Selective Capture of Anti-N-glucosylated NTHi Adhesin Peptide Antibodies by a Multivalent Dextran Conjugate

Abstract

Tentacle-like polymers decorated with several copies of peptide antigens can be interesting tools for increasing the ability to capture circulating antibodies in patient sera, using cooperative effects for stronger avidity. We previously showed that antibodies from multiple sclerosis (MS) patient sera preferentially recognize hyperglucosylated adhesin protein HMW1ct of non-typeable Haemophilus influenzae (NTHi). We selected the C-terminal HMW1ct(1347-1354) minimal epitope and prepared the diglucosylated analogue Ac-KAN(Glc)VTLN(Glc)TTG-K(N₃ )-NH₂ to graft a 40 kDa dextran scaffold modified with glycidyl-propargyl moieties to perform a copper catalyzed alkyne-azide coupling reaction (CuAAC). Quantitative NMR measurements allowed the characterization of the peptide loading (19.5 %) on the multivalent dextran conjugate. This novel polymeric structure displayed optimal capturing properties of both IgG and, more interestingly, IgM antibodies in MS sera. Specific antibodies from a representative MS serum, were successfully depleted using a Sepharose resin bearing the new glucosylated multivalent conjugate, as confirmed by ELISA. These results may offer a promising proof-of-concept for the selective purification of high affinity autoantibodies from sera of autoimmune patients, in general, and of specific high affinity antibodies against a minimally glcosylated epitope Asn(Glc) from sera of multiple sclerosis (MS) patients, in particular.

Keywords: ELISA; antibody caption; dextran conjugates; multivalence; peptides.

Scheme 1

Functionalization of 40 kDa dextran with glycidyl‐propargyl moieties.

Figure 1

¹H‐NMR overlaid spectra of the commercially available 40 kDa dextran (blue spectrum) and GPE functionalized Dex40‐GP (red spectrum): new signals appearance that were assigned to the corresponding protons of the GP‐functionalized glucose units are indicated by colored circles.

Scheme 2

CuAAC promoted reaction between the diglucosylated peptide 1′_A,BGK(ϵN₃) and Dex40‐GP, leading to the multivalent peptide‐dextran conjugate Dex40‐Peptide 1.

Figure 2

¹H‐NMR of Dex40‐Peptide 1’. Signals and areas assignments characterizing the novel multivalent peptide‐dextran conjugate are reported in colored circles.

Figure 3

Schematic representation of the novel conjugate Dex40‐Peptide 1’, carrying HMW1ct(1347–1354) diglucosylated epitopes shared by Peptide 1’_A,B. On the right, main features of dextran and dextran derivatives, based on NMR characterization, are summarized.

Figure 4

Competitive ELISA obtained coating the hyperglucosylated protein antigen HMW1ct(N‐Glc). Inhibition curves of anti‐HMW1ct(N‐Glc) IgG (A) and IgM (B) antibodies using Dex40, its alkyne‐functionalized analogue Dex40‐GP, and the novel peptide‐dextran conjugate Dex40‐Peptide 1’. Results show the inhibition activity % (ordinate axis) of a representative serum MS1 for IgG (A) and of a representative serum MS4 for IgM (B) vs antigen concentrations on a logarithmic scale (abscissas axis).

Figure 5

SP‐ELISA to detect IgG (A) and IgM (B) antibody titers in 5 representative Multiple Sclerosis sera samples using the diglucosylated Peptide 1’_AB (red) and Dex40‐Peptide 1’ (blue) as antigens. Mean absorbance values were evaluated at 405 nm; standard deviation (SD) for n=3 independent measures evaluated at the same conditions are reported as error bars.

Figure 6

SP‐ELISA to detect IgG and IgM antibody titers after immunoaffinity purification of representative MS5 sample serum, on Sepharose column functionalized with Dex40‐Peptide 1’. The multivalent peptide‐dextran conjugate Dex40‐Peptide 1’ was coated on the ELISA plate. Mean absorbances (±standard deviation for n=3 independent experiments) of IgG and IgM antibodies of the different fractions are plotted. MS5 (sample serum), FT1 (diluted sample serum), and FT2 (final flow‐through) were tested using same dilution ratio. Eluted antibodies were approximately 5 times more concentrated.