma (0.5 ml) was applied to the pA-Si column with 0.1 M phosphate, pH 7.5 (1 ml / min.) As a mobile phase.
IgG3 och större delen av IgM och IgA (> 90%) gick oretarderat i fronten. de fraktionen på pA- dient ( Den adsorbera- Si eluerades ut med hjälp av en linjär pH-gra- 0,1M citrat) från pH 5,0 till pH 2,5. Ett karakteristiskt fraktionsmönster av immunoglobuliner (i kombination med eventuel- la antigener) erhölls. Vid analys med specifika antisera visade det sig att IgG2 eluerades vid pH 4,6, IgG1 vid pH 4,3 samt ett flertal mindre immunoglobulinfraktioner i pH-omrâdet 3,8-2,6. Vad dessa representerar är ännu inte klarlagt, möjligen rör det sig om aggregerade immunoglobuliner alternativt immunkomplex. IgG4 är ännu inte identifierad. Upplösningen är tillfredsställande vid den 5 459 095 här snabba analystiden, ca 40 minuter. Analystiden kan reduceras till ca 10-15 minuter utan att “eperformance" nämnvärt försämras.IgG3 and most of IgM and IgA (> 90%) went undefeated in the front. de fraction on pA- dient (The adsorber- Si was eluted using a linear pH-gra- 0.1M citrate) from pH 5.0 to pH 2.5. A characteristic fraction pattern of immunoglobulins (in combination with any antigens) was obtained. Analysis with specific antisera revealed that IgG2 eluted at pH 4.6, IgG1 at pH 4.3 and a number of smaller immunoglobulin fractions in the pH range 3.8-2.6. What these represent is not yet clear, possibly they are aggregated immunoglobulins or immune complexes. IgG4 has not yet been identified. The resolution is satisfactory at the fast analysis time here, about 40 minutes. The analysis time can be reduced to about 10-15 minutes without "performance" significantly deteriorating.
Beträffande alternativa kopplingsmetoder för immobiliseringen av den biologiskt aktiva substansen, exempelvis protein A, till de- aktiverad kisel eller glas hänvisas till K. Mosbach, Methods in ENZYMOLOGY, vol. XLIV, 1976. Exempel på sådana metoder kan vara 1) Direkt reaktion, där protein A binds in till matrisens epoxi- grupper via proteinets aminogrupper. 2) I ett första steg hydrolyseras epoxiglaset till motsvarande di- ol, som i sin tur bromcyanidaktiveras. Den aktiverade gelen bin- der sedan protein A:s aminogrupper. 3) Diolgelen (framställd enligt 2, ovan) kan antingen direkt eller efter perjodatoxidation tosyleras. Den tosylaktiverade gelen kan sedan reagera med protein Azs aminogrupper. 4) Diolgelen (enligt 3, ovan) kan aktiveras med karbonyldiimidazol, som sedan kopplar till amiñogrupper. 5) Diolgelen (enligt 3, ovan) kan efter aktivering med cyanurklo- rid eller derivat därav binda protein A:s aminogrupper. 6) Epoxi-, diol- eller aldehyd-(perjodatoxidation av diolgel)-ba- serad matris kan modifieras med förlängingsarmar (spacers) av olika funktionaliteter, t.ex. amino, karboxyl, tio. Dessa mat- tiser med spacers kan sedan användas för immobilisering, där ett antal etablerade kopplingsmetoder står till förfogande.For alternative coupling methods for the immobilization of the biologically active substance, for example protein A, to deactivated silicon or glass, see K. Mosbach, Methods in ENZYMOLOGY, vol. XLIV, 1976. Examples of such methods may be 1) Direct reaction, in which protein A binds to the epoxy groups of the matrix via the amino groups of the protein. 2) In a first step, the epoxy glass is hydrolyzed to the corresponding diol, which in turn is activated by bromocyanide. The activated gel then binds the amino groups of protein A. 3) The diol gel (prepared according to 2, above) can be tosylated either directly or after periodate oxidation. The tosyl activated gel can then react with the amino groups of protein Az. 4) The diol gel (according to 3, above) can be activated with carbonyldiimidazole, which then binds to amino groups. 5) The diol gel (according to 3, above) can, after activation with cyanuric chloride or derivatives thereof, bind the amino groups of protein A. 6) Epoxy, diol or aldehyde (periodate oxidation of diol gel) -based matrix can be modified with spacers of different functionalities, e.g. amino, carboxyl, thio. These mats with spacers can then be used for immobilization, where a number of established coupling methods are available.
Framställning av protein A immobiliserad på titanoxidpartiklar: 10 g porös titanoxid (1000 Å, 20-SO pm) omskakades i 100 ml 1% SnCl2 i 2 h vid 40oC. Efter avfiltrering på glasfilter tvättades bäraren med 3 x 50 ml H20. 5 g av den ovan aktiverade titanoxiden suspenderades i 0,1M Na-fosfat, pH 7,5, innehållande 25 mg pro- tein A. Blandningen omskakades på vippbord vid 35oC i 24 h. Pro- tein A-titanoxid-partiklarna filtrerades och tvättades med 3 x 1 port. 0,5M NaCl resp. H20. 459 095 6 Industriell användbarhet Uppfinningen är användbar för HBLAC-teknik i immunologiska sam- omfattar härvid såväl analy- exempelvis metoder för diag- dling av sjukdomsförlopp och ätska, isynnerhet en blod- manhang. Uppfinningens användbarhet tiska som preparativa applikationer, nostik och monitoring och/eller behan šeparering av immunoglobuliner i en v vätska, exempelvis plasma.Preparation of protein A immobilized on titanium oxide particles: 10 g of porous titanium oxide (1000 Å, 20-50 μm) was shaken in 100 ml of 1% SnCl 2 for 2 hours at 40 ° C. After filtration on a glass filter, the support was washed with 3 x 50 ml H 2 O. 5 g of the above-activated titanium oxide was suspended in 0.1M Na-phosphate, pH 7.5, containing 25 mg of protein A. The mixture was shaken on a tilting table at 35 ° C for 24 hours. The protein A-titanium oxide particles were filtered and washed. with 3 x 1 port. 0.5M NaCl resp. H20. 459 095 6 Industrial usability The invention is useful for HBLAC technology in immunological fields. This includes both analytical and, for example, methods for diagnosing the course of disease and eating, in particular a blood condition. The utility of the invention as preparative applications, nostics and monitoring and / or treatment of immunoglobulins in a liquid, for example plasma.
Claims (2)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
SE8104876A SE459095B (en) | 1981-08-17 | 1981-08-17 | Fixed biologically active materials |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
SE8104876A SE459095B (en) | 1981-08-17 | 1981-08-17 | Fixed biologically active materials |
Publications (2)
Publication Number | Publication Date |
---|---|
SE8104876L SE8104876L (en) | 1983-02-28 |
SE459095B true SE459095B (en) | 1989-06-05 |
Family
ID=20344376
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
SE8104876A SE459095B (en) | 1981-08-17 | 1981-08-17 | Fixed biologically active materials |
Country Status (1)
Country | Link |
---|---|
SE (1) | SE459095B (en) |
-
1981
- 1981-08-17 SE SE8104876A patent/SE459095B/en not_active IP Right Cessation
Also Published As
Publication number | Publication date |
---|---|
SE8104876L (en) | 1983-02-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Rodriguez et al. | Affinity chromatography: A review of trends and developments over the past 50 years | |
Robbins et al. | Purification of antibodies with immunoadsorbents prepared using bromoacetyl cellulose | |
US4672040A (en) | Magnetic particles for use in separations | |
US3652761A (en) | Immunochemical composites and antigen or antibody purification therewith | |
EP0764048B1 (en) | Chromatography adsorbents utilizing mercapto heterocyclic ligands | |
AU709290B2 (en) | IgG separation medium and novel protein A variant | |
US7326776B2 (en) | Preparation and use of mixed mode solid substrates for chromatography adsorbents and biochip arrays | |
WO1988006632A1 (en) | Magnetic particles for use in separations | |
EP0235526A2 (en) | Activated polymer solids and process for their manufacture | |
JPS6158811A (en) | Chromatography filler stabilized with metal oxide | |
Wilson et al. | The covalent coupling of proteins to periodate-oxidized sephadex: a new approach to immunoadsorbent preparation | |
Hearn et al. | Application of 1, 1′-Carbonyldiimidazone-activated agarose for the purification of proteins: II. The use of an activated matrix devoid of additional charged groups for the purification of thyroid proteins | |
JPH059440B2 (en) | ||
JPS63500002A (en) | Method for purifying biologically active substances by biologically specific adsorption and adsorbent therefor | |
US20050029196A1 (en) | Packing materials for separation of biomolecules | |
JP4443764B2 (en) | Method for immobilizing biological molecules on a support surface | |
SE466521B (en) | REAGENTS OF ANALYZE CONSISTING OF AN ENZYM AND ANOTHER LIGAND, WHICH ARE COVALENTLY BONDED TO A WATER INSULATED PARTICLE WITH A DIAMETER OF LESS THAN 500 AA | |
JP5550109B2 (en) | Method for measuring the amount of immunoglobulin in solution | |
US5112952A (en) | Composition and method for isolation and purification of Immunoglobulin M | |
Smith et al. | Cross-linked filamentous phage as an affinity matrix | |
EP0153763A2 (en) | Affinity chromatography matrix with built-in reaction indicator | |
SE459095B (en) | Fixed biologically active materials | |
Peng et al. | Stability of antibody attachment in immunosorbent chromatography | |
CA1332598C (en) | Polyethyleneimine matrixes for affinity chromatography | |
Hobbs | Solid-phase immunoassay of serum antibodies to peptides covalent antigen binding to adsorbed phenylalanine-lysine copolymers |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
NAL | Patent in force |
Ref document number: 8104876-1 Format of ref document f/p: F |
|
NUG | Patent has lapsed |
Ref document number: 8104876-1 Format of ref document f/p: F |