SAA1 Recombinant Monoclonal Antibody

What is SAA1 and why is it significant in research?

SAA1 is a major acute-phase protein secreted by the liver during inflammatory conditions and microbial infections. It plays a crucial role in the body's defense mechanisms by modulating immune responses and tissue repair. SAA1 is primarily found in the high-density lipoprotein (HDL) fraction of plasma and serves as a precursor to amyloid A protein, which is a key component of fibril deposits associated with reactive amyloidosis. The SAA gene family includes SAA1, SAA2, and SAA4, located on human chromosome 11p15.1, exhibiting high homology that reflects its evolutionary significance . Research on SAA1 is valuable for understanding inflammation, immune response mechanisms, and amyloid-related diseases.

What are the main applications of SAA1 recombinant monoclonal antibodies?

SAA1 recombinant monoclonal antibodies are primarily used in Western blotting (WB), immunoprecipitation (IP), and sandwich ELISA for detecting human SAA in research samples . These applications enable researchers to investigate SAA1's role in various physiological and pathological contexts. Mouse monoclonal SAA1 antibodies have been validated for these applications with human samples and recombinant full-length protein . Additionally, anti-SAA1 antibodies have been utilized in mechanistic studies to neutralize SAA1 activity in vivo, as demonstrated in models of allergen-driven type 2 immunity .

How does SAA1 function as a pattern recognition receptor?

SAA1 functions as a soluble pattern recognition receptor (sPRR) that can interact with specific molecular patterns. Research has revealed that SAA1 can interact with fatty acid-binding proteins (FABPs) from house dust mites, such as Der p 13 and Blo t 13. This interaction has been demonstrated through immunoblotting, native PAGE analysis showing altered electrophoretic mobility, and chemical cross-linking experiments. The SAA1-FABP interaction appears to trigger SAA1 dissociation from hexameric to monomeric forms, which subsequently leads to the release of IL-33 from airway epithelial cells and promotes type 2 immune responses . This mechanism highlights SAA1's role in allergen recognition and immune response initiation.

How should I validate the specificity of an SAA1 monoclonal antibody?

Validating the specificity of an SAA1 monoclonal antibody requires a multi-step approach:

• Western blot analysis with recombinant SAA1 protein alongside acute phase plasma samples to confirm size-appropriate recognition

• Comparison with known SAA1-positive and negative controls

• Testing cross-reactivity with related proteins (SAA2, SAA4)

• Immunoprecipitation followed by mass spectrometry to confirm target identity

• Using SAA1 knockout samples as negative controls when available

Evidence from antibody validation studies shows that qualified SAA1 antibodies should detect bands of approximately 12-14 kDa in Western blots of human samples and should distinguish between different isoforms of SAA . Additionally, antibodies may recognize both native and denatured forms of SAA1 depending on the epitope, so validation in both reducing and non-reducing conditions is advisable.

What are the optimal conditions for using SAA1 antibodies in Western blotting?

Based on experimental validation, the following optimization parameters are recommended for Western blotting with SAA1 antibodies:

These conditions have been validated with acute phase plasma samples and recombinant SAA proteins, showing clear and specific detection of the target protein . It's important to note that experimental conditions may need optimization based on specific sample types and the particular antibody clone being used.

How can I optimize SAA1 antibody usage for immunoprecipitation experiments?

For optimal immunoprecipitation (IP) of SAA1, consider the following methodological approach:

• Use antibody concentrations of 2-5 μg per 200-500 μg of total protein lysate

• Pre-clear samples with protein A/G beads to reduce non-specific binding

• Incubate antibody with sample overnight at 4°C to ensure complete antigen capture

• Wash extensively with buffers containing low concentrations of detergent (0.1% Triton X-100 or NP-40)

• Elute under mild conditions to preserve protein-protein interactions if studying complexes

IP experiments with SAA1 antibodies have been successfully used to investigate SAA1's interactions with other proteins, including its binding to fatty acid-binding proteins from house dust mites . This technique is particularly valuable for studying the molecular mechanisms of SAA1's functions in various biological contexts.

How can SAA1 antibodies be used to investigate the oligomeric states of SAA1?

SAA1 exists in multiple conformational states (monomer, dimer, hexamer) that affect its biological functions. To investigate these oligomeric states:

• Use native PAGE rather than SDS-PAGE to preserve oligomeric structures

• Apply chemical cross-linking with glutaraldehyde prior to SDS-PAGE to stabilize complexes

• Employ SAA1 antibodies that recognize epitopes accessible in different oligomeric forms

• Compare antibody binding under different conditions that favor specific oligomeric states

• Utilize size-exclusion chromatography followed by immunoblotting to confirm oligomer size

Research has shown that SAA1 hexamers (60-80 kDa) can dissociate into dimers and monomers upon interaction with ligands such as house dust mite FABPs. This structural transition appears functionally important, as the dissociated forms of SAA1 show enhanced ability to trigger IL-33 release from epithelial cells . Anti-SAA1 antibodies targeting the C-terminal region (aa 89-104) have shown altered binding patterns when SAA1 interacts with ligands, suggesting conformational changes that expose this region .

What is the significance of SAA1 structure-function relationship in immune responses?

The structure-function relationship of SAA1 is critical for understanding its role in immune responses:

Research has demonstrated that introducing a Trp53Ala mutation in the hydrophobic core of SAA1 results in significantly higher IL-33 release after house dust mite (HDM) stimulation compared to wild-type SAA1. Similarly, depleting lipophilic molecules that stabilize the hexameric form enhances HDM-induced IL-33 release . These findings suggest that the structural transitions of SAA1 are mechanistically linked to its immune-modulatory functions.

How do bacterial contaminants affect the biological activities attributed to recombinant SAA1?

This question addresses a critical issue in SAA1 research:

• Recombinant SAA1 expressed in E. coli can be contaminated with bacterial products like lipopolysaccharides, lipoproteins, and formylated peptides

• These contaminants can confound biological assays by activating TLR-mediated pathways

• Purification by RP-HPLC to homogeneity is necessary to remove these contaminants

• Pure homogeneous rSAA1 (hrSAA1) lacks most cell-activating properties previously attributed to SAA1

• FPR2-mediated effects (like leukocyte recruitment and monocyte survival) remain preserved in pure hrSAA1

Research has shown that treatment of E. coli-expressed SAA1 with lipoprotein lipase causes a dose-dependent decline in its cytokine-inducing capacity in PBMCs and neutrophils. In contrast, SAA1 expressed in mammalian HEK293T cells (which lacks bacterial contaminants) does not induce inflammatory cytokine expression . This highlights the importance of using properly purified or mammalian-expressed SAA1 for accurate functional studies.

How can I verify that my recombinant SAA1 preparation is free from bacterial contaminants?

Ensuring the purity of recombinant SAA1 preparations is crucial for accurate experimental results:

• Perform the limulus amebocyte lysate (LAL) assay to determine endotoxin levels

• Conduct RP-HPLC coupled to mass spectrometry to identify and remove bacterial contaminants

• Use ion trap mass spectrometry to confirm the identity and purity of SAA1 fractions

• Compare biological activities between purified hrSAA1 and the original preparation

• Test for TLR-dependent activities as a functional indicator of contamination

Research has demonstrated that purification using C8 Aquapore RP-300 HPLC columns with a gradually increasing acetonitrile gradient effectively removes bacterial contaminants from recombinant SAA1. After purification, fractions containing SAA1 should be lyophilized and reconstituted with PBS supplemented with human serum albumin for stabilization . This purified hrSAA1 can then be reliably used for investigating the intrinsic biological activities of SAA1.

What controls should be included when using SAA1 antibodies for neutralization experiments?

When designing neutralization experiments with SAA1 antibodies:

• Include isotype-matched control antibodies to account for non-specific effects

• Use concentration-matched irrelevant target antibodies as negative controls

• Prepare F(ab')2 fragments of the antibody to eliminate Fc-mediated effects if necessary

• Include SAA1 knockout or knockdown controls when available

• Perform dose-response experiments to establish specificity of neutralization

Research has employed SAA1 neutralizing antibodies administered locally in the lungs of mice to investigate the contribution of SAA1 to allergen-driven type 2 immunity. These studies demonstrated reduced numbers of HDM-induced lung IL-13+ ILC2s following antibody-mediated neutralization . Proper controls are essential to distinguish between specific neutralization of SAA1 and non-specific effects of antibody administration.

How can I distinguish between SAA1 and other SAA isoforms in my experiments?

Distinguishing between different SAA isoforms requires careful experimental design:

• Select antibodies with validated specificity for SAA1 versus SAA2 and SAA4

• Use recombinant proteins of each isoform as positive controls

• Consider employing isoform-specific PCR for expression analysis at the mRNA level

• Utilize mass spectrometry-based approaches for definitive protein identification

• When possible, use samples from individuals with known SAA genotypes

How should I interpret changes in SAA1 oligomeric states in experimental samples?

Interpreting changes in SAA1 oligomeric states requires careful analysis:

• Compare native PAGE patterns between control and experimental conditions

• Quantify the relative proportions of monomers, dimers, and hexamers

• Correlate structural changes with functional readouts (e.g., IL-33 release)

• Consider the influence of lipid content in experimental media

• Assess the impact of potential ligands that may promote dissociation

Research has shown that untreated bronchial epithelial cells secrete SAA1 as a lipid-free oligomer of approximately 60-80 kDa (hexamer) into the supernatant. Following house dust mite treatment, this hexameric SAA1 rapidly dissociates into dimers and monomers, correlating with increased IL-33 release. Similarly, increasing concentrations of the mite FABP Blo t 13 trigger a concentration-dependent decrease in SAA1 hexamer formation, associated with increasing IL-33 concentrations . These patterns suggest that SAA1 oligomeric state transitions can serve as indicators of functional activation.

What is the significance of epitope accessibility in different SAA1 conformational states?

Understanding epitope accessibility in different SAA1 conformations provides valuable insights:

• The C-terminal tail (aa 89-104) of SAA1 may be differentially exposed in various conformational states

• Antibodies targeting this region show altered binding patterns when SAA1 interacts with ligands

• This suggests that ligand binding leads to conformational changes in SAA1

• Changes in epitope accessibility correlate with functional activation of SAA1

• Epitope mapping can help identify antibodies suitable for detecting specific conformational states

Research with sequence-specific antisera against the C-terminal tail of human SAA1 has shown altered electrophoretic mobility and stronger binding when SAA1 interacts with the mite FABP Blo t 13. This suggests that ligand binding leads to conformational changes that make the C-terminal tail more accessible for antibody binding . Such findings highlight how antibody binding patterns can reveal important structural transitions associated with SAA1 activation.

How can I differentiate between TLR-mediated and FPR2-mediated effects of SAA1 in my experimental system?

Distinguishing between different receptor-mediated effects of SAA1 requires specific experimental approaches:

• Use highly purified hrSAA1 to eliminate TLR-activating contaminants

• Compare responses between wild-type cells and those deficient in specific receptors

• Employ selective antagonists for FPR2 (e.g., WRW4 peptide) or TLRs

• Assess receptor-specific downstream signaling events

• Measure distinct functional outcomes (chemotaxis for FPR2, cytokine production for TLRs)

Research has demonstrated that pure homogeneous rSAA1 retains FPR2-mediated functions like leukocyte recruitment and monocyte survival, while lacking TLR-mediated activities such as cytokine induction and ROS production. This indicates that intrinsic SAA1 activities primarily involve FPR2 activation, whereas TLR-related effects observed with E. coli-expressed rSAA1 are likely due to bacterial contaminants . These findings highlight the importance of using properly purified SAA1 and appropriate receptor-specific controls for accurate functional characterization.