2.6. Isotype DeterminationThe isoty

2.6. Isotype Determination

The isotype of monoclonal antibodies were
determined by isotyping kit goat anti mouse antibody for
IgG1, IgG2a, IgG2b, IgG3, IgM and mouse anti goat
HRP (Sigma Co.). Briefly, 100 μL of hybridoma
supernatant was added to each rOMPw pre-coated 96 wells plate and incubated for 30 min at 37°C. After three usual washing, 100 μL of each of above mentioned
antibodies (diluted 1/1000) was added to respective
wells and incubated for further 30 min at 37°C. They washed
again and 100 μL mouse anti goat HRP (diluted
1/100000) were added to each well. Plates were
incubated for further 30 min at 37°C and washed again.
Substrate (TMB) was added in 100 μL volume,
incubated in dark room for 25 min. After that stopper
solution was added and absorbance was read at 450
using Multiscan ms Labsystem.

Hasil (Bahasa Indonesia) 2:

2.6. Penentuan isotipe The isotype antibodi monoklonal yang ditentukan oleh isotyping kit kambing antibodi anti tikus untuk IgG1, IgG2a, IgG2b, IgG3, IgM dan mouse anti kambing HRP (Sigma Co.). Secara singkat, 100 uL hibridoma supernatan ditambahkan ke setiap rOMPw pra-dilapisi 96 sumur piring dan diinkubasi selama 30 menit pada suhu 37 ° C. Setelah tiga cuci biasa, 100 uL masing-masing disebutkan di atas antibodi (diencerkan 1/1000) ditambahkan ke masing-masing sumur dan diinkubasi selama lebih 30 menit pada 37 ° C. Mereka dicuci lagi dan 100 uL tikus anti kambing HRP (diencerkan 1/100000) ditambahkan ke masing-masing dengan baik. Pelat diinkubasi selama lebih 30 menit pada 37 ° C dan dicuci lagi. Substrat (TMB) ditambahkan dalam 100 mL Volume, diinkubasi di ruang gelap selama 25 menit. Setelah itu stopper solusi ditambahkan dan absorbansi dibaca pada 450 menggunakan Multiscan ms Labsystem.