Increased antibody levels to stage-specific Epstein-Barr virus antigens in systemic autoimmune diseases reveal a common pathology.

The immune responses to antigens from different stages of the Epstein-Barr virus (EBV) life cycle were investigated in systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Sjögren's syndrome (SS), and systemic sclerosis (SSc) to gain knowledge of EBV's involvement in the etiology of systemic autoimmune diseases (SADs) and for an overview of the humoral immune responses against EBV. Investigations were performed by the use of ELISA. IgM, IgA, and IgG antibody binding to 11 EBV antigens: EBNA1, EBNA2, BALF5, EAD, BALF2, EA/R, VCA p18, VCA p23, gB, gp350, and gp42 were examined in serum pools from SAD patients and healthy controls (HCs). Increased antibody levels against the 11 EBV antigens in the SAD pools were seen compared to the HC pool. Specifically, SLE was characterized by strongly increased IgA to EAD both compared to HCs and other SADs, and RA was characterized by increased IgM levels to several EBV antigens. The SADs may be partly distinguished by their differential immune responses to various antigens in the EBV life cycle. All together, these findings support an association between EBV infection and SADs.

Fluorescent Molecularly Imprinted Polymer Layers against Sialic Acid on Silica-Coated Polystyrene Cores-Assessment of the Binding Behavior to Cancer Cells.

Sialic acid (SA) is a monosaccharide usually linked to the terminus of glycan chains on the cell surface. It plays a crucial role in many biological processes, and hypersialylation is a common feature in cancer. Lectins are widely used to analyze the cell surface expression of SA. However, these protein molecules are usually expensive and easily denatured, which calls for the development of alternative glycan-specific receptors and cell imaging technologies. In this study, SA-imprinted fluorescent core-shell molecularly imprinted polymer particles (SA-MIPs) were employed to recognize SA on the cell surface of cancer cell lines. The SA-MIPs improved suspensibility and scattering properties compared with previously used core-shell SA-MIPs. Although SA-imprinting was performed using SA without preference for the α2,3- and α2,6-SA forms, we screened the cancer cell lines analyzed using the lectins Maackia Amurensis Lectin I (MAL I, α2,3-SA) and Sambucus Nigra Lectin (SNA, α2,6-SA). Our results show that the selected cancer cell lines in this study presented a varied binding behavior with the SA-MIPs. The binding pattern of the lectins was also demonstrated. Moreover, two different pentavalent SA conjugates were used to inhibit the binding of the SA-MIPs to breast, skin, and lung cancer cell lines, demonstrating the specificity of the SA-MIPs in both flow cytometry and confocal fluorescence microscopy. We concluded that the synthesized SA-MIPs might be a powerful future tool in the diagnostic analysis of various cancer cells.

Molecularly imprinted polymers in biological applications.

Molecularly imprinted polymers (MIPs) are currently widely used and further developed for biological applications. The MIP synthesis procedure is a key process, and a wide variety of protocols exist. The templates that are used for imprinting vary from the smallest glycosylated glycan structures or even amino acids to whole proteins or bacteria. The low cost, quick preparation, stability and reproducibility have been highlighted as advantages of MIPs. The biological applications utilizing MIPs discussed here include enzyme-linked assays, sensors, in vivo applications, drug delivery, cancer diagnostics and more. Indeed, there are numerous examples of how MIPs can be used as recognition elements similar to natural antibodies.

Determination of Autoantibodies by Line Immunoblotting.

Autoantibody detection is a useful and obligatory tool for clinicians and researchers in diagnosing autoimmune diseases. Line blotting is a simple, sensitive, and flexible technique for fast semiquantitative detection of multiple antibodies. Line blotting enables the detection of antibodies on membrane strips coated with thin parallel lines of several purified, biochemically characterized antigens, which are fixed onto a synthetic support. Each strip can contain more than ten antigens, thus allowing simultaneous screening for multiple antibodies. Here, we describe the principle of line blotting and how the buffer composition can interfere with the results of autoantibody detection.

EBNA1 IgM-Based Discrimination Between Rheumatoid Arthritis Patients, Systemic Lupus Erythematosus Patients and Healthy Controls.

Epstein-Barr Virus (EBV) has been associated with development of rheumatic connective tissue diseases like rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) in genetically susceptible individuals. Diagnosis of RA and SLE relies on clinical criteria in combination with the presence of characteristic autoantibodies. In addition, antibodies to several EBV antigens have been shown to be elevated in patients with these diseases compared to healthy controls (HC). Here, we elaborated improved enzyme-linked immunosorbent assays for antibodies (IgM, IgA, IgG) to the EBV proteins Epstein-Barr Virus nuclear antigen (EBNA)1 and early antigen diffuse (EAD) in order to determine their potential diagnostic role. We showed that especially EBNA1 IgM distinguished RA from SLE and HCs and also distinguished SLE from HCs. EBNA1 IgA was almost as effective in differentiating RA from SLE and HC, while EAD IgG and IgA were able to discern SLE patients from RA patients and HCs. Collectively, these findings illustrate the potential diagnostic use of antibodies to EBV proteins to diagnose RA and to differentiate SLE from RA.

Efficient evaluation of humoral immune responses by the use of serum pools.

BACKGROUND: Collection and testing of individual serum samples are often used in research to gain knowledge about e.g. the humoral response against bacteria or virus. This is a valid but time-consuming method and might be a waste of valuable serum samples for inefficient research. So far, no study has considered using serum pools as a quick and efficient screening method to confirm or deny hypotheses. METHODS: We created serum pools from four different patient groups (systemic lupus erythematosus n=85, rheumatoid arthritis n=77, Sjögren's syndrome n=91, systemic sclerosis n=66) and one healthy control group (n=67). Each serum pool was analyzed using three well-known immunoassays: enzyme-linked immunosorbent assay (ELISA), line blot, and immunofluorescence microscopy (anti-nuclear antibody (ANA) screening). The presence of Epstein-Barr virus (EBV) EA/D-, EBNA-1-, VCA p23-, and gp350-directed antibodies was used to validate serum pools as an efficient tool for further investigations by comparison to previous findings in this area. RESULTS: The presence of EBV EA/D-, EBNA-1-, VCA p23-, and gp350-directed antibodies in each pool was consistent within the obtained ELISA and line blot results, as increased titers of IgG against the four antigens were found in all patient serum pools and also in individual sera regarding gp350. These results correspond to previous findings on individual samples from patients with these diseases. The presence of ANAs was observed in all four patient serum pools and not in the HC pool by both line blots and immunofluorescence microscopy, which corresponds with the expectations and further corroborate the application of serum pools for screenings. CONCLUSION: We developed and validated the use of serum pools that reliably and rapidly can confirm or deny hypotheses, which enables a more efficient research concentrating on the most evident factors.