Hrp donkey anti guinea pig antibody

A sandwich enzyme-linked immunosorbent assay (ELISA) was performed to
detect rotavirus antigen in mouse feces as previously described (Zhang et al., 2014 (link)). Briefly, mouse Fecal Rotavirus
shedding was analyzed via Sandwich Elisa. Ninety-six well EIA/RIA plates
(Costar, 3590) were coated with Rabbit Anti-Rotavirus Group-A (Biorad, AHP1360)
capture antibody at 1:1000 dilution in PBS overnight RT, and subsequently
blocked by 1% BSA PBS. Mouse fecal homogenates were prepared at 100 mg/mL
concentration and centrifuged at 10,000g to remove debris. Supernatants of the
homogenates were then incubated in test tubes with serial dilutions of stock
mouse Rotavirus utilized as control. Hyperimmune guinea pig anti-RRV serum
(Prfal) at 1:1000 dilution in 1% BSA PBS was incubated as detection antibody
followed by the incubation of HRP Donkey Anti-Guinea Pig Antibody (Jackson
ImmunoResearch, 706-035-148) secondary antibody at 1:10000 dilution in 1% BSA
PBS. All incubation steps following capture antibody were at one hour at room
temperature. TMB ELISA Substrate Solution (Invitrogen, 00420156) was utilized to
develop the signal, followed by addition of TMB Stop Solution (KPL, 50-85-04)
after ten minutes of substrate incubation. OD readings were taken at 450nm with
540nm OD subtracted as a correction (Zhang et
al., 2014).