96 well immunosorbent plates

To define HIV-1 particle containing fractions and control purification efficiency, we performed a semi-quantitative p24 ELISA on the fractions using the Alliance HIV-1 p24 ELISA kit (Perkin Elmer). For this assay, we boiled 50 μl of each fraction at 95°C for 5 minutes prior to transfer to immunosorbent 96-well plates (NUNC, Denmark). After sealing the plates were incubated overnight at 4°C. Next, we removed the supernatant and blocked non-specific signal with 5% donkey serum for 30 minutes at room temperature. The subsequent experimental procedures and plate reading were performed following the manufacturer’s instructions.

H. pylori strain P12, or its isogenic cagT deletion mutant, were pre-incubated with DARPins at 5 μM, cisplatin at 100 μM, or left untreated, as described above. Subsequently, AGS cells were infected with the pre-incubation mixtures at an MOI of 60, or left uninfected. Supernatants were collected after 4 h of co-incubation at 37°C and 5% CO₂, and centrifuged to remove unbound bacteria. Aliquots of the cell supernatants were added to immunosorbent 96-well plates (Nunc MaxiSorp) that had been coated overnight with a monoclonal anti-human IL-8 capture antibody (BD Pharmingen 554716; 3 μg/ml), washed 4 times with PBS/0.05% Tween 20, and blocked for 2 h at room temperature with PBS/10% FCS. After overnight incubation at 4°C, plates were extensively washed, then incubated with a biotinylated IL-8 detection antibody (BD Pharmingen 554718; 0.5 μg/ml) for 1 h at room temperature, and washed 4 times with PBS/0.05% Tween 20. For detection, a Vectastain ABC Kit (Biozol) was used together with a TMB substrate reagent set (BD Biosciences), according to the manufacturers’s protocols. Absorbance was measured at 450 nm in a Clariostar reader, and IL-8 concentrations were calculated using a standard curve obtained with an IL-8 standard (BD Pharmingen).

Sporozoite lysate was prepared by pelleting MAF purified sporozoites and flash freezing, before diluting in PBS and using to coat 96-well immunosorbent plates (NUNC MaxiSorp) overnight (1,500 sporozoites per well). Subsequently, liquid was removed and wells allowed to air dry before blocking with 1% BSA in PBS. Wells were subsequently incubated with mouse serum with starting dilutions of 1:50 or 1:100 in 0.01% Tween-PBS. Anti-mouse IgG secondary antibody conjugated to AP (Sigma-Aldrich) was added after five washes in 0.01% Tween-PBS. AP was quantified after five washes in 0.01% Tween-PBS using 4-nitrophenyl phosphate disodium salt hexahydrate (Sigma-Aldrich) measured at 405 nm absorbance.