Characterization of Two Monoclonal Antibodies to Human Epidermal Keratohyalin: Reactivity With Filaggrin and Related Proteins

Two monoclonal antibodies (ARH1 and AKH2) were elicited with partially purified human filaggrin and characterized by immunohistochemistry on normal and abnormal skin biopsies, immunoblotting techniques, and antigen purification. Both antibodies react strongly with the granular cell layer consistent with the distribution of keratohyalin and show a more diffuse reaction with the stratum corneum in normal skin biopsies. Reaction in cultured human keratinocytes is limited to immunofluorescent granules in flattened, well-differentiated cells in confluent cultures, in which we have previously demonstrated keratohyalin. On immunoblots AKH1 reacts with filaggrin (37 kD) and profilaggrin (400 kD), while AKH2 primarily stains bands of 150 and 300 kD. The AKH2 antigens were identified in the cationic protein fraction used for immunization and were purified by gel permeation and high-performance liquid chromatography. Amino acid composition of these proteins differs only slightly from filaggrin. Immunohistochemical staining patterns of the two antibodies are very similar in the genetic disorders of keratinization tested, except for ichthyosis vulgaris, and reflect the presence and distribution of keratohyalin. In ichthyosis vulgaris, AKH1 staining is weak, consistent with the morphology and with biochemical absence of profilaggrin/filaggrin; however, AKH2 staining is positive, although weaker than normal, suggesting the presence of the AKH2 antigens even when keratohyalin is absent or abnormal. Antibodies AKH1 and AKH2 may be useful as differentiation markers for keratinization in tissues and for cells in culture. Antibody AKH1 can be used specifically for detection of profilaggrin/filaggrin in tissues, cultured keratinocytes, and extracts