MOAB-2 detects preparations enriched in U-, O-, F-Aβ42, and U-Aβ40 by dot-blot, and is thus a pan-specific Aβ antibody. However, MOAB-2 is selective for the more neurotoxic Aβ42 compared to Aβ40. Indeed, MOAB-2 demonstrated a titration against antigen concentration, and detects Aβ40 at 2.5 pmol but U-, O- and FAβb42 at antigen concentrations as low as ~ 0.1 pmol {Youmans. KL et al 2012}. MOAB-2 does not detect APP (Amyloid precursor protein).
Application Information:
Western Blotting (WB), Immunohistochemistry (IH), Immunohistochemistry/paraffin embedded IH(P), Immunoprecipitation (IP), Immunofluorescence (IF), ELISA.

Antibody has been tested in WB using purified synthetic beta-amyloid preparations and from transgenic mouse brain formic acid extracts (see figure 1). Formic acid extraction/concentration is required for western blot detection from extracts. MOAB-2 antibody is specific for beta-amyloid and does not detect APP. Suggested dilution of 1:2000-1:5,000 for WB, standard ECL detection systems.

Tissue samples for the detection of beta-amyloid should be prepared as detailed in K.L. Youmans et al. {Journal of Neuroscience Methods 196 (2011) 51–59} for best results. Detection of beta-amyloid 40/42 in direct westerns can be difficult; Dot-blots of prepared samples are recommended as detailed in Youmans. KL et al 2012.

IR or fluorescent detection systems not yet tested, they but are expected to work well with higher primary antibody dilutions because of the increased sensitivity of the detection methods.

Suggested dilutions for IHC are 1:50-1:1,000. Fresh frozen, 4% paraformaldehyde fixed frozen, or formalin fixed paraffin embedded tissues are all suitable. Optimal dilutions must be determined by the end user. Antigen retrieval is required in fixed tissues for optimal staining.

Antibody was tested on 4% paraformaldehyde/0.1% glutaraldehyde fixed frozen tissue from 3xTg and 5xFAD mice. MOAB-2 antibody detects intraneuronal and extracellular beta-amyloid in IHC and does not detect APP {Youmans KL et al 2012}.

The antibody also reacts with archival formalin-fixed, paraffin-embedded tissue samples with antigen Heat Induced Epitope Retrieval (HIER): Recommended Citrate, pH 6.0 buffer for HIER. Signal was weak without antigen retrieval. Immunoreactively was expressed in intraneural-amyloid deposition (plaque) in Alzheimer’s brain. MoAB-2 was found to be extremely clean and with an excellent signal to noise ratio with no neuro-cellular diffusive staining.

In addition MOAB-2 demonstrated no significant differences in Aβ detection using paraffin fixed, free-floating sections {Youmans KL et al 2012}. Formic acid (FA) treatment resulted in optimal detection of both intraneuronal and extracellular Aβ compared to without FA (incubated in 88% FA 8 min, Youmans KL et al 2012). Free floating tissue sections were permeabilized in TBS containing 0.25% Triton X-100 (TBSX; 3 × 10 min), blocked with 3% horse serum in TBSX (3 × 10 min) followed by 1% horse serum in TBSX (2 ×10 min) and incubated with appropriate primary antibodies diluted in TBSX containing 1% horse serum overnight. See Youmans KL et al 2012 for full IH(P) protocol and method details.

For IF, suggested dilution is 1:100-1:500. The antibody was tested on 4% PFA fixed frozen tissue. Fixed tissues were washed in TBS (3 × 10 min), then incubated in 88% FA (8 min), and then permeabilized in TBSX (3 × 10 min), and blocked in TBSX containing 5% bovine serum albumin (BSA; 1 hr). Sections were subsequently incubated with appropriate primary antibodies diluted in TBSX containing 2% BSA overnight on an oscillatory rotator. Detection was via fluorescently labelled absorbed secondary antibodies {Youmans KL et al 2012}.

For IP, the suggested dilution is 1:200 to 1:1,000 for labeled beta-amyloid using Protein A/G conjugated beads as the capture vehicle {Youmans KL et al 2012}.

In an ELISA, a dilution of 1:50-1:1000 is suggested. The antibody has been tested in ELISAs on synthetic beta-amyloid and tissue homogenates from beta-amyloid-Tg mice. Biosensis recommends optimal dilutions/concentrations should be determined by the end user for all applications. Dilutions provided are only meant to serve as a basic guide.