Example 2
For the initial hybridoma fusion (Fusion #1), six female Balb/c mice were immunized two times with 75 μg of His-AmdR1R2, in the Sigma Adjuvant System (Sigma, Cat. No. S6322) by intraperitoneal injection at seven-week intervals. Two of the mice with the highest titers in ELISA on immobilized His-AmdR1R2 were selected for hybridoma fusion. Each mouse received a final immunization of 350 μg of His-AmdR1R2, i.p., four days prior to sacrifice and hybridoma fusion.
For the second hybridoma fusion (Fusion #2), Balb/c mice were immunized two times: first dose with 120 μg of His-AmdR1R2-B from GenScript (Lot Number 222933505/P20011303) in Sigma Adjuvant System (Sigma, Cat. No. S6322), and a second immunization with 100 μg of His-AmdR1R2-B conjugated with Keyhole limpet hemocyanin (KLH) (Imject EDC mcKLH Spin Kit; Thermo Scientific; Cat #77671) at twelve-week intervals. Two of the mice with the highest titers in ELISA on immobilized His-AmdR1R2 were selected for hybridoma fusion. Each mouse received a final immunization of 100 μg of His-AmdR1R2, i.p., four days prior to sacrifice and hybridoma fusion.
Hybridomas were prepared from splenocytes by conventional methods.