Glycopeptide Based Antibody Detection In Multiple Sclerosis Biology Essay

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Magnetic resonance imaging (MRI) is up to now the most reliable technique not only for MS diagnosis, but also in prognosis because no gold standard simple immunoassays are available. However, it is evident that MRI cannot be considered a routine technique when the clinical symptoms are not still visible to guide a targeted MRI checkup.

The target antigens in MS are not definitively defined because of their difficult localization. Growing evidences indicate that post-translational modifications, either native or aberrant, may play a fundamental role for specific autoantibody recognition in autoimmune diseases. [5] MS patients often produce multiple types of autoantibodies which can be present in tissues and biological fluids. Antibodies can be identified as biomarkers and used to set up diagnostic/prognostic tools. [6] , [7] Moreover, antibody fluctuation with disease exacerbations or remissions can be measured for monitoring the efficacy of a therapeutic treatment. [8] , [9] but the results are constrastion, and no single test has definitely entered the clinical routine.

In particular, in our laboratories, it was demonstrated that the synthetic glucosylated myelin oligodendrocyte glycoprotein fragment [Asn31(Glc)hMOG(30-50)] was able to detect autoantibodies in MS patients' sera by enzyme-linked immunosorbent assay (ELISA). [10] The ability of the glucosylated sequence to detect autoantibodies in Multiple Sclerosis patients' sera was correlated to the N-linked glucosyl moiety. [11] Hence, the recognition properties of the molecule were optimized through the design and screening of focused libraries of glycopeptides by a "Chemical Reverse Approach". The specific antigenic probe CSF114(Glc) was developed to best identify autoantibodies as biomarkers of Multiple Sclerosis in ELISA correlating with disease activity. The structure-based designed glucosylated peptide CSF114(Glc), first Multiple Sclerosis Antigenic Probe, [12] , [13] is able to measure accurately high affinity autoantibodies in sera of a statistically significant patients' population. The glycopeptide is characterised by a β-turn structure bearing as minimal epitope a β-D-glucopyranosyl moiety linked to an Asn residue on the tip of the turn [14] , [15] possibly reproducing an aberrant N-glucosylation of myelin proteins fundamental for autoantibody recognition., [16] ELISA diagnostic/prognostic test MSPepKit, [17] based on CSF114(Glc), has been developed to recognize specific autoantibodies in MS patients' sera and follow up disease activity.

ELISA is a simple and relatively inexpensive technique offering advantages such as simultaneous analyses of a large number of samples. However, non-reproducible or non-interpretable results due to operator-dependent procedures, non-specific 'matrix effects', or failure or heightened detection of low-affinity, background antibodies are some accepted disadvantages of the assay. [18] Consequently, there is a need for sensitive and more consistent techniques, particularly for quantitative autoantibody determination to follow up disease activity.

Biosensor technology based on surface plasmon resonance (SPR) has become increasingly popular for monitoring binding interactions. [19] , [20] SPR technique is extremely interesting in biological and clinical assays because it has the potential to directly visualize biomolecular interactions in real-time. [21] , [22] In this optical method the ligand is covalently linked on the biosensor surface and the specific analyte is perfused recognizing and binding ligand quickly. [23] Binding on the biosensor surface is expressed graphically in sensorgrams that depict accumulation of mass over time providing instantaneous data. Other advantages of SPR technology include the ability to reuse sensor chips for serial analysis and eradicate the need for labelled reagents providing a rapid one-step analytical methodology. Optical label-free devices have been infrequently used for detection of disease specific antibodies directly in patients' sera. Although published results in this field are fundamentally based on spiked serum samples or on a competitive assay set-up, [24] , [25] , [26] analyses can also be performed directly on crude serum samples. [27] , [28] 

Herein we present the evaluation of the feasibility of a glycopeptide-based biosensor to detect MS specific antibodies in sera by a SPR assay. The glycopeptide CSF114(Glc) has been immobilized on a gold sensor chip and used for the screening of healthy blood donors' and MS patients' serum samples.



Glycopeptide antigen CSF114(Glc) was prepared by microwave-assisted solid phase peptide synthesis. The glycopeptide was purified to homogeneity by solid phase extraction and high-pressure liquid chromatography (HPLC), and further characterized by mass spectrometry and analytical HPLC as described elsewhere. [29] 

Sensor chip CM5 and the running buffer HBS-EP+ 10Ã- (0.1 M HEPES, 1.5 M NaCl, 30 mM EDTA and 0.5% v/v Surfactant P20; yielded pH 7.4 when diluted) were purchased from Biacore AB (GE Healthcare, Uppsala, Sweden). The amine coupling reagents N-Hydroxysuccinimide (NHS), 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and 1 M Ethanolamine hydrochloride-NaOH pH 8.5 were provided by Biacore AB (GE Healthcare, Uppsala, Sweden). Sodium acetate was purchased from Carlo Erba (Milano, Italy). Sodium hydroxide was provided by Honeywell-Riedel deHaen (Seelze, Germany). All analyses were performed in a Biacore T100 instrument (GE Healthcare). All experiments were made at 25 °C using HBS-EP+ as running buffer.

Serum collection

121 human serum samples were obtained for diagnostic purposes from patients and healthy blood donors who had given their informed consent. Each serum sample was aliquoted and stored at -20 °C until use. Patients' sera were obtained from a group of 61 MS patients after a diagnostic lumbar puncture. CSF and MRI examinations were performed for diagnostic purposes.

Enzyme linked immunosorbent assay (ELISA)

The two panels of serum samples were tested in ELISA to check the presence of specific antibodies using 96-well plates (NUNC) coated with the glycopeptide antigen CSF114(Glc) according to the method previously described.

Surface plasmon resonance (SPR)

A stock solution of glycopeptide CSF114(Glc) was prepared in pure water (1µg/µL) and stored at +4 °C. Immediately prior to immobilization procedure, peptide stock solution was diluted to a concentration of 0.01 µg/mL in 0.1 mM sodium acetate pH 5.5. Standard amine coupling procedure was employed for glycopeptide immobilization, essentially according to standard Biacore procedures. The appropriate flow cell of the sensor chip surface was activated by injecting an EDC/NHS (50:50) mixture at a flow rate 10 µL/min during 420 sec. CSF114(Glc) was injected at 10 µL/min using the aim of immobilization procedure to give a final immobilization level of 800 ± 100 Resonance Units (RU). Unreacted groups on sensor chip surface were blocked by injecting 60 sec-pulses of 1M ethanolamine at pH 8.5 until complete deactivation. Reference channel was activated and subsequently blocked with ethanolamine.

All analyses were performed in triplicate at a flow rate of 30 µL/min. Human serum samples were diluted 1:100 and/or 1:50 in running buffer. Samples were injected for 240 sec in both active and control channels followed by 60 sec of buffer injection to allow dissociation. Interaction of samples with sensor chip flow cells were monitored as separate sensorgrams and measurements were taken 15 sec after the end of each injection. The antibody responses were measured in RU units as a signal difference between active channel and reference channel. After each measurement, surface was regenerated injecting two pulses of a solution 100mM NaOH during 60 sec.


To determinate MS specific antibody reactivity against the CSF114(Glc)-based biosensor in a BIAcore T100 instrument 60 healthy blood donors (BD) and 61 MS patients' sample sera were tested. For this purpose glycopeptide CSF114(Glc) was reproducibly immobilized to the sensor chip surface following the amino coupling chemistry under optimized conditions. Biosensor was used for the screening of high positive control sera at dilution 1:100 and 1:50. The analytical variability of the assay was checked repeating the same test (2 sera, 15 runs each) or in different experiments (2 sera, 12 runs performed once a week). The within-assay and between-assay coefficients of variation (SE/mean) were bellow 10% for sample dilution 1:100 and below 5% for dilution 1:50. Further serological analysis had been performed at sample dilution 1:50, which presented lower signal average. For each measurement a sample volume of 150 µL was employed, thus small amount of 3 µL of patient serum was required for the assay.

Label-free serodiagnosis of Multiple Sclerosis

Specific antibodies were detectable in some patients' sera. A typical sensorgram obtained when both healthy control and MS patient' sera were injected over the glycopeptide CSF114(Glc) is illustrated in Figure 1.

Figure 1. Sensorgram obtained for binding of a MS positive sample and a healthy blood donor sample to the CSF114(Glc)-modified sensor surface. A corresponds to the diluted serum start injection point, which pass through the sensor chip during 240 sec. B corresponds to the serum end injection point followed by a buffer wash. C corresponds to the evaluation point 15 sec after the injection.

The column scatter of the data is reported in Figure 2. The differences between the MS and BD medium values were significant, observing the higher values in MS subjects.

Figure 2. Column scatter and mean values of anti-CSF114(Glc) antibodies (dilution 1:50) in Multiple Sclerosis (MS, n=61) and blood donors (BD, n=60). The lines indicate the median value of each group.

A receiver operating characteristic (ROC)-based analysis has been employed comparing different cut-off values as sensitivity, specificity and likelihood ratios. [30] ROC curve for anti-CSF114(Glc) activity was constructed based on 61 cases with Multiple Sclerosis versus 60 controls (Figure 3).

Figure 3. ROC curve analysis of antibodies to CSF114(Glc) in MS versus BD determined by SPR. The area under the curve is 0.82 (p<0.0001). Cut-off was set at 105 RU with a sensitivity of 36%, a specificity of 94%, and a positive likelihood ratio of 7.21.

A discriminative power for the anti-CSF114(Glc) antibodies was found (area under the curve 0.82). The ROC analysis established that the optimal diagnostic cut-off value for the method was 105 RU, obtaining a sensitivity of 36%, a specificity of 94%, and a positive likelihood ratio of 7.21. The incidence of an increased antibody level in sera detected was 22 (36%) of the 61 MS samples versus 3 (5%) of the BD samples.

Comparision Biacore/ELISA analyses

The panel of MS and BD sera was also tested against the synthetic peptide CSF114(Glc) following the previously validated ELISA procedure. Results obtained in ELISA were compared with surface plasmon resonance data. As shown in Figure 4, a correlation was obtained between the SPR and ELISA data. Although ELISA exhibited results similar to those from the SPR system, some differences has been observed.

The new CSF114(Glc)-based biosensor scored as positives nine MS patients further screened as negative in ELISA (both IgM and IgG type). Furthermore, the SPR biosensor was capable of distinguish as positives seven cases in which ELISA recognized only IgG type antibodies, and two positive cases of IgM positive in ELISA.

In the other hand, The SPR-biosensor did not detect specific antibody responses whereas the ELISA recognized ten positive cases (IgM and/or IgG).

Antibody levels were found to be significantly higher in MS than in healthy subjects both in SPR and ELISA. False positive cases (4%) scored in SPR-assay were similar to those observed in ELISA.

Although results suggested that screening immunoassays employed were detecting the same type of antibodies, there were some particularly cases in which only one of the assays was able to recognize specific antibodies. The hypothesis put forward could be that the biacore assay detects different molecules because of its ability to detect low-affinity antibodies.

The use of both the SPR-assay and the ELISA for screening purposes in clinical studies to support specific Multiple Sclerosis antibody detection is justified because both screening tests detected positive samples in only one assay type.

Figure 4. Correlation of glycopeptide CSF114(Glc) reactivity between BIAcore and enzyme-linked immunosorbent assay (ELISA). IgG (â- ) and IgM (â-²) antibodies observed in ELISA against Biacore for MS patients (upper) and BD (lower).


Herein we developed a label-free method for the screening of disease-specific antibodies which would offer a protocol with sensitivity and high specificity obtaining a response within a few minutes. CSF114(Glc)-biosensor protocol employed only small volumes of blood serum. In contrast to ELISA, in which information is provided by the use of one specific secondary antibody, the SPR assay yield data directly as the antibody binding to the glycopeptide CSF114(Glc). Moreover, the sensor surface can be regenerated and reused for many measurements saving the costs of the method.

The presented glycopeptide-based biosensor could be useful in patient diagnosis and accurate monitoring of patient serum antibody levels in response to treatments.


Ente Cassa Risparmio di Firenze, PRIN 2008, and ANR Chaire d'Excellence PepKit 2009-2013 (France) are gratefully acknowledged.