Sequencing, cloning and expression of the panfilovirus glycoprotein specific recombinant mAb 8C12F11 in a CHO mammalian cell line
Bibliographic record
Abstract
Abstract Background Point-of-care diagnostics are a pivotal component of the medical counter-measures against natural outbreaks or bioterror attacks of Class A pathogens. We previously identified and patented conserved B cells of filovirus glycoproteins for diagnostic, therapeutic and subunit vaccine R & D. In recent in-vitro studies, we validated three mAbs as components of an effective sandwich combo for the capture & detection of zaire ebolavirus (EBOV) species. This work describes the gene sequences and methods for the cloning and expression of a recombinant anti-peptide 2 mAb (8C12F11) in CHO mammalian cells towards faster recombinant in-bulk production in-vitro relative to mice-hybridoma generation in-vivo. Methods Mice hybridoma were prepared & cultured on modified dulbecco's eagle’s medium (MDEM) Total RNA was extracted using RNA-easy isolation reagent (Vazyme), and reverse transcribed into cDNA using SMARTScribe reverse transcriptase. Antibody fragments of heavy (H) and light (L) chains were amplified by rapid amplification of cDNA ends (RACE) and cloned into standard vectors for screening in 5 colonies. Inserts of matching phenotypic colonies were sequenced by Sanger capillary sequencing and aligned by IMGT analysis of VDJ. Isotyping was done by constant region analysis. Results Consensus sequences of the coding DNA of the heavy and light chains of an isotype IgG1/kappa recombinant mice mAb (Genbank accession #s OP966766 and OP966767, respectively) are presented. The biophysical profile of the full expressed recombinant mAb demonstrates a single protein of approximately 142 kda under non reducing conditions and tetramer of duplexes of 23ka and 48kda, respectively. The average area under sec-HPLC curve at a wavelength of 280 nm is 2490.55 Conclusions This work summarizes the first ever gene sequencing, cloning, and expression of the recombinant panfilovirus mAb 8C12F11 in a CHO mammalian cell line
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.000 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.002 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".