Mouse monoclonal antibody isotyping kit

The recombinant FXYD6 proteins antigen was produced by bacteria, and purified from the soluble cell lysate fractions by nickel affinity chromatography. The anti-human FXYD6 mAb of FD10 was generated from mouse. The mAb was purified from mice ascites and the isotype was IgG2 determined by a mouse monoclonal antibody isotyping kit (Sigma) according to the manufacturer’s instructions.

All hybridoma clones were screened for reactivity to the respective unconjugated peptide that was used for immunization by enzyme-linked immunosorbent assay (ELISA). MaxiSorp plates (Thermo Scientific, Waltham, MA) were coated with 1 μg/ml peptide in PBS and blocked with 5% FBS/PBS. Media from the hybridomas was applied to plates, which were then incubated at room temperature. Next, the plates were washed with PBS and incubated with goat anti-mouse secondary antibody conjugated to horseradish peroxidase (HRP; Jackson Immuno Research Labs, West Grove, PA) at room temperature. Then, plates were washed and TMB substrates (Pierce, Rockford, IL) were applied until color changes were observed. Reactions were then quenched with 1 M HCl and absorbance was measured at 450 nm. Clones that were positive by ELISA were transferred to larger culture plates as needed. The positive clones were next screened by immunohistochemistry of a human AD autopsy case with abundant tau pathology.
Antibody clones were isotyped with the mouse monoclonal antibody isotyping kit purchased from Sigma-Aldrich (St. Louis, MO).

Both the class and subclass of the mAbs against Ag85A produced were determined using a mouse monoclonal antibody isotyping kit (Sigma-Aldrich, USA). Protocols were carried out according to the manufacturer’s instructions.
ELISA plates (Nunc, Denmark) were coated with purified rHis-Ag85A protein (1 µg/ml) and left overnight at 4°C. The next morning, plates were washed three times with PBST and blocked with phosphate-buffered saline (PBS) containing 2% bovine serum albumin (BSA) at 37°C for 2 h. Following the blocking step, the plates were washed three times with PBST. Plates were then incubated with serially diluted culture supernatant and ascites. Plates were incubated with primary antibody for 1 h at 37°C. HRP-conjugated goat anti-mouse IgG antibody (1:8,000 dilution) (Sigma-Aldrich, USA) was then added to each well (100 µl/well) and incubated for 1 h at 37°C. Finally, the TMB substrate was then added to the plates and incubated for 10 min, after which the plates were read at 450 nm.