Alkaline phosphatase (AP)-conjugated anti-mouse antibodies are enzyme-linked immunoreagents designed for sensitive detection of mouse immunoglobulins and antigens in immunoassays. As with other enzyme-conjugated anti-species reagents, these products are predominantly secondary antibodies—anti-mouse immunoglobulins raised in a different host species (most commonly goat, rabbit, sheep, or donkey) and covalently linked to AP. They bind to mouse primary antibodies or to mouse immunoglobulins present in a sample and provide an enzymatic signal for detection. AP-conjugated primary antibodies are commonly used in enzyme-driven applications, such as Western blotting, ELISA, immunohistochemistry, and others, with detection achieved through chemiluminescent or colorimetric AP substrates.

Applications

AP-conjugated anti-mouse antibodies are validated for a broad range of immunoassay applications.

ELISA. These conjugates are extensively used as secondary detection reagents in enzyme-linked immunosorbent assays. The goat anti-mouse IgG (H&L)-AP conjugate is recommended for ELISA at 1:15,000. AP-conjugated antiserum produces the most sensitive and convenient ELISA for the murine viruses examined, using the substrate p-nitrophenyl phosphate, which produces a water-soluble yellow reaction product. In a representative protocol, the appropriate dilution of anti-mouse immunoglobulins conjugated with alkaline phosphatase is prepared in conjugate buffer and added to each well.

Western Blotting. AP conjugates are used with chemiluminescent or colorimetric substrates for detection of mouse proteins transferred to membranes. Recommended dilutions for Western blot range from 1:500 to 1:20,000 depending on the product and application. The detection limit with AP conjugates was 4 ng/mL for mouse IgG.

Immunohistochemistry. AP-conjugated anti-mouse antibodies are suitable for immunohistochemical staining of frozen and paraffin-embedded tissue sections, with working dilutions typically ranging from 1:200 to 1:20,000.

Immunocytochemistry and Flow Cytometry. The AP-conjugated donkey anti-mouse IgG secondary antibody is suitable for immunomicroscopy and flow cytometry. Goat anti-mouse IgA + IgG + IgM antibody (AP) reacts with the heavy and light chains of mouse IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA as demonstrated by ELISA, FLISA, and/or flow cytometry.

ELISPOT and FLISA. AP-conjugated anti-mouse antibodies are also validated for ELISPOT and fluorescent immunoassay applications, including detection of mouse IgE and kappa light chains.