Horseradish peroxidase (HRP) is a 44 kDa heme-containing glycoprotein that catalyzes the oxidation of substrates such as TMB or DAB in the presence of hydrogen peroxide, producing a chromogenic or chemiluminescent signal. For directly conjugated primary antibodies, HRP is covalently linked to the immunoglobulin, typically through amine-reactive or carbohydrate-directed chemistries, and the resulting conjugate is characterized by its enzyme-to-protein molar ratio. For a representative anti-rat IgG2a HRP conjugate, this ratio is 4:1. Direct HRP labeling eliminates the secondary antibody incubation step, reducing assay time and avoiding cross-reactivity from anti-rat secondary reagents—a particular advantage in rat-on-rat applications where endogenous immunoglobulins would otherwise produce high background.

Applications

Directly HRP-conjugated anti-rat primary antibodies are validated across several immunoassay platforms. The anti-rat CD11b monoclonal antibody (clone TJL-163) is documented for flow cytometry, immunohistochemistry, and immunohistochemistry on frozen sections. The anti-rat C5b-9 monoclonal antibody (clone TJL-64) is validated for Western blot (1:100–1:1,000), immunohistochemistry (1:50–1:500), and ELISA (1:30–1:3,000). For broader anti-rat immunoglobulin reagents that are sometimes classified as primary antibodies, working dilutions range from 1:2,000–1:20,000 for immunoblotting, 1:200–1:2,000 for immunohistochemistry and immunocytochemistry, and 1:10,000–1:100,000 for ELISA.