Despite its overall simplicity, protein blotting or Western blotting has been proven to be a powerful procedure for the immunodetection of proteins, especially those that are of low abundance, following electrophoresis. The usefulness of this procedure stems from its ability to provide simultaneous resolution of multiple immunogenic antigens within a sample for detection by specific antibodies. Protein blotting has evolved greatly since its inception and researchers have a variety of ways and means to carry out this transfer. This procedure is used in combination with other important antibody-based detection methods such as enzyme-linked immunosorbant assay and immunohistochemistry to provide confirmation of results both in research and diagnostic testing. Specificity of antibodies used for immunohistochemistry is of critical importance and therefore Western blot is a "must" to address antibodies' specificity.
Our previous work showed that immunization of rabbits with 4-hydroxy 2-nonenal-modified Ro60 (HNE-Ro60) accelerates autoimmunity. We extended this model into mice, hypothesizing that the severity of autoimmunity would be dependent on degree of HNE-modification of Ro60. Five groups of BALB/c mice (ten/group) were used. Group I was immunized with Ro60. Groups II to IV were immunized with Ro60 modified with 0.4 mM (low), 2 mM (medium) and 10 mM (high) HNE respectively. Group V controls received Freund's adjuvant. A rapid abrogation of tolerance to Ro60/La antigens occurred in mice immunized with HNE-modified Ro60, especially in the low and medium HNE-Ro60 groups. Lymphocytic infiltration and significantly high decrement in salivary flow (37%) compared to controls was observed only in high HNE-Ro60 group, suggesting induction of a SS-like condition in this group. Anti-dsDNA occurred only in mice immunized with medium HNE-Ro60. This group did not have significant decrement in salivary flow, suggesting induction of SLE-like manifestation in this group. Significantly high antibodies to Ro60 were found in saliva of mice in low and medium HNE-Ro60, and Ro60 groups as well as anti-HNE Ro60 in low and medium HNE-Ro60 groups. Understanding the mechanism of this differential induction may help delineate between these two autoimmune diseases.
Previous studies have showed that immunization with peptides from Ro 60 results in Sjögren's syndrome (SS)-like condition in BALB/c mice. We hypothesized that oral feeding with Ro 60 peptide or Ro 60 would prevent the disease. Four groups (each consisting of 10) of BALB/c mice were used. Group I-III were immunized with Ro 274 peptide. Group IV mice were administered adjuvant only. Group II mice were fed orally with Ro 274 peptide and Group III with Ro 60 for 5 days before immunization. There was a significant reduction in the binding of sera from both Group II and Group III mice to most of the Ro multiple antigenic peptides bound by Group I mice. In Group III mice, salivary flow was maintained above that of the Group I mice (average: 117.5 versus 58.6 ml; t ¼ 2.7; P ¼ 0.02). Salivary infiltrates were drastically decreased in the Ro peptide or Ro 60-fed groups, compared to non-tolerized group. Two of eight mice in Group II and 3/6 mice in Group III had no infiltrates, whereas all eight mice studied in Group I had a significant number of infiltrates. Thus, epitope spreading was prevented, lymphocytic infiltration was blocked and saliva flow was restored by means of oral feeding of either Ro 274 or Ro 60 in this animal model of SS.
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