Hrp conjugated polyclonal antibody goat anti rabbit

Tail blood diluted with assay buffer was kept on ice until the end of sampling, then stored at −20°C until LH assay by the University of Virginia Ligand Assay and Analysis Core (Steyn et al., 2013 (link)). The capture monoclonal antibody (anti-bovine LHß subunit, 518B7) is provided by Janet Roser, University of California, Davis. The detection polyclonal antibody (rabbit LH antiserum, AFP240580Rb) is provided by the National Hormone and Peptide Program (NHPP). HRP-conjugated polyclonal antibody (goat anti-rabbit) is purchased from DakoCytomation (Glostrup, Denmark; D048701-2). Mouse LH reference prep (AFP5306A; NHPP) is used as the assay standard. The limit of quantitation (functional sensitivity) was 0.016 ng/mL, defined as the lowest concentration that demonstrates accuracy within 20% of expected values. Coefficient of variation (%CV) was determined from serial dilutions of a defined sample pool. Intraassay CV was 2.2%; interassay CVs were 7.3% (low QC, 0.13 ng/mL), 5.0% (medium QC, 0.8 ng/mL), and 6.5% (high QC, 2.3 ng/mL).

Eight different Hb-Hp complexes were formed by incubating Hb and Hp (1 : 1 molar ratio) for 15 min at 25°C and then incubating with HDL for one hour. H₂O₂ (1 : 5 molar excess) was added to Hb-Hp complexes for 30 min at 37°C and the samples were passed through a PD-10 column, in order to remove H₂O₂. The samples were diluted to a final concentration of 500 nM and were loaded on SDS-PAGE gels and transferred onto a PVDF membrane. In order to prevent nonspecific antigen binding, the membrane was incubated with 1% milk for 30 min in 25°C. A rabbit anti-Hb antibody (DAKO, dilution 1 : 2000) was added overnight: incubation was performed at 4°C. After washing the membrane for 10 min × 3 times with TBST, polyclonal goat anti-rabbit HRP-conjugated antibody (DAKO, dilution 1 : 2000) was added for 30 min at 25°C. Proteins were identified and quantified using an ECL substrate. The membrane was washed with a reblot buffer purchased from Mercury Scientific & Industrial Products Ltd. (Rosh Ha-ayin, Israel), and the procedure was repeated with rabbit anti-Hp antibody. The potential role of Hb-induced tyrosine radicals in generating Hb-HDL cross-links was tested by studying the effect of different concentrations (10–400 nM) of hydroxyurea on HDL Hb cross-linking.