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Preactivation Crosslinking - An Efficient Method for the Oriented Immobilization of Antibodies

  • Crosslinking of proteins for their irreversible immobilization on surfaces is a proven and popular method. However, many protocols lead to random orientation and the formation of undefined or even inactive by-products. Most concepts to obtain a more targeted conjugation or immobilization requires the recombinant modification of at least one binding partner, which is often impractical or prohibitively expensive. Here a novel method is presented, which is based on the chemical preactivation of Protein A or G with selected conventional crosslinkers. In a second step, the antibody is added, which is subsequently crosslinked in the Fc part. This leads to an oriented and covalent immobilization of the immunoglobulin with a very high yield. Protocols for Protein A and Protein G with murine and human IgG are presented. This method may be useful for the preparation of columns for affinity chromatography, immunoprecipitation, antibodies conjugated to magnetic particles, permanent and orientedCrosslinking of proteins for their irreversible immobilization on surfaces is a proven and popular method. However, many protocols lead to random orientation and the formation of undefined or even inactive by-products. Most concepts to obtain a more targeted conjugation or immobilization requires the recombinant modification of at least one binding partner, which is often impractical or prohibitively expensive. Here a novel method is presented, which is based on the chemical preactivation of Protein A or G with selected conventional crosslinkers. In a second step, the antibody is added, which is subsequently crosslinked in the Fc part. This leads to an oriented and covalent immobilization of the immunoglobulin with a very high yield. Protocols for Protein A and Protein G with murine and human IgG are presented. This method may be useful for the preparation of columns for affinity chromatography, immunoprecipitation, antibodies conjugated to magnetic particles, permanent and oriented immobilization of antibodies in biosensor systems, microarrays, microtitration plates or any other system, where the loss of antibodies needs to be avoided, and maximum binding capacity is desired. This method is directly applicable even to antibodies in crude cell culture supernatants, raw sera or protein-stabilized antibody preparations without any purification nor enrichment of the IgG. This new method delivered much higher signals as a traditional method and, hence, seems to be preferable in many applications.zeige mehrzeige weniger

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Metadaten
Autoren/innen:Barbara Schroeder, Hoa Le Xuan, Jule L. Völzke, Michael G. Weller
Dokumenttyp:Zeitschriftenartikel
Veröffentlichungsform:Verlagsliteratur
Sprache:Englisch
Titel des übergeordneten Werkes (Englisch):Methods and Protocols
Jahr der Erstveröffentlichung:2019
Organisationseinheit der BAM:1 Analytische Chemie; Referenzmaterialien
1 Analytische Chemie; Referenzmaterialien / 1.5 Proteinanalytik
Veröffentlichende Institution:Bundesanstalt für Materialforschung und -prüfung (BAM)
Verlag:MDPI
Verlagsort:Basel, Switzerland
Jahrgang/Band:2
Ausgabe/Heft:2
Erste Seite:1
Letzte Seite:14
DDC-Klassifikation:Naturwissenschaften und Mathematik / Chemie / Analytische Chemie
Technik, Medizin, angewandte Wissenschaften / Ingenieurwissenschaften / Ingenieurwissenschaften und zugeordnete Tätigkeiten
Freie Schlagwörter:Affinity chromatography; Antibody coating; Bioconjugation; Biosensor; IgG; Immunoaffinity extraction; Immunoassay; Immunocapture; Immunoglobulins; Immunoprecipitation; Immunosensor; Oriented immobilization; Protein A; Protein G; Proximity-enhanced reaction; Regeneration
Themenfelder/Aktivitätsfelder der BAM:Material
Analytical Sciences
Analytical Sciences / Strukturanalytik und Materialographie
Material / Nanoskalige Materialien und deren Eigenschaften
DOI:https://doi.org/10.3390/mps2020035
URN:urn:nbn:de:kobv:b43-479786
URL:https://www.mdpi.com/2409-9279/2/2/35
ISSN:2409-9279
Verfügbarkeit des Dokuments:Datei für die Öffentlichkeit verfügbar ("Open Access")
Datum der Freischaltung:20.05.2019
Referierte Publikation:Nein
Schriftenreihen ohne Nummerierung:Wissenschaftliche Artikel der BAM