Epitope Identification and Affinity Determination of an Inhibiting Human Antibody to Interleukin IL8 (CXCL8) by SPR- Biosensor–Mass Spectrometry Combination

Abstract

The polypeptide chemokine Interleukin-8 (IL8) plays a crucial role in inflammatory processes in humans. IL8 is involved in chronic inflammatory lung diseases, rheumatoid arthritis, and cancer. Previous studies have shown that the interaction of IL8 with its natural receptors CXCR1 and CXCR2 is critical in these diseases. Antibodies have been used to study the receptor interaction of IL8; however, the binding epitopes were hitherto unknown. Identification of the antibody epitope(s) could lead to a molecular understanding of the inhibiting mechanism and development of improved inhibitors. Here, we report the epitope identification and the affinity characterization of IL8 to a monoclonal anti-human IL8 antibody inhibiting the receptor binding by a combination of surface plasmon resonance (SPR) biosensor analysis and MALDI-mass spectrometry. SPR determination of IL8 with the immobilized antibody revealed high affinity (K D, 82.2 nM). Epitope identification of IL-8 was obtained by proteolytic epitope-extraction mass spectrometry of the peptide fragments upon high pressure trypsin digestion, using an affinity microcolumn with immobilized anti-IL-8 antibody. MALDI-MS of the affinity-bound peptide elution fraction revealed an assembled (discontinuous) epitope comprising two specific peptides, IL8 [12–20] and IL8 [55–60]. Identical epitope peptides were identified by direct MALDI-MS of the eluted epitope fraction from the immobilized anti-IL8 antibody on the SPR chip. SPR determination of the synthetic epitope peptides provided high affinities confirming their binding specificity. The previously reported finding that the anti-Il8 antibody is inhibiting the IL8–CXCR1 interaction is well consistent with the overlapping region of epitope interactions identified in the present study.

Identification and Affinity Determination of Protein-Antibody and Protein-Aptamer Epitopes by Biosensor-Mass Spectrometry Combination

“…Following removal of excess supernatant [ 38 ] at pH 7, no unbound peptides were found in the last washing fraction. Subsequent elution performed at slightly acidified solution and MALDI-MS analysis of the elution fractions from the IL8 tryptic digestion mixture revealed two distinct epitope peptides, IL8 (12–20) (TYSKPFHPK) and IL8 (55–60) (ENWVQR) [ 53 ]. These epitope sequences ascertained an assembled (discontinuous) epitope structure comprising two spatially adjacent IL8 sequences ( Figure 5 ).…”

“… Identification of the interleukin-8 epitope to a monoclonal anti-IL8 antibody by proteolytic extraction MALDI-MS, upon elution of the bound epitopes from a microaffinity column ( A ), and elution from an SPR chip ( B ). The antibody was immobilized by NHS/EDC coupling as described [ 53 ]. Epitope elution provided a discontinuous epitope comprising IL-8 sequences (12–20) and (55–60).…”

Antibody Epitope and Affinity Determination of the Myocardial Infarction Marker Myoglobin by SPR-Biosensor Mass Spectrometry

“…The antimyoglobin antibody was immobilized on a self-assembled monolayer (SAM) of the activated surface of an SPR chip, using amine coupling as previously described. , SAM chips were washed three times with alternating water and 70% aqueous ethanol and then immersed for 15 s in piranha solution (H 2 SO 4 /H 2 O 2 , 2:1). This step was repeated three times, and the final washing was performed with deionized water and 70% ethanol.…”

“…The instrumental combination of SPR with ESI-MS comprises valves that enable affinity isolation, mass spectrometric identification, and quantification of affinity-bound ligands from a protein-ligand complex immobilized on the gold chip. The SPR-MS interface provides sample concentration and in situ desalting for direct MS analysis of the ligand eluate. , Both ESI-ion trap and triple quad-MS systems can be coupled to the SPR-MS interface. Applications of the SPR-MS combination were previously described in studies of a mixed-disulfide antibody epitope of the rheumatoid target protein, HLA-B27, and the epitope identification of chaperone complexes of lysosomal enzymes. ,, …”

Development of a New Highly Selective Monoclonal Antibody against Preferentially Expressed Antigen in Melanoma (PRAME) and Identification of the Target Epitope by Bio-Layer Interferometry

“…In the case of PRAME, the epitope analysis of a panel of mAbs [20] and the development of a polyclonal antibody against the predicted extracellular PRAME 310-331 peptide [16] have been recently described. Capture experiments of protein fragments with antibody-immobilized sensor chips can also be performed in this instance using, for example, label-free devices that provide real-time evidence of the capture and of the release of the bound epitope [21,22]. Instruments working in flow are well suited for these purposes; however, dilution of the sample and difficulties in collecting the bound fractions may represent an important limitation.…”