A modified cold osmotic shock protocol was applied to release expressed antibody fragments from the periplasmic space [20 (link)]. After cells were pelleted by centrifugation at 16,000 ×g for 1 min, for every two OD600 cells, the pellet was resuspended in 350 μl periplasmic buffer (200 mM Tris-HCl, pH 7.5, 20% sucrose, 30 U/μl lysozyme), and incubated at RT for 5 min. 350 μl ice-cold DDW was then added for osmotic shock and incubation on ice for 10 min. The resulting suspensions were centrifuged at 16,000 ×g for 2 min and clarified supernatants were subjected to Western blotting. Either anti-His-HRP (Abcam) or anti-Fab-HRP (Sigma-Aldrich) was used as the second antibody. Signals were developed with chemiluminescent substrate (Thermo Scientific) and analyzed with an imager (Bio-Rad).
Anti fab hrp
Manufactured by Merck Group
Anti-Fab-HRP is a laboratory reagent used for the detection and quantification of antibodies in biological samples. It consists of an anti-Fab antibody conjugated to the enzyme horseradish peroxidase (HRP). This reagent can be used in various immunoassay techniques, such as ELISA, to measure the presence and concentration of antibodies in a sample.
Automatically generated - may contain errors
3 protocols using anti fab hrp
1
Periplasmic Antibody Fragment Release Protocol
2
SARS-CoV-2 Omicron RBD Antibody Binding Assay
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His-tagged WT and Omicron RBD at 100 ng per well was coated in 96 well half-area microplate (Corning #3690) over night at 4 °C. The antigen coated plate was blocked with PBS containing 5% bovine serum albumin (BSA) for 1 h at 37 °C and washed by three times of PBST (PBS with 0.05% Tween 20). 50 μL of three-fold serially diluted antibody in PBS or plasma from vaccinees at a dilution of 1:10 was added and incubated at 37 °C for 1.5 h. The plate was washed with PBST for three times. The anti-Flag-HRP (Sigma-Aldrich) was used for detection of n3130v and n3113v and anti-Fab-HRP (Sigma-Aldrich) for IgG antibody (S309, CB6, P2B-2F6, CR3022) and plasma. The plate was washed with PBST for five times and the absorbance at 405 nm was measured after incubation with ABTS substrate (Invitrogen) for 10 min. The data was plotted using Graphpad Prism and the antibody concentration or plasma dilution was transformed into log[concentration]/[dilution] for four parameters nonlinear regression fitting. The EC50 (concentration for 50% of maximal effect) and ED50 (median effective dose) were calculated.
3
OVA-Specific Antibody Binding Assay
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96-well plates were coated with 0.1 µM OVA in PBS overnight at 4°C, blocked in 10% FBS/PBS, and 25 µl OB1 antibody at 50 ng/ml or Fab at 500 ng/ml was added in the presence of 25 µl of increasing concentrations of peptides diluted in 10% FBS/PBS as described in the Results and incubated for 2 h at room temperature. Plates were washed three times with PBS-T and then incubated with anti–IgG1-HRP (SouthernBiotech) or anti–Fab-HRP (Sigma-Aldrich) at a 1:1,000 dilution in 10% FBS/PBS for 1 h and developed with an OptEIA TMB substrate reagent kit (BD), and the reaction was stopped with 1M sulfuric acid and plate read at 450-nm absorbance.
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