Dear Gerald,

The attached paper entitled " Anti-CD40 Monoclonal Antibody Synergizes with CTLA-4 Ig in Promoting Long-Term Graft Survival in Murine Models of Transplantation" describes the requested protocol:

Competitive binding and affinity assays

The M12 B cell lymphoma cell line was generously provided by N. Iwakoshi (Emory University, Atlanta, GA). To compare the binding affinities of the antibodies, M12 B cells were incubated at 4°C for 30 minutes with 8 pg/mL to 1 mg/mL of 7E1-G1, 7E1-G2b, isotype matched control or FGK4.5. Isotype control antibodies and FGK4.5, a rat IgG2a isotype (42), were purchased from BioXCell (West Lebanon, NH). Cells were washed with FACS buffer (phosphate buffered saline (PBS) supplemented with 0.5% bovine serum albumin and 1 mM EDTA, pH 7.2) and bound antibody was then detected with PE-conjugated F(ab')2 goat anti-rat IgG (Jackson ImmunoResearch, West Grove, PA) at 4°C for 30 minutes. For the competitive binding assay, cells were incubated with the same titrations of antibodies as noted above, along with 10 μg/mL of FLAG-tagged soluble recombinant mouse CD154 (Axxora, San Diego, CA) at 4°C for 30 minutes. Cells were washed with FACS buffer and bound CD154 was detected with FITC-conjugated anti-FLAG M2 antibody (Sigma-Aldrich, St. Louis, MO) at 4°C for 30 minutes. Nonviable cells were excluded by the addition of 7AAD 10 minutes prior to sample acquisition (BD Biosciences, San Jose, CA).

Hoping this will be helpful,

Rafik

 Thank you very much Rafik!

Gerald, here is another method:

Competition AlphaLISA

Affinity of CTLA4-Ig variants to ligand was measured by an amplified

luminescent proximity homogeneous assay. Abatacept (Bristol-Myers

Squibb, Princeton, NJ) was biotinylated with sulfo-NHS-biotin using

standard methods and dialyzed against PBS. AlphaLISA acceptor beads

were conjugated with goat anti-human Cl Ab by following the manufacturer’s

instructions (“AlphaLISA assay development guide,” PerkinElmer, Waltham,

MA). The binding assay was performed in a 384-well AlphaPlate (Perkin-

Elmer) in assay buffer (1% BSA, 0.01% Tween 20 in PBS [pH 7.1]). For

CD80 binding assays, 5 ml 1:4 serially diluted unlabeled CTLA4-Ig variant

protein was added to each well followed by the addition of 5 ml 2.5 nM

biotinylated abatacept. Then, a mixture of 5 ml 0.39 mg/ml CD80-Cl and

5 ml 25 mg/ml goat anti-human Cl-conjugated acceptor beads was added.

Each data point of the dilution curve was duplicated in the assay plate. The

plate was incubated in the dark for 1 h at room temperature. AlphaScreen

streptavidin donor beads (PerkinElmer) were subsequently added to each

well at 5 mg/ml to be 25 ml total reaction volume. The plate was incubated

in the dark at room temperature for an additional 30 min. As a result, the

final concentration of each component per sample was 40 nM competitor

variant protein to start with, 0.5 nM biotinylated abatacept, 0.078 mg/ml

CD80-Cl, and 5 mg/ml each type of beads. For CD86 binding assays, the

final concentration of each component per sample was 100 nM competitor

variant protein to start with, 8 nM biotinylated abatacept, 0.1 mg/ml CD86-

Cl, and 10 mg/ml each type of beads. Fluorescence of the plates was read

on an EnVision multilabel reader (PerkinElmer). Data were fitted using

nonlinear regression with the software GraphPad Prism (GraphPad Software,

San Diego, CA) and reported as IC50 wild-type/IC50 mutant.

Thank you very much! This is very helpful and greatly appreciated! Best regards, Gerald