Association of anti‐gangliosides antibodies and anti‐CMV antibodies in Guillain–Barré syndrome

Abstract Introduction Numerous types of infection were closely related to GBS, mainly including Campylobacter jejuni, Cytomegalovirus, which may lead to the production of anti‐gangliosides antibodies (AGA). Currently, although there are increased studies on the AGA and a few studies of anti‐CMV antibodies in GBS, the association between them remains poorly documented. Therefore, our research aims to analyze the correlation of anti‐CMV antibodies and AGA in GBS. Methods A total of 29 patients with GBS were enrolled in this study. The CMV antibodies were tested by the electrochemiluminescence immunoassay “ECLIA” (Roche Diagnostics GmbH). The serum gangliosides were determined by The EUROLINE test kit. Results Of the 29 patients with GBS, 9 (31%) were AGA‐seropositive, in which 22 were CMV‐IgG positive in CSF at the same time, but all 29 samples were CMV‐IgM negative in both serum and CSF. In the AGA‐positive group, the rate of both serum and CSF positive was 87.5% (7/8), higher than 50% (7/14) of the negative group, although no statistical significance was found. In addition, we found that there was a trend of higher ratio of men, a younger age onset, less frequent preceding infection, a higher level of CSF proteins, and less frequent cranial nerve deficits, although the data did not reach a statistical significance. Conclusion In spite of no statistical significance association was found between serum AGA and CMV‐IgG in serum and CSF. However, we found that there was a trend of high positive rate of both serum and CSF‐CMV‐IgG in AGA‐positive than the negative group. So we should further expand the sample size to analyze the association between AGA and CMV or other neurotropic virus antibodies in various diseases, to observe whether they could be serological marker of these diseases (especially GBS) or the underlying pathogenesis.

Gangliosides are a family of sialylated glycosphingolipids located in higher density in nervous system, especially in axons of neuron (Ledeen & Yu, 1982;Schuster & Haller, 1990). It consists of several subtypes depending on the number and position of sialic acids, the number of glucose molecules, and their synthetic pathways, for example, GM1, GM2, GM3, GD1a, GD1b, GT1b, and GQ1b and so on (Asthana et al., 2016;Yuki, 2012). It is reported that the GBS is associated with various types of infection (such as Campylobacter jejuni, Cytomegalovirus, Epstein-Barr virus, Mycoplasma pneumoniae, and hepatitis E virus) which lead to a cross-reaction with nervous system, demyelination of neurons, and finally initiation of nervous signs and symptoms by stimulating immune system Taheraghdam et al., 2014). Simultaneously, accumulating evidence has indicated that the antecedent infection with C. jejuni enteritis may trigger the generation of AGA (Nyati & Nyati, 2013).
Moreover, previous studies have shown that Cytomegalovirus (CMV), a member of the β herpes family may lead to the incidence of GBS and is second only to C. jejuni enteritis (Orlikowski et al., 2011;Taheraghdam et al., 2014). Currently, although there are a number of studies on the AGA and a few studies of anti-CMV antibodies in GBS, the association between them remains poorly documented (Annunziata, Figura, Galli, Mugnaini, & Lenzi, 2003;McCombe, Wilson, & Prentice, 1992;Taheraghdam et al., 2014). Therefore, our own research aims to analyze the correlation of anti-CMV antibodies and AGA in the GBS.

| Detection of anti-gangliosides antibodies
We detected auto-antibodies of the IgG and IgM class to the seven gangliosides GM1, GM2, GM3, GD1a, GD1b, GT1b, and GQ1b in serum by The EUROLINE test kit. By using a combination of different antigens on one strip, multiple auto-antibodies against gangliosides can be investigated in one sample simultaneously. The test kit contains test strips coated with parallel lines of purified antigens ( Figure 1). The patient samples for analysis are diluted 1:51 with ready for use diluted sample buffer. Because of the special membrane used in the present EUROLINE, a pretreatment of the test strips is not necessary. Detailed steps are as follows: (1) Fill each channel with 1.5 ml of the diluted samples and incubate for 120 min at room temperature (+18°C to +25°C) on a rocking shaker with the test strips fully covered with liquid and not float on top; (2) Aspirate off the liquid from each channel and wash 3 × 5 min each with 1.5 ml working strength wash buffer on a rocking shaker; (3) Pipette 1.5 ml diluted enzyme conjugate (alkaline phosphatase conjugated anti-human IgG/IgM) into each channel and incubate for 60 min at room temperature (+18°C to +25°C) on a rocking shaker; (4) Aspirate off the liquid from each channel and wash as described above; (5) Pipette 1.5 ml substrate solution into the channels of the incubation tray and incubate for 10 min at room temperature (+18°C to +25°C) on a rocking shaker; (6) Aspirate off the liquid from each channel and wash each strip 3 × 1 min with deionized or distilled water; (7) Place test strip on the evaluation protocol, air dry, and evaluate.

| Detection of anti-CMV antibodies
The electrochemiluminescence immunoassay "ECLIA" is used for the measurement of CMV-IgG/IgM. The first step: 20 μl of sample, F I G U R E 1 GBS was grouped by AGA positive and negative. Median serum CMV-IgG levels were 389.41 and 386.1 U/ml for the groups of AGA positive group and negative group, respectively, and there were no significant differences between them (p > .05)

| Statistical analysis
Statistical analysis was performed using SPSS 20.0. With respect to the clinical features of the patients with GBS, differences in the proportions between groups were tested using the chi-square test or Fisher's exact test, and differences in medians were tested using the t-test or nonparametric test. The level of statistical significance was set at p < .05.

| DISCUSSION
GBS, known as a common cause of acute flaccid paralysis, typically occurs after an antecedent infection. Thereafter, it will produce the AGA against the bacterial lipo-oligosaccharide which cross-react with gangliosides at nerve membranes, finally leading to demyeliniza- F I G U R E 2 GBS was grouped by AGA positive and negative. Median CSF CMV-IgG levels were 8.61 and 6.71 U/ml for the groups of AGA positive group and negative group, respectively, and there were no significant differences between them (p > .05)
(immunoglobulin M-type and G-type AGA were considered together in this figure) jejuni is the most common reason to cause GBS and a second infection associated with the GBS is CMV. Elevated researches showed that Campylobacter jejuni was closely related to the GBS (Koga, Yuki, & Hirata, 1999;Odaka, Koga, Yuki, Susuki, & Hirata, 2003;Ogawara et al., 2000;Zhang et al., 2010Zhang et al., , 2015. Furthermore, serials of investigations indicated that AGA can be found in the serum of patients with CMV-IgG positive (Caudie et al., 2002;Sivadon et al., 2005;Yuki, Yoshino, Sato, & Miyatake, 1990). Meanwhile, Simanek AM and co-workers provided evidence that the CMV reaction had the relationship with chronic inflammation (Simanek et al., 2011). Therefore, we investigated the relationship between AGA and CMV-IgG in patients with GBS in our study.
Our results showed that all 29 patients with GBS was CMV- Hao Q, Aliakbar T and their colleagues respectively (Hao et al., 1998;Taheraghdam et al., 2014). But 31% (9/29) of AGA was found in patients involved in our study, which is higher than the results of them, Moreover, Irie et al. (1996) found that a lower rate of CMV infections in their patients, in which they obtained the serum samples at rather a long time after neurological onset. Additionally,  also showed that anti-GM2 antibodies can be found in some patients with GBS with C. jejuni infections, yet, the frequency was significantly lower than in patients infected with CMV. Interestingly, some AGA specificities are associated with the GBS subtypes such as anti-GM1 is closely related to the AMAN and GQ1b antibody are notably associated with MFS, characterized by ophthalmoplegia, ataxia, and areflexia Mori, Kuwabara, & Yuki, 2012). Researchers have shown that approximately up to 80% GQ1b antibody was found in patients with MFS (Ito et al., 2008;Mori et al., 2012). However, in our study, no GQ1b antibody was measured, al- Except where specified otherwise, the data are n (%) or mean ± SD values.
occur as a result of the detection method used by us. Hashemilar et al. (2014) suggested that EUROLINE method could be used instead of the ELISA method except for the anti-GQ1b antibody.
In this study, we confirmed that there was a trend of higher ratio of men, a younger age onset, less frequent preceding infection, a higher level of CSF proteins, less frequent cranial nerve deficits, although the data did not reach a statistical significance. Moreover, a higher positive rate of CMV-IgG both in the serum and CSF was found in AGApositive group than the negative group, but no statistical significance was found. This result may probably because the small samples are to observe whether they could be serological marker of these diseases or the underlying pathogenesis.