CML26 Antibody

What is CML26 antibody and what epitope does it recognize?

CML26 is a monoclonal antibody specifically designed to recognize human carboxymethyl-lysine (CML), which is a well-characterized glycoxidation product and a major antigenic AGE compound . The antibody binds to the CML domain formed through the oxidation of both carbohydrates and lipids, making it valuable for detecting this biomarker of general oxidative stress . Unlike some antibodies that may cross-react with various AGE epitopes, CML26 demonstrates specificity for the CML modification, allowing for targeted detection of this particular post-translational modification in various experimental contexts .

What is the biological significance of CML detection in research?

CML is a glycoxidation product that accumulates in tissues with age, and its rate of accumulation is accelerated in diabetes and other conditions characterized by oxidative stress . The detection of CML has significant implications for understanding the pathogenesis of several chronic conditions:

• CML serves as a biomarker of general oxidative stress due to its formation through both carbohydrate and lipid oxidation pathways

• The accumulation of CML in long-lived tissue such as skin collagen reflects oxidative stress over an extended period

• CML levels have been shown to be elevated in patients with diabetic complications compared to those without complications

• Oxidative stress and protein modification involving CML have been implicated in the pathogenesis of chronic complications of diabetes, including nephropathy and atherosclerosis

• CML detection can help monitor disease progression and evaluate the efficacy of therapeutic interventions in conditions characterized by increased oxidative stress

How does CML formation occur in biological systems?

CML (carboxymethyl-lysine) formation occurs through non-enzymatic processes primarily involving glycation and oxidation reactions. CML is formed through the oxidation of both carbohydrates and lipids in biological systems . The process typically begins with the non-enzymatic glycation of proteins, where reducing sugars react with free amino groups on proteins to form Schiff bases and Amadori products . Subsequently, these intermediate products undergo irreversible oxidation (hence the term "glycoxidation") to form stable CML adducts on lysine residues of proteins . CML represents a subset of advanced glycation end-products (AGEs) that are formed by this nonenzymatic glycation followed by irreversible oxidation of proteins . The rate of CML formation is accelerated under conditions of increased oxidative stress, which explains its elevated levels in diabetes, aging tissues, and various inflammatory conditions .

What are the validated research applications for CML26 antibody?

The CML26 monoclonal antibody has been validated for several research applications focusing on the detection and quantification of carboxymethyl-lysine. Based on the available information, validated applications include:

• Immunofluorescence (IF): The antibody has been specifically tested for immunofluorescence applications. The recommended protocol involves fixation in 2% phosphate-buffered glutaraldehyde solution, post-fixation in 1% osmium tetroxide, dehydration through a graded series of ethanol, and cutting of 0.5-3.0 mm-thick sections with a glass knife .

• ELISA (Enzyme-Linked Immunosorbent Assay): Both direct and competitive ELISA formats have been validated for CML detection using monoclonal antibodies like CML26 . These assays enable quantitative measurement of CML in biological samples.

• Detection of CML in clinical samples: The antibody has been used to detect and quantify circulating levels of CML in biological fluids of patients with chronic kidney disease (CKD) and potentially other conditions characterized by increased oxidative stress .

• Investigation of oxidative stress and protein modification: CML26 can be utilized to study oxidative stress-related protein modifications in various tissues and experimental models .

How can I optimize ELISA protocols for CML detection using CML26 antibody?

For optimal detection of CML using CML26 antibody in ELISA formats, researchers should consider the following methodological approaches:

Direct ELISA Format:

• Coating: Immobilize CML-protein (10 μg/mL) onto 96-well plates and incubate at 4°C overnight

• Blocking: Saturate non-specific binding sites with 2% casein for 60 minutes

• Primary antibody: Incubate with CML26 antibody (1 μg/mL) for 60 minutes at 37°C

• Secondary antibody: Incubate with HRP-conjugated anti-mouse IgG for an additional 60 minutes

• Detection: Add substrate solution (1,2-phenylenediamine dihydrochloride) for 20 minutes, stop the reaction with 1.0 M sulfuric acid, and measure absorbance at 490 nm

Competitive ELISA Format (for serum samples):

• Coating: Coat plates overnight with CML-protein (10 μg/mL) in carbonate buffer pH 9.6

• Blocking: Block with 2% casein in PBS

• Sample competition: Incubate human sera (diluted 1:10) in the presence of 0.1 μg/mL of CML26 antibody for 120 minutes at 37°C

• Detection: Follow with HRP-conjugated anti-mouse IgG and substrate as in direct ELISA

• Standard curve: Use CML-HSA at various concentrations (0.4–200 ng/mL) for quantification

For both formats, thorough washing with PBS (pH 7.4) containing 0.1% Tween 20 between steps is essential for reducing background and improving assay sensitivity .

What considerations should be made when using CML26 for immunofluorescence detection?

When employing CML26 antibody for immunofluorescence detection of CML in tissues, researchers should consider the following protocol considerations:

• Fixation protocol: The validated approach involves initial fixation in 2% phosphate-buffered glutaraldehyde solution, followed by post-fixation in 1% osmium tetroxide . This two-step fixation helps preserve both tissue morphology and antigenicity of CML epitopes.

• Tissue processing: Proper dehydration through a graded series of ethanol is critical for maintaining tissue integrity while allowing antibody penetration .

• Section thickness: The recommended thickness for sections is 0.5-3.0 mm, cut with a glass knife . This range allows for adequate tissue visualization while maintaining sufficient antigen accessibility.

• Antigen retrieval considerations: While not explicitly mentioned in the available data, researchers should evaluate whether antigen retrieval steps might improve CML detection, particularly in heavily fixed tissues.

• Controls: Include appropriate positive and negative controls to validate staining specificity. The use of non-glycated proteins or tissues from models with reduced CML formation can serve as negative controls.

• Signal amplification: For tissues with low CML content, consider signal amplification techniques compatible with immunofluorescence to enhance detection sensitivity.

How does CML26 antibody compare to other anti-CML antibodies in specificity and sensitivity?

The specificity and sensitivity of CML26 antibody can be evaluated in comparison to other anti-CML antibodies based on several criteria:

Epitope Recognition:
CML26 specifically recognizes the carboxymethyl-lysine (CML) domain, which is a major antigenic AGE compound . In direct ELISA testing, CML26 demonstrates the ability to recognize the CML domain while showing no cross-reactivity with native carrier proteins . This specificity for modified versus unmodified proteins is crucial for accurate detection of glycoxidation products.

Comparative Performance:
When compared to other monoclonal antibodies targeting CML, such as clone 3G3 mentioned in the research, CML26 (specifically subclone 2D6G2) exhibited higher capacity to secrete specific antibodies . This suggests potentially superior sensitivity for CML detection compared to some alternative antibodies.

Validation in Biological Samples:
CML26 has been validated for use in detecting CML in biological fluids of patients with chronic kidney disease, indicating its applicability to clinical research samples with complex protein matrices .

Immunoassay Performance:
The antibody performs well in both direct and competitive ELISA formats, with demonstrated ability to detect CML-modified proteins at concentrations ranging from 0.4–200 ng/mL in competitive assays . This range is suitable for detecting physiologically relevant concentrations of CML in biological samples.

What are the known limitations or potential pitfalls when using CML26 antibody?

Researchers should be aware of several limitations and potential pitfalls when working with CML26 antibody:

Cross-reactivity considerations: While CML26 shows specificity for CML domains, researchers should validate the absence of cross-reactivity with other AGE structures or modified amino acids in their specific experimental systems . This is particularly important when working with complex biological samples containing multiple AGE species.

Sample preparation effects: The detection of CML can be influenced by sample preparation methods. Oxidation during sample processing might artificially increase CML levels, leading to false-positive results. Conversely, masking of CML epitopes through protein-protein interactions or conformational changes might reduce antibody binding and lead to underestimation of CML content .

Standardization challenges: There is no internationally recognized standard unit of measurement to express AGE levels, making comparison of results between different laboratories extremely difficult . This limitation necessitates the inclusion of appropriate internal standards and controls in each experiment.

Stability considerations: The stability of both the antibody and CML-modified proteins during storage and handling should be considered. Repeated freeze-thaw cycles or prolonged storage might affect antibody performance or the integrity of CML epitopes.

How can CML26 be used to investigate the relationship between CML formation and disease pathogenesis?

CML26 antibody offers valuable opportunities for investigating the relationship between CML formation and disease pathogenesis through several methodological approaches:

Tissue-specific accumulation studies:
CML26 can be used to map the tissue-specific accumulation of CML in various disease models. Since CML accumulation in long-lived tissue like skin collagen reflects oxidative stress over extended periods of the lifespan, immunohistochemical or immunofluorescence studies using CML26 can help identify organs and tissues particularly susceptible to glycoxidation damage .

Longitudinal monitoring of CML levels:
By establishing standardized ELISA protocols with CML26, researchers can monitor CML levels in patient cohorts over time. This approach is particularly valuable for conditions like diabetes, where CML accumulation has been shown to be greater in patients with complications than those without complications . Such longitudinal studies can help identify whether CML serves as a predictive biomarker for disease progression.

Intervention studies:
CML26 can be employed to evaluate the efficacy of interventions designed to reduce oxidative stress or inhibit AGE formation. By quantifying CML levels before and after treatment, researchers can assess whether therapeutic approaches effectively reduce this specific glycoxidation product .

Correlation with functional outcomes:
Combining CML detection using CML26 with functional assays allows researchers to investigate potential causal relationships between CML accumulation and cellular or tissue dysfunction. This approach can help distinguish whether CML is merely a biomarker or an active contributor to pathological processes .

Integration with other oxidative stress markers:
Since CML is known to be formed from the oxidation of both carbohydrates and lipids, parallel analysis of CML (using CML26) alongside other oxidative stress markers can provide a more comprehensive understanding of the oxidative damage profile in specific disease states .

What analytical techniques can be combined with CML26 antibody for comprehensive CML analysis?

For comprehensive analysis of CML in research contexts, CML26 antibody can be integrated with several complementary analytical techniques:

Mass Spectrometry-Based Approaches:
Liquid chromatography tandem mass spectrometry (LC-MS/MS) offers high specificity for CML detection and quantification. The method described in the research uses hydrophilic interaction liquid chromatography (HILIC) separation on a Luna amino column followed by positive electrospray ionization and multiple reaction monitoring (MRM) . This approach allows for absolute quantification of CML with high sensitivity and can be used to validate immunoassay results obtained with CML26 antibody.

High-Resolution Mass Spectrometry (HRMS):
For identifying CML degradation products and metabolites, high-resolution mass spectrometry using instruments like the Exactive Orbitrap HRMS can be employed . This technique enables the tentative identification of novel CML-related compounds that may not be detectable by antibody-based methods alone.

Combined Immunoprecipitation and MS Analysis:
CML26 antibody can be used for immunoprecipitation of CML-modified proteins, followed by mass spectrometry analysis to identify specific proteins carrying the CML modification. This combinatorial approach helps map the CML-modified proteome in different biological contexts.

Western Blotting:
While not explicitly mentioned in the search results, Western blotting using CML26 antibody would allow for the detection of CML-modified proteins separated by molecular weight, providing insights into which specific proteins carry this modification.

Imaging Mass Spectrometry:
Combining immunohistochemistry using CML26 with imaging mass spectrometry could provide spatial information about CML distribution in tissues while confirming the identity of the modification through mass spectrometric verification.

How can researchers standardize CML quantification across different experimental systems?

Standardizing CML quantification across different experimental systems presents several challenges, as noted in the literature: "no gold standard method is available for detection and quantification of AGEs... there is no internationally recognized standard unit of measurement to express AGEs levels" . To address these challenges, researchers can implement the following standardization approaches:

Internal Standards and Reference Materials:

• Utilize isotopically labeled internal standards such as d4-CML for mass spectrometry-based quantification

• Develop and share well-characterized reference materials with defined CML content for calibration across laboratories

• Express results relative to stable reference proteins or as molar ratios (e.g., mmol CML/mol lysine) rather than absolute concentrations

Method Validation Parameters:

• Establish and report key analytical performance characteristics including robustness, sensitivity, reproducibility, repeatability, linearity, accuracy, and matrix effects

• Determine method-specific limits of detection and quantification for CML in different sample matrices

• Conduct inter-laboratory comparison studies to assess method transferability

Sample Preparation Standardization:

• Develop standardized protocols for sample collection, storage, and preparation to minimize artifactual CML formation during processing

• Include antioxidants or other stabilizers during sample preparation to prevent ex vivo oxidation

• Document and control pre-analytical variables that might influence CML levels

Calibration Approaches:

• Use competitive ELISA formats with CML26 antibody and standardized CML-HSA preparations at defined concentrations (0.4–200 ng/mL) for immunoassay calibration

• Implement multi-point calibration curves rather than single-point calibrations

• Verify calibration stability throughout analytical runs

What considerations should be made when interpreting CML data in the context of oxidative stress research?

When interpreting CML data in oxidative stress research, several important considerations should be addressed:

Biological Half-Life and Turnover Rates:
CML accumulates in tissues with age, particularly in long-lived tissues such as skin collagen . Therefore, CML levels reflect oxidative stress over an extended period rather than acute oxidative events. Researchers should consider the turnover rate of the specific tissue or protein being analyzed when interpreting CML data. Rapidly turned-over proteins may show different CML accumulation patterns compared to long-lived structural proteins.

Multiple Pathways of Formation:
CML is formed from the oxidation of both carbohydrates and lipids . This dual origin means that elevated CML levels could result from increased oxidative stress affecting either carbohydrate metabolism, lipid peroxidation, or both. Additional markers specific to each pathway may help distinguish the predominant source of oxidative stress.

Context-Specific Normal Ranges:
The "normal" range of CML levels varies across different tissues, age groups, and physiological states. Researchers should establish appropriate control groups matched for relevant variables such as age, as CML naturally accumulates during aging even in the absence of pathology .

Relationship to Other AGE Species:
CML is one of many advanced glycation end-products. A comprehensive assessment of oxidative stress may require analysis of multiple AGE species, as different AGEs may form under specific oxidative conditions or have distinct biological effects .

Functional Consequences vs. Biomarker Status:
Distinguish between CML as a biomarker of oxidative stress and as a potential mediator of cellular dysfunction. Elevated CML levels indicate increased oxidative stress but do not necessarily establish causality in disease processes. Functional studies are needed to determine whether CML actively contributes to the observed pathology or simply serves as a marker .

How can CML26 antibody be used to study the relationship between diet, gut microbiota, and CML metabolism?

CML26 antibody offers unique opportunities for investigating the complex relationships between dietary CML intake, gut microbiota activity, and systemic CML levels:

Dietary CML Assessment:
CML26 antibody can be used to quantify CML content in various food items, particularly in processed and heat-treated foods that are known to contain elevated levels of advanced glycation end-products . This allows researchers to correlate dietary CML intake with systemic CML levels and potential health outcomes.

Tracking CML Metabolism by Gut Microbiota:
Recent research has demonstrated that intestinal bacteria can metabolize CML under anaerobic conditions, with significant variability between individuals based on their microbiota composition . CML26 antibody can be used to:

• Quantify CML degradation in fecal samples or in vitro gut fermentation models

• Identify individuals with high or low capacity for CML degradation

• Monitor changes in CML metabolism following dietary interventions or probiotic treatments

Microbiota-Dependent CML Transformation:
The study of CML degradation products by gut bacteria has identified carboxymethylated biogenic amines and carboxylic acids as potential metabolites . CML26 antibody, in combination with mass spectrometry, can help track the parent compound while specialized analytical methods identify its transformation products.

Correlation with Microbial Taxa:
Research has identified specific bacterial taxa, including Oscillibacter and Cloacibacillus evryensis, that may be involved in anaerobic CML degradation . CML26 antibody can be used in studies correlating the abundance of these bacteria with CML degradation capacity and systemic CML levels.

Intervention Studies:
CML26 antibody enables the assessment of how dietary or microbiome-targeted interventions affect CML metabolism and systemic levels. This includes studying:

• Effects of prebiotics or probiotics on CML degradation

• Impact of dietary patterns on both CML intake and microbial CML metabolism

• Potential for microbial modulation as a strategy to reduce AGE burden in high-risk populations

What role does CML play in the pathogenesis of chronic kidney disease, and how can CML26 be used to investigate this?

CML has emerged as an important factor in chronic kidney disease (CKD) pathogenesis, and CML26 antibody provides valuable tools for investigating these connections:

Biomarker Validation in CKD:
CML26 antibody has been specifically validated for detecting and quantifying circulating levels of CML in biological fluids of CKD patients . This enables researchers to assess whether CML levels correlate with disease severity, progression rates, or response to treatments in CKD.

Mechanistic Studies:
Using CML26 for immunohistochemical analysis of kidney tissues can help localize CML accumulation to specific renal structures, potentially identifying vulnerable cell types and providing insights into pathogenic mechanisms. This approach can determine whether CML deposition precedes structural damage or develops as a consequence of initial kidney injury.

Correlation with Renal Function:
By establishing standardized ELISA protocols with CML26, researchers can investigate correlations between CML levels and various markers of renal function (e.g., estimated glomerular filtration rate, proteinuria). Such analyses can determine whether CML serves as a predictive biomarker for functional decline in CKD.

Therapeutic Target Identification:
If CML is established as a pathogenic factor rather than a mere biomarker, CML26 antibody can be used to screen for interventions that effectively reduce CML accumulation in renal tissues or circulation. This could lead to novel therapeutic approaches targeting CML formation or enhancing its clearance.

Uremic Toxin Assessment:
CML has been described as a uremic toxin in the context of CKD . CML26 antibody enables the quantification of this specific toxin among the complex mixture of retained solutes in CKD, potentially helping to delineate which uremic toxins contribute most significantly to CKD-associated complications.

How can researchers investigate the relationship between CML accumulation and aging using CML26 antibody?

CML accumulation has been established as a feature of normal aging, and CML26 antibody provides researchers with tools to investigate this relationship in greater depth:

Age-Related Tissue Distribution Mapping:
CML26 can be employed in immunohistochemical studies to map the tissue-specific patterns of CML accumulation across different age groups. This approach can identify which tissues show the most pronounced age-related increases in CML content and whether this correlates with functional decline in those tissues.

Long-lived Protein Analysis:
CML accumulates particularly in long-lived proteins such as skin collagen . Using CML26 antibody, researchers can compare CML accumulation rates in proteins with different turnover rates to better understand how protein longevity contributes to AGE burden during aging.

Interventional Gerontology Studies:
CML26 antibody enables the evaluation of interventions proposed to slow aging processes, such as caloric restriction, exercise programs, or geroprotective compounds. By quantifying CML levels before and after such interventions, researchers can assess whether these approaches effectively reduce this specific marker of aging.

Correlation with Healthspan Metrics:
Beyond chronological age, CML levels detected using CML26 antibody can be correlated with functional measures of biological age and healthspan. This might include physical performance metrics, cognitive assessments, or other biomarkers of aging to determine whether CML serves as a reliable indicator of biological versus chronological age.

Comparative Biology Approaches: CML26 can be used to compare CML accumulation patterns across species with different lifespans, provided there is sufficient epitope conservation. Such comparative studies might reveal whether differences in CML accumulation rates correlate with species longevity and could identify natural protective mechanisms against AGE accumulation.