ES2325391B1 - PROCEDURE FOR THE COVALENT ORIENTED IMMOBILIZATION OF ANTIBODIES, ANTIBODIES SO OBTAINED AND THEIR APPLICATIONS. - Google Patents
PROCEDURE FOR THE COVALENT ORIENTED IMMOBILIZATION OF ANTIBODIES, ANTIBODIES SO OBTAINED AND THEIR APPLICATIONS. Download PDFInfo
- Publication number
- ES2325391B1 ES2325391B1 ES200701782A ES200701782A ES2325391B1 ES 2325391 B1 ES2325391 B1 ES 2325391B1 ES 200701782 A ES200701782 A ES 200701782A ES 200701782 A ES200701782 A ES 200701782A ES 2325391 B1 ES2325391 B1 ES 2325391B1
- Authority
- ES
- Spain
- Prior art keywords
- support
- antibody
- antibodies
- immobilization
- aldehyde
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 238000000034 method Methods 0.000 title claims abstract description 34
- 239000000427 antigen Substances 0.000 claims abstract description 7
- 108091007433 antigens Proteins 0.000 claims abstract description 7
- 102000036639 antigens Human genes 0.000 claims abstract description 7
- 238000001514 detection method Methods 0.000 claims abstract description 6
- 238000011138 biotechnological process Methods 0.000 claims abstract description 4
- 238000002360 preparation method Methods 0.000 claims description 8
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 7
- 125000003172 aldehyde group Chemical group 0.000 claims description 7
- 229920000936 Agarose Polymers 0.000 claims description 6
- 125000001931 aliphatic group Chemical group 0.000 claims description 6
- 239000012279 sodium borohydride Substances 0.000 claims description 6
- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 5
- 230000007935 neutral effect Effects 0.000 claims description 5
- 239000004471 Glycine Substances 0.000 claims description 4
- 125000001176 L-lysyl group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C([H])([H])C([H])([H])C([H])([H])C(N([H])[H])([H])[H] 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 4
- 239000003638 chemical reducing agent Substances 0.000 claims description 4
- 230000004913 activation Effects 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 3
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 claims description 3
- 239000002122 magnetic nanoparticle Substances 0.000 claims description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 2
- 230000002209 hydrophobic effect Effects 0.000 claims description 2
- 239000000377 silicon dioxide Substances 0.000 claims description 2
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 claims 2
- 238000001042 affinity chromatography Methods 0.000 claims 1
- 150000002433 hydrophilic molecules Chemical class 0.000 claims 1
- 238000004587 chromatography analysis Methods 0.000 abstract description 3
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 abstract description 2
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 abstract description 2
- 235000018102 proteins Nutrition 0.000 description 25
- 102000004169 proteins and genes Human genes 0.000 description 24
- 108090000623 proteins and genes Proteins 0.000 description 24
- 238000001179 sorption measurement Methods 0.000 description 10
- 102000013415 peroxidase activity proteins Human genes 0.000 description 9
- 108040007629 peroxidase activity proteins Proteins 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 239000002245 particle Substances 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 235000018977 lysine Nutrition 0.000 description 4
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 3
- 239000004793 Polystyrene Substances 0.000 description 3
- 150000001299 aldehydes Chemical class 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 229920002223 polystyrene Polymers 0.000 description 3
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 150000002009 diols Chemical group 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 125000003700 epoxy group Chemical group 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- -1 for example Substances 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 229940072221 immunoglobulins Drugs 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000002427 irreversible effect Effects 0.000 description 2
- 150000002669 lysines Chemical class 0.000 description 2
- 229920002521 macromolecule Polymers 0.000 description 2
- 239000006249 magnetic particle Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 1
- 235000019750 Crude protein Nutrition 0.000 description 1
- 239000004593 Epoxy Substances 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 244000088415 Raphanus sativus Species 0.000 description 1
- 235000006140 Raphanus sativus var sativus Nutrition 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—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
- G01N33/54326—Magnetic particles
-
- 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
- G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
-
- 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
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
- G01N33/548—Carbohydrates, e.g. dextran
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
Procedimiento para la inmovilización covalente orientada de anticuerpos, anticuerpos así obtenidos y sus aplicaciones.Procedure for covalent immobilization oriented antibodies, antibodies thus obtained and their Applications.
La invención describe un procedimiento para la inmovilización covalente orientada de anticuerpos sobre un soporte activado con grupos glioxil que dirigen la inmovilización por la región de la superficie del anticuerpo opuesta al fragmento Fab del anticuerpo. Este anticuerpo inmovilizado obtenido por el procedimiento de la invención puede ser utilizado en distintos procesos biotecnológicos como por ejemplo en cromatografía de afinidad o de detección de antígenos utilizado como inmunosensor.The invention describes a process for covalent immobilization of antibodies on a support activated with glyoxyl groups that direct immobilization by region of the antibody surface opposite the Fab fragment of the antibody. This immobilized antibody obtained by the procedure of the invention can be used in different biotechnological processes such as chromatography of affinity or antigen detection used as immunosensor
Description
Procedimiento para la inmovilización covalente orientada de anticuerpos, anticuerpos así obtenidos y sus aplicaciones.Procedure for covalent immobilization oriented antibodies, antibodies thus obtained and their Applications.
La aplicación es la preparación de columnas de afinidad o inmunosensores, con posible aplicación en Biotecnologia, Biocatálisis, Fermentación, Purificación de proteínas o enzimas Biomedicina, Medio-ambiente, Deteción preventiva de toxinas, Alimentación, Control de aguas y Bioseguridad.The application is the preparation of columns of affinity or immunosensors, with possible application in Biotechnology, Biocatalysis, Fermentation, Purification of proteins or enzymes Biomedicine, Environment, Preventive detection of toxins, food, water control and biosecurity.
La alta especificidad de los anticuerpos les ha convertido en herramientas fundamentales en procedimientos de inmunoafinidad y en el desarrollo de biosensores. No obstante, para ambas aplicaciones, los anticuerpos han de estar inmovilizados y la etapa de inmovilización puede definir las posibles aplicaciones de los anticuerpos inmovilizados (ver referencias 1-7 y 10-11).The high specificity of the antibodies has become fundamental tools in procedures immunoaffinity and in the development of biosensors. However, for both applications, the antibodies must be immobilized and the immobilization stage can define the possible applications of immobilized antibodies (see references 1-7 and 10-11).
Un sistema óptimo para inmovilizar anticuerpos para cualquiera de sus posibles aplicaciones debería de reunir una serie de requisitos, dependiendo del tipo de sistema al que se quiera aplicar:An optimal system to immobilize antibodies for any of your possible applications you should gather a series of requirements, depending on the type of system to which want to apply:
1.- La inmovilización no debe de provocar grandes distorsiones del centro de reconocimiento del anticuerpo,1.- The immobilization must not cause major distortions of the recognition center of the antibody,
2.- La zona de reconocimiento debe de estar lo más expuesta posible al medio. Esto será clave si se pretenden utilizar en el reconocimiento de bio-macromoléculas o células, o si se pretende realizar la lectura mediante la utilización de un segundo anticuerpo o biomacromolécula, ya que sólo las moléculas cuyo centro activo estén expuestas al medio serán capaces de adsorber las macromoléculas, de forma que el porcentaje de moléculas bien orientadas determinarán la velocidad de adsorción del analito (biosensores) o la capacidad de carga de la columna,2.- The recognition zone must be as exposed as possible to the environment. This will be key if they are intended use in the recognition of bio-macromolecules or cells, or if reading is intended by use of a second antibody or biomachromolecule, since Only molecules whose active center are exposed to the environment they will be able to adsorb macromolecules, so that the percentage of well oriented molecules will determine the speed of adsorption of the analyte (biosensors) or the carrying capacity of the column,
3.- La superficie final del soporte debe de ser lo más inerte posible, para evitar adsorciones inespecificas de otras moléculas sobre el soporte, de forma que compliquen los análisis finales. En columnas de bioafinidad, la adsorción inespecífica de cualquier otro compuesto reduciría drásticamente el potencial uso de este tipo de técnicas de purificación. En el caso de biosensores, una adsorción inespecífica podrá también disminuir las posibles aplicaciones del mismo, pero se debería de conseguir especialmente que no se adsorban de forma inespecífica ninguno de los componentes implicados en la lectura de la señal (p.e., anticuerpos marcados con proteínas).3.- The final surface of the support must be as inert as possible, to avoid nonspecific adsorption of other molecules on the support, so that they complicate the final analyzes In bioaffinity columns, adsorption nonspecific of any other compound would drastically reduce the potential use of this type of purification techniques. If of biosensors, a nonspecific adsorption may also decrease the possible applications of it, but it should be achieved especially that none of the components involved in the reading of the signal (e.g., protein labeled antibodies).
Recientemente, se ha descrito un protocolo que permite inmovilizar anticuerpos según estas características (5 y 6). El procedimiento está basado en la inmovilización del anticuerpo a través de las cadenas glicosiladas oxidadas con periodato: el anticuerpo se inmoviliza covalentemente sobre soportes aminados, que son capaces de adsorber proteínas por intercambio aniónico, y para evitarlo se procede a una etapa de bloqueo con dextrano-aspártico-aldehído, requiriéndose un control muy estricto de esta etapa para evitar la modificación química de las zonas de reconocimiento del anticuerpo. Este método permite recuperar anticuerpos con hasta un 60-70% de funcionabilidad y sin que el soporte adsorba proteínas de crudos de diferentes microorganismos. Sin embargo, el procedimiento es complejo cuando se pretende escalar a nivel industrial y complicado si se pretende recubrir el soporte de anticuerpos debido a la relativa lentitud de la reacción covalente azúcar oxidado-aminos primarios.Recently, a protocol has been described that allows to immobilize antibodies according to these characteristics (5 and 6). The procedure is based on the immobilization of the antibody through oxidized glycosylated chains with periodate: the antibody is covalently immobilized on amine supports, which are capable of adsorbing proteins by an anion exchange, and to avoid it we proceed to a stage of lock with dextran-aspartic-aldehyde, requiring a very strict control of this stage to avoid chemical modification of the antibody recognition zones. This method allows to recover antibodies with up to a 60-70% functionality and without the support adsorbs crude proteins from different microorganisms. Without However, the procedure is complex when it is intended to escalate to industrial level and complicated if you want to cover the support of antibodies due to the relative slowness of the covalent reaction oxidized sugar-primary aminos.
Un objeto de la invención lo constituye un procedimiento para la inmovilización covalente orientada de anticuerpos, en adelante procedimiento de la invención, basado en que se lleva a cabo sobre un soporte activado que dirigen la inmovilización por la región de la superficie del anticuerpo más rica en grupos Lys y porque comprende las siguientes etapas:An object of the invention is constituted by a procedure for covalently oriented immobilization of antibodies, hereinafter method of the invention, based on which is carried out on an activated support that direct the immobilization by the surface region of the antibody plus Rich in Lys groups and because it includes the following stages:
i) activación de un soporte con grupos aldehídos alifáticos, preferentemente grupos glioxil, yi) activation of a support with aldehyde groups aliphatic, preferably glyoxyl groups, and
ii) una reducción final de la preparación en baja concentración con un agente reductor de aldehídos o de bases schif, preferentemente con borohidruro sódico.ii) a final reduction of the preparation in low concentration with an aldehyde or base reducing agent schif, preferably with sodium borohydride.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
Otro objeto de la invención lo constituye el anticuerpo inmovilizado obtenido por el procedimiento de la invención.Another object of the invention is the immobilized antibody obtained by the procedure of the invention.
Otro objeto de la invención lo constituye el uso del anticuerpo inmovilizado en un proceso biotecnológico perteneciente al siguiente grupo: proceso de cromatografía de afinidad o de detección de antígenos utilizado como inmunosensor, por ejemplo en mezclas con partículas en suspensión, por ejemplo, membranas o células.Another object of the invention is the use of the immobilized antibody in a biotechnological process belonging to the following group: chromatography process of affinity or antigen detection used as immunosensor, for example in mixtures with suspended particles, for example, membranes or cells.
La presente invención se basa en que los inventores han observado que la inmovilización covalente orientada de un anticuerpo puede llevarse a cabo sobre un soporte activado con grupos aldehido alifaticos implicando principalmente las cadenas pesadas del anticuerpo. Más concretamente, este procedimiento de inmovilización covalente orientada está caracterizado por el uso de soportes activados con grupos aldehído alifáticos tipo glioxil que dirigen la inmovilización del anticuerpo a pH alcalino.The present invention is based on the fact that inventors have observed that covalently oriented immobilization of an antibody can be carried out on an activated support with aliphatic aldehyde groups mainly involving heavy chains of the antibody. More specifically, this oriented covalent immobilization procedure is characterized by the use of activated supports with aldehyde groups glyoxyl-type aliphatics that direct the immobilization of the antibody at alkaline pH.
Cada enlace individual amino-aldehído es muy débil (incluso considerando los grupos amino más reactivos) y el anticuerpo (u otra cualquier proteína) no se inmovilizan sobre estos soportes a pH neutro y sobre soportes poco activados. Únicamente cuando se usan soportes muy activados y se realiza la inmovilización a pH alcalino (muchas lisinas reactivas en la superficie de la proteína) se produce una inmovilización muy rápida e irreversible por unión covalente multipuntual entre las regiones más ricas en lisina de la proteína y estos soportes muy activados. Este método de inmovilización es único ya que no involucra los residuos amino más reactivos de la superficie de la proteína (como hacen todos los métodos de inmovilización irreversible vía grupos amino) sino que involucra únicamente la región o regiones más ricas en lisinas situadas en la superficie de las proteínas. Como puede observarse en la Figura 1, en el caso de las inmunoglobulinas, estas regiones ricas en lisina se encuentran en la proximidad de las cadenas glicosiladas en la región opuesta al fragmento Fab del anticuerpo. Es decir, la región más adecuada para una correcta inmovilización de anticuerpos el soporte.Each individual link amino-aldehyde is very weak (even considering the most reactive amino groups) and the antibody (or any other protein) are not immobilized on these supports at neutral pH and on poorly activated supports. Only when brackets are used highly activated and immobilization is performed at alkaline pH (many reactive lysines on the surface of the protein) a very fast and irreversible immobilization by covalent union multipuntual among the richest regions of protein lysine and these supports very activated. This method of immobilization is unique since it does not involve the most reactive amino residues of the protein surface (as do all methods of irreversible immobilization via amino groups) but involves only the region or regions richest in lysines located in the protein surface. As can be seen in Figure 1, in the case of immunoglobulins, these regions rich in lysine they are in the proximity of the glycosylated chains in the region opposite the Fab fragment of the antibody. That is, the region more suitable for a correct immobilization of antibodies the support.
En estas condiciones la inmovilización se produce por la región de la superficie de la proteína más rica en grupos lisina (Lys) capaces de reaccionar por su accesibilidad con una superficie plana (referencias 12 y 13) que se encuentran precisamente en la zona opuesta al centro de reconocimiento del anticuerpo. Este tipo de anticuerpos inmovilizados y orientados conservan su actividad biológica y adsorben muy rápidamente y específicamente sus correspondientes antígenos (ver Ejemplos). Tras reducir el anticuerpo inmovilizado (p.e. con borohidruro sódico), los aldehídos se transforman en inertes hidroxilos (referencia 13). Si se desea aumentar la hidrofilicidad o polaridad de la superficie del soporte, es posible incubar la preparación de anticuerpo inmovilizada (antes de reducir), con 1 M de glicina durante 1 hora y reducir la preparación posteriormente, quedando así la superficie del soporte, recubierta de estos aminoácidos, inerte para la adsorción inespecífica de cualquier otro tipo de proteínas (albúmina, inmunoglobulinas, extractos crudos de Escherichia coli, etc.). De esta forma estas superficies son tanto química como físicamente inertes, con los que ninguna proteína, por ejemplo, de E. coli. o suero bovino se adsorbe a las mismas en incubación a pH neutro y moderada fuerza fónica (10-100 mM fosfato sódico).Under these conditions, the immobilization is produced by the region of the surface of the protein richest in lysine groups (Lys) capable of reacting by its accessibility with a flat surface (references 12 and 13) that are precisely in the area opposite the center of antibody recognition. These types of immobilized and oriented antibodies retain their biological activity and adsorb very rapidly and specifically their corresponding antigens (see Examples). After reducing the immobilized antibody (eg with sodium borohydride), the aldehydes are transformed into inert hydroxyls (reference 13). If it is desired to increase the hydrophilicity or polarity of the surface of the support, it is possible to incubate the immobilized antibody preparation (before reducing), with 1 M glycine for 1 hour and reduce the preparation subsequently, thus leaving the surface of the support, coated of these amino acids, inert for the nonspecific adsorption of any other type of proteins (albumin, immunoglobulins, crude extracts of Escherichia coli , etc.). In this way these surfaces are both chemically and physically inert, with which no protein, for example, from E. coli . or bovine serum is adsorbed thereto in incubation at neutral pH and moderate phonic strength (10-100 mM sodium phosphate).
Este procedimiento de inmovilización se puede llevar a cabo sobre todo tipo de soportes sólidos pre-existentes y para todo tipo de aplicaciones de anticuerpos inmovilizados. Por un lado, la inmovilización de anticuerpos sobre geles de agarosa es útil para la preparación de soportes de inmunoafinidad para la adsorción selectiva de enzimas y proteínas. Por otro lado, la inmovilización de anticuerpos sobre partículas magnéticas sería muy útil para la detección de trazas de células, proteínas y cualquier analitos de interés presentes en mezclas complejas (sangre, alimentos, aguas residuales, etc.).This immobilization procedure can be carry out on all types of solid supports pre-existing and for all types of applications immobilized antibodies On the one hand, the immobilization of antibodies on agarose gels is useful for the preparation of immunoaffinity supports for selective adsorption of enzymes and proteins On the other hand, the immobilization of antibodies on Magnetic particles would be very useful for trace detection of cells, proteins and any analytes of interest present in complex mixtures (blood, food, sewage, etc.).
Así, un objeto de la invención lo constituye un procedimiento para la inmovilización covalente orientada de anticuerpos, en adelante procedimiento de la invención, basado en que se lleva a cabo sobre un soporte activado que dirigen la inmovilización por la región de la superficie del anticuerpo más rica en grupos Lys y porque comprende las siguientes etapas:Thus, an object of the invention constitutes a procedure for covalently oriented immobilization of antibodies, hereinafter method of the invention, based on which is carried out on an activated support that direct the immobilization by the surface region of the antibody plus Rich in Lys groups and because it includes the following stages:
i) activación de un soporte con grupos aldehídos alifáticos, preferentemente grupos glioxil, yi) activation of a support with aldehyde groups aliphatic, preferably glyoxyl groups, and
ii) una reducción final de la preparación en baja concentración con un agente reductor de aldehidos o de bases schif, preferentemente con borohidruro sódico.ii) a final reduction of the preparation in low concentration with an aldehyde or base reducing agent schif, preferably with sodium borohydride.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
Por otro lado, cuando el soporte a utilizar es hidrofóbico, por ejemplo, partículas magnéticas de poliestireno, el procedimiento de la invención debe someterse a una etapa intermedia a i) y ii) de hidrofilización de la superficie del soporte mediante la incubación del anticuerpo inmovilizado con un compuesto hidrofílico con un grupo amino primario, preferentemente con carga neta 0 a pH neutro (e.g., gly, mezclas Lys/Asp, etc.)On the other hand, when the support to be used is hydrophobic, for example, polystyrene magnetic particles, the procedure of the invention must undergo an intermediate stage a i) and ii) hydrophilization of the surface of the support by incubation of the immobilized antibody with a compound hydrophilic with a primary amino group, preferably charged net 0 at neutral pH (e.g., gly, Lys / Asp mixtures, etc.)
Un objeto particular de la invención lo constituye el procedimiento de invención en el que el soporte utilizado es poroso, por ejemplo, agarosa, vidrio, silica o epoxi-acrílico; o no porosa, como por ejemplo una nanopartícula magnética no porosa.A particular object of the invention is constitutes the process of invention in which the support used is porous, for example, agarose, glass, silica or epoxy acrylic; or not porous, such as a non-porous magnetic nanoparticle.
Otro objeto de la invención lo constituye el anticuerpo inmovilizado obtenido por el procedimiento de la invención.Another object of the invention is the immobilized antibody obtained by the procedure of the invention.
Otro objeto de la invención lo constituye el uso del anticuerpo inmovilizado en un proceso biotecnológico perteneciente al siguiente grupo: proceso de cromatografía de afinidad o de detección de antígenos utilizado como inmunosensor, por ejemplo en mezclas con partículas en suspensión, por ejemplo, membranas o células.Another object of the invention is the use of the immobilized antibody in a biotechnological process belonging to the following group: chromatography process of affinity or antigen detection used as immunosensor, for example in mixtures with suspended particles, for example, membranes or cells.
Figura 1.- Imágenes de IgG (obtenida con el programa PyMOL v0.99) a partir del fichero 1IGY correspondiente al anticuerpo anti-peroxidasa obtenido en conejo y depositado en el banco de datos de proteínas (PDB). En la Figura 1A se observa una visión vertical de la IgG con las cadenas pesadas coloreadas en verde y las cadenas ligeras coloreadas en naranja. Las regiones de reconocimiento del antígeno (Fab) se encuentran en las partes superiores derecha e izquierda (en el extremo de la intersección entre cadenas ligeras y pesadas). En la Figura 2 se observa la región con mayor densidad en grupos lisina, coloreados en color azul, (la región que reacciona con los soportes glioxil). Desde esta perspectiva se observan muy próximas las cadenas glicosiladas (coloreadas de amarillo) y ya descritas como buena orientación para la inmovilización de IgG. Desde esta visión no se observan las cadenas ligeras confirmándose que efectivamente se está intentando inmovilizar el anticuerpo por una región muy alejada de la región Fab y, por tanto, con una orientación muy adecuada para un posterior reconocimiento de antígenos sobre los anticuerpos inmovilizados.Figure 1.- Images of IgG (obtained with the PyMOL v0.99 program) from the 1IGY file corresponding to the anti-peroxidase antibody obtained in rabbit and deposited in the protein data bank (PDB) . Figure 1A shows a vertical view of IgG with heavy chains colored in green and light chains colored in orange. The antigen recognition regions (Fab) are found in the upper right and left parts (at the end of the intersection between light and heavy chains). Figure 2 shows the region with the highest density in lysine groups, colored in blue, (the region that reacts with glyoxyl supports). From this perspective, the glycosylated chains (colored yellow) and already described as a good orientation for the immobilization of IgG are very close. From this view, the light chains are not confirmed confirming that it is actually trying to immobilize the antibody by a region very far from the Fab region and, therefore, with a very suitable orientation for a subsequent recognition of antigens on immobilized antibodies.
10 mg de anticuerpo anti-peroxidasa se disolvieron en 100 mL de tampón bicarbonato a pH 10 y 4ºC. A continuación se añadieron 10 g de glioxil agarosa. Tras 3 horas bajo agitación suave, todo el anticuerpo se había inmovilizado. En ese momento se añadieron 100 mg de borohidruro sódico. Tras 30 minutos, el derivado se lavó abundantemente con tampón pH 7 y agua destilada.10 mg of antibody anti-peroxidase was dissolved in 100 mL of buffer bicarbonate at pH 10 and 4 ° C. Then 10 g of glyoxyl agarose. After 3 hours under gentle agitation, all the antibody had been immobilized. At that time 100 were added mg of sodium borohydride. After 30 minutes, the derivative was washed abundantly with buffer pH 7 and distilled water.
A continuación, a 1 g de este anticuerpo se ofreció 10 ml de un solución que contenía 100 mg de un extracto de proteínas de E. coli conteniendo 0.2 mg de peroxidasa de rábano en fosfato 10 mM a pH 7. Tras 1 hora, con agitación continua, el 100% de la actividad peroxidasa había sido adsorbida por el soporte. En un experimento similar en el que el anticuerpo había sido sustituido por 1 g de glioxil-agarosa reducida, no se apreció que la peroxidasa se adsorbiera al soporte. A continuación, se recuperó el anticuerpo inmovilizado con la peroxidasa adsorbida, se lavó abundantemente con agua y se sometió a una electroforesis en SDS, las únicas bandas visibles correspondieron a la cadena ligera del anticuerpo y a la peroxida.Then, 1 ml of this antibody was offered 10 ml of a solution containing 100 mg of an extract of E. coli proteins containing 0.2 mg of horseradish peroxidase in 10 mM phosphate at pH 7. After 1 hour, with stirring Continuous, 100% of the peroxidase activity had been adsorbed by the support. In a similar experiment in which the antibody had been replaced by 1 g of reduced glyoxyl agarose, it was not appreciated that the peroxidase adsorbed to the support. Then, the immobilized antibody was recovered with the adsorbed peroxidase, washed thoroughly with water and subjected to an SDS electrophoresis, the only visible bands corresponded to the antibody light chain and peroxidase.
De esta forma se confirmó que el anticuerpo era plenamente funcional y que no se producía ninguna adsorción inespecífica de proteínas sobre los mismos.In this way it was confirmed that the antibody was fully functional and that no adsorption occurred nonspecific protein on them.
1 g de nano-partículas magnéticas de poliestireno conteniendo grupos epóxido se incubó durante 12 horas en 100 ml de ácido sulfúrico 250 mM para transformar los grupos epóxido en grupos diol. Las partículas se lavaron repetidas veces con agua destilada hasta que el pH era neutro, y se añadió 100 mL de periodato sódico 10 mM para generar grupos aldehido por oxidación de los dioles.1 g of nano-particles Polystyrene magnetic containing epoxide groups was incubated for 12 hours in 100 ml of 250 mM sulfuric acid for transform epoxy groups into diol groups. The particles are repeatedly washed with distilled water until the pH was neutral, and 100 mL of 10 mM sodium periodate was added to generate aldehyde groups by oxidation of the diols.
50 mg de anti-peroxidasa se disolvieron en 100 mL de tampón bicarbonato a pH 10 y 4ºC. A continuación, se añadieron 1 g de nanopartículas-aldehído. Tras 6 horas bajo agitación continua, todo el anticuerpo se había inmovilizado. En ese momento, se eliminó el sobrenadante y se resuspendió durante 1 hora en 100 mL de 1 M de glicina. Pasado este tiempo, se añadieron 100 mg de borohidruro sódico. Tras 30 minutos, el derivado se lavó abundantemente con tampón pH 7 y agua destilada.50 mg of anti-peroxidase is dissolved in 100 mL of bicarbonate buffer at pH 10 and 4 ° C. TO then 1 g of nanoparticles-aldehyde. After 6 hours low continuous stirring, all antibody had been immobilized. In that time, the supernatant was removed and resuspended for 1 hour in 100 mL of 1 M glycine. After this time, they were added 100 mg of sodium borohydride. After 30 minutes, the derivative was washed abundantly with buffer pH 7 and distilled water.
A continuación, a 10 mg de este anticuerpo inmovilizado se ofreció 100 ml de un solución que contenía 100 mg de un extracto de proteínas de E. coli conteniendo 1 mg de peroxidasa de rábano en fosfato 50 mM a pH 7. Tras 1h, el 100% de la actividad peroxidasa había sido adsorbida por el soporte. En un experimento similar en el que el anticuerpo inmovilizado había sido sustituido por 10 mg de partículas aldehido-glicina reducidas no se apreció que la peroxidasa se adsorbiera al soporte. A continuación, se recuperó el anticuerpo inmovilizado con la peroxidasa adsorbida, se lavó abundantemente con agua y se sometió a una electroforesis en SDS, las únicas bandas visibles correspondieron a la cadena ligera del anticuerpo y a la peroxidasa.Then, at 10 mg of this immobilized antibody, 100 ml of a solution containing 100 mg of an extract of E. coli proteins containing 1 mg of 50 mM phosphate radish peroxidase at pH 7 was offered. After 1 h, 100 % of the peroxidase activity had been adsorbed by the support. In a similar experiment in which the immobilized antibody had been replaced by 10 mg of reduced aldehyde-glycine particles it was not seen that the peroxidase adsorbed to the support. Then, the immobilized antibody was recovered with the adsorbed peroxidase, washed thoroughly with water and subjected to an SDS electrophoresis, the only visible bands corresponded to the antibody light chain and peroxidase.
De esta forma se confirmó que el anticuerpo era plenamente funcional y que no se producía ninguna adsorción inespecífica de proteínas sobre el soporte.In this way it was confirmed that the antibody was fully functional and that no adsorption occurred non-specific protein on the support.
1.- Ahluwalia A., DeRossi D., Schirone A., Serra C.. 1991. A comparative study of protein immobilization techniques for optical immunosensors. Biosens. Bioelectron. 7: 207-214.1.- Ahluwalia A., DeRossi D., Schirone A., Serra C .. 1991 . A comparative study of protein immobilization techniques for optical immunosensors. Biosens Bioelectron 7: 207-214.
2.- Anderson G.P., Jacoby M.A., Ligler F.S., King K.D.. 1997. Effectiveness of protein A for antibody immobilization for a fiber optic biosensor. Biosens. Bioelectron 12: 39-336.2.- Anderson GP, Jacoby MA, Ligler FS, King KD. 1997 Effectiveness of protein A for antibody immobilization for a fiber optic biosensor. Biosens Bioelectron 12: 39-336.
3.- Babacam S, Pivarnik P., Lecther S., Rand AG, 2000. Evaluation of antibody immobilization methods for piezoelectric biosensor application. Biosens. Bioelectron. 15: 615-6213.- Babacam S, Pivarnik P., Lecther S., Rand AG, 2000 . Evaluation of antibody immobilization methods for piezoelectric biosensor application. Biosens Bioelectron 15: 615-621
4.- Danczyk R., Krieder B., North T., Webster T., Hogenesch H., Rundell A. 2003 Comparison of antibody functionality using different immobilization methods. Biotechnol. Bioeng. 84: 215-223.4.- Danczyk R., Krieder B., North T., Webster T., Hogenesch H., Rundell A. 2003 Comparison of antibody functionality using different immobilization methods. Biotechnol Bioeng 84: 215-223.
5.- Fuentes, Manuel, Cesar Mateo, J.M. Guisán* and Roberto Fernández-Lafuente 2005 "PREPARATION OF INERT MAGNETIC NANO-PARTICLES FOR THE DIRECTED IMMOBILIZATION OF ANTIBODIES" Biosen. Bioelec. 20: 1380-13875.- Fuentes , Manuel, Cesar Mateo , JM Guisán * and Roberto Fernández-Lafuente 2005 "PREPARATION OF INERT MAGNETIC NANO-PARTICLES FOR THE DIRECTED IMMOBILIZATION OF ANTIBODIES" Biosen. Bioelec 20: 1380-1387
6.- Fuentes, García, Manuel, César Mateo González, Roberto Fernández Lafuente, Jose Manuel Guisán Seijas, Carmen Force Redondo, Ignacio Rodríguez Galván, Pedro Tartaj Salvador, Carlos Serna Pereda "Inmovilización de anticuerpos en partículas magnéticas para detección de trazas de proteínas en fluidos biológicos" Patente española 2002027516.- Fuentes, García , Manuel, César Mateo González , Roberto Fernández Lafuente , Jose Manuel Guisán Seijas , Carmen Force Redondo , Ignacio Rodríguez Galván , Pedro Tartaj Salvador , Carlos Serna Pereda "Immobilization of antibodies in magnetic particles for detection of protein traces in biological fluids "Spanish Patent 200202751
7.- Lu B, Smy MR, O'Kennedy R., 1996. Oriented immobilization of antibodies and its application in immunoassays and immunosensors. Analyst 121: 29R-32R.7.- Lu B, Smy MR, O'Kennedy R., 1996 . Oriented immobilization of antibodies and its application in immunoassays and immunosensors. Analyst 121: 29R-32R.
8.- Mateo, C. Fernández-Lorente G., Abian O.; Fernández-Lafuente R., Guisán J.M.. 2000. Multifunctional epoxy- supports. A new tool to improve the covalent immobilization of proteins: the promotion of physical adsorption of proteins: the promotion of physical adsorptions of proteins on the supports before their covalent linkage. Biomacromolecules 1: 739-745.8.- Mateo , C. Fernández-Lorente G., Abian O .; Fernández-Lafuente R., Guisán JM. 2000 Multifunctional epoxy- supports. A new tool to improve the covalent immobilization of proteins: the promotion of physical adsorption of proteins: the promotion of physical adsorptions of proteins on the supports before their covalent linkage. Biomacromolecules 1: 739-745.
9.- Mateo C., Torres R., Fernández G., Ortiz C., Fuentes M., Hidalgo A., Lopez-Gallego F., Betancor L., Pessela B.C.C., Aminati A., Fernández-Lafuente R., Guisán J.M.. 2003. Epoxy-amino sepabeads: a new support for immobilization of proteins under mild conditions. Biomacromolecules 4: 772-777.9.- Mateo C., Torres R., Fernández G., Ortiz C., Fuentes M., Hidalgo A., Lopez -Gallego F., Betancor L., Pessela BCC, Aminati A., Fernández -Lafuente R., Guisán JM. 2003 Epoxy-amino sepabeads: a new support for immobilization of proteins under mild conditions. Biomacromolecules 4: 772-777.
10.- Weetall H H, Lee M J. 1989. Antibodies immobilized on Inorganic supports. Appl Biochem. Biotech. 22: 311-330.10.- Weetall HH, Lee M J. 1989 . Antibodies immobilized on inorganic supports. Appl Biochem. Biotech 22: 311-330.
11.- Zull JE, Reed-Mundell J, Lee YW, Vezenov D, Ziats NP, Anderson Jm, Sukenik CN, 1994. Problems and approaches in covalent attachment of peptides and proteins to inorganic surfaces for biosensors applications. J. Indust. Microbiol. 13: 137-143.11.- Zull JE, Reed -Mundell J, Lee YW, Vezenov D, Ziats NP, Anderson Jm, Sukenik CN, 1994 . Problems and approaches in covalent attachment of peptides and proteins to inorganic surfaces for biosensors applications. J. Indust. Microbiol 13: 137-143.
12.- Mateo, C., Abian, O., Bernedo, M., et al 2005 "SOME SPECIAL FEATURES OF GLYOXYL SUPPORTS TO IMMOBILIZE PROTEINS" Enzyme Microb. Technol. 37, 456-462.12.- Mateo , C., Abian , O., Bernedo , M., et al 2005 "SOME SPECIAL FEATURES OF GLYOXYL SUPPORTS TO IMMOBILIZE PROTEINS" Enzyme Microb. Technol 37, 456-462.
13.- Mateo, C., Palomo, J.M., Fuentes, M. et al 2006 "Glyoxyl-agarose: a fully inert hydrophilic support for immobilization and high stabilization of proteins" Enzyme Microb. Technol. 39. 274-280.13.- Mateo , C., Palomo , JM, Fuentes , M. et al 2006 "Glyoxyl-agarose: a fully inert hydrophilic support for immobilization and high stabilization of proteins" Enzyme Microb. Technol 39. 274-280.
Claims (9)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ES200701782A ES2325391B1 (en) | 2007-06-26 | 2007-06-26 | PROCEDURE FOR THE COVALENT ORIENTED IMMOBILIZATION OF ANTIBODIES, ANTIBODIES SO OBTAINED AND THEIR APPLICATIONS. |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ES200701782A ES2325391B1 (en) | 2007-06-26 | 2007-06-26 | PROCEDURE FOR THE COVALENT ORIENTED IMMOBILIZATION OF ANTIBODIES, ANTIBODIES SO OBTAINED AND THEIR APPLICATIONS. |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| ES2325391A1 ES2325391A1 (en) | 2009-09-02 |
| ES2325391B1 true ES2325391B1 (en) | 2010-06-17 |
Family
ID=41044982
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| ES200701782A Expired - Fee Related ES2325391B1 (en) | 2007-06-26 | 2007-06-26 | PROCEDURE FOR THE COVALENT ORIENTED IMMOBILIZATION OF ANTIBODIES, ANTIBODIES SO OBTAINED AND THEIR APPLICATIONS. |
Country Status (1)
| Country | Link |
|---|---|
| ES (1) | ES2325391B1 (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002033412A1 (en) * | 2000-10-14 | 2002-04-25 | Macrogen Inc. | Bio-support and preparing method of the same |
-
2007
- 2007-06-26 ES ES200701782A patent/ES2325391B1/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002033412A1 (en) * | 2000-10-14 | 2002-04-25 | Macrogen Inc. | Bio-support and preparing method of the same |
Non-Patent Citations (3)
| Title |
|---|
| FUENTES, M. et al. Preparation of Inert Magnetic Nano-particles for the Directed Immobilization of Antibodies. Biosensors and Bioelectronics. 2005. Vol. 20, páginas 1380-1387. En particular páginas 1384-1386. * |
| GRAZU, V. et al. Glyoxil Agarose a New Cromatographic Matrix. Enzyme and Microbial Technology. 2006. Vol. 38, páginas 960-966. En particular página 960. * |
| MATEO, C. et al. Glyoxil Agarose: A Fully Inert and Hydrophilic Support for Immobilization and High Stabilization of Proteins. Enzyme and Microbial Technology. 2006. Vol. 39, páginas 274-280. * |
Also Published As
| Publication number | Publication date |
|---|---|
| ES2325391A1 (en) | 2009-09-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Turkova | Oriented immobilization of biologically active proteins as a tool for revealing protein interactions and function | |
| CN115639365B (en) | Sideflow device and its usage | |
| CN109996606B (en) | Lateral flow test device | |
| JP2018141017A5 (en) | ||
| Batalla et al. | Covalent immobilization of antibodies on finally inert support surfaces through their surface regions having the highest densities in carboxyl groups | |
| CN102411051A (en) | Immunodetection method for organophosphorus pesticide multi-residue | |
| CN102998444A (en) | Stereotactic fixing method of IgG antibody on surface of polystyrene carrier | |
| ES2325391B1 (en) | PROCEDURE FOR THE COVALENT ORIENTED IMMOBILIZATION OF ANTIBODIES, ANTIBODIES SO OBTAINED AND THEIR APPLICATIONS. | |
| WO2004090542A1 (en) | Protein array and process for producing the same | |
| JPS62209363A (en) | Composition selectively coupling biological active substanceproper to production of antibody and manufacture thereof | |
| CN103865916B (en) | Magnetic drives immobilized enzyme, preparation method and the application in large water body catalysis thereof | |
| CN111406215B (en) | Lateral flow measuring device | |
| ES2214136B1 (en) | NEW IMMOBILIZATION METHOD OF ENZYMES AND OTHER BIO-MACROMOLECULES ON SUPPORTS ACTIVATED WITH EPOXIDE GROUPS CONTAINING IONIZED GROUPS IN THE SPACER ARM THAT JOINS EACH EPOXIDE GROUP TO THE SUPPORT SURFACE. | |
| Hall et al. | A molecular biology approach to protein coupling at a biosensor interface | |
| WO2008077984A1 (en) | Method for oriented immobilisation of antibodies on solid media, resulting devices and uses thereof | |
| ES2402024T3 (en) | Molecular assembly comprising gold and a linker for detection of biochemical entities | |
| ES2352779B1 (en) | COVALENT COMPLEXES OF LIPASES WITH PROTEINS, DNA PROBES, COFACTORS OR OTHER BIOMOLECULES | |
| JP2008044917A (en) | Protein immobilization method | |
| JP5392683B2 (en) | Activating carrier for preparing immobilized protein | |
| Santana | Magnetic nanoparticles for biocatalysis and bioseparation | |
| Vasu | DEVELOPMENT OF RECOMBINANT ANTIGEN FABRICATED SURFACE FOR DETECTING PESTE DES PETITS RUMINANTS VIRUS SPECIFIC ANTIBODIES | |
| SU1500670A1 (en) | Method of immobilized proteins | |
| CN118126976A (en) | A method for large-scale directional labeling of HRP | |
| JPH05345022A (en) | Physiologically active substance immobilized carrier and its preparation | |
| Viswanath | Site-directed and random enzyme catalysis on functionalized membranes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EC2A | Search report published |
Date of ref document: 20090902 Kind code of ref document: A1 |
|
| FG2A | Definitive protection |
Ref document number: 2325391B1 Country of ref document: ES |
|
| FD2A | Announcement of lapse in spain |
Effective date: 20180912 |