TK1 Recombinant Monoclonal Antibody
Description
Definition and Molecular Targets
TK1 Recombinant Monoclonal Antibodies are produced using hybridoma or phage display technologies to target specific epitopes on TK1. Key epitopes include:
-
C-terminal sequences: GEAVAARKLF (SEQ ID NO: 2), NCPVPGKPGEAV (SEQ ID NO: 3) .
-
Conformation-dependent epitopes: Formed by 3D folding of TK1’s C-terminal domain .
These antibodies bind diverse forms of TK1, including:
-
Cellular TK1: Cytosolic enzyme in proliferating cells.
-
Serum TK1: Released into circulation during cell death, a biomarker for malignancies .
-
Recombinant TK1 (rTK1): Lab-produced protein for standardization .
Key Innovations
-
Epitope Diversity: Antibodies targeting six distinct regions of tetrameric TK1 (e.g., 8G2, 3B4, 7H2) enhance detection sensitivity across isoforms .
-
Phage Display Libraries: Used to isolate high-affinity clones (e.g., hTK1-IgY-rmAb#5 with KD = 3.95 × 10⁻¹⁰ mol/L) .
-
Species Specificity: Antibodies like Mab-2 (canine-specific) enable veterinary diagnostics .
Validation Methods
-
Western Blot: Confirmed binding to recombinant and native TK1 .
-
siRNA Knockdown: Reduced TK1 expression in cancer cells validated antibody specificity .
-
Flow Cytometry: Detected membrane-associated TK1 in lung, breast, and colon cancers .
Key Assays
Performance Metrics
Therapeutic Potential
-
Antibody-Dependent Cytotoxicity (ADCC):
-
Targeted Delivery: Membrane-associated TK1 on cancer cells enables antibody-drug conjugates .
Research Advancements
-
Dual Antibody Systems: AroCell TK 210 ELISA uses Ar-1 and Ar-2 antibodies for robust TK1 quantification in hematological malignancies .
-
IHC Utility: hTK1-IgY-rmAb#5 showed >50% labeling index in ovarian serous adenocarcinoma tissues .
-
Cross-Species Reactivity: Mab-1 binds feline, equine, and human TK1, unlike species-specific Mab-2 .
Challenges and Future Directions
-
Batch Consistency: Recombinant antibodies (e.g., hTK1-IgY-rmAb#5) reduce variability (SD < 2.5%) compared to polyclonal pools .
-
Epitope Accessibility: Conformation-dependent antibodies require native TK1 folding for binding .
-
Therapeutic Optimization: Humanization of rabbit/chicken antibodies to reduce immunogenicity .
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- STK1p has been identified as a potential proliferative biomarker for the early detection of individuals at risk of developing or already exhibiting pre-malignancies or diseases associated with the risk of malignancy. PMID: 29689706
- Evidence suggests that serum thymidine kinase 1 (S-TK) activity could be a useful parameter for monitoring the efficacy of neoadjuvant therapy (nTx). PMID: 29189449
- Our research provides the first evidence that serum TK1 activity as early as 2 weeks after CDK4/6 inhibitors is highly correlated with tumor cell proliferation response in patients with early-stage HR+ breast cancer. PMID: 29162134
- TK1 may be involved in the development and progression of pancreatic ductal adenocarcinoma (PDAC) by regulating cell proliferation. PMID: 29266545
- The combined detection of TK1 with cytokeratin-19 fragment (CYFRA21-1), carcinoembryonic antigen (CEA), or neuron-specific enolase (NSE) enhanced the diagnostic value of TK1 for lung squamous cell carcinoma, adenocarcinoma, and small cell lung cancer, respectively. PMID: 29247745
- These findings have implications for the mechanism by which fragile histidine triad (FHIT) regulates TK1 mRNA and, more broadly, for its modulation of multiple functions as a tumor suppressor/genome caretaker. PMID: 28093273
- Results indicate that positive expression of CK19 mRNA and TK1 protein is closely associated with poor prognosis in advanced gastrointestinal cancer. PMID: 27625087
- While TK1 expression was an independent prognostic factor for relapse, but not for survival, TK1 is a more informative expression than Ki-67 for local invasion (LI), relapse, and overall survival rates. Therefore, when combined with MDACC grading, pTNM staging, and lymph node metastasis, immunohistochemical (IHC) determination of TK1 expression may improve the overall prediction of prognosis in patients with ovarian cancer. PMID: 28651488
- Data reveals that the median thymidine kinase (TK1) levels found in sera from breast cancer patients with T1 to T4 stage disease were 0.31, 0.46, 0.47, and 0.55 ng/ml, respectively. These levels differed significantly from healthy controls. PMID: 27079872
- STK1 is a reliable biomarker for identifying individuals with malignant tumors in cancer screening. PMID: 27002755
- This study examined serum Thymidine Kinase 1 Activity Following Nephrectomy for Renal Cell Carcinoma and Radiofrequency Ablation of Metastases to Lung and Liver. PMID: 27069161
- TK1 expression is significantly different in invasive urothelial carcinoma and benign urothelium, suggesting its potential as a diagnostic marker. PMID: 26231311
- This research investigated the Protein expression of BIRC5, TK1, and TOP2A in malignant peripheral nerve sheath tumors—a prognostic test after surgical resection. PMID: 25769404
- High Thymidine Kinase expression is associated with adenocarcinoma in Non-small Cell Lung Cancer. PMID: 25921119
- These results demonstrate that there are differences in the specific activities and the subunit compositions of STK1 in hematological malignancies compared with breast and prostate cancer. PMID: 25881026
- A regression analysis showed that only TK1 levels were significant (relative risk (RR)=1.03 for each unit, confidence interval (CI)=1-1.05; p=0.015) for diagnosing a true transformation. PMID: 25964590
- We propose that a chip including DPYD, TYMS, TYMP, TK1, and TK2 genes is a potential tool for predicting response in Liver-directed Radioembolization (LARC) following fluoropyrimidine-based chemo-radiotherapy. PMID: 24455740
- Nuclear TK1 expression in early grade cervical intraepithelial neoplasia predicts risk for progression to malignancy. PMID: 23693054
- 3'-deoxy-3'-[18F]-fluorothymidine (18F-FLT) kinetics correlates with thymidine kinase-1 expression and cell proliferation in newly diagnosed gliomas. PMID: 23229746
- The magnitude of maximum fluorodeoxyglucose uptake in primary tumors and the serum TK1 level in patients with metastatic non-small cell lung cancer (NSCLC) were found to be independent prognostic predictors of overall survival. PMID: 23116493
- Increased serum level of thymidine kinase 1 correlates with metastatic site in patients with malignant melanoma. PMID: 23179401
- Results suggest that the serum TK1 protein differs from cellular or recombinant forms, is more active in high molecular weight complexes, and is sensitive to reducing agents. PMID: 22741536
- Data suggests that serum thymidine kinase 1 (TK1) levels may assist in refining risk assessment in the current immunotherapy era. PMID: 22263569
- The crystal structure of the T163S-mutated HuTK1 reveals a less ordered conformation of the ligand thymidine triphosphate compared with the wild-type structure. PMID: 22385435
- TK1 may be involved in poor survival in patients with pT1 of lung adenocarcinoma. PMID: 22143937
- Frequencies of polymorphic mutations in HIV-1 (subtype B) were compared between patients detected with the 69 insertion (n = 17), Q151M (n = 29), >/=2 thymidine analogue mutations (TAM) 1 (n = 400) or >/=2 TAM 2 (n = 249). PMID: 22027876
- Data demonstrates that the Flt3L/TK gene therapeutic approach can induce systemic immunological memory capable of recognizing a brain tumor neoantigen in a model of recurrent glioblastoma multiforme (GBM). PMID: 21505426
- (18)F-FLT uptake and retention within cells may be influenced by a variety of factors, including but not limited to TK1 enzymatic activity. PMID: 21764789
- Elevated serum thymidine kinase 1 is associated with pre/early cancerous progression. PMID: 21545220
- High levels of HER2 and Ki-67 or TK1 expression are correlated with an increase in tumor grades and tumor recurrence in meningiomas. PMID: 20450760
- Serological thymidine kinase 1 is a useful marker for prognosis in patients with esophageal, cardial, and lung carcinomas. PMID: 20479645
- Serum TK1 correlates with clinical stages and clinical reactions and monitors the effect of tumor therapies, not only in controlled clinical trials, but also in routine clinical settings. PMID: 20354751
- This research suggests a direct involvement of the G-quadruplex motif in transcription of TK1. PMID: 20849417
- The nucleoside recognition mechanisms for TK1 and TK2 are significantly different. Nonpolar nucleosides are likely to be active in the nucleotide salvage pathway in human cells. PMID: 20560637
- Results suggest that higher thymidine levels in TK- cells caused by a defect in thymidine salvage to dTTP provide protection against UV irradiation. PMID: 20544518
- The expression of TK1 in tumor tissues correlated with pathological stages and clinical grades of carcinomas (ca) of esophagus, lung, and in premalignancy of breast ductal ca. STK1p could monitor the outcome of tumor therapy. PMID: 20544519
- Increased dTTP synthesis via TK1 occurs after genotoxic insults in tumor cells, improving DNA repair during G(2) arrest. PMID: 20554529
- The cell cycle regulation of TK1 in normal tubule cells differs from that in other types of normal and malignant renal cells. PMID: 19957115
- Thymidine kinase-1 and thymidylate synthase expression was markedly different between cancer types, suggesting that response to TAS-102 may differ. PMID: 20372850
- Thymidine kinase plays a role in the progression of lung cancer. PMID: 20592392
- Serum TK1 may hold a reference value in the evaluation of treatment and prognosis of non-Hodgkin's lymphoma following chemotherapy. PMID: 20140744
- The TK1 model presented supports both K and k positive cooperativity. Three-parameter mass action models can and should replace the 3-parameter Hill model. PMID: 20003201
- TK1 gene expression, along with TS, TP, and DPD gene expression, may play significant roles in influencing the malignant behavior of epithelial ovarian cancer. PMID: 11992400
- This study investigated Mutation analysis in the coding sequence of thymidine kinase 1 in breast and colorectal cancer. PMID: 12699056
- Long-term treatment of H9 human lymphoid cells in the presence of dideoxycytidine down-regulated TK1 gene expression and reduced the expression and activity of TK in resistant cells. PMID: 14659972
- The enzymatic function at the G2/M phase of TK1 depends on its quaternary structure. PMID: 14697231
- Activation of the APC/C-Cdh1 complex during mitotic exit controls the timing of TK1 destruction. PMID: 14701726
- This research examined the Activity of thymidine kinase, thymidine phosphorylase, and thymidilate synthase in human cancer xenografts to investigate the contribution of these enzymes to the sensitivity of TAS-102. PMID: 14719072
- The importance of valine 106 for the structure and function of TK1. PMID: 15153115
- Activation of TK1 may be crucial in modulating radiation-induced cell death and cell cycle progression in irradiated K562 cells. PMID: 15353126
Q&A
What are the structural and functional characteristics of TK1 recombinant monoclonal antibodies that make them preferable to polyclonal versions in proliferation studies?
TK1 recombinant monoclonal antibodies exhibit three defining characteristics: 1) Precision epitope targeting through single-chain variable fragment (scFv) phage display libraries, 2) Batch-to-batch consistency due to recombinant production methods, and 3) Enhanced sensitivity thresholds down to 0.01 pM in chemiluminescence assays . Unlike polyclonal antibodies, which recognize multiple epitopes on the 31-peptide sequence (195GQPAGPDNKE...225), rmAbs like hTK1-IgY-rmAb#5 demonstrate monovalent binding to a conserved region critical for cell cycle regulation. This specificity reduces cross-reactivity with degradation products, as evidenced by Western blot analyses showing no binding in TK1-negative 143B cell lines .
How does the affinity maturation process for TK1 recombinant monoclonal antibodies impact experimental reproducibility?
The described phage display methodology achieved a binding affinity of mol/L through four-stage affinity panning . This process:
-
Eliminates clones with off-target binding to peptide fragments
-
Selects for conformational epitope recognition using full-length recombinant TK1
-
Verifies structural stability via SDS-PAGE analysis of heavy (66 kDa) and light chains (25 kDa)
Researchers should validate affinity constants through surface plasmon resonance (SPR) rather than indirect ELISA to avoid matrix effects.
What validation protocols are essential when implementing TK1 recombinant monoclonal antibodies in novel assay platforms?
A three-tier validation framework is recommended:
Table 1: Validation Parameters for TK1 rmAbs
| Parameter | Acceptance Criteria | Methodological Approach |
|---|---|---|
| Specificity | No binding in TK1-negative cell lines | Western blot (143B vs. HT29 cells) |
| Sensitivity | LOD ≤0.05 pM | Dose-response curve slope ≥89.98 |
| Inter-batch consistency | CV <2.5% across 4 production lots | Pearson correlation (r >0.95) |
Data from 292 serum samples showed 85.7% concordance between automated chemiluminescence and manual ECL platforms when using validated protocols .
How can researchers optimize TK1 recombinant monoclonal antibody performance in multiplexed biomarker panels?
Integrate TK1 rmAbs with complementary proliferation markers using:
-
Orthogonal validation: Compare TK1 IHC results (5% labelling index) with Ki-67 staining in ovarian adenocarcinoma tissues
-
Algorithmic weighting: Assign TK1 a 0.78 coefficient in prognostic models based on ROC AUC values of 0.96 from 35,365 patient samples
-
Platform synchronization: Utilize the biotin-streptavidin (BSA) sandwich architecture to enable simultaneous detection of TK1 with PD-L1 or EGFR
What strategies resolve apparent contradictions between TK1 immunoassay results and clinical outcomes?
A four-step discrepancy analysis is recommended:
-
Pre-analytical audit: Verify serum collection protocols (storage at -80°C prevents TK1 degradation)
-
Threshold calibration: Reassess the 2.0 pM risk cutoff using local population baselines
-
Longitudinal profiling: Monitor STK1p levels at 3-month intervals rather than single measurements
-
Platform cross-check: Compare automated chemiluminescence results (CV=1.8%) with ECL dot blot controls
The 132-month longitudinal study demonstrated that 24.2% of subjects with elevated STK1p developed malignancies versus 6.1% in the low-STK1p cohort .
How should researchers adapt TK1 recombinant monoclonal antibodies for single-cell proliferation analysis?
Implement a modified protocol:
-
Cell fixation: 4% paraformaldehyde with 0.1% Triton X-100 permeabilization
-
Antibody conjugation: Label rmAbs with Alexa Fluor 647 (Ex/Em 650/665 nm)
-
Signal amplification: Tyramide-based deposition system with 30-second substrate exposure
-
Quantification: High-content imaging analysis of nuclear/cytoplasmic TK1 localization
Validation data from tonsil tissues showed 92.3% agreement between fluorescent IHC and conventional DAB staining .
What experimental controls are critical when observing atypical TK1 staining patterns?
Include these controls in every assay batch:
Table 2: Essential Experimental Controls
| Control Type | Purpose | Acceptance Criteria |
|---|---|---|
| TK1-negative cell line | Specificity verification | 0% staining in 143B cells |
| Serum spike recovery | Matrix effect assessment | 85-115% recovery at 2.2-20 pM |
| Epitope competition | Confirmation of target engagement | ≥80% signal inhibition with 31-peptide |
The original study achieved 98.8% inter-method concordance using these controls across 90 serum samples .
How can researchers mitigate batch-specific variability when scaling TK1 assays?
Adopt a unified conjugation protocol:
-
Standardize biotinylation at 4:1 molar ratio (antibody:EZ-Link NHS-Biotin)
-
Implement real-time stability monitoring via differential scanning fluorimetry
-
Use master cell banks for recombinant antibody production with <5% LC/HC ratio variation
Batch consistency data showed SD <2.5% across four production lots in automated platforms .
What novel applications are enabled by the 0.01 pM detection limit of TK1 recombinant monoclonal antibodies?
Three emerging applications warrant exploration:
-
Liquid biopsy refinement: Correlate ultra-low STK1p levels (0.01-0.1 pM) with premalignant lesions
-
Therapeutic monitoring: Detect early response signals in immunotherapy cohorts
-
Spatial biology mapping: Integrate with CODEX/MIBI-TOF for tumor microenvironment analysis
The 31-peptide immunization strategy described in the foundational study provides a template for developing isoform-specific rmAbs against TK1 splice variants .
How can the research community improve standardization of TK1 proliferation assays?
Propose a consortium-led initiative with three components:
-
Reference material repository (NIBSC-style TK1 calibrators)
-
Interlaboratory proficiency testing program
-
Open-source algorithm repository for STK1p data normalization
The original automated platform achieved 85.7% inter-platform concordance, suggesting achievable standardization .
