An Effective and Rapid Method for Functional Characterization of Immunoadsorbents Using POROS Beads and Flow Cytometry

Abstract

To facilitate the construction, functional characterization, and use of immunoadsorbents, we have developed a flow cytometry method that allows rapid assessment of large numbers of particle-bound antibodies. Protein G derivitized POROS beads were used to bind affinity-purified antibodies specific for synthetic peptides designed from human plasma proteins. The antibodies were covalently coupled to the beads and used to capture and release synthetic peptides that had been labeled at the C-terminus with the fluorochrome Alexa Fluor 488. Antibody coupling and specificity of antigen binding and release were measured by analysis of the POROS affinity beads by flow cytometry. The affinity-capture matrixes were also used through several antigen-binding and release cycles without loss of peptide binding efficiency. The ability to produce and characterize extremely small amounts of POROS affinity matrices will facilitate their use in protein microchemical procedures such as protein chip technology, monoclonal antibody screening and mass spectrometry, applications where analytes are limiting or present in low abundance in complex mixtures.

Microfluidic enzymatic reactors for proteome research

“…are now commercially available or have been specifically developed for immobilization processes [8,9].…”

Optimization of antibody immobilization for on‐line or off‐line immunoaffinity chromatography

“…These methods routinely use 2 M Na 2 SO 4 during immobilization to get the antibodies to partially precipitate on the matrix surface, thus improving immobilization dramatically [9], but also making crosslinks between the antibodies themselves possible. Finally, site-directed immobilization of antibodies may be performed in a two-stage approach by using a Protein G-derivatized matrix [9,11] (method 4) followed by chemical crosslinking of antibody and Protein G. Site-directed methods appear generally to result in IA-matrices with increased capacities [25]. An overview of the methods is shown in Table 1.…”

“…Covalent crosslinking of antibody and Protein G was accomplished according to Anderson et al [11]. Briefly, 125 lL of 10% POROS Protein G or an Protein G-agarose bead slurry was washed three times in 1.125 mL of 10 mM phosphate buffer, pH 7.2, 0.15 M NaCl; followed by addition of 1.25 mg antibody solution and PBS, 0.15 M NaCl, pH 7.4, to a total volume of 1.6 mL.…”

Targeting Peptide Termini, a Novel Immunoaffinity Approach to Reduce Complexity in Mass Spectrometric Protein Identification

“…Immunoaffinity material was prepared according to the protocol published by Anderson et al (22) using POROS G 20 beads (Applied Biosystems, Foster City, CA) and purified anti-AMTR and anti-LGYR antibodies. Affinity columns were prepared in 10 ϫ 4.0-mm PEEK columns with 2-m PEEK frits and polytetrafluoroethylene sealing gaskets (Alltech Grom GmbH, Rottenburg, Germany) using a syringe pump.…”