High-affinity binding of remyelinating natural autoantibodies to myelin-mimicking lipid bilayers revealed by nanohole surface plasmon resonance

Abstract

Multiple sclerosis is a progressive neurological disorder that results in the degradation of myelin sheaths that insulate axons in the central nervous system. Therefore promotion of myelin repair is a major thrust of multiple sclerosis treatment research. Two mouse monoclonal natural autoantibodies, O1 and O4, promote myelin repair in several mouse models of multiple sclerosis. Natural autoantibodies are generally polyreactive and predominantly of the IgM isotype. The prevailing paradigm is that because they are polyreactive, these antibodies bind antigens with low affinities. Despite their wide use in neuroscience and glial cell research, however, the affinities and kinetic constants of O1 and O4 antibodies have not been measured to date. In this work, we developed a membrane biosensing platform based on surface plasmon resonance in gold nanohole arrays with a series of surface modification techniques to form myelin-mimicking lipid bilayer membranes to measure both the association and dissociation rate constants for O1 and O4 antibodies binding to their myelin lipid antigens. The ratio of rate constants shows that O1 and O4 bind to galactocerebroside and sulfated galactocerebroside, respectively, with unusually small apparent dissociation constants (K(D) ≈ 0.9 nM) for natural autoantibodies. This is approximately one to 2 orders of magnitude lower than typically observed for the highest affinity natural autoantibodies. We propose that the unusually high affinity of O1 and O4 to their targets in myelin contributes to the mechanism by which they signal oligodendrocytes and induce central nervous system repair.

Figure 1

Assessment of oligodendrocyte phenotype by immunocytochemistry. (a) This diagram represents the morphological and antigen expression changes that occur as oligodendrocytes (OL) differentiate from a simple bipolar proliferative precursor cell to a mature complex post-proliferative cell capable of synthesizing myelin membrane. A2B5 is a mouse IgM that recognizes several complex gangliosides and is often used to define oligodendrocyte precursor cells. The binding of O4 to cell surface Sulf indi cates the cell’s commitment to the oligodendrocytes phenotype. The loss of A2B5 binding and the expression of Sulf is associated with the cessation of proliferation. The binding of O1 to surface GalC and the subsequent expression of MBP and MOG are indicative of mature post-mitotic oligodendrocytes. (b and c) An example of O4 binding to the surface of a live immature rat oligodendrocyte (b) and of O1 binding to the surface of a live mature oligodendrocyte (c). (d) ELISA of O1 and O4 binding to purified myelin.

Figure 2

(a) A schematic illustration of the nanohole fabrication process using nanoimprint lithography. (b) A scanning electron micrograph (SEM) of a nanohole array. The hole diameter and periodicity are 200 nm and 500 nm, respectively. The inset to Figure 2(b) shows a photograph of the fabricated nanohole SPR chip. The nanohole array is patterned in a ~cm² area in the middle of a gold-coated glass slide. (c) Schematic representation of a SLB on a gold nanohole array. The pentameric structures represent antibodies (IgM Abs), such as O1 and O4.

Figure 3

FRAP of SLBs on SiO₂-coated gold nanohole arrays. (a) Four frames showing, from left to right, SLB fluorescence before photobleaching, the same SLB immediately after photobleaching, and at 20 and 80 seconds after photobleaching . (b) Fluorescence recovery curves for SLBs composed solely of Egg PC and Egg PC with 2 % GalC or 2 % Sulf.

Figure 4

(a) The optical transmission spectra before SLB formation (red), after SLB formation (green) and after IgM antibody binding (blue). (b) A full sensorgram from formation of a SLB containing 2 % Sulf to IgM antibody binding to the SLB surface . The spectral position in minimum transmission around 700 nm was monitored to track the changes on the surface. A 2.7 M MgCl₂ solution was used to regenerate for serial kinetic measurements on the same membrane surface.

Figure 5

SPR kinetic curves for IgM autoantibodies binding to SLBs. (a) O4 binding to SLBs containing 2 % Sulf. (b) O1 binding to SLBs with 2 % GalC. (c) Negative controls showing that neither O1 nor O4 bind to SLBs composed solely of egg PC, and that neither O1 nor O4 bind to BSA-blocked, silica-coated nanohole arrays. (d) Kinetic curves for O4 binding to a SLB containing 6 % Sulf and 16 % GalC, which mimics the lipid composition of myelin. The dark lines superimposed upon all of the kinetic curves are exponential fits to the data.