ES2297955B1 - HAPTEN-CARRIER MULTIVALENT COMPLEXES WITH DENDRIMEROS AS EMULATORS OF THE CARRIER PROTEIN. - Google Patents
HAPTEN-CARRIER MULTIVALENT COMPLEXES WITH DENDRIMEROS AS EMULATORS OF THE CARRIER PROTEIN. Download PDFInfo
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- ES2297955B1 ES2297955B1 ES200302737A ES200302737A ES2297955B1 ES 2297955 B1 ES2297955 B1 ES 2297955B1 ES 200302737 A ES200302737 A ES 200302737A ES 200302737 A ES200302737 A ES 200302737A ES 2297955 B1 ES2297955 B1 ES 2297955B1
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- Prior art keywords
- carrier
- hapten
- multivalent
- complexes
- emulators
- 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.)
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Links
- 102000014914 Carrier Proteins Human genes 0.000 title claims abstract description 6
- 108010078791 Carrier Proteins Proteins 0.000 title claims abstract description 6
- 239000003814 drug Substances 0.000 claims abstract description 8
- 229940079593 drug Drugs 0.000 claims abstract description 7
- 239000000126 substance Substances 0.000 claims abstract description 6
- 238000003018 immunoassay Methods 0.000 claims abstract description 5
- 229920002521 macromolecule Polymers 0.000 claims abstract description 4
- 238000004458 analytical method Methods 0.000 claims abstract description 3
- 239000000412 dendrimer Substances 0.000 abstract description 15
- 229920000736 dendritic polymer Polymers 0.000 abstract description 15
- 238000000338 in vitro Methods 0.000 abstract description 4
- 230000000890 antigenic effect Effects 0.000 abstract description 3
- 239000007787 solid Substances 0.000 abstract description 3
- 206010020751 Hypersensitivity Diseases 0.000 abstract description 2
- 239000003782 beta lactam antibiotic agent Substances 0.000 abstract description 2
- 238000012631 diagnostic technique Methods 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 239000002132 β-lactam antibiotic Substances 0.000 abstract description 2
- 229940124586 β-lactam antibiotics Drugs 0.000 abstract description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 8
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 6
- 239000007790 solid phase Substances 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 229920000729 poly(L-lysine) polymer Polymers 0.000 description 4
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 4
- 235000017557 sodium bicarbonate Nutrition 0.000 description 4
- 238000000034 method Methods 0.000 description 3
- 229940056360 penicillin g Drugs 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- HDFXRQJQZBPDLF-UHFFFAOYSA-L disodium hydrogen carbonate Chemical compound [Na+].[Na+].OC([O-])=O.OC([O-])=O HDFXRQJQZBPDLF-UHFFFAOYSA-L 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 235000019371 penicillin G benzathine Nutrition 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 238000004833 X-ray photoelectron spectroscopy Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 150000001414 amino alcohols Chemical class 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 150000001913 cyanates Chemical class 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000008241 heterogeneous mixture Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- IQPQWNKOIGAROB-UHFFFAOYSA-N isocyanate group Chemical group [N-]=C=O IQPQWNKOIGAROB-UHFFFAOYSA-N 0.000 description 1
- 150000002542 isoureas Chemical class 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 1
- 238000005406 washing Methods 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
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
Abstract
La presente invención trata de la producción de complejos multivalentes hapteno-portador, en los que la proteína portadora está emulada por un dendrímero o molécula dendrimérica, que actúa como macromolécula portadora sobre la que se une de forma covalente el hapteno único o combinado de diferentes fármacos o estructuras químicas. Estos complejos multivalentes hapteno-portador se pueden utilizar, acoplados a fases sólidas o en fase soluble, como estructuras antigénicas para la captación de anticuerpos IgE específicos de antibióticos beta-lactámicos y de otros fármacos, permitiendo su aplicación en técnicas diagnósticas in vitro que evalúen las reacciones alérgicas, siendo asimismo aplicables en sistemas de análisis tipo inmunoensayo.The present invention relates to the production of multivalent hapten-carrier complexes, in which the carrier protein is emulated by a dendrimer or dendrimeric molecule, which acts as a carrier macromolecule on which the single or combined hapten of different drugs covalently binds. or chemical structures. These multivalent hapten-carrier complexes can be used, coupled to solid or soluble phase, as antigenic structures for the uptake of IgE antibodies specific for beta-lactam antibiotics and other drugs, allowing their application in in vitro diagnostic techniques that evaluate the allergic reactions, also being applicable in immunoassay type analysis systems.
Description
Complejos multivalentes hapteno-portador con dendrímeros como emuladores de la proteína portadora.Multivalent complexes hapten carrier with dendrimers as emulators of the carrier protein
La presente invención se refiere a la utilización de dendrimeros funcionalizados en su superficie con medicamentos, para emular in vitro a los conjugados hapteno-portador generados in vivo, responsables de los procesos de reconocimiento de anticuerpos IgE dirigidos a estos fármacos. La exactitud de la estructura química del conjugado hapteno-dendrímero, en cuanto al número de haptenos anclados en su superficie y la disposición tridimensional, mejoran la calidad de la técnica para la detección de anticuerpos IgE con respecto al empleo de otras macromoléculas tradicionalmente empleadas.The present invention relates to the use of functionalized dendrimers on its surface with medicaments, to emulate in vitro hapten-carrier conjugates generated in vivo , responsible for the recognition processes of IgE antibodies directed to these drugs. The accuracy of the chemical structure of the hapten-dendrimer conjugate, in terms of the number of haptens anchored on its surface and the three-dimensional arrangement, improve the quality of the technique for the detection of IgE antibodies with respect to the use of other macromolecules traditionally employed.
Para la detección in vitro de anticuerpos
IgE dirigidos a medicamentos se usan conjugados
Hapteno-Portador (en adelante H-P)
sintéticos en los que la proteína portadora es emulada por Albúmina
Humana (en adelante HSA) o
poli-L-lisina (en adelante PLL).
Estos conjugados adolecen de dos factores imprescindibles para que
el proceso de reconocimiento molecular sea reproducible y sensible:
no presentan una densidad de Hapteno por unidad de portador precisa
en sus estructuras y, además, las moléculas de Hapteno se
encuentran distribuidas de forma aleatoria en la amplia superficie
de la proteína o el polipéptido, lo que entraña la eventual
inaccesibilidad de los anticuerpos específicos. La HSA presenta la
limitación intrínseca de poder soportar una densidad de
H-P baja, además de la dificultad de poder
establecer la estructura exacta de estos conjugados. Por otro lado,
los conjugados con PLL, aunque con una mayor densidad
H-P, actualmente consisten en una mezcla de
estructuras químicas de diferentes tamaños, ya que la PLL comercial
es siempre una mezcla heterogénea de péptidos de un determinado
rango de pesos moleculares. Como consecuencia,
estos
conjugados se caracterizan por una baja reproducibilidad estructural
y consecuentemente una baja fiabilidad.For in vitro detection of drug-directed IgE antibodies, synthetic Haptene-Carrier (hereinafter HP) conjugates are used in which the carrier protein is emulated by Human Albumin (hereinafter HSA) or poly-L-lysine (hereinafter PLL) . These conjugates suffer from two essential factors so that the molecular recognition process is reproducible and sensitive: they do not have a Haptene density per precise carrier unit in their structures and, in addition, Hapteno molecules are distributed randomly throughout the surface of the protein or polypeptide, which implies the eventual inaccessibility of specific antibodies. The HSA has the intrinsic limitation of being able to withstand a low HP density, in addition to the difficulty of establishing the exact structure of these conjugates. On the other hand, PLL conjugates, although with a higher HP density, currently consist of a mixture of chemical structures of different sizes, since commercial PLL is always a heterogeneous mixture of peptides of a certain range of molecular weights. Due,
These conjugates are characterized by low structural reproducibility and consequently low reliability.
La presente invención trata de la producción de complejos multivalentes H-P, en los que la proteína portadora está emulada por un dendrímero o molécula dendrimérica, que actúa como macromolécula portadora sobre la que se une de forma covalente el hapteno único o combinado de diferentes fármacos o estructuras químicas.The present invention concerns the production of multivalent H-P complexes, in which the protein carrier is emulated by a dendrimer or dendrimer molecule, which acts as a carrier macromolecule on which it joins covalent the single or combined hapten of different drugs or chemical structures
Estos conjugados H-P, preparados utilizando dendrímeros para simular la proteína portadora (en adelante H-D), son estructuras macromoleculares de composición química perfectamente definidas. El empleo de dendrímeros de diferentes generaciones permite obtener conjugados H-D con diferentes, pero precisos, números de Haptenos unidos por enlaces de tipo covalente a la superficie del dendrímero. El empleo de las técnicas convencionales de determinación estructural, como la Resonancia Magnética Nuclear de Protón y Carbono trece, la Espectrometría de Masas de tiempo de vuelo con ionozación por láser asistida por matriz, la espectroscopia de fotoelectrones de rayos-X y otras, permiten analizar y establecer de forma univoca las estructuras de estos conjugados H-D.These H-P conjugates, prepared using dendrimers to simulate the carrier protein (in H-D), are macromolecular structures of Chemical composition perfectly defined. The employment of dendrimers of different generations allows to obtain conjugates H-D with different, but precise, numbers of Join us by covalent bonds to the surface of the dendrimer The use of conventional techniques of structural determination, such as the Nuclear Magnetic Resonance of Proton and Carbon thirteen, the Mass Spectrometry of time of flight with matrix-assisted laser ionozation, the X-ray photoelectron spectroscopy and others, allow to analyze and establish univocally the structures of these H-D conjugates.
Los conjugados H-D se pueden utilizar, acoplados a fases sólidas o en fase soluble, como estructuras antigénicas para la captación de anticuerpos IgE específicos de antibióticos beta-lactámicos y de otros fármacos, permitiendo su aplicación en técnicas diagnósticas in vitro que evalúen las reacciones alérgicas, siendo asimismo aplicables en sistemas de análisis tipo inmunoensayo.HD conjugates can be used, coupled to solid or soluble phase, as antigenic structures for the uptake of IgE antibodies specific for beta-lactam antibiotics and other drugs, allowing their application in in vitro diagnostic techniques that evaluate allergic reactions, being also applicable in immunoassay type analysis systems.
Para la realización de las determinaciones de inmunoensayos en los que el conjugado H-D se encuentre en fase soluble, al dendrímero, de la generación elegida, se le une de forma covalente el medicamento, empleando los reactivos de acoplamiento adecuados en cada caso y se puede purificar empleando las técnicas de cromatografía convencionales.For the realization of the determinations of immunoassays in which the H-D conjugate is find in the soluble phase, the dendrimer, of the chosen generation, the medication is covalently bound, using the suitable coupling reagents in each case and you can purify using chromatography techniques conventional.
Para la realización de las determinaciones de inmunoensayos en los que el conjugado H-D se encuentre acoplado a fases sólidas, las superficies de las fases sólidas, como celulosa, sílice o cualquier otro análogo, se activan con los reactivos adecuados, como bromuro de cianógeno, alfa-halocloruros de ácidos o agentes de acoplamiento similares, para enlazar los dendrímeros, se tratan con amino-alcoholes para neutralizar los grupos activados libres y se funcionalizan con el hapteno capaz de ser reconocido por las IgE's.For the realization of the determinations of immunoassays in which the H-D conjugate is find coupled to solid phases, the surfaces of the phases solids, such as cellulose, silica or any other analog, are activated with the appropriate reagents, such as cyanogen bromide, alpha-halochlorides of acids or agents Similar coupling, to bind dendrimers, are treated with amino alcohols to neutralize the groups activated free and functionalized with the hapten capable of being recognized by the IgE's.
A continuación se presenta un modo de realización preferida para la obtención de estos conjugados H-D.Below is a mode of preferred embodiment for obtaining these conjugates H-D
Discos de celulosa se activan, por ejemplo, con bromuro de cianógeno para transformar los grupos hidroxilos en los derivados cianatos. Después del tiempo de reacción, los discos se lavan con bicarbonato sódico 0.1 M y con concentraciones crecientes de acetona, y se hacen reaccionar con los grupos amino-terminales del dendrímero, para dar las isoureas derivadas a través de las cuales el dendrímero se ancla sobre la fase sólida. Las fases sólidas se filtran, se lavan con disolución de bicarbonato sódico 0.1 M y se hacen reaccionar con etanolamina durante 1 hora, para desactivar los grupos isocianatos que permanezcan libres. Una posterior filtración y lavado secuencia) con una disolución de bicarbonato sódico 0.1 M, una disolución tampón ácido acético-acetato sódico 0.1 M de pH 4 y finalmente una disolución carbonato sódico-bicarbonato sódico 0.05 M de pH 10.2, nos permite obtener una superficie modificada químicamente, en la que la unión covalente de los dendrímeros a esta fase sólida puede utilizarse para inmovilizar de forma covalente una variedad de analitos de significación clínica.Cellulose discs are activated, for example, with cyanogen bromide to transform hydroxyl groups into cyanate derivatives. After the reaction time, the discs are wash with 0.1 M sodium bicarbonate and with increasing concentrations of acetone, and are reacted with the groups amino terminal of the dendrimer, to give the derived isoureas through which the dendrimer is anchored On the solid phase. The solid phases are filtered, washed with 0.1 M sodium bicarbonate solution and reacted with ethanolamine for 1 hour, to deactivate isocyanate groups Let them remain free. A subsequent filtration and washing sequence) with a 0.1 M sodium bicarbonate solution, a solution 0.1 M acetic acid-sodium acetate buffer pH 4 and finally a carbonate solution sodium-sodium bicarbonate 0.05 M pH 10.2, we allows to obtain a chemically modified surface, in which the covalent bonding of dendrimers to this solid phase can be used to covalently immobilize a variety of analytes of clinical significance.
Como ejemplo, esta fase sólida sobre la que hemos fijado el dendrímero, se suspende en un tampón carbonato sódico-bicarbonato sódico 0.05 M de pH 10.2 y se hace reaccionar con bencilpenicilina (Penicilina G) durante 48 horas a 4ºC, se filtra pasado este tiempo y se lava con una disolución de bicarbonato sódico 0.1 M. De esta forma, se obtiene una fase sólida sobre la que se encuentra fijado de forma covalente, un conjugado H-D peniciloilado en su superficie, que contiene el determinante antigénico mayor (BPO) de los anticuerpos IgE dirigidos a bencilpenicilina. Los discos que contienen el conjugado H-D con antígenos BPO, se guardan en disolución tampón PBS a 4ºC.As an example, this solid phase on which we have fixed the dendrimer, it is suspended in a carbonate buffer sodium-sodium bicarbonate 0.05 M pH 10.2 and reacts with benzylpenicillin (Penicillin G) for 48 hours at 4 ° C, filtered after this time and washed with a solution of 0.1 M sodium bicarbonate. In this way, a solid phase is obtained. on which a conjugate is fixed covalently H-D penicilloylated on its surface, which contains the major antigenic determinant (BPO) of IgE antibodies directed to benzylpenicillin. The disks containing the conjugate H-D with BPO antigens, stored in solution PBS buffer at 4 ° C.
Claims (2)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ES200302737A ES2297955B1 (en) | 2003-11-21 | 2003-11-21 | HAPTEN-CARRIER MULTIVALENT COMPLEXES WITH DENDRIMEROS AS EMULATORS OF THE CARRIER PROTEIN. |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ES200302737A ES2297955B1 (en) | 2003-11-21 | 2003-11-21 | HAPTEN-CARRIER MULTIVALENT COMPLEXES WITH DENDRIMEROS AS EMULATORS OF THE CARRIER PROTEIN. |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| ES2297955A1 ES2297955A1 (en) | 2008-05-01 |
| ES2297955B1 true ES2297955B1 (en) | 2009-03-16 |
Family
ID=39316056
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| ES200302737A Expired - Fee Related ES2297955B1 (en) | 2003-11-21 | 2003-11-21 | HAPTEN-CARRIER MULTIVALENT COMPLEXES WITH DENDRIMEROS AS EMULATORS OF THE CARRIER PROTEIN. |
Country Status (1)
| Country | Link |
|---|---|
| ES (1) | ES2297955B1 (en) |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6121056A (en) * | 1993-02-24 | 2000-09-19 | Dade Behring Inc. | Random detection of antigens with antibodies immobilized on soluble submicron particles |
| KR100407822B1 (en) * | 2001-12-04 | 2003-12-01 | 한국전자통신연구원 | Electrochemical immune-sensor, and kit and method for detecting biochemical analyte using the same |
-
2003
- 2003-11-21 ES ES200302737A patent/ES2297955B1/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| SANCHEZ-SANCHO F., PEREZ-INESTROSA E., SUAU R., MAYORGA C., TORRES M.J., BLANCA M., "Dendrimers as carrier protein mimetics for IgE Antibody recognition. Synthesis and characterization of densely penicilloylated dendrimers"{} Bioconjugate Chem. 2002 vol. 13 páginas 647-653. * |
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| Publication number | Publication date |
|---|---|
| ES2297955A1 (en) | 2008-05-01 |
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