Abstract BACKGROUND Many studies have shown that dysregulation of protein phosphorylation plays an important role in the development of cancer. Aberrantly phosphorylated peptides can be derived from dysregulated cell signaling pathways in various cancers and may serve as tumor-specific antigens and might be promising targets for cancer immunotherapy. Several techniques are available to purify immunoglobulins (IgG) from plasma or other body fluids using affinity to immobilized binders In contrast, Melon Gel resin retains non-IgG proteins and hence allows enrichment of IgG directly from the sample without affinity binding. We have developed a technique for the detection of auto-antibodies in HGG. MATERIAL AND METHODS To perform an Ab-peptide binding assay, we used a phospho-peptide library (a mixture of phospho-peptides obtained after phospho-enrichment of enzymatically digested fresh-frozen GBM tissue) and three plasma samples from HGG patients and a healthy donor as source of IgG. After mixing of the plasma samples with the phospho-peptide library the IgG purification was performed using the Melon Gel resin. We separated possible unbound peptides using a 30 kDa-molecular-weight (MW) filter device. The high MW fraction, containing IgGs and IgG-peptide complexes were acidified and precipitated to release Ab-bound peptides. Subsequently, the IgG-bound peptides were measured and identified in a mass spectrometer. RESULTS Performing the developed Ab-peptide binding assay using the HGG and healthy plasma samples combined with the GBM phospho-peptide library, we detected EGFR phospho-peptide GSHQIS[+80]LDNPDYQQDFFPK (S1166) in three of four IgG-bound fractions of samples incubated with HGG plasma and not in the corresponding unbound fractions. This result indicates the presence of auto-antibodies against EGFR phospho-peptide S1166 in the plasma of the three HGG patients. A contrasting result was obtained in the donor sample, where the EGFR peptide was detected almost exclusively in the unbound fraction. With the use of Avastin as negative control instead of plasma, the EGFR peptide S1166 was solely found in the unbound fraction. CONCLUSION An antibody-peptide-antigen complex enrichment with Melon Gel was successfully applied to find auto-antibodies that recognize disease-related phospho-peptides. This method has the potential to detect the putative presence of auto-antibodies without knowledge of the antigen. We demonstrated in a proof of concept the presence of auto-antibodies against an EGFR phospho-peptide in plasma of HGG patients. This antibody-peptide binding assay could potentially be further applied to other types of cancer to find targets for therapeutic interventions.