TNNI3 Antibody, HRP conjugated
Description
Introduction to TNNI3 Antibody, HRP Conjugated
TNNI3 antibody, HRP conjugated is a specialized immunological reagent consisting of an antibody that specifically binds to cardiac troponin I (TNNI3) protein, chemically linked to horseradish peroxidase (HRP) enzyme. This conjugation creates a powerful detection tool that enables direct visualization of TNNI3 in various experimental and diagnostic applications without requiring secondary antibody steps .
The cardiac-specific nature of TNNI3 makes these conjugated antibodies particularly valuable for cardiovascular research and cardiac injury assessment. Unlike other troponin isoforms, TNNI3 is expressed exclusively in cardiac tissue, providing exceptional specificity for heart-related investigations . The HRP enzyme component generates a colorimetric, chemiluminescent, or fluorescent signal when exposed to appropriate substrates, allowing for sensitive detection of the target protein .
Physiological Role
Cardiac troponin I plays a crucial role in the regulation of heart muscle contraction. It forms part of the troponin complex alongside troponin T and troponin C, which together regulate the calcium-dependent interaction between actin and myosin filaments in the sarcomere .
When calcium levels are low, TNNI3 binds to the thin filament and blocks the interaction between thick and thin filaments necessary for muscle contraction. An increase in calcium levels causes structural changes in troponin C, triggering the troponin complex to detach from the thin filament, thus allowing heart muscle contraction .
Clinical Significance
Mutations in the TNNI3 gene are associated with several cardiac conditions, most notably familial hypertrophic cardiomyopathy, which is characterized by thickening of the cardiac muscle . TNNI3 gene mutations are found in less than 5% of people with this condition, but all affected individuals have an increased risk of heart failure and sudden death . Recent research has also implicated TNNI3 in restrictive cardiomyopathy, particularly in pediatric cases .
Principles of Conjugation
Horseradish peroxidase (HRP) conjugation to antibodies involves the chemical coupling of the enzyme to the antibody while preserving the functional properties of both molecules. Traditional methods utilize sodium meta-periodate to generate aldehyde groups by oxidation of carbohydrate moieties on the HRP molecule, which then react with amino groups on the antibody .
Enhanced Conjugation Methods
Recent advancements in conjugation technology have significantly improved the efficiency and sensitivity of HRP-antibody conjugates. One notable innovation is the incorporation of a lyophilization step after HRP activation, which enhances the binding capacity of antibodies to HRP molecules .
According to research by Sharma et al., this modified method dramatically improves the sensitivity of detection:
| Parameter | Classical Method | Lyophilization-Enhanced Method | P-value |
|---|---|---|---|
| Working Dilution | 1:25 | 1:5000 | <0.001 |
| HRP Binding Capacity | Standard | Enhanced | <0.001 |
| Storage Stability | Limited | Extended at 4°C | N/A |
The enhanced method reduces reaction volume without changing the amounts of reactants, allowing for more efficient coupling through increased molecular collision opportunities, as explained by collision theory principles .
Recombinant Production Approaches
Beyond chemical conjugation, recombinant DNA technology offers another approach for producing HRP-antibody conjugates. Research demonstrates the successful production of recombinant conjugates of HRP with Fab antibody fragments in Pichia pastoris expression systems . These recombinant conjugates offer advantages of homogeneity, strictly determined stoichiometry, and retained functional activity of both the marker protein and the antibody .
Application-Specific Dilution Recommendations
Different applications require specific antibody dilutions for optimal results:
| Application | Recommended Dilution Range | Reference |
|---|---|---|
| Western Blot (WB) | 1:300-1:5000 | |
| ELISA | 1:500-1:1000 | |
| Immunohistochemistry - Paraffin (IHC-P) | 1:200-1:400 | |
| Immunohistochemistry - Frozen (IHC-F) | 1:100-1:500 |
Detection of Cardiac Injury
TNNI3 antibody, HRP conjugated products are invaluable for detecting cardiac troponin I in heart tissue and serum samples, serving as critical tools for studying heart injury and disease models . The high specificity for cardiac tissue makes these antibodies particularly useful in discriminating between cardiac and skeletal muscle damage .
Cardiomyopathy Research
These conjugated antibodies play a significant role in research on various cardiomyopathies, including:
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Hypertrophic cardiomyopathy: Studies investigating TNNI3 mutations and their effects on cardiac function
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Restrictive cardiomyopathy: Research on engineered heart tissue models using induced pluripotent stem cells from patients with TNNI3 mutations
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Ischemia/reperfusion injury: Investigations into the role of TNNI3K (TNNI3-interacting kinase) in cardiac damage
Immunoassay Development
TNNI3 antibody, HRP conjugated products are essential components in developing sensitive immunoassays for cardiac troponin detection. The direct conjugation of HRP eliminates the need for secondary antibody steps, reducing background and improving specificity . These antibodies can be used in various immunoassay formats, including:
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Direct ELISA for quantification of TNNI3 levels
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Western blotting for protein expression analysis
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Immunohistochemistry for tissue localization studies
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Flow cytometry for cellular analysis
Signal Enhancement Techniques
Several strategies can improve the performance of TNNI3 antibody, HRP conjugated in experimental settings:
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Substrate Selection: Using enhanced chemiluminescent substrates can significantly increase detection sensitivity
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Signal Amplification: Employing tyramide signal amplification (TSA) can boost signal intensity by up to 100-fold
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Incubation Optimization: Extending primary antibody incubation time at 4°C improves specific binding while reducing background
Sample Preparation Considerations
Proper sample preparation is crucial for optimal results with TNNI3 antibody, HRP conjugated:
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Tissue Samples: Complete fixation and proper antigen retrieval are essential for immunohistochemistry applications
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Protein Extracts: Use of cardiac-specific lysis buffers with protease inhibitors preserves TNNI3 integrity
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Serum Samples: Appropriate dilution and blocking steps minimize matrix effects in ELISA applications
Comparative Analysis with Other Detection Methods
TNNI3 antibody, HRP conjugated offers several advantages over alternative detection methods:
| Feature | HRP-Conjugated Antibody | Two-Step Detection System | Fluorescent Detection |
|---|---|---|---|
| Sensitivity | High | Moderate to High | Very High |
| Signal Stability | Hours to Days | Hours | Minutes to Hours |
| Background Noise | Low | Moderate | Low to Moderate |
| Protocol Complexity | Simple (One-Step) | Complex (Two-Step) | Complex |
| Cost | Moderate | Higher | Higher |
| Equipment Needs | Basic | Basic | Specialized |
The direct conjugation of HRP to TNNI3 antibodies simplifies workflows and reduces potential sources of variability compared to indirect detection methods that require secondary antibodies .
Recent Discoveries
Recent research using HRP-conjugated antibodies has revealed important insights into TNNI3 biology:
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Studies demonstrating that gene correction and overexpression of TNNI3 improve impaired relaxation in engineered heart tissue models of pediatric restrictive cardiomyopathy
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Investigations showing that TNNI3K, a cardiac-specific kinase that interacts with TNNI3, plays a regulatory role in cardiac pathophysiology and represents a potential therapeutic target
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Research exploring the post-translational modifications of TNNI3 and their impact on cardiac function
Emerging Applications
Novel applications for TNNI3 antibody, HRP conjugated are being developed:
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Point-of-care diagnostics: Development of rapid tests for cardiac injury assessment
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Therapeutic antibody development: Use in screening and characterizing activity-modulating antibodies similar to approaches used for other targets
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Engineered heart tissue models: Applications in testing cardiac tissue constructs derived from stem cells
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High-throughput screening: Use in drug discovery platforms targeting cardiac diseases
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Research suggests that while incident atrial fibrillation (AF) occurrence groups had similar baseline troponin I (TnI) levels, they exhibited higher troponin T (TnT) levels [Review and Meta-Analysis]. PMID: 29631448
- Studies indicate that in patients with end-stage renal disease (ESRD), elevated cardiac-specific troponin T (cTnT) was more prevalent than elevated troponin I (cTnI) [Review]. PMID: 28545334
- The frequency of h-FABP positivity among acute myocardial infarction patients was higher than that of hs-TnI, which would have missed six of them; however, hs-TnI area under curve was superior to that of h-FABP. PMID: 28650717
- Reversible Covalent Reaction of Levosimendan with Cardiac Troponin C in Vitro and in Situ. PMID: 29558109
- The QT interval demonstrates a strong positive linear correlation with cardiac troponin-I levels in Non-ST-elevation myocardial infarction. PMID: 28366473
- Apelin-12 influences troponin I levels during the acute phase of STEMI, while during the non-acute phase, low apelin levels were associated with a high rate of MACE. PMID: 28728608
- In clinically stable patients without known cardiovascular disease, a comprehensive chest pain history combined with hs-TnI testing can identify a significant low-risk group. PMID: 28031149
- A study revealed that in patients undergoing liver transplantation, elevated preoperative high-sensitivity cardiac troponin I level was associated with 1-year and 30-day mortality. PMID: 28542299
- Serial measurement of troponin I revealed persistent elevation in individuals with diabetes mellitus type 2. PMID: 28246236
- Plasma troponin C1 (cTnI) is the preferred biomarker for diagnosing acute myocardial infarction (AMI) due to its high specificity for myocardial tissue damage. Data suggest the "best cut-off" for plasma cTnI is 0.014 micrograms/L in AMI. These studies were conducted in the emergency department of a university hospital in Italy using point-of-care testing in patients presenting with chest pain, ages 18-101. PMID: 28377153
- NT-proBNP and hs-cTnI levels were higher in systemic sclerosis patients compared to controls. Both NT-proBNP and hs-cTnI were associated with the presence of echocardiographic abnormalities. PMID: 27601074
- The value of cTnI level assessed 24 hours post-surgery proved to be a reliable predictor of death following liver transplantation, with an optimal cut-off value of 0.215 ng/mL. The surgery time was identified as the most significant predictor of cTnI elevation. PMID: 28455997
- Elevated cTnI levels are common in Fabry disease patients, indicative of cardiac involvement. PMID: 27322070
- This report introduces a novel troponin I rule-out value below the upper reference limit for acute myocardial infarction. PMID: 27067356
- cTnI determined in hemodynamically stable patients with suspected AMI and wide QRS complex using optimized diagnostic thresholds improves rule-in and rule-out accuracy with respect to the presence of significant obstructive CAD. PMID: 27148734
- 83 preterm infants with Bronchopulmonary dysplasia born at <28-wk gestation and still inpatients at 36-wk corrected age received an echocardiogram and blood tests of B-type natriuretic peptide (BNP), troponin I, and YKL-40. PMID: 27760764
- Serum cardiac troponin I was elevated in septic patients with myocardial depression compared to those without myocardial depression. PMID: 27238916
- Elevated BNP and hs-cTnI after kidney transplantation identify candidates for targeted risk reduction. PMID: 26910333
- These perturbed biophysical and biochemical myofilament properties are likely to significantly contribute to the diastolic cardiac pump dysfunction observed in patients suffering from a restrictive cardiomyopathy associated with the cTnI R145W mutation. PMID: 27557662
- Epigenetic modification causing cTnI expression decrease is a potential cause of reduced cTnI level and diastolic dysfunction in older mouse hearts. PMID: 27184165
- Among hospitalized patients with cardiac troponin I values above 30 ng/L, the majority will experience myocardial injury. Cardiac nonischemic conditions are associated with very high troponin concentrations, but the outcome is generally favorable. Conversely, myocardial injury related to noncardiac or multiple conditions carries a poor long-term prognosis. PMID: 26763756
- The troponin I carboxy terminal mobile domain and linker sequence play a role in regulating cardiac contraction. PMID: 26971468
- The last 5 C-terminal residues of cTnI influence the binding of cTnI with cTnC and cTnT and affect the Ca(2+) dependence of filament sliding. PMID: 26919894
- A study found that N-terminal pro-brain natriuretic peptide (NT-proBNP) and high-sensitivity cardiac troponin I are independently associated with incident dementia, and NT-proBNP with incident Alzheimer's disease. PMID: 28039523
- Clones were selected using microtiter plates coated with human cardiac troponin I (hcTnI). Hybridoma cells that bind with high affinity to human cardiac troponin I were chosen. PMID: 27556913
- Sex, age, and systolic blood pressure are among the strongest determinants of hs-cTnI in healthy adults. PMID: 27535138
- This review summarizes recent proteomic data on amino acid sequences of cTnT and cTnI in various species, as well as selected analytical characteristics of human cardiac troponin high-sensitivity assays. PMID: 26876101
- In stable coronary artery disease patients successfully treated with PCI, pre-procedural cTnI levels, within the upper limits of the normal range, are associated with hard cardiac endpoints. PMID: 25405803
- Calcium channel blockers and adrenergic beta antagonists significantly reduced hs-TnI levels both at rest and during exercise in atrial fibrillation patients/. PMID: 27142292
- Compromised interactions of K206I with actin and hcTnC may lead to impaired relaxation and HCM. PMID: 26553696
- hsTnI at the time of presentation followed by early advanced coronary CTA assessment improves risk stratification and diagnostic accuracy for acute coronary syndromes. PMID: 26476506
- These findings indicate that a double heterozygous mutation in the TNNI3 gene is involved in the pathogenesis of hypertrophic cardiomyopathy via haploinsufficiency. PMID: 26506446
- The incidence of adverse cardiovascular events was significantly higher in patients with troponin elevation after carotid endarterectomy, primarily due to silent non-ST segment elevation MIs occurring in the early postoperative phase. PMID: 26553374
- Four novel missense variants were identified in TNNI3. PMID: 26169204
- Letter/Case Report: acute decompensated heart failure with troponin I elevation in hereditary hemochromatosis. PMID: 25916738
- In this pilot study, the addition of CACS to hsTnI improves the identification of low-risk subjects where CTA might be avoided. PMID: 26049777
- Exclusion of acute myocardial infarction 2h after presentation in emergency patients with possible acute coronary syndrome can be achieved using hs-cTnT or hs-cTnI assays. PMID: 24316100
- Hybrid coronary revascularization is associated with lower postoperative cTn release compared to off-pump coronary artery bypass surgery. PMID: 25217621
- Carotid endarterectomy is followed by a high incidence of asymptomatic cTnI increase that is associated with late cardiac events. PMID: 25601178
- Mutations underlying restrictive cardiomyopathy are all marked by right-sided cardiac manifestations in South African patients. PMID: 25940119
- Circulating levels of sensitive cTnI and NT-proBNP are related to LV function and infarct size in patients with stable CAD after revascularization. PMID: 25788439
- Serum TnI detected significant myocardial necrosis in a majority of patients with chronic HF due to LVSD, and when measured serially, provided independent risk information for poor CV outcomes and deleterious LV remodeling. PMID: 25777344
- The elevation of Tn I after PCI in patients with normal initial level is a more significant predictor of early (30-day) mortality compared to later (within 12 months) mortality. PMID: 25617100
- AF patients, both with and without CAD, showed similar cTnI concentrations at admission. A second validation of cTnI is mandatory for all patients. PMID: 25653186
- Cardiac troponin T or troponin I compared to creatine kinase in patients with revascularized acute myocardial infarction. PMID: 25381953
- Even a single elevated Troponin I value increased the risk of myocardial infarction. PMID: 25195101
- Abbott high-sensitivity cardiac-TnI levels were determined in a total of 3314 Korean patients with chest pain. PMID: 25887868
- Absolute delta performed significantly better than relative delta at all time intervals to measure changes in troponin I for early diagnosis of myocardial infarction. PMID: 25261587
- The high accordance with LGE, reflecting cardiac dysfunction, suggests that cTNI-elevation can be a useful laboratory parameter for assessing myocardial damage in FD. PMID: 24626231
- Using an overall 99th percentile for cTnI does not appear to increase the prevalence of myocardial injury or lead to further hospital admissions from the emergency department. PMID: 26185217
Q&A
Basic Research Questions
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What is TNNI3 and why is it an important target for antibody detection in cardiac research?
TNNI3 (Troponin I Type 3, Cardiac Type) is a 210 amino acid protein with a mass of approximately 24 kDa that is predominantly expressed in heart muscle and testis. It belongs to the Troponin I protein family and plays a crucial role in cardiac tissue development and regulation of intracellular calcium . The protein undergoes important post-translational modifications, particularly phosphorylation, which affects its function in cardiac muscle contraction . TNNI3 detection is especially valuable because, unlike TNNT2 (cardiac Troponin T), TNNI3 expression is restricted to cardiomyocytes throughout human development, making it a more specific cardiac marker . The gene has been associated with cardiomyopathies, including restrictive and hypertrophic variants, highlighting its clinical significance beyond basic research applications .
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What are the optimal sample preparation methods for TNNI3 detection using HRP-conjugated antibodies?
For optimal TNNI3 detection using HRP-conjugated antibodies, tissue or cell lysate preparation should be performed under reducing conditions. Based on validated protocols, heart tissue samples should be lysed thoroughly and loaded at approximately 0.5 mg/mL for optimal detection . For Western blot applications, electrophoresis should be conducted on 5-20% SDS-PAGE gels (70V for stacking gel, 90V for resolving gel) for 2-3 hours, followed by protein transfer to nitrocellulose membranes at 150 mA for 50-90 minutes . When working with paraffin-embedded tissue sections, immersion fixation followed by antigen retrieval is crucial for preserving epitope accessibility . For cardiac tissue specifically, approximately 30 μg of protein per lane provides clear band detection at the expected 24-29 kDa range under standard reducing conditions using Immunoblot Buffer Group 1 .
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How do different detection methods compare when using TNNI3 antibodies?
TNNI3 antibodies can be utilized across multiple detection platforms with varying sensitivity and application-specific advantages:
For HRP-conjugated antibodies specifically, the signal development typically employs enhanced chemiluminescent (ECL) detection systems for Western blot and chromogenic substrates like DAB (3,3'-Diaminobenzidine) for immunohistochemistry . The sandwich ELISA format provides particularly quantitative results for TNNI3 detection in biological fluids, where the target is captured between matched antibody pairs and signal is generated through enzyme-substrate reactions .
