Supplemental Methods
Immunohistochemistry (IHC) for detection of converted IgG-k/l deposition in
mouse organs.
KAS-6/1 cells were infected with MOI=1 of MV-lambda for 4 h, wash once and
resuspended at a high density of 4x106 per ml in serum-free medium SFM-1 (Invitrogen)
supplemented with 3 ng/ml IL-6. After 3 days of incubation supernatants from infected
and non-infected control cells were collected and inactivated for 30 min at 56C.
Myeloma IgG was isolated by affinity chromatography using Protein G columns
according to the producer’s recommendations (Pierce). Fractions were desalted on Zeba
desalting columns (Pierce) and concentration of the marker was measured by ELISA as
described in Materals and Methods.
Groups of 2 female nude mice were injected intraperitoneally with purified KAS-6/1 IgG
in PBS. Animals were bled for serum and sacrificed on day 2 and 4. Organs were frozen
and 6 m sections on microscopic slides were prepared for IHC analysis. Human
immunoglobulin concentration in the serum was measured by ELISA.
Tissue sections were fixed in methanol and incubated in 0.2-0.5% H2O2 for 5-10 min to
inactivate endogenous peroxidase activity. Samples were blocked for 20 min with 5%
normal goat serum in PBS, washed and incubated for 30-60 min with 1:100-200 diluted
(in PBS with 5% normal goat serum) HRPO conjugated goat antibody specific for heavy
and light chains of human IgG (Bethyl Laboratories). Slides were washed and visualized
using peroxidase substrate (Vector). Hematoxilin or fast nuclear red (Vector) were used
for counterstaining.
2
KAS-6/1 and Jurkat cells were used as positive and negative control of the assay.