PENGEMBANGAN KONJUGAT BIOTIN DAN HORSERADISH PEROXIDASE TERHADAP ANTIBODI MONOKLONAL AMILOID-ß42 DAN APLIKASI PADA TEKNIK ELISA

Abstract

Alzheimer disease is one of cause of dementia that could make the patient lose their memories until they can’t do normal activities. It is caused by accumulation of protein in the brain called Amyloid-ß42 (Aß42), that often used as biomarker to detect alzheimer by enzyme-linked immunosorbent assay (ELISA). To increase the sensitivity and accuracy, capture antibodies are conjugated with labels such as HRP and Biotin. The yield of the antibody conjugation with labels HRP and Biotin is 97% and 47%. The molecular mass of the conjugated antibodies was characterized with SDS-PAGE, result shows that the pre-conjugated antibody has 150 kDa bands, conjugated antibody with label Biotin has more mass than the pre-conjugated one, while conjugated antibody with label HRP has more mass than pre-conjugated and one with label Biotin, all of it are due to the addition of each label. Conjugated antibody was dilluted 7 times, tested with tetramethylbenzidine (TMB), then analyzed by ELISA reader. The measurement precision decided with %CV value. Label with HRP shows dillution from 10-1 to 10-4 has %CV less than 10%, while label with Biotin is from 10-1 to 10-5. Optimization was done by reading fixed antigen with the conjugated antibody, considering with the %CV, the optimal concentration for HRP is at 10-3 while biotin is at 10-4. Stability of the conjugated antibody determined by storage the conjugated antibody with the dilution at 0?, and measured with ELISA coated with antigen Aß42 once a month until the reading is unstable. HRP measurement started to unstable around second month and third month, while biotin start to unstable around fourth month, this shows that the biotin is more stable than the HRP.