Goat anti mouse igm

Antibody concentrations in mouse plasma were measured with standard ELISA assays and determined as μg anti-Aβ42 IgG/ml plasma. Aβ peptides were purchased from rPeptide, AnaSpec, Bachem, and and were used at concentrations of 2 μg/ml in coating buffer. Antibody isotypes binding to Aβ42 were measured with biotinylated goat anti-mouse IgG1, and goat anti-mouse IgG2a and IgG2c (Balb/c mice possess the Igh1-a allele, B6/129F2 mice carry both alleles, Igh1-a and Igh1-b, 33 (link), 34 (link)), goat anti-mouse IgG2b, and goat anti-mouse IgM secondary antibodies (Southern Biotechnology) followed by Avidin-HRP incubation and TMB detection.
IgA antibodies were assayed with two secondary anti-mouse IgA antibodies: goatanti-mouse IgA (Southern Biotechnology) and rat-anti-mouse IgA (Biolegend).

ELISA and ELISPOT were performed as previously described (25 (link)). For detection of total serum IgM and IgG, serum Ig was captured using goat anti-mouse IgM or IgG(H+L) (1 μg/ml) and detected using polyclonal HRP-conjugated Abs to IgM, IgG1, IgG2a, IgG2b, or IgG3 (SouthernBiotech) at a dilution of 1:3000. For PC ELISA, plates were coated with PC-2–BSA or BSA alone (5 μg/ml), and sera were added at 1:5 starting dilution and serially diluted 2-fold. Titers were calculated by subtracting the BSA signal from the PC BSA signal. NP-binding IgM was captured using plates coated with NP-29–BSA (1 μg/ml) in PBS. For ELISPOT, wells were coated with goat anti-mouse IgM (10 μg/ml). BM and spleen cells were plated in duplicate at 5 × 10⁵ cells per well and 2.5 × 10⁵ cells per well, respectively, and serially diluted 2-fold. Spots were detected with goat anti-mouse IgM-biotin (1:10,000), followed by SA–alkaline phosphatase (1:1,000), and developed with BCIP/NBT Substrate.

An SDS-PAGE assay (Thermo Fisher Scientific) was carried out to examine the protein profile of AGS cell lysates, AGS membranes, and AGS-NPs. Specifically, all samples were adjusted to equivalent total protein concentrations in lithium dodecyl sulfate (LDS) loading buffer. The samples were then separated on a 4–12% Bis-Tris 17-well minigel in MOPS running buffer using a Novex Xcell SureLock Electrophoresis System. The protein bands were stained with InstantBlue Protein Stain (Expedeon) for observation according to manufacturer’s protocol. Western blotting was conducted to identify membrane proteins on AGS-NPs. Specifically, gels from the SDS-PAGE study were transferred onto a nitrocellulose membranes (Thermo Scientific) and probed with primary antibodies including mouse anti-human CD29 (Biolegend), mouse anti-human Blood Group Lewis b (Santa Cruz Biotechnology), and mouse anti-human CD66e (CEACAM5, Santa Cruz Biotechnology). Horseradish peroxidase (HRP)-conjugated secondary antibodies including goat anti-mouse IgG (Biolegend) and goat anti-mouse IgM (Southern Biotech) were used as secondary staining based on the isotype of the primary antibodies. The nitrocellulose membrane was then incubated with ECL western blotting substrate (Pierce) and developed with the Mini-Medical/90 Developer (ImageWorks).

The synthetic TLR4 agonist, E6020, was provided by Eisai, Inc. (Cambridge, MA) and reconstituted in endotoxin-free saline (E6020)^{(11 (link))}. Sterile fluid thioglycollate was purchased from Remel (ThermoFisher, Dover, DE). The hapten 4-Hydroxy-3-nitrophenylacetyl (NP) conjugates, NP₂₈-bovine serum albumin (BSA), NP₄₄-Ficoll, and NP₁₇-Ovalbumin (Ova), were purchased from Biosearch Technologies (Novarto, CA) and reconstituted in endotoxin-free water from Quality Biological (Gaithersburgh, MD). NP-Ficoll and NP-Ova were twice purified over endotoxin removal columns from Pierce Biotechnologies (ThermoFisher, Dover, DE). HRP-conjugated secondary antibodies include goat-anti-mouse IgM, IgG1, IgG2b, IgG2c, and IgG3 that had been purchased as adsorbed against all other mouse immunoglobulin isotypes as well as against human sera to limit cross-reactivity from Southern Biotech (Birmingham, AL).