K. Handono, K.N. Benita, F.U. Habibah, E.S. Dewi
Gold standard diagnosis for Systemic Lupus Erythematosus (SLE) is ANA and anti-dsDNA antibody test using ELISA. We are developing rapid test kit for early detection of SLE which is affordable and ease by blotting antigen into membrane since 2015, yet the previous trial did not produce good result. Our recent trials aim to optimize antigen isolation and its attachment to membrane. This was trial-and-error experiment using healthy unused human transfusion blood and manual (non-kit) isolation methods. We compare 2 methods each for leukocyte isolation, nuclear antigen isolation, dsDNA extraction and antigen attachment to membrane to produce colour differences in the final kit. Leukocyte buffy coat is thicker using single sample human transfusion blood compared to pool multiple sample, and the quantity was comparable in both isolation methods. More nuclear protein yield was achieved through the use of Nuclear (NE) and Cytoplasmic Extract (CE) buffer. From 15 cc of whole blood, can be produced 3-5 μL/μLprotein. Modified salting out method was using high concentration salt and extra 0.05 mg/mL Proteinase-K result in more yield, 700-800 μg/μL of dsDNA. Attaching dsDNA was feasible using PVDF membrane, by applying 200 μg antigen in each blot and BSA 3% blocking. © Published under licence by IOP Publishing Ltd.
Clinical Pathology Department, Medical Faculty of Brawijaya University, Malang, Indonesia; Doctor Profession Study Program, Medical Faculty of Brawijaya University, Malang, Indonesia; Nursing Department, Medical Faculty of Brawijaya University, Malang, Indonesia
