Antibodies induced by vaccination were measured by ELISA. Briefly, 96 well Nunc-Immuno Maxisorp plates were coated for 1 hour at room temperature with recombinant GFP (Millipore) at a concentration of 2 µg/ml diluted in bi-carbonate buffer (Sigma Aldrich). After blocking with 1% BSA in 0.5% Tween-20 PBS, serum was incubated for 2 hours prior to detection of bound antibodies with alkaline phosphatase-conjugated goat anti-mouse IgG (whole molecule) (Sigma Aldrich) diluted 1∶5000 and development with NPP substrate (Sigma Aldrich). Serum antibody endpoint titres were taken as the x-axis intercept of the dilution curve at an absorbance value 2× standard deviations greater than the OD405 for naïve mouse serum (typical cut-off OD405 for positive sera = 0.15). Serum from naïve mice were pooled and used as controls for all the ELISAs and were always below the level of detection.
Recombinant gfp
Manufactured by Merck Group
Sourced in Sweden
Recombinant GFP is a fluorescent protein derived from the jellyfish Aequorea victoria. It exhibits bright green fluorescence when exposed to blue or ultraviolet light. The core function of Recombinant GFP is to serve as a reporter molecule and a tool for visualizing various cellular and molecular processes.
Automatically generated - may contain errors
3 protocols using recombinant gfp
1
Quantifying Antibody Responses by ELISA
2
Detection of Anti-GFP Antibodies in Macaques
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Serum was collected prior to dose; at day 7, day 15, day 30, day 60, day 90, and month 6 post-injection; and at necropsy. Anti-GFP antibodies were detected by enzyme-linked immunosorbent assay (ELISA). Plates were coated with 2 μg/mL of recombinant GFP (Millipore) in 0.1M carbonate coating buffer at 4°C overnight. The plates were blocked with PBS-Tween 0.1% gelatin 1% at 37°C. Monkey sera were serially diluted and incubated for 2 h at 37°C. The positive control consisted of a commercial anti-GFP antibody (Millipore). Plates were then incubated with a goat anti-rhesus IgG horseradish peroxidase (HRP) antibody (Southern Biotech) for 1 h at 37°C. Substrate (tetramethylbenzidine [TMB]; BD Biosciences) was added, and plates were incubated for 5 min at RT. The reaction was stopped using 1M phosphoric acid. Plates were read in a spectrometer (Multiscan GO; Thermo Fisher Scientific) at 450 nm with a correction at 570 nm. Threshold of positivity was determined using 22 negative sera obtained from naive macaques as mean of optic density (OD) for each dilution + 2∗SD. IgG titers for experimental animals were defined as the last serum dilution for which OD remained above the threshold.
3
Measuring Optical Density and GFP Fluorescence
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Samples for measurement of optical density at OD600 (using Ultraspec 1000, Pharmacia Biotech, UK) and the biomass dry weight were taken from the bioreactor intermittently through a steamed pipe. Diluted un-centrifuged GFP samples were measured in a fluorimeter (Fluostar Galaxy, BMG Labtechnologies GmbH, Offenburg, Germany) at excitation/emission wavelengths of 470/515 nm in 1-mL samples. As reference was used un-induced culture media. The fluorescence units of the fluorimeter were calibrated vs. a GFP standard solution (recombinant GFP; Millipore AB, Solna, Sweden).
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