Inmovilización orientada de anticuerpos sobre superficies inertes para su uso como biosensores

1. Inmovilización orientada de anticuerpos sobre superficies inertes para su uso como biosensores Roberto Fernández-Lafuente Departamento de biocatalisis Instituto de Catálisis. CSIC. Madrid.

2. One step concentration Magnetic particles begin end of analytical protocol “Design of biosensors: new protocols to achieve a direct covalent immobilization of bio-molecules (antibodies and DNA probes) on paramagnetic particles” • Cell separation • Immunoassay • Isolation of viruses • Isolation of organelles • Molecular genetics • Biocatalysis ??? (insoluble substrates) purification DETECTION OF TRACES DE ANALYTES

3. + + + + “New general protocols for the conjugation of biomacromolecules to magnetic microspheres” An ideal tailor-made immobilization protocol • Correct orientation: Good specific biorecognization • Stable attachment: Uni- o multipoint attachment • Unspecific adsorption: Second antibody, target molecule, enzyme,… • Spacer arms: Steric hindrances

4. Correct orientation • Different immobilization protocols: • Simple • Universal • High loading capacity • Very inert surface of magnetic particles Conjugation of antibodies to magnetic particles

5. Immobilization of Antibodies on magnetic particles • Different Immobilization protocols: • ●Random Immobilization through amino groups • ●His- rich regions • ●Sugar chains

6. NH2 NH2 NH2 NH2 NH2 NH2 NH2 NH2 Immobilization of Antibodies on magnetic particles: ramdon immobilization through amino groups Br Br Primary amino groups ■Terminal amino groups ■Lys Br Magnetic particles activated with Cyanogenbromide • not correctly oriented ???? • unstable activated supports

7. Cu+2 NH His His COO- COO- Immobilization of Antibodies on magnetic particles: His-rich regions o Highly activated magnetic particles with IDA – Cu +2 His moieties • Pre-teatment of IgG not necessary • Correctly oriented ???? • High loading capacity • Rapid and simple immobilization • Mild conditions

8. .. NH2 PGA 1M fosfato pH 7.0 Bloqueo con pH 7,8 Método estándar de inmovilización a soportes epóxido pH 7.0 Inmovilización covalente Adsorción física (hidrofóbica)

9. His His His His COO - COO - Co +2 COO - COO - His His His His

10. ¨ NH2 + NH - + ¨ NH3 ¨ NH2 + ¨ NH3 ¨ NH2 O O C C H H ¨ NH2 ¨ + NH3 ¨ NH2 Immobilization of Antibodies on magnetic particles: Sugar chains Very mild oxidation of IgG Very rapid immobilization Highly activated magnetic particles with controlled pK amino groups • Correctly immobilized antibody ??? • Oxidation of sugar chains present in Fc region • Unaltered immunoactivity

11. Immobilization of Antibodies on magnetic particles RESULTS Immunoactivity expressed • Cyanogenbromide decreases immunoactivity • IDA-Cu chelates-epoxide correctly orientates and mantains immunoactivity • Sugar-chain correctly orientates and mantains immunoactivity

12. Sugar chain % Immunoactivity IDA-Cu Time (hours) Immobilization of Antibodies on magnetic particles • Unaltered inmunoactivity • Better rate was achieved using sugar than IDA- Cu +2 • Spacer arms: Sugar chain

13. + + + + + + + Immobilization of Antibodies on magnetic particles Primary amino groups in magnetic particles surface • Excellent ionic exchanger • Approximately 70% proteins • are ionically adsorbed Charged amino in magnetic particles Unspecific adsorption

14. muestra Analito de interés Soporte altamente activado Adsorción inespecífica de otras biomoléculas presentes en la muestra Inmovilización de anticuerpos a partículas magnéticas Adsorción inespecífica Tratamiento con polímeros???

15. + + - - + + + + + + + - - + + + Inmovilización de anticuerpos a partículas magnéticas Colapso de las IgG inmovilizadas sobre superficies activadas

16. Resultados O O O O C C C C H H H H NH3+ NH2 NH2 O O O O = = = = O O = = NH2 NH2 NH2 NH2 NH2 NH3+ NH3+ + + + + - - - - COO- COO- COO- COO- Inmovilización Covalente y Orientada de Anticuerpos sobre Nanopartículas magnéticas aminadas Superficie inerte Tratamiento con polímeros + +

17. Resultados Inmovilización Covalente y Orientada de Anticuerpos sobre Nanopartículas magnéticas aminadas Estabilidad de los anticuerpos inmovilizados sobre nanopartículas magnéticas aminadas Dextrano-aspártico-aldehído Tampón fosfato sódico 25 mM pH 7

18. C N - + + + _ _ _ ¨ ¨ ¨ + + + NH3 NH3 NH3 - C N - - Immobilization of Antibodies on magnetic particles Inert surface Primary amino magnetic particles • Covalent attachment • Blocks positive charges Aspartic-aldehyde-dextran • Heterobifunctional polymer (different molecular weights, different percentage of carboxylic groups) • A first Ionic Adsorption plus an additional covalent attachment • Not significant adsorption of protein

19. Resultados Inmovilización Covalente y Orientada de Anticuerpos sobre Nanopartículas magnéticas aminadas Detección de trazas de HRP con anti-HRP inmovilizandose nanopartículas magnéticas + proteína diana Incubación Anticuerpo inmovilizado sobre partículas magnéticas otras proteínas Generador campo magnético lavados Electroforesis o determinación actividad HRP (100μl)

20. Resultados IgG Cadena pesada HRP IgG Cadena ligera Calle1 Calle 2 Calle 3 Inmovilización Covalente y Orientada de Anticuerpos sobre Nanopartículas magnéticas aminadas

21. Xarxa Temàtica de Nanopartícules Magnètiques. Barcelona/Octubre 2.006 Biótica, S.L. Bacteria LIBRE Bacterias capturadas en partículas IgG anti E.coli Partícula magnética

22. Immobilization of antibodies on magnetic particles Results • Simple and controlled microspheres surface activation • Correctly oriented antibody immobilized • Universal and simple protocol • High yield immobilization • High loading binding • Inert surface: Hetero-bifunctional polymer Excellent immunobiosensor 0.01 μg peroxidase in 5 liters was fully adsorbed on 1 μg of magnetic beads containing anti-peroxidase

23. Institute of Catalysis. CSIC. Madrid. Spain Prof. Jose Manuel Guisán Dr. Roberto Fernández-Lafuente Dr. Cesar Mateo Dr Manuel Fuentes

24. Institute of Catalysis. CSIC. Madrid . Spain