We have developed a method to detect oligoclonal IgG bands in unconcentrated CSF from patients with MS or guinea pigs with chronic relapsing experimental allergic encephalomyelitis, by isoelectric focusing (IEF) in polyacrylamide gel and silver staining. Five to 10 μl of CSF was sufficient. The bands were identified as IgG in immunofixation after IEF.
We subjected cerebrospinal fluid (CSF) from 20 patients with multiple sclerosis and 20 patients with other neurological diseases to agarose gel ( Panagel ) electrophoresis followed by staining with silver. Ten microliters of unconcentrated CSF from multiple sclerosis patients containing 0.4 to 0.8 microgram of immunoglobulin G was found to be optimum for detection of oligoclonal IgG bands, so identified by immunofixation. The band patterns for unconcentrated CSF stained with silver were almost identical to those for the same CSF concentrated 40-fold and stained with Coomassie Brilliant Blue. Silver staining thus enables the clinical laboratory to electrophorese unconcentrated CSF on commercially prepared ( Panagel ) plates.
Persons with DS (35 years and older) have neuropathological changes characteristic of Alzheimer disease (AD). Many of them show dementia of the Alzheimer type. The amyloid-beta (Abeta) peptides are cleaved from a much larger precursor protein by several enzymes. N-terminally truncated pyroglutamate Abeta starting at position 3 represents a dominant fraction of Aβ peptides in post-mortem brains of AD and DS. Studies showed that pE3-Aβ is plaque specific, and mouse monoclonal antibody to pE3-Aβ showed promise as an agent for immunotherapy (DeMattos et al., Neuron, 2012). There are no published reports of the quantitation of pE3-Aβ in plasma of DS. We examined plasma pE3-Aβ levels in DS (N=35; mean±SD; 47.5±8.7 years old), controls (N=32; 48.3±8.5 years old) and non-DSDD (N=27; 45±10 years old) using high affinity rabbit antibodies specific to pE3-Aβ in sandwich ELISA. The groups were compared with each constituent using Kruskal-Wallis ANOVA. The rabbit antisera to pE3-Aβ were specific to pE3-Aβ, and showed no reactivity to Aβ40 or Aβ42. In sandwich ELISA the antibodies detected pE3-Aβ concentrations of 100 pg/ml. Plasma pE3-Aβ levels were higher in DS (2.5±4.1 ng/ml) vs controls (.29±.72 ng/ml, p<.004), and non-DSDD (.56±1.03 ng/ml, p<.026). pE3-Aβ levels in controls and non-DSDD were similar (p<.253). There was no association of age, sex or apolipoprotein E isoform with pE3-Aβ levels in either group. The higher levels of pE3-Aβ found in DS plasma are consistent with those found in DS and AD brains. Longitudinal studies will determine whether there is a relation between plasma pE3-Aβ levels and cognitive decline in persons with DS accompanying dementia, and in patients with mild cognitive impairment and probable AD.
Abstract We previously analyzed unconcentrated cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS) and other neurologic diseases by isoelectric focusing in agarose gel. We have now developed an immunoblot method for detection of oligoclonal IgG bands in unconcentrated MS CSF. The oligoclonal IgG band patterns seen after immunoblotting were compared with those of conventional immunofixation. Although immunoblotting was found to be rapid the resolution and intensity of oligoclonal IgG bands were somewhat better after immunofixation. Since immunofixation is simpler than immunoblotting, we recommend that clinical laboratories use immunofixation after isoelectric focusing to detect oligoclonal IgG bands in CSF.
May 7, 2019April 9, 2019Free AccessA Highly Sensitive Electrochemiluminescence (ECL) Immunoassay for Measurement of Amyloid Beta1–42 peptide in Human Plasma (P3.1-027)Pankaj Mehta, David Miller, Bruce Patrick, and Thomas WisniewskiAuthors Info & AffiliationsApril 9, 2019 issue92 (15_supplement)https://doi.org/10.1212/WNL.92.15_supplement.P3.1-027 Letters to the Editor
Secreted soluble amyloid-β 1–37 (Aβ 37 ) peptide is one of the prominent Aβ forms next to Aβ 40 , and is found in cerebrospinal fluid (CSF) and blood. Recent studies have shown the importance of quantitation of CSF Aβ 37 levels in combination with Aβ 38 , Aβ 40 , and Aβ 42 to support the diagnosis of patients with probable Alzheimer’s disease (AD), and the value of antibody to Aβ 37 to facilitate drug discovery studies. However, the availability of reliable and specific monoclonal antibody to Aβ 37 is very limited. Our aims were: 1) to generate and partially characterize rabbit monoclonal antibody (RabmAb) to Aβ 37 , and 2) to determine whether the antibody detects changes in Aβ 37 levels produced by a γ-secretase modulator (GSM). Our generated RabmAb to Aβ 37 was found to be specific to Aβ 37 , since it did not react with Aβ 36 , Aβ 38 , Aβ 39 , Aβ 40 , and Aβ 42 in an ELISA or immunoblotting. The epitope of the antibody was contained in the seven C-terminal residues of Aβ 37 . The antibody was sensitive enough to measure CSF and plasma Aβ 37 levels in ELISA. Immunohistological studies showed the presence of Aβ 37 -positive deposits in the brain of AD, and Down syndrome persons diagnosed with AD. Our studies also showed that the antibody detected Aβ 37 increases in CSF and brains of rodents following treatment with a GSM. Thus, our antibody can be widely applied to AD research, and in a panel based approach it may have potential to support the diagnosis of probable AD, and in testing the effect of GSMs to target AD.
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