Enhancement of Immunocomplex Detection and Application to Assays for DNA Adduct of Benzo[<i>a</i>]pyrene
Bibliographic record
Abstract
The stability of antibody and formation of immunocomplexes are essential to high-sensitivity capillary electrophoresis immunoassays (CEIA). However, little attention has been paid to enhancing or maintaining immunocomplex formation and antibody stability to improve the performance of CEIA. We report here the use of nonspecific proteins, such as bovine serum albumin (BSA) and rabbit immunoglobulin (rIgG), to enhance immunocomplex formation and to stabilize antibodies and immunocomplexes for immunoassays. Complexes between DNA adducts of benzo[a]pyrenediol epoxide (BPDE) and their antibodies were examined using capillary electrophoresis with laser-induced fluorescence detection (CE-HF). A tetramethylrhodamine (TMR)-labeled single-stranded oligonucleotide (16-mer) containing a single BPDE adduct was used as a fluorescent probe to study its immunocomplexes with a monoclonal antibody (8E11). To examine the formation of larger complexes, a TMR-labeled secondary antibody (anti-mouse), a primary antibody (mouse monoclonal antibody 5D11), and BPDE adducts in cellular DNA were used. We demonstrate that the use of nonspecific proteins stabilized the antibody and greatly enhanced the formation and stability of the immunocomplexes, resulting in substantial improvements in the detection limit (10-fold) and the reproducibility of the analysis. Another advantageous consequence of the stabilization was a 150-fold reduction of the concentration of the antibody needed for the immunoassay, resulting in reduced background and cost. We successfully applied this technique to the determination of DNA adducts of BPDE using a competitive immunoassay. The results from both small complexes (between a primary antibody and an oligonucleotide) and larger complexes (among a secondary antibody, a primary antibody, and cellular DNA) indicate that the technique can be extended to other immunoassays. We suggest that nonspecific proteins may assist the formation and stabilization of antibody-antigen complexes by maintaining the correct conformation of the antibody and antigen for optimum binding.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| 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 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 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 teacher head, 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".