Table 2: Demonstration of pathogenicity of antibodies directed towards type VII collagen.

MethodProtocolReadoutReference

Neutrophil activation in vitroIncubation of isolated neutrophils with immune complexes of anti-COL7 antibodies and COL7(i) ROS release
(ii) Elastase release
[61]

Neutrophil activation ex vivoIncubation of cryosections of human/mouse skin with antibodies against COL7 (serum, IgG, affinity-purified IgG from patients, monoclonal anti-COL7 antibodies, or rabbit anti-COL7 IgG)(i) Deposition of formazan (ROS detection)
(ii) Extent of dermal-epidermal separation
[60, 62]

Anti-COL7 IgG transfer into mice(a) Repetitive injection of rabbit anti-mouse COL7 IgG into C57Bl/6, BALB/c, or BALB/cnude mice(i) Body surface area affected by skin lesions
(ii) Several secondary endpoint measurements
[44]
(b) Repetitive injection of rabbit anti-human COL7 IgG into SKH1 miceSame as above[63]
(c) Repetitive injection of human anti-human COL7 IgG into SKH1 miceSame as above[64]
(d) Repetitive injection of human affinity-purified (CMP) anti-human COL7 IgG into SKH1 miceSame as above[57]
(e) Repetitive injection of human affinity-purified (Fn3-like domain) anti-human COL7 IgG into SKH1 miceSame as above[65]
(f) Repetitive injection of rabbit anti-mouse COL7 IgG (vWFA2-like specific) into several in- or outbred miceSame as above[66]
(g) Repetitive injection of rabbit anti-human COL7 IgG into COL7-humanized miceSame as above[67]

Immunization of mice with COL7(a) Repetitive immunization of SJL/J, BALB/c, and Fc gamma receptor IIB-deficient mice (antigen located within the Fn3-like repeats)(i) Autoantibody production
(ii) Body surface area affected by skin lesions
(iii) Several secondary endpoint measurements
[45]
(b) Single immunization of SJL/J, B6.SJL-H2s, C57Bl/10.s, and MRK/MpJ mice (antigen located within the Fn3-like repeats)Same as above[68, 69]
(c) Single immunization of SJL/J, B6.SJL-H2s, mice (antigen: vWFA2-like domain)Same as above[66]

Abbreviations used: COL7: type VII collagen, ROS: reactive oxygen species.