Cell-based immunofluorescence test applying recombinant laminin 332 for the serological differential diagnosis of pemphigoid
S.Goletz1,  C. Probst2, L. Komorowski2, W. Schlumberger2, D. Zillikens3, W. Stöcker2, and E. Schmidt1,3
1Lübeck Institute for Experimental Dermatology (LIED), University of Lübeck, Lübeck, 
 Germany
2 Institute of Experimental Immunology, EUROIMMUN AG, Lübeck, Germany
3Department of Dermatology, University of Lübeck, Lübeck, Germany

A subgroup of patients with mucous membrane pemphigoid (MMP) exhibits autoimmune reactions against laminin 332 (Lam332) and can be identified by the presence of the corresponding autoantibodies. Nearly 30% of patients with anti-Lam332 MMP develop solid cancers, yet no commercial diagnostic assay for anti-Lam332 autoantibodies is widely available. In this study, recombinant Lam332 or fragments of it were used in a cell-based immunofluorescence (IF) test applying a heterotrimer of alpha3, beta3, and gamma2 as well as the chains separately expressed in HEK293 cells.
Subsequently, sera from 93 anti-Lam332 positive MMP patients defined by Western blot reactivity with extracellular matrix of cultured human keratinocytes were tested. Controls included sera from patients with non-anti-Lam332 MMP (n=153) serologically defined by lack of dermal site reactivity with salt-split skin and/or by reactivity against BP180 and/or BP230. Furthermore, sera from patients with bullous pemphigoid (n=20), pemphigus vulgaris (n=20), non-inflammatory dermatoses (n=22), and from blood donors (n=100) were included. 
The IF test with the heterotrimer full length laminin 332 as antigen substrate revealed a sensitivity of 69%, with the alpha3 chain 43%, the beta3 chain 34%, and the gamma2 chain 10% at a specificity of 100% with each of the substrates. The combination did not significantly increase the sensitivity. Of the 93 MMP sera that were reactive with the cell-derived Lam332 by Western blotting, 72 reacted with the dermal site of human salt-split skin. The sensitivity in this clearer defined MMP group with the Lam 332 heterotrimer was 86%. 
The novel IF-based assay with recombinant Lam332 as a substrate is beneficial for the serological diagnosis of anti-Lam332 MMP.