Usefulness of immunoblotting using purified laminin 5 in the diagnosis of anti-laminin 5 cicatricial pemphigoid.
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 alpha3, beta3 and gamma2 subunits. Recent studies have suggested that autoantibodies of anti-laminin 5 CP recognize the G domains of alpha3 subunit.


OBJECTIVE
We examined the reactivity of anti-laminin 5 CP by immunoblotting using purified laminin 5 and recombinant proteins of alpha3 subunit.


METHODS
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 alpha3 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 alpha3 subunit, while 8 sera reacted with beta3 subunit and one serum reacted with gamma2 subunit. Two sera reacted with both alpha3 and beta3 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 alpha3 subunit. However, in the studies using recombinant proteins of G domains of alpha3 subunit, none of the CP sera, including the sera reactive with alpha3 subunit in purified laminin 5, reacted with any recombinant proteins. The reason for this negative reactivity with the recombinant proteins is not clear.


CONCLUSIONS
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.
Hisamatsu
Yoshiko
Y
Department of Dermatology, Kurume University School of Medicine, 67 Asahimachi, Fukuoka 830-0011, Kurume, Japan.
Nishiyama
Toshio
T
Amano
Satoshi
S
Matsui
Chihiro
C
Ghohestani
Riza
R
Hashimoto
Takashi
T
D001483:Base Sequence; D015415:Biomarkers; D015815:Cell Adhesion Molecules / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers; D006801:Humans; D015151:Immunoblotting; D008958:Models, Molecular; D010390:Pemphigoid, Benign Mucous Membrane / Q000175:diagnosis* / Q000473:pathology; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D000097794:Kalinin
D015415:Biomarkers; D015815:Cell Adhesion Molecules; D017931:DNA Primers; D000097794:Kalinin