Author: Andrabi, Raiees; Pallesen, Jesper; Allen, Joel D.; Song, Ge; Zhang, Jinsong; de Val, Natalia; Gegg, Gavin; Porter, Katelyn; Su, Ching-Yao; Pauthner, Matthias; Newman, Amanda; Bouton-Verville, Hilary; Garces, Fernando; Wilson, Ian A.; Crispin, Max; Hahn, Beatrice H.; Haynes, Barton F.; Verkoczy, Laurent; Ward, Andrew B.; Burton, Dennis R.
Title: The Chimpanzee SIV Envelope Trimer: Structure and Deployment as an HIV Vaccine Template Document date: 2019_5_21
ID: 1ni7949q_50
Snippet: ELISA binding experiments were performed as described previously with minor modification . ELISA binding with SOSIP.664 trimer proteins with mAbs was carried out by either capturing the trimer proteins onto the anti-His capture antibodies or on the streptavidin coated plates through biotinylated trimers. For trimer biotinylation, the SOSIP.664 proteins were randomly biotinylated using a 2:1 molar ratio of biotin reagent to trimer using the EZ-lin.....
Document: ELISA binding experiments were performed as described previously with minor modification . ELISA binding with SOSIP.664 trimer proteins with mAbs was carried out by either capturing the trimer proteins onto the anti-His capture antibodies or on the streptavidin coated plates through biotinylated trimers. For trimer biotinylation, the SOSIP.664 proteins were randomly biotinylated using a 2:1 molar ratio of biotin reagent to trimer using the EZ-link-NHS-PEG4-Biotin kit (Thermo Fisher Scientific, 21324). MaxiSorp plates (Thermo Fisher Scientific) were coated overnight at 4C with 2 ug/mL of anti-His Ab (Thermo Fisher Scientific) or 2 ug/mL streptavidin (Thermo Fisher Scientific). Plates were blocked for 1 hr with 3% BSA and washed three times with 0.05% Tween 20-PBS (PBS-T) (pH 7.4). Anti-His or Streptavidin-coated plates were incubated with biotinylated trimers in 1%BSA plus PBST for 1.5 hr and washed three times with PBST. 3-fold serially diluted mAbs or sera were added starting at a maximum concentration of 10 ug/mL (100ug/ml for iGL Abs) (sera at 1:100 dilution) in 1% BSA plus PBST, and incubated at room temperature (RT) for 1.5 hr. Plates were washed three times with PBST. Alkaline-phosphatase-conjugated goat anti-human IgG Fc secondary antibody (Jackson ImmunoResearch Laboratories) was diluted 1:1000 in 1% BSA PBST and added to plates for 1 hr at RT. Plates were washed three times with PBST and incubated with phosphatase substrate (Sigma) for 15 mins and the absorbance at 405 nm recorded. The 50% binding (EC 50 ) was recorded as the half of the maximum binding activity and was calculated by linear regression method using Prism 6 Software.
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