Quantifying Serum Antibodies Using AlphaLISA

AlphaLISA was used to quantify the serum antibodies against the purified proteins. The α-luminescent photon counts represent the serum antibody levels (35 (link), 40 (link)). The AlphaLISA assay was performed in 384-well microtiter plates (white opaque OptiPlate; PerkinElmer, Waltham, MA, USA). Each well contained 2.5 μL serum (1:100 dilution) and 2.5 μL GST or GST fusion protein (10 μg/mL) in AlphaLISA buffer (25 mM HEPES [pH 7.4], 0.1% (w/v) casein, 0.5% (w/v) Triton X-100, 1 mg/mL Dextran-500, and 0.05% (w/v) Proclin-300). The mixture was then incubated at 25°C for 8 h. Then 2.5 μL of 40 μg/mL anti-human IgG-conjugated acceptor beads and 2.5 μL of 40 μg/mL glutathione-conjugated donor beads were added and the mixture was incubated in the dark at 25°C for 7–21 d. Chemical emission was measured in an EnSpire Alpha microplate reader (PerkinElmer) as previously described. The reactions were calculated by subtracting the Alpha counts for the GST control from those for the GST fusion proteins.
The levels of serum squamous cell carcinoma antigen (SCC-Ag) (41 (link)) and p53 antibody (p53-Abs) (42 (link)) were evaluated as previously described. The serum SCC-Ag and p53-Abs cutoff values were 1.5 ng/mL and 1.3 IU/mL, respectively.