Malays. Appl. Biol. (1997) 26(1): 81-86
OPTIMISATION OF THE IMMUNOPEROXIDASE METHOD FOR THE DETECTION OF KERATIN USING POLYCLONAL ANTIKERATIN ANTIBODY
WAN ZURINAH WAN NGAH1*, NG MEE LIAN1, JANAGEE BAI SINNADURAI1 and NOR ARIPIN SHAMAAN2
1Department of Biochemistry, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur 2Department of Biochemistry and Microbiology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor
ABSTRACT
Human keratin from the stratum corneum of the soles of feet was purified and used to raise polyclonal antibodies in rabbits. The antibodies were purified by ammonium sulphate fractionation and affinity chromatography and found to be suitable for the detection of keratin by immunoperoxidase staining. Commercial antikeratin antibodies were initially used to establish the procedure subsequently adopted for the immunohistochemical localization of keratin. Our antibodies were comparable to the commercial antikeratin antibodies with respect to specificity of staining. A comparison of the quality ol staining between two immunoperoxidase methods, peroxidase-antiperoxidase (PAP) method and the indirect method, showed that the former was superior to the latter. Optimum antibody dilution was established at 1:100 while the optimum incubation time was between 1-1.5 hr.
ABSTRAK
Keratin dari stratum korneum tapak kaki manusia diekstrak dan ditulenkan dan digunakan untuk menghasilkan antibodi poliklon dalam arnab. Antibodi poliklon yang dihasilkan ditulenkan melalui pemfraksian amonium sulfat dan kromatografi keafinan dan didapati sesuai digunakan dalam kaedah pewarnaan imunoperoksidase untuk mengesan keratin secara imunohistokimia. Antibodi antikeratin komersial telah digunakan pada mulanya untuk kaedah pengesanan keratin secara imunohistokimia. Kaedah ini kemudiannya digunakan untuk antibodi antikeratin poliklon yang ditulenkan dan kajian perbandingan di antara dua kaedah imunoperoksidase menunjukkan yang kaedah peroksidase-antiperoksidase (PAP) adalah lebih berkesan berbanding kaedah tidak langsung. Antibodi antikeratin poliklon yang dihasilkan didapati setara dengan antibodi antikeratin komersial dari segi kespesifikan pewarnaan. Pencairan optimum antibodi antikeratin poliklon ialah pada 1:100 dan masa eraman optimum ialah di antara 1-1.5 jam.
Key words: Antikeratin, immunoperoxidase, polyclonal
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