Usefulness of immunoblotting using purified laminin 5 in the diagnosis of anti-laminin 5 cicatricial pemphigoid

Abstract

Background: Anti-laminin 5 cicatricial pemphigoid (CP) is a mucosal-dominant subepithelial blistering disease characterized by IgG anti-basement membrane zone autoantibodies, that bind to dermal side of 1 M NaCl split skin and immunoprecipitate laminin 5. Laminin 5 is an epidermis-specific extracellular matrix consisting of α3, β3 and γ2 subunits. Recent studies have suggested that autoantibodies of anti-laminin 5 CP recognize the G domains of α3 subunit. Objective: We examined the reactivity of anti-laminin 5 CP by immunoblotting using purified laminin 5 and recombinant proteins of α3 subunit. Method: We first examined the reactivity of anti-laminin 5 CP by immunoblotting using purified laminin 5. To further investigate the epitopes in the G domains of α3 subunit, we produced recombinant proteins of G1–2, G1–3, G2–3, G3–5 domains, that covered entire G domain, and examined the reactivity of anti-laminin 5 CP sera with these recombinant proteins by immunoblotting. Results: By immunoblotting using purified laminin 5, 7 of 21 anti-laminin 5 CP sera reacted with α3 subunit, while 8 sera reacted with β3 subunit and one serum reacted with γ2 subunit. Two sera reacted with both α3 and β3 subunits, while seven sera did not show positive reactivity. This result indicates that the reactivity of anti-laminin 5 CP sera is much more heterogeneous, although the previous studies suggested that most sera reacted with α3 subunit. However, in the studies using recombinant proteins of G domains of α3 subunit, none of the CP sera, including the sera reactive with α3 subunit in purified laminin 5, reacted with any recombinant proteins. The reason for this negative reactivity with the recombinant proteins is not clear. Conclusion: The immunoblotting using purified laminin 5 should be useful technique for the diagnosis of anti-laminin 5 CP, although the sensitivity was less than conventional immunoprecipitation analysis.