Anti human igg hrp conjugate

Using a vacuum (150 mm Hg), 10μL of 125ng MTP peptide in sodium phosphate buffer (pH 8) was applied onto a wet nitrocellulose membrane placed on a Hoefer PR600 Slot Blot filtration manifold (Fisher Scientific). The membrane was dried at 37 °C for 1 hr and washed with phosphate buffered saline (PBS) containing 5 % skim milk, pH 7.2 (Blocking buffer) with gentle shaking for 1 hr at 37 °C. This was followed by another wash with PBS containing 10% Tween 20 (PBST) for 1 hr at 37 °C. PBS and human serum albumin (HSA) were negative controls and Tuberculin was a positive control.
A 50 μL volume of a 5-fold dilution series of serum/plasma samples in 1×PBS (pH 7.2), ranging from 1 in 5 to 1 in 3125 was applied in duplicate and incubated overnight at 4 °C. After 3 washes with PBST for 1hr, the membrane was incubated with anti-Human IgG-HRP conjugate (ThermoScientific) secondary antibody (1:1000) in blocking buffer, at room temperature for 1 hr with shaking. Following 3 washes in PBST for 1 hr, the membrane was exposed to One Step Ultra TMB blotting substrate (ThermoScientific) in the dark for 5 min. The reaction was stopped by distilled water and the results were documented in the Syngene Imager (Anatech) (iris setting = 3.1). CorelDraw X7 software was used to standardise the intensity and contrast of the images.