Milr1 Antibody

Basic Research Questions

• What is MILR1 and what is its functional significance in immunology research?

  MILR1 (mast cell immunoglobulin-like receptor 1), also known as Allergin-1, is a cell surface immunoreceptor that plays a crucial role in suppressing immunoglobulin E (IgE)-mediated, mast cell-dependent responses in both mice and humans . This receptor functions as an inhibitory regulator in the immune system, particularly in allergic reactions and inflammatory responses.

  MILR1 is located on chromosome 17q23.3 in humans, which is one of the linkage regions for atopy defined by skin prick testing . The protein exhibits an inhibitory role in mast cell degranulation and negatively regulates IgE-mediated mast cell activation, thereby suppressing type I immediate hypersensitivity reactions .

  Functionally, studies have demonstrated that coligation of MILR1 and FcεRI suppresses IgE-mediated degranulation of bone marrow-derived cultured mast cells. Furthermore, mice deficient in MILR1 develop enhanced passive systemic and cutaneous anaphylaxis, confirming its immunosuppressive role .

• What antibody options are available for MILR1 detection and what applications are they validated for?

  Various antibody options for MILR1 detection include:

  Antibody Type	Host	Applications	Reactivity	Reference
  Polyclonal	Rabbit	WB, ELISA, ICC, IHC	Human
  Monoclonal	Mouse	ELISA, Flow Cytometry	Mouse
  Polyclonal	Rabbit	WB, ELISA, IF, IHC	Human

  Commercially available antibodies are often validated for Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), immunocytochemistry (ICC), immunohistochemistry (IHC), and flow cytometry applications . The choice of antibody depends on the specific experimental requirements, including the species being studied and the desired application.

  When selecting an antibody, researchers should consider the clonality (monoclonal vs. polyclonal), host species, and validated applications to ensure optimal results for their specific experimental system .

• How are MILR1 antibodies typically purified and what formulations are available?

  MILR1 antibodies are typically purified using affinity chromatography methods to ensure high specificity and minimal cross-reactivity . Available formulations include:

  • Unconjugated antibodies for standard applications

  • Fluorophore-conjugated antibodies (FITC, PE, AbBy Fluor® 350, AbBy Fluor® 647) for flow cytometry and imaging

  • Enzyme-conjugated antibodies (HRP) for ELISA and Western blotting

  • Biotin-conjugated antibodies for amplification strategies

  Most commercial preparations are supplied in PBS buffer with stabilizers such as 0.09% sodium azide and 2% sucrose . For storage, manufacturers typically recommend keeping antibodies at -20°C, with stability generally guaranteed for 12 months from the date of receipt when stored properly .

  Purification quality is crucial for experimental success; antibodies purified by affinity chromatography generally provide the highest specificity for MILR1 detection .

• What are the key considerations for experimental design when using MILR1 antibodies?

  When designing experiments using MILR1 antibodies, researchers should consider:

  • Antibody validation: Confirm specificity using positive and negative controls. For MILR1, K562 human leukemia cell lines and human embryonic kidney 293T cell lines have been used for expression validation .

  • Application-specific protocols: Different applications require specific antibody concentrations. For Western blotting, manufacturers typically provide recommended dilutions .

  • Species specificity: Ensure the antibody is reactive to the species being studied. Some antibodies are specific to human MILR1, while others detect mouse MILR1 .

  • Cross-reactivity: Consider potential cross-reactivity with related proteins, particularly other immunoglobulin-like receptors.

  • Sample preparation: For tissue samples, proper preparation is critical. Studies examining intestinal immune cells have used specific isolation protocols involving EDTA treatment followed by collagenase digestion .

  • Antibody storage and handling: Follow manufacturer recommendations to maintain antibody integrity and activity .

  Proper controls, including isotype controls for flow cytometry and immunohistochemistry, are essential for accurate interpretation of results .

Advanced Research Questions

• How do genetic polymorphisms affect MILR1 expression and what methods are used to study these effects?

  Research has identified several polymorphisms in the MILR1 gene, with rs6504230 being particularly significant in affecting MILR1 expression levels and susceptibility to atopic conditions .

  Methodologies for studying MILR1 polymorphisms include:

  1. Mutation screening: In one study, researchers amplified the 5'-flanking region and coding region of MILR1 by PCR with genomic DNA from 146 unrelated Japanese subjects, followed by direct sequencing using the Big Dye Terminator Cycle Sequencing Ready Reaction Kit on an ABI 3130 autosequencer .

  2. Luciferase reporter assays: These have been used to determine how polymorphisms affect gene expression. For example, researchers constructed plasmids encompassing various regions of the human MILR1 promoter relative to the translation initiation site, using site-directed mutagenesis to introduce specific variants (e.g., rs6504230:C) .

  3. Expression quantitative trait loci (eQTL) analysis: This approach has demonstrated that the C allele of rs6504230 is associated with increased expression levels of MILR1 (P<0.0001) .

  4. Genotyping: Large-scale studies have used genotyping of identified polymorphisms in population cohorts (e.g., 1505 individuals from the general Japanese adult population) to establish associations with phenotypes such as atopy .

  Results from these studies show that the C allele of rs6504230 has protective effects against atopy (P=0.002), with luciferase reporter assays revealing that this allele is associated with increased expression of MILR1 .

• What techniques are employed to study MILR1's role in intestinal barrier function and microbiota regulation?

  Research on MILR1's role in intestinal function employs several specialized techniques:

  1. Mouse models: Allergin-1-deficient (Milr1−/−) mice on C57BL/6N background provide a valuable model. Both specific pathogen-free (SPF) and germ-free (GF) versions of these mice have been utilized to study different aspects of MILR1 function .

  2. DSS-induced colitis model: Researchers have administered 3.5% dextran sulfate sodium in drinking water to WT and Milr1−/− mice to assess MILR1's role in intestinal inflammation. Monitoring includes body weight, survival, colon length measurement, and histological analysis .

  3. Co-housing experiments: 4-week-old Milr1−/− female weanlings placed with age-matched WT mice help determine whether microbiota differences are transferable .

  4. Flow cytometry: For analyzing Allergin-1 expression on intestinal immune cells, tissues are processed through EDTA treatment, collagenase digestion, and staining with specific antibodies including anti-mouse Allergin-1 (TX98) .

  5. 16S rRNA gene sequencing analysis: This technique has revealed that Bifidobacterium, Turicibacter, and Allobaculum were significantly increased in Milr1−/− mice compared to WT mice .

  6. Microbiota transfer experiments: Transfer of B. pseudolongum to GF mice helps determine the specific effects of this bacterium on intestinal permeability .

  7. Antibiotic treatment studies: Treatment with neomycin or ampicillin has demonstrated differential effects on intestinal barrier integrity in Milr1−/− mice .

  These methods have revealed that MILR1 plays an important role in maintaining intestinal barrier integrity by regulating dysbiosis, with gene expressions of Tnfα, Ifnβ, Saa1, and Nos2 tending to be higher in Milr1−/− mice than in WT mice .

• How can researchers effectively use MILR1 antibodies for investigating trans-kingdom immune interactions with pathogens?

  MILR1 antibodies are valuable tools for studying trans-kingdom immune interactions, particularly between hosts and fungal pathogens. Research has shown that fungal pathogens can deploy small silencing RNAs (milRNAs) that affect host immunity . Effective investigation strategies include:

  1. RNA immunoprecipitation (RIP) assays: Using antibodies against host ARGONAUTE 1 (AGO1) to detect association of fungal milRNAs with host RNA-induced silencing complexes (RISC). This approach demonstrated that Beauveria bassiana-derived milR1 (bba-milR1) binds to mosquito AGO1 .

  2. Target verification assays: Dual-luciferase reporter assays can verify interactions between fungal milRNAs and predicted host immune-related target genes. This approach revealed that bba-milR1 can suppress Spz4 and induce CLIPB9 expression in mosquito hosts .

  3. In vivo validation: Injection of synthetic milRNA agomirs (chemically modified double-stranded microRNAs) into host organisms, followed by qRT-PCR to examine transcript levels of target genes, can confirm in vivo effects .

  4. Pathogenicity assays: Using MILR1 antibodies to track expression changes during pathogen infection can help understand the role of MILR1 in host defense. Studies with Fusarium oxysporum have shown that its milRNA (Fol-milR1) acts as a virulence factor by suppressing host immunity .

  5. Immunoprecipitation with specific antisera: This technique has revealed that Fol-milR1 interferes with host immunity by binding to tomato ARGONAUTE 4a (SlyAGO4a) .

  These approaches have demonstrated that pathogen-derived small RNAs can target host immune pathways, including those involving MILR1, representing a novel virulence strategy .

• What are the optimal protocols for quantifying MILR1 expression levels in different tissues and cell types?

  Quantifying MILR1 expression requires tailored protocols depending on the tissue or cell type being studied:

  1. RT-PCR and qRT-PCR: For mRNA expression analysis, extraction protocols using TRIzol reagent followed by cDNA synthesis with High-Capacity RNA-to-cDNA Kit have been effective. Expression levels can be normalized against internal controls such as TATA-binding protein (Tbp) . For analyzing MILR1 expression in cell lines, researchers have used K562 human leukemia cell lines and human embryonic kidney 293T cell lines .

  2. Flow cytometry: For cell surface protein expression, particularly in immune cells, flow cytometry with fluorophore-conjugated anti-MILR1 antibodies is the method of choice. Proper sample preparation is critical:

     • For intestinal tissues: Longitudinal opening, washing with PBS, cutting into segments, incubation in EDTA/DTT solution, followed by collagenase digestion

     • For blood cells: Standard protocols for peripheral blood mononuclear cell (PBMC) isolation

     Analysis is typically performed on instruments such as BD LSR Fortessa Cell Analyzer with FlowJo software .

  3. Western blotting: For protein expression quantification, standard protocols using commercially available antibodies at recommended dilutions are effective. The predicted protein size for human MILR1 is 36 kDa .

  4. Immunohistochemistry: For tissue localization studies, paraffin-embedded or frozen sections can be stained with anti-MILR1 antibodies. Various commercial antibodies have been validated for IHC applications .

  5. Expression quantitative trait loci (eQTL) analysis: For studying how genetic variants affect expression, researchers have used human leukocytes and analyzed correlations between genotypes (e.g., rs6504230) and MILR1 expression levels .

  For all methods, appropriate positive and negative controls (including isotype controls for flow cytometry) are essential for reliable quantification .

• How does MILR1 signaling integrate with other immune pathways in allergic disease models?

  MILR1 signaling integrates with several key immune pathways in allergic disease models:

  1. FcεRI pathway interaction: MILR1 has been shown to inhibit IgE-mediated activation through FcεRI. Research has demonstrated that coligation of MILR1 and FcεRI suppresses IgE-mediated degranulation of bone marrow-derived cultured mast cells . This interaction represents a critical regulatory mechanism in allergic responses.

  2. Toll-like receptor (TLR) signaling: MILR1 inhibits TLR2 and TLR4 signaling, which are important sensors of microbiota. In Milr1−/− mice, increased expressions of Tnfα, Ifnβ, Saa1, and Nos2 suggest that MILR1 normally suppresses these pro-inflammatory pathways .

  3. Type I IFN pathway: Evidence suggests that MILR1 regulates Bifidobacterium pseudolongum expansion by inhibiting TLR-induced IFN-β production, as type I IFN has been shown to enhance colonization of Bifidobacterium species .

  4. Intestinal barrier regulation: MILR1 maintains intestinal barrier integrity through interactions with the inflammatory cascade. Nos2 (inducible nitric oxide synthase) expression, which is enhanced in Milr1−/− mice, is mediated by TLR signaling and linked to LPS-induced intestinal permeability .

  5. Anaphylaxis models: MILR1-deficient mice develop enhanced passive systemic and cutaneous anaphylaxis, demonstrating the receptor's role in limiting severe allergic reactions .

  Understanding these pathway intersections is crucial for developing therapeutic strategies targeting allergic and inflammatory conditions. Research approaches include comparing wild-type and MILR1-deficient mice in various disease models, co-immunoprecipitation studies to identify interaction partners, and signaling pathway analyses using phospho-specific antibodies .

• What are the best practices for validating novel MILR1 antibodies for research applications?

  Validating novel MILR1 antibodies requires a comprehensive multi-step approach:

  1. Sequence verification: Confirm that the immunogen sequence used for antibody generation matches the target species' MILR1 sequence. Some commercially available antibodies use synthetic peptides directed towards specific regions, such as the C-terminal region of Human MILR1 .

  2. Expression systems validation: Test antibodies on cells with known MILR1 expression. K562 human leukemia cell lines and human embryonic kidney 293T cell lines have been used to validate MILR1 expression and antibody reactivity .

  3. Western blot analysis: Confirm specificity by detecting a band at the expected molecular weight (approximately 36 kDa for human MILR1) .

  4. Positive and negative controls:

     • Positive controls: Tissues known to express MILR1 (e.g., mast cells, certain leukocyte populations)

     • Negative controls: MILR1-knockout tissues or cells, isotype control antibodies

  5. Cross-reactivity assessment: Test against closely related proteins or in species other than the intended target to confirm specificity.

  6. Application-specific validation:

     • For flow cytometry: Compare staining patterns with isotype controls

     • For IHC/ICC: Include absorption controls using the immunizing peptide

     • For ELISA: Establish standard curves with recombinant MILR1

  7. Reproducibility testing: Confirm consistent results across different lots and experimental conditions.

  8. Functional validation: For certain applications, confirm that the antibody can modulate MILR1 function in predictable ways (e.g., triggering or blocking signaling).

  9. Knockout validation: The gold standard is testing on MILR1-knockout samples to confirm absence of signal. Allergin-1-deficient (Milr1−/−) mice have been generated for such validation purposes .

  Documentation of these validation steps is essential for publication-quality research and reproducibility across different laboratories studying MILR1 biology .