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Preactivation crosslinking – An efficient method for the oriented immobilization of antibodies
(2019)
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 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.
Screening of one-bead-one-compound (OBOC) libraries is a proven procedure for the identification of protein-binding ligands. The demand for binders with high affinity and specificity towards various targets has surged in the biomedical and pharmaceutical field in recent years. The traditional peptide screening involves tedious steps such as affinity selection, bead picking, sequencing, and characterization. Herein, we present a high-throughput “all-on-one chip” system to avoid slow and technically complex bead picking steps. On a traditional glass slide provided with an electrically conductive tape, beads of a combinatorial peptide library are aligned and immobilized by application of a precision sieve. Subsequently, the chip is incubated with a fluorophore-labeled target protein. In a fluorescence scan followed by matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry, high-affinity binders are directly and unambiguously sequenced with high accuracy without picking of the positive beads. The use of an optimized ladder sequencing approach improved the accuracy of the de-novo sequencing step to nearly 100%. The new technique was validated by employing a FLAG-based model system, identifying new peptide binders for the monoclonal M2 anti-FLAG antibody, and was finally utilized to search for IgG-binding peptides. In the present format, more than 30,000 beads can be screened on one slide.