Establishment Of The Sandwich ELISA Methods For Aβ Detection

Objective:Alzheimer’s disease (AD) is an age-related neurodegenerative disease. However, the pathogenesis of AD has been poorly understood and there is no effective treatment.The β-amyloid peptide (Aβ) is considered the initiator of AD and Aβ has become one of the important early biomarkers for AD diagnosis. The aim of this study is to establish the double antibody sandwich ELISA (DAS-ELISA) method for Aβ detection.Methods:(I) Identification of the Aβ standards. The soluble Aβ standards were assembled in vitro, and the molecular size was detected by sodium dodecyl sul-fate-polyacrylamide gel electrophoresis (SDS-PAGE). The Ap standards were nega-tively stained and detected via transmission electron microscopy (TEM). The immu-noreactivity was measured using indirect ELISA. (II) Preparation and identification of candidate antibodies (AC-1, AC-2, AN-1, and ACE-1). Hybridoma cell lines (estab-lished before in our group) were thawed and cultured for induction of ascites in mice, and then the monoclonal antibodies were prepared and purified via affinity chroma-tography. At the same time, rabbit serums anti-AP40/42 was producted as the backup. The titer, sensitivity and specificity of the antibodies were measured by indirect ELISA. (III) Monoclonal antibody labeling. The antibodies were conjugated with bio-tin or horseradish peroxidase (HRP), and the bioactivity was tested through indirect ELISA. (IV) The antibody pairing and the optimization of DAS-ELISA. The formau-lation of coating buffer, concentrations of capture and detection antibodies were opti-mized for antibody pairs screening. The sensitivities of DAS-ELISA were determined.Results:(I) The data of SDS-PAGE analysis suggested that the bands of Aβ standards assembled in vitro ranged about 7-17kD. TEM images showed that the assembled soluble Aβ standards looked spherical or like string of beads via negative staining. AP42 standards instead of Aβ40 standards were exclusiviely recognized by 12F4, a kind of commercial monoclonal antibody which is Aβ42 specific. (II) Anti-body AC-2 recognized Aβ42, while AC-1 recognized Aβ40 specifically; The titers of monoclonal antibodies, AC-1, AC-2, AN-1, and ACE-1, were 1:512000,1:1024000, 1:2048000 and 1:256000, and that of the both of polyclonal antibodies, BJD-002 and BJD-004, was 1:2048000; The sensitivities of monoclonal antibodies, AC-1, AC-2, AN-1, and ACE-1, were 31.2 ng/mL,125 ng/mL,62.5 ng/mL, and 20 ng/mL, and the sensitivities of polyclonal antibodies, BJD-002 and BJD-004, were 31.2 ng/mL and 250 ng/mL respectively; (III) Four conjugated antibodies, AC-l-HRP, AC-2-biotin, AN-1-biotin, and ACE-1-HRP, were with bioactivities for antibody pairing. (IV) The optimized coating buffer was Tris-HCl (pH7.4), and the best concentration of capture antibody was 5 μg/mL; Four antibody pairs were obtained for DAS-ELISA. ACE-1/AC-l-HRP recognized Aβ40, while AN-1/AC-2-biotin recognized Aβ42 specifically.Conclusion:In this study, the antibody pairs which specifically recognized dif-ferent components of AP were successfully found out, and the parameters of DAS-ELISA for Aβ detection were optimized, which would provide support for fu-ther studies in AD diagnosis.