Increased levels of antigen-bound ß-amyloid autoantibodies in serum and cerebrospinal fluid of Alzheimer's disease patients.

Recent studies have suggested a protective role of physiological ß-amyloid autoantibodies (Aß-autoantibodies) in Alzheimer's disease (AD). However, the determination of both free and dissociated Aß-autoantibodies in serum hitherto has yielded inconsistent results regarding their function and possible biomarker value. Here we report the application of a new sandwich enzyme-linked immunosorbent assay (ELISA) for the determination of antigen-bound Aß-autoantibodies (intact Aß-IgG immune complexes) in serum and cerebrospinal fluid (CSF) of a total number of 112 AD patients and age- and gender-matched control subjects. Both serum and CSF levels of Aß-IgG immune complexes were found to be significantly higher in AD patients compared to control subjects. Moreover, the levels of Aß-IgG complexes were negatively correlated with the cognitive status across the groups, increasing with declining cognitive test performance of the subjects. Our results suggest a contribution of IgG-type autoantibodies to Aß clearance in vivo and an increased immune response in AD, which may be associated with deficient Aß-IgG removal. These findings may contribute to elucidating the role of Aß-autoantibodies in AD pathophysiology and their potential application in AD diagnosis.

Antigen-bound and free ß-amyloid autoantibodies in serum of healthy adults.

Physiological ß-amyloid autoantibodies (Aß-autoantibodies) are currently investigated as potential diagnostic and therapeutic tools for Alzheimer's disease (AD). In previous studies, their determination in serum and cerebrospinal fluid (CSF) using indirect ELISA has provided controversial results, which may be due to the presence of preformed Aß antigen-antibody immune complexes. Based on the epitope specificity of the Aß-autoantibodies, recently elucidated in our laboratory, we developed (a) a sandwich ELISA for the determination of circulating Aß-IgG immune complexes and (b) an indirect ELISA for the determination of free Aß-autoantibodies. This methodology was applied to the analysis of serum samples from healthy individuals within the age range of 18 to 89 years. Neuropsychological examination of the participants in this study indicated non-pathological, age-related cognitive decline, revealed especially by tests of visual memory and executive function, as well as speed-related tasks. The ELISA serum determinations showed significantly higher levels of Aß-IgG immune complexes compared to free Aß-autoantibodies, while no correlation with age or cognitive performance of the participants was found.

Interaction structure of the complex between neuroprotective factor humanin and Alzheimer's ß-amyloid peptide revealed by affinity mass spectrometry and molecular modeling.

Humanin (HN) is a linear 24-aa peptide recently detected in human Alzheimer's disease (AD) brain. HN specifically inhibits neuronal cell death in vitro induced by ß-amyloid (Aß) peptides and by amyloid precursor protein and its gene mutations in familial AD, thereby representing a potential therapeutic lead structure for AD; however, its molecular mechanism of action is not well understood. We report here the identification of the binding epitopes between HN and Aß(1-40) and characterization of the interaction structure through a molecular modeling study. Wild-type HN and HN-sequence mutations were synthesized by SPPS and the HPLC-purified peptides characterized by MALDI-MS. The interaction epitopes between HN and Aß(1-40) were identified by affinity-MS using proteolytic epitope excision and extraction, followed by elution and mass spectrometric characterization of the affinity-bound peptides. The affinity-MS analyses revealed HN(5-15) as the epitope sequence of HN, whereas Aß(17-28) was identified as the Aß interaction epitope. The epitopes and binding sites were ascertained by ELISA of the complex of HN peptides with immobilized Aß(1-40) and by ELISA with Aß(1-40) and Aß-partial sequences as ligands to immobilized HN. The specificity and affinity of the HN-Aß interaction were characterized by direct ESI-MS of the HN-Aß(1-40) complex and by bioaffinity analysis using a surface acoustic wave biosensor, providing a K(D) of the complex of 610 nm. A molecular dynamics simulation of the HN-Aß(1-40) complex was consistent with the binding specificity and shielding effects of the HN and Aß interaction epitopes. These results indicate a specific strong association of HN and Aß(1-40) polypeptide and provide a molecular basis for understanding the neuroprotective function of HN.

Differential epitope identification of antibodies against intracellular domains of alzheimer's amyloid precursor protein using high resolution affinity-mass spectrometry.

Several polypeptides comprising the carboxy-terminal domain of the 1-amyloid precursor protein (cAPP) were prepared by solid phase peptide synthesis, and employed as antigens for the determination of the epitopes recognised by anti-cAPP antibodies. Selective proteolytic epitope-excision and -extraction on the immobilised immune complexes, in combination with high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) were used as major methods for epitope identification. The epitope recognised by a polyclonal anti-cAPP antibody (36-BO) was identified as APP(727-737), a sequence close to the APP transmembrane region. In contrast, the epitope recognised by a monoclonal anti-cAPP antibody (Jonas-mAb) was identified at APP(740-747) to be located more remote from the transmembrane region. The two adjacent, yet distinct epitopes recognised by two different antibodies should provide efficient tools for (i), molecular diagnostic applications, and (ii), the study of intracellular processing pathways of APP relevant to Alzheimer's disease, utilising suitable mass spectrometric and molecular imaging approaches.

Mass spectrometric approaches for elucidation of antigenantibody recognition structures in molecular immunology.

Mass spectrometric approaches have recently gained increasing access to molecular immunology and several methods have been developed that enable detailed chemical structure identification of antigen-antibody interactions. Selective proteolytic digestion and MS-peptide mapping (epitope excision) has been successfully employed for epitope identification of protein antigens. In addition, "affinity proteomics" using partial epitope excision has been developed as an approach with unprecedented selectivity for direct protein identification from biological material. The potential of these methods is illustrated by the elucidation of a beta-amyloid plaque-specific epitope recognized by therapeutic antibodies from transgenic mouse models of Alzheimer's disease. Using an immobilized antigen and antibody-proteolytic digestion and analysis by high resolution Fourier transform ion cyclotron resonance mass spectrometry has lead to a new approach for the identification of antibody paratope structures (paratope-excision; "parex-prot"). In this method, high resolution MS-peptide data at the low ppm level are required for direct identification of paratopes using protein databases. Mass spectrometric epitope mapping and determination of "molecular antibody-recognition signatures" offer high potential, especially for the development of new molecular diagnostics and the evaluation of new vaccine lead structures.