| Multiple sclerosis (MS) is an autoimmune inflammatory disease characterized by demyelination and neurodegeneration within the central nervous system (CNS). Because MS is one of the most common demyelinative diseases and its highest incidence is in young adults, it attracts many clinician's attention. MS is a clinically heterogeneous disease that varies according to the location of plaques or lesions in the CNS, so it is difficult to diagnose early only by clinical manifestations. Establishment of effective auxiliary examinations is becoming more important in this situation. Intrathecal synthesis of immunoglobulins indicates there exists humoral immune response in the CNS. It can be measured by IgG synthesis rate (IgG Syn) and IgG index (IgG I) quantitatively, and can also be measured by the detection of oligoclonal bands (OCBs) qualitatively. Whereas, there are different detection rates in MS patients of different populations, and their diagnostic value varies a lot. The purpose of this investigation is to evaluate the significance of detecting OCBs, IgG Syn and IgG I in the diagnosis of MS in our area. Since isoelectric focusing (IEF) is superior to zone electrophoresis, it has increasingly been used to the studies of proteomics. For this reason, IEF combined with silver staining was applied to detect OCBs in the study, and in the meantime we evaluated its stability and reproducibility. Part 1. The establishment of detecting oligoclonal bands by isoelectric focusing Objective: The study is to establish the technique applying IEF to detect OCBs, and to evaluate its stability and reproducibility. We are also intend to classify cerebrospinal fluid (CSF) and matched serum based on IgG band patterns and explicate the standard of positive result. Methods: From October 2003 to November 2004, 92 patients were chosen for the research. They were divided into three different diagnostic groups: multiple sclerosis (MS) 30 cases, neurological inflammatory diseases (NID) 40 cases and neurological noninflammatory diseases (NNID) 22 cases, according to corresponding diagnostic criteria. All CSF and serum samples were divided into aliquots and stored at -20℃until assayed. IgG in serum and CSF was quantified by rate nephelometry method on an automated Beckman Array 360 System. The CSF and matched serum IgG concentrations were adjusted to the same level by adding phosphate buffered saline. Ampholine polyacrylamide gel plates, pH3.5 to 9.5, were used. The anode solution was 1M phosphoric acid, and the cathode solution 1M sodium hydroxide. Samples (20μl) were applied on 5×10mm pieces of filter paper around the pH5.7 portion of the IEF gel. Focusing was performed for 3 hours at 4℃with 5 to 6watts. After focusing, the bands were visualized by silver staining, including fixing, rinse, silver reaction, developing and stop. Using IEF and silver staining method, human IgG molecules were distributed in pH region 6.5 to 9.5. To assess the system's stability and determine the most appropriate sample loading, we compared the gels of different times and different sample loadings. Two or more discrete bands of immunoglobulins present in CSF that are not present in corresponding serum were considered abnormal and demonstrative of OCBs. Results: 1. The numbers and positions of protein bands in the relative alkaline region between gels performed at different times but with the same sample were identical. 2. The proper sample loading was 0.6～2.0μg of IgG in this approach. 3. There were four types of band patterns: ⑴same polyclonal bands in serum and CSF; ⑵OCBs restricted to CSF; ⑶OCBs restricted to CSF plus some identical bands in CSF and serum; ⑷identical OCBs in CSF and serum. Among the four types, only patterns 2 and 3 represent local synthesis of IgG within the CNS. 4. Of 92 patients, 36 displayed OCBs: 19/30 MS (63.3%), 16/40 NID (40.0%) and 1/22 NNID (4.5%). Conclusions: The study use IEF combined with silver staining to demonstrate the restricted heterogeneous immunoglo-bulins in CSF, and successfully establish the method of detecting...
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