AlligBAITOR™ Affinity Purification for Pan-Ig Capture and Serum Ig Depletion
Application
Single-resin affinity platform capturing all Immunoglobulin isotypes from serum, enabling high-resolution proteomic analysis of Ig and non-Ig fractions.
Pan-Ig capture from human serum enables selective isolation of immunoglobulins, improving analytical performance in downstream proteomic workflows by reducing sample complexity and enhancing spectral clarity. By capturing all Ig isotypes, a single-resin approach provides a reproducible substrate for immune profiling and antibody characterization. In parallel, the Ig-depleted serum fraction may retain informative low-abundance proteins and potential biomarkers that are otherwise masked by the presence of immunoglobulins, offering additional value for discovery-driven proteomic or functional analyses.
Here, we demonstrate AlligBAITOR Affinity Purification as an efficient and broadly applicable pan-Ig binding platform for bottom-up proteomics, enabling simultaneous isolation and denaturation of the immunoglobulin fraction for Ig profiling, while generating an informative Ig-depleted fraction also suitable for downstream functional analyses.
Pooled donor human serum was incubated with AlligBAITOR Affinity Purification resin for 30 minutes at room temperature. The flow-through fraction containing non-immunoglobulin proteins was collected, and the resin was subsequently washed in binding buffer and high-salt/urea buffer. Immunoglobulins were eluted using 8 M guanidinium hydrochloride. All fractions were then denatured, reduced, alkylated, and enzymatically digested with trypsin and LysCERATOR™. The resulting peptides were analyzed by RP-LC-MS/MS and searched against the UniProt/Swiss-Prot database.
Approximately 25% (rel.) of the serum sample was determined to be immunoglobulins. Using AlligBAITOR Affinity Purification, we can demonstrate highly efficient capture of the full range of Ig isotypes, resulting in a final sample containing approximately 90% (rel.) Ig species when eluted from the Affinity Puification column. The efficient Ig binding capability of AlligBAITOR Affinity Purification is further evidenced by the minimal Ig content (<0.5% rel.) observed in the flow-through fraction. We demonstrate highly efficient capture of immunoglobulins from human serum using AlligBAITOR Affinity Purification, targeting the full spectrum of Ig isotypes as the dominant component of the eluted fraction. In addition, we generate an Ig-depleted serum flow-through fraction for further downstream analysis.
Capture of all Ig isotypes from complex sample matrix
This robust binding performance enables reliable and reproducible isolation of antibodies from complex biological matrices in a single workflow step. Elution in 8 M guanidinium hydrochloride ensures complete release of bound immunoglobulins while simultaneously providing an integrated denaturation step that is directly compatible with downstream proteolytic digestion and LC-MS-based proteomics workflows. Although this conditions the material for analytical rather than functional applications, it significantly streamlines sample preparation for high-resolution mass spectrometry.
Ig profiling with improved spectral clarity, sensitivity, and proteome coverage
In parallel, the native-state non-immunoglobulin (flow-through) fraction is depleted of high-abundance antibodies to <0.5% (rel.), reducing sample complexity and improving access to lower-abundance serum proteins. This facilitates the detection and analysis of potential biomarker candidates and biological signals that may otherwise be masked by abundant immunoglobulins.
Improved identification of low-abundant proteins in Ig depleted serum
Together, this dual-fraction approach provides a powerful platform for comprehensive serum analysis, combining highly efficient immunoglobulin capture with enhanced discovery potential from the depleted serum fraction.
