Inhibitory Effects of Horse Serum on Immunoassay of Horse Ferritin.

Binding of Mammalian and Avian Ferritins with Biotinylated Hemin: Demonstration of Preferential Binding of the H Subunit to Heme

“…After washing, 100-µl aliquots of ELISA buffer containing ALP-conjugated NeutrAvidin at a concentration of 1 µg/ml were added to each well of the plate and then incubated at 37°C for 1 hr. After washing, the ALP enzyme detection reaction was performed using p-nitrophenyl phosphate as described previously [20]. For the inhibition test, hemin was added such that the resulting solution of biotinylated hemin was 54 µM in concentrated NaOH.…”

“…This study demonstrates that avian ferritins as well as mammalian ferritins bind biotinylated hemin and that the H subunit preferentially binds heme. Ferritin and apoferritin preparation: Horse spleen ferritin monomers were purified from commercial horse spleen ferritin as described previously [20]. Horse spleen, bovine spleen, and canine liver ferritin monomers were purified from pieces of frozen horse spleen, bovine spleen and canine liver, respectively, as described previously [13,17,18].…”

Development of a Sandwich Enzyme-Linked Immunosorbent Assay to Detect and Measure Serum Levels of Canine Ferritin

“…Plates were incubated at 37°C for 3 hr. After three times washing with PBST, color development was performed as previously described [11], and the absorbance at 405 nm was measured (iMark, Bio Rad, Tokyo, Japan). The standard curve for this ELISA is shown in Fig.…”

Binding Analysis of Ferritin with Heme Using α-Casein and Biotinylated-Hemin: Detection of Heme-Binding Capacity of Dpr Derived from Heme Synthesis-Deficient <i>Streptococcus mutans</i>

“…After washing, 100 µl of a solution containing 2 µg/ml of ALP-labeled avidin (Thermo Fisher Scientific Inc.) and 1 µg/ml of ALP-labeled goat anti-rabbit IgG antibody (Bethyl Laboratories, Montgomery, TX, U.S.A.) in ELISA buffer was added to the wells of the plate for the detection of biotinylated antibody and IgG antibody bound to the wells, respectively, and the plate was incubated at 37°C for 1 hr. After washing, the enzyme reaction was carried out with disodium p-nitrophenyl phosphate as previously described [17]. The concentration of p-nitrophenol produced by the ALP reaction was determined by measuring the absorbance at 405 nm as the α-casein-binding activity.…”