Design and expression of a QconCAT protein to validate Hi3 protein quantification of influenza vaccine antigens
Bibliographic record
Abstract
UNLABELLED: Quantification of the antigens hemagglutinin and neuraminidase in influenza vaccines has been reported using an antibody-free liquid chromatography-mass spectrometry (LC-MS) based method known as MS(E) "Hi3". This approach is based on the average signal intensity of the three most intense tryptic peptides relative to a primary standard. This strategy assumes that the Hi3 signal responses are consistent for all proteins, and therefore comparable to a spiked reference for absolute quantification. This method is much faster than the current standard methods; however, the results can vary significantly which brought the method's accuracy into question. To address this question we generated synthetic proteins comprising a concatenation of the peptides used to quantify the proteins of interest (QconCAT). Complete tryptic digestion of a QconCAT protein produces equal molar peptide amounts, allowing verification of equal signal response of Hi3 peptides for the proteins of interest. The generation of an intact, stable, QconCAT protein that digest completely is challenging. We have designed and analyzed five QconCAT proteins with unique design elements to address these challenges. We conclude that a suitable QconCAT protein can be produced and that the results obtained reinforce the validity of the Hi3 approach for quantifying proteins in annual influenza vaccine formulations. SIGNIFICANCE: The advances in quantitative proteomics have allowed the adaptation and application of these methods to numerous fields. In this paper we have validated a Hi3 approach to augment the antigen quantification for influenza vaccines injected into many millions annually. This methodology allows analysis of multiple antigens simultaneously without the need to generate antibodies. Key circumstances where this is advantageous are for quantitation of very similar antigens, such as the new quadravalent products and when time is critical such as in a flu pandemic.
Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.
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 | 0.001 | 0.001 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.001 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.001 | 0.000 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
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".