The ECM thus extracted was saponified with 2 N NaOH at pH 2 for 24 h to release the ester- and ether-linked phenolics, neutralized with 2 N HCl, and then re-extracted as above and pooled together for quantification as well as reverse-phase high-pressure liquid chromatography (RP-HPLC) analysis.
In addition, even though endogenous RVLP levels in mAb 1 HCCF may be lower than spiked X-MuLV, the amount of both viruses in mAb 1 HCCF and Protein A pools are above the assay quantification level.
In Fig. 2d, a quantification of the pooled data from these experiments is reported.
CD4+ T-cell responses were evaluated by in vitro stimulation with a pool of HPV peptides followed by quantification by cytokine flow cytometry.
Standard curves of serial dilutions ranging from 3 × to 0.005 × of a pool of cDNA were used for quantification, and slopes generated by the standard curves ranged from 3.3 to 3.6.
The amplified samples were pooled together without exact quantification and the pool was purified with Agencourt AMPure XP beads (Beckman Coulter, cat. no A63881) twice using 0.8 × volume of beads compared to the sample pool volume, which removes effectively primer dimers from the sample pool.
The eluate was collected in 10 fractions of 1 ml and fractions 4 to 6 were pooled and used for quantification.
Due to high signal-to-noise, we could not reliably quantify A3G3F1 at N64, A3G3F2 and A4G4F1 at N187, and A3G3F1 at N453; quantitative data obtained on the remaining structures for both HPX and CFH closely resemble those obtained by XIC-based quantification on pooled samples and validate the results.
A standard curve generated from a pool of all cDNA samples was used for quantification.
AMPure SPRI beads were used to purify amplified templates before pooling based on quantification using an Agilent Bioanalyser chip.
