A2M Antibody

What is Alpha-2-Macroglobulin and why is it important in research?

Alpha-2-macroglobulin (A2M) is a large plasma protein (observed molecular weight of 185 kDa) that is abundant in vertebrate plasma and plays diverse roles in biological systems. It functions primarily as a broad-spectrum protease inhibitor, but also demonstrates important activities in immune modulation, cytokine transport, and inflammation regulation. The protein consists of 1474 amino acids and has a calculated molecular weight of 163 kDa, though it is typically observed at 185 kDa in experimental settings . A2M has gained significant research interest due to its involvement in numerous physiological and pathological processes including inflammation, immunity, and defense against invading microorganisms .

What are the primary applications of A2M antibody in research?

A2M antibody (such as the 13545-1-AP product) can be utilized in multiple experimental applications:

The antibody has been validated for ELISA applications as well, showing reactivity with human samples . These diverse applications make A2M antibody a versatile tool for researchers investigating A2M expression, localization, and interactions in various experimental contexts.

In which sample types has A2M antibody been validated?

The A2M antibody has been tested and validated in multiple human tissue and cell types:

This validation across diverse sample types ensures researchers can confidently apply the antibody in various experimental systems . It is worth noting that while predominantly tested in human samples, there have also been citations reporting reactivity with Plasmodium falciparum, expanding its potential applications in infectious disease research .

How can A2M-protease complexes be quantified using ELISA methods?

Quantification of A2M-protease complexes, particularly elastase-A2M complexes (EMC), can be accomplished using a modified ELISA technique. This methodology leverages a crucial insight: pre-incubation of anti-A2M antibody with EMC in solution significantly enhances the immunological detection of A2M-bound elastase.

The procedure involves:

• Incubating EMC with anti-A2M antibody

• Extracting the complex using solid-phase bound rabbit anti-elastase antibody

• Performing a standard ELISA detection protocol

This approach has demonstrated a lower detection limit of 0.5 ng bound elastase per ml and can distinguish between serum and plasma levels of EMC (28 vs. 21 ng/ml, p < 0.05) . The critical factor enabling reliable detection is the initial pre-incubation of anti-A2M antibody with EMC in solution, which likely causes conformational changes that expose epitopes on the bound elastase.

What role does A2M play in cytokine and growth factor regulation?

A2M serves as a crucial carrier molecule for multiple cytokines and growth factors, with significant implications for immune regulation and tissue repair. Research has identified several binding patterns:

• A2M can bind transforming growth factor-beta (TGF-β), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, and vascular endothelial growth factor (VEGF) .

• Binding sites are conformation-dependent, where:

  • FGF competes with TGF-β, but not PDGF, for binding to native A2M

  • PDGF isoforms (AA, BB & AB) compete with each other but not with TGF-β1, TGF-β2, TNF-α, FGF2, IL-1β and IL-6 for native A2M

  • On transformed A2M (A2M**), PDGF competes with TGF-β1 and FGF2

• The functional consequences of these interactions are complex:

  • TGF-β and transformed A2M (A2M**) synergistically promote smooth muscle cell proliferation

  • A2M can reduce TGF-β2's ability to inhibit lung cell proliferation

  • A2M** reduces FGF-2–induced endothelial cell proliferation

These findings demonstrate that A2M not only binds cytokines but can actively modulate their biological activities, suggesting important immunoregulatory functions.

How can mass spectrometry be used alongside A2M antibodies for biomarker analysis?

Advanced biomarker analysis can be performed by combining A2M antibodies with mass spectrometry (MALDI-MS). This approach utilizes the unique properties of A2M-trypsin interactions to generate characteristic peptides that can be quantified using isotopically labeled internal standards .

The methodology involves:

• Isolating A2M from serum using size exclusion chromatography (HiLoad 26/60 Superdex 200 pg column)

• Monitoring elution through optical density at 280 nm

• Collecting fractions with A2M tetramers (retention volume 100-150 ml)

• Analyzing fractions using SDS-PAGE and colloidal Coomassie staining

• Concentrating A2M-containing fractions

• Performing metal-affinity chromatography for further purification

• Using MALDI-MS for protein identification through the MASCOT system and Swiss Prot database

This technique is particularly valuable because conventional antibody-based methods often cannot distinguish between free A2M and its reaction products, whereas mass spectrometry provides this differentiation while also accounting for conformational changes in A2M that might affect antibody binding .

What are the key considerations for optimizing Western Blot protocols with A2M antibody?

For optimal Western Blot results with A2M antibody, researchers should consider:

• Dilution optimization: While the recommended dilution range is 1:5000-1:20000, the optimal dilution should be determined empirically for each experimental system .

• Sample preparation: A2M is detected at approximately 185 kDa, significantly higher than its calculated molecular weight of 163 kDa, likely due to post-translational modifications. Ensure your gel and transfer system can accommodate this size range effectively .

• Sample selection: The antibody has been validated in human blood tissue, brain tissue, HepG2 cells, and human plasma. Consider these positive controls when establishing your protocol .

• Blocking and washing: Use standard blocking with 5% non-fat milk or BSA in TBST, followed by thorough washing steps to reduce background.

• Exposure time: Due to the abundance of A2M in plasma and some tissues, shorter exposure times may be needed to prevent oversaturation.

Each experimental system may require specific adjustments, and it is recommended to titrate the reagent to obtain optimal results in your specific testing system .

How do A2M conformational changes affect antibody binding and experimental outcomes?

A2M undergoes significant conformational changes when interacting with proteases and other molecules, which can profoundly affect antibody binding and experimental interpretations:

• Native A2M versus activated forms: When A2M binds proteases or is chemically modified (e.g., with methylamine), it transitions from the native form to activated forms (A2M* or A2M**) with distinct conformational properties .

• Epitope accessibility: These conformational changes can expose or mask epitopes, affecting antibody recognition. For example, the binding of anti-A2M antibody to elastase-A2M complexes (EMC) enhances the subsequent detection of elastase, suggesting that antibody binding induces further conformational changes that expose epitopes on the bound elastase .

• Differential binding properties: The various A2M conformations exhibit different affinities for cytokines and growth factors. For instance, modified forms of A2M (such as "macroglobulin activated for cytokine binding" or MAC) demonstrate increased binding affinity for pro-inflammatory cytokines like TNF-α and IL-1β .

• Methodological implications: When designing experiments, researchers should consider which A2M conformation they are targeting and select or validate antibodies accordingly. Some antibodies may preferentially recognize specific conformations, potentially leading to biased results if not accounted for .

Understanding these conformational dynamics is crucial for accurate experimental design and interpretation, particularly in studies investigating A2M-protease or A2M-cytokine interactions.

How is A2M being studied in inflammatory diseases and potential therapeutics?

A2M has emerged as a significant target in inflammatory disease research, with several innovative approaches being explored:

• Modified A2M forms as anti-inflammatory agents:

  • Researchers have developed "macroglobulin activated for cytokine binding" (MAC) by consecutively treating A2M with cross-linking reagents and methylamine

  • MAC demonstrated increased binding affinity for pro-inflammatory cytokines TNF-α and IL-1β

  • In mouse models, MAC administration prior to lipopolysaccharide (LPS) challenge increased survival rates

  • MAC also suppressed inflammation in peripheral nerve injury models

• Oxidation effects on A2M function:

  • Oxidation of A2M or A2M** by hypochlorite (released by neutrophils) increases their affinity for certain molecules

  • This suggests A2M may have distinct functions in oxidative inflammatory environments

• A2M in specific inflammatory conditions:

  • A2M levels may vary considerably in different diseases, suggesting potential diagnostic applications

  • Both immunological and enzymatic methods are used to determine A2M concentration, though these approaches have distinct limitations in detecting different conformational states

These findings suggest that manipulating A2M conformations and binding properties could yield novel therapeutic strategies for inflammatory conditions, while measurements of A2M and its complexes may serve as valuable diagnostic markers.

What is the relationship between A2M and the complement system in immunity?

Recent research has revealed complex interactions between A2M and the complement system, particularly the lectin pathway:

These findings indicate that A2M may play a regulatory role in complement activation, potentially modulating innate immune responses to pathogens and contributing to the clearance of damaged proteins. This relationship provides new perspectives on A2M's functions beyond protease inhibition and cytokine transport, pointing to an integrated role in coordinating different arms of the immune system.

What are the recommended storage conditions for A2M antibody, and how do they affect experimental outcomes?

Proper storage of A2M antibody is crucial for maintaining reactivity and ensuring reproducible experimental results:

• Storage recommendations:

  • Store at -20°C

  • Stable for one year after shipment

  • Aliquoting is unnecessary for -20°C storage

  • 20μl sizes contain 0.1% BSA

  • Preserved in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Stability considerations:

  • Repeated freeze-thaw cycles should be avoided as they may degrade antibody quality

  • The presence of sodium azide prevents microbial contamination but may interfere with some enzyme-based detection systems

  • The glycerol component helps prevent freezing damage to the antibody structure

• Impact on experimental outcomes:

  • Degraded antibody can lead to reduced sensitivity, higher background, or complete loss of signal

  • Contaminated antibody preparations may produce inconsistent results

  • Changes in antibody conformation due to improper storage may affect epitope recognition, particularly important when studying conformationally variable targets like A2M

Following these storage guidelines will help ensure optimal antibody performance and experimental reproducibility when working with A2M antibody.

What controls should be included when working with A2M antibody in various applications?

Proper controls are essential for validating experimental results with A2M antibody:

• For Western Blot:

  • Positive controls: Include human blood tissue, brain tissue, HepG2 cells, or human plasma samples, which have been validated to express A2M

  • Negative controls: Include samples known not to express A2M or use primary antibody omission

  • Loading controls: Use established housekeeping proteins appropriate for your experimental system

  • Molecular weight marker: Ensure it covers the 185 kDa range where A2M is typically detected

• For Immunoprecipitation:

  • Input control: Save a portion of the pre-IP lysate to confirm target protein presence

  • Negative control: Use non-specific IgG of the same species as the A2M antibody

  • HepG2 cells have been validated for IP with this antibody

• For Immunohistochemistry/Immunofluorescence:

  • Positive tissue controls: Human liver and colon tissues have been validated

  • Antigen retrieval method controls: Compare TE buffer pH 9.0 with citrate buffer pH 6.0

  • Antibody omission control: Process sections without primary antibody

  • Absorption control: Pre-absorb antibody with immunizing peptide if available

• For ELISA and complex detection:

  • Standard curve: Generate using purified A2M or A2M-protease complexes

  • For A2M-protease complexes: Consider including phenyl methyl sulfonyl fluoride (PMSF) in assay buffer, which has been shown to produce comparable results in EMC detection

Including these controls enables confident interpretation of results and helps troubleshoot potential experimental issues.