Goat anti rabbit igg peroxidase

EXAMPLE 8

For the ELISA assessing the species specificity of three different subclones, 96-well microtiter plates were coated with 0.5 μg/ml human CD95L-T4 (black) or 0.5 μg/ml Macaca fascicularis CD95L-T4 (dark grey) or 0.5 μg/ml Mus musculus CD95L-T4 (light grey). After blocking with StartingBlock, wells were incubated with subclones 119-4, 103-7 and 145-12 at a final dilution of 1:200. Binding of the rabbit monoclonal antibodies was detected by incubation with goat anti rabbit IgG-Peroxidase (Sigma; dilution 1:5000) and subsequent detection of the converted Peroxidase-substrate TMBone at a wavelength of 450 nm in an ELISA reader (FIG. 8).

Clone 119-4 shows strong binding to CD95L from all tested species. Antibodies from clone 103-7 and 119-4 showed strong binding to human and monkey CD95L and only weak binding to CD95L derived from mouse.

EXAMPLE 14

For the ELISA assessing the epitope of clone 145-12, 96-well microtiter plates were coated with 2 μg/ml human CD95L (APG296) or 2 μg/ml human LIGHT (APG707) or peptides immobilized to BSA or ovalbumin that comprise a part of the extracellular amino acid sequence of human CD95L. After blocking with StartingBlock, wells were incubated with clone 145-12 at a concentration of 2 μg/ml. Binding of the rabbit monoclonal antibody was detected by incubation with goat anti rabbit IgG-Peroxidase (Sigma; dilution 1:5000) and subsequent detection of the converted Peroxidase-substrate TMBone at a wavelength of 450 nm in an ELISA reader (FIG. 14).

Antibody 145-12 shows specific binding to APG296. However, binding to the tested peptides is weak or even absent (peptide “C-YMRNSKY” of SEQ IDS NO: 68). Although the antibody shares the same epitope as clone 119-4 (as shown by peptide-array) it is conceivable that other possibly structural components of CD95L are required to define the full epitope of the 145-12 antibody.