Our Anti-Amyloid beta peptide (A-beta 40/42) mouse monoclonal primary antibody detects human and rat Amyloid beta peptide (A-beta 40/42), and is IgG. It is validated for use in ELISA, ICC, IHC-Frozen, IHC-Paraffin-embedded, IP, WB.
Immunohistochemical detection of amyloid plaques in the brain of 9 months old WT and APP/PS1 mice. Brain tissues were fixed with 4% paraformaldehyde and dehydrated in 30% sucrose solution. 30 µm frozen sections were prepared and blocked in 5% Normal horse serum at room temperature for 1h. Then, sections were incubated with biotinylated MOAB-2 antibody (1:1000, red), overnight at 4°C. MOAB-2 binding was visualized with a streptavidin-Cy3 conjugate (1:1000, 1h). Cell nuclei were counterstained with DAPI dye. Figure courtesy of Dr C.S. Ruan, University of South Australia.
The amyloid beta peptide is derived from the cleavage of the Amyloid precursor protein (APP) and varies in length from 39 to 43 amino acids. However, the form(s) of amyloid-beta peptide (Aβ associated with the pathology characteristic of Alzheimer's disease (AD) remains unclear. In particular, the neurotoxicity of intraneuronal Aβ accumulation is an area of considerable research and controversy principally because antibodies thought to be specific for Aβ have been shown to actually detect intraneuronal APP and not Aβ exclusively.
MOAB-2 (mouse IgG2b) is a pan-specific, high-titer antibody to Aβ residues 1-4 as demonstrated by biochemical and immunohistochemical analyses (IHC), and is highly specific just to amyloid beta peptide. MOAB-2 did not detect APP or APP-CTFs in cell culture media/lysates (HEK-APPSwe or HEK APPSwe/BACE1) or in brain homogenates from transgenic mice expressing 5 familial AD (FAD) mutation (5xFAD mice).
Using IHC on 5xFAD brain tissue, MOAB-2 immunoreactivity co-localized with C-terminal antibodies specific for Aβ40 and Aβ42. MOAB-2 did not co-localize with either N- or C-terminal antibodies to APP. In addition, no MOAB-2-immunreactivity was observed in the brains of 5xFAD/BACE-/- mice, although significant amounts of APP were detected by N- and C-terminal antibodies to APP, as well as by 6E10. In both 5xFAD and 3xTg mouse brain tissue, MOAB-2 co-localized with cathepsin-D, a marker for acidic organelles, further evidence for intraneuronal Aβ, distinct from Aβ associated with the cell membrane. MOAB-2 demonstrated strong intraneuronal and extra-cellular immunoreactivity in 5xFAD and 3xTg mouse brain tissues.
Biosensis now offers biotinylated MOAB-2antibody allowing more flexibility in experimental design by using the biotin-avidin/streptavidin detection method. Biotinylated MOAB-2 antibody may also help to reduce background staining in difficult-to-stain tissues and increase detection sensitivity. The ability of biotinylated MOAB-2 antibody to detect amyloid beta has been validated by IHC.
Purified, non-biotinylated MOAB-2 antibody is available here.
IgG
Monoclonal
MOAB-2
ELISA, ICC, IHC, IP, WB
Mouse
Recombinant human amyloid beta protein 42 (Aβ42): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA
Human
Human, Rat
Spin vial briefly before opening. Reconstitute in 50 µL sterile-filtered, ultrapure water to give a concentration of 1 mg/mL. Centrifuge to remove any insoluble material. Final buffer is PBS, pH 7.4 without preservative.After reconstitution keep aliquots at -20°C to -70°C for a higher stability. At 2-8°C keep up to one week; use sterile methods and pipettes. Highly purified glycerol (1:1) may be added for an additional stability. Avoid repetitive freeze/thaw cycles. Keep tightly closed when not in use and protected from light.
Lyophilized
Antibody was purified from cell culture supernatant by Protein G chromatography, biotinylated and buffer-exchanged into PBS, pH 7.4 buffer
Lyophilized from PBS buffer, pH 7.4; contains no preservative.
The biotinylated MOAB-2 antibody has been tested by IHC and is also expected to work in applications validated for the unlabelled antibody (M-1586-100) at same or higher dilutions: Western Blotting (WB), Immunohistochemistry (IHC), Immunohistochemistry/paraffin embedded IHC(P), Immunoprecipitation (IP), Immunofluorescence (IF), ELISA.
Western Blotting:
MOAB-2 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. Standard ECL detection systems.
Tissue samples for the detection of beta-amyloid should be prepared as detailed in Youmans KL et al., 2011 (Journal of Neuroscience Methods 196: 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.
Immunohistochemistry:
IHC:` Fresh frozen, 4% paraformaldehyde fixed frozen, or formalin fixed paraffin embedded tissues are all suitable. 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 buffer for HIER is citrate, pH 6.0. Signal was weak without antigen retrieval. Immunoreactivity was observed 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 A-beta 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 A-beta 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 x 10 min), blocked with 3% horse serum in TBSX (3 x 10 min) followed by 1% horse serum in TBSX (2 x10 min) and incubated with appropriate primary antibodies diluted in TBSX containing 1% horse serum overnight. See Youmans KL et al., 2012, for full IHC(P) protocol and method details.
Immunofluorescence:
IF: The antibody was tested on 4% PFA fixed frozen tissue. Fixed tissues were washed in TBS (3 x 10 min), then incubated in 88% FA (8 min), and then permeabilized in TBSX (3 x 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).
Immunoprecipitation:
Labelled beta-amyloid using SA-coated beads as the capture vehicle, similar to the protocols employed by Youmans KL et al., 2012.
ELISA:
The antibody has been tested in ELISAs on synthetic beta-amyloid and tissue homogenates from beta-amyloid-Tg mice.
Unconjugated
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 F-Aβb42 at antigen concentrations as low as ~ 0.1 pmol (Youmans. KL et al., 2012; PMID: 22423893). MOAB-2 does not detect APP (Amyloid Precursor Protein). Human, rat, other species not yet tested. By Dot Blot, MOAB-2 detected rat Aβ40 and human Aβ40, albeit with less affinity than for Aβ42 (Youmans KL et al., 2012).
