Abstract
We have established high-throughput lectin-antibody ELISAs to measure different glycans on transferrin (Tf) in cerebrospinal fluid (CSF) using lectins and an anti-transferrin antibody (TfAb). Lectin blot and precipitation analysis of CSF revealed that PVL (Psathyrella velutina lectin) bound an unique N-acetylglucosamine-terminated N-glycans on “CSF-type” Tf whereas SSA (Sambucus sieboldiana agglutinin) bound α2,6-N-acetylneuraminic acid-terminated N-glycans on “serum-type” Tf. PVL-TfAb ELISA of 0.5 μL CSF samples detected “CSF-type” Tf but not “serum-type” Tf whereas SSA-TfAb ELISA detected “serum-type” Tf but not “CSF-type” Tf, demonstrating the specificity of the lectin-TfAb ELISAs. In idiopathic normal pressure hydrocephalus (iNPH), a senile dementia associated with ventriculomegaly, amounts of the SSA-reactive Tf were significantly higher than in non-iNPH patients, indicating that Tf glycan analysis by the high-throughput lectin-TfAb ELISAs could become practical diagnostic tools for iNPH. The lectin-antibody ELISAs of CSF proteins might be useful for diagnosis of the other neurological diseases.
Highlights
cerebrospinal fluid (CSF), which circulates within the ventricles of the brain and subarachnoid space, reflects the physiological and pathological conditions of the central nervous system [1]
CSF proteins are used as biomarkers to diagnose neurological diseases such as idiopathic normal pressure hydrocephalus [2,3,4] and Alzheimer’s disease (AD) [5, 6] and may predict the disease onset in a preclinical stage [7]
To establish high throughput lectin-transferrin antibody (TfAb) ELISAs that distinguish “CSF-type” Tf-1 and “serum-type” Tf-2, we first examined whether PVL and SSA detect glycans on Tf-1 and Tf-2, respectively, by lectin-blotting
Summary
CSF (cerebrospinal fluid), which circulates within the ventricles of the brain and subarachnoid space, reflects the physiological and pathological conditions of the central nervous system [1]. Since iNPH and AD show similar phenotypes such as dementia and ventriculomegaly, it is difficult to distinguish the two diseases especially in elderly patients. We previously designated Tf-1 and Tf-2 as two isoforms of transferrin in CSF which are separable by SDS-PAGE and showed that the Tf-2/Tf-1 ratio is higher in iNPH patients than in non-iNPH patients [3, 4]. In that study we used immunoblotting method to detect Tf-1 and Tf2, but the low throughput of immunoblotting makes it impractical for clinical use. Sandwich ELISA (enzyme-linked immunosorbent assay) or latex photometric immunoassay is high throughput, Tf-1 and Tf-2 cannot be distinguished by these methods because Tf-1 and Tf-2 are different only in their glycan portion, and the antibodies
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