HLA-H Antibody

What is HLA-H and why is it important for antibody research?

HLA-H is a protein encoded by a gene in the major histocompatibility complex (MHC) class I region that was initially categorized as a pseudogene but has since been recognized as transcriptionally active with potential functional significance. HLA-H plays a crucial role in iron metabolism, with mutations in this gene being strongly associated with hereditary hemochromatosis (HH), an autosomal recessive disorder leading to excessive iron storage in the liver and other organs .

Between 83-100% of HH patients have been found homozygous for the Cys-282 → Tyr mutation in the HLA-H gene, which is believed to disrupt protein function by altering a critical disulfide bridge . Antibodies against HLA-H are essential research tools for understanding its expression patterns, subcellular localization, and potential roles in immune regulation. Recent research has revealed that certain HLA-H alleles, particularly H*02:07, encode functional membrane-bound proteins capable of inhibiting human effector cell activity .

What methods are used to generate and characterize HLA-H-specific antibodies?

Generating high-quality antibodies against HLA-H typically involves several key steps:

• Peptide selection and synthesis: Researchers commonly synthesize peptides corresponding to specific regions of HLA-H, such as the C-terminal amino acids. This targeting is strategic, as demonstrated in studies where a 16 C-terminal amino acid peptide was chosen specifically because it had minimal sequence overlap with related proteins (only 4 amino acids in common with HLA-A2, 1 with HLA-G, and 2 with human neonatal Fc receptor) .

• Conjugation to carrier proteins: The synthesized peptide is coupled to carrier proteins like pig thyroglobulin using bifunctional reagents such as disuccinimidyl suberate to enhance immunogenicity .

• Immunization protocol: The conjugated peptide-protein complex is administered to animals (typically rabbits) following established immunization schedules. For example, protocols may involve initial subcutaneous injection of 300 μg protein in complete Freund's adjuvant, followed by subsequent injections with incomplete Freund's adjuvant at 4-week intervals .

• Antibody purification: Affinity purification techniques using peptide-coupled resins (such as C-terminal HLA-H peptide–Affigel 10) are employed to isolate specific IgG antibodies, which are then stored appropriately (e.g., in 50% glycerol at −20°C) .

• Specificity testing: Validation of antibody specificity through multiple approaches, including peptide-specific blocking of bands identified on Western blots, comparative reactivity with other antibodies targeting different HLA-H domains, lack of cross-reactivity with related proteins, and positive reactivity with cells expressing HLA-H cDNA but not vector-only controls .

This methodological approach ensures the generation of antibodies with high specificity for HLA-H, critical for accurate research applications.

How does the subcellular distribution of HLA-H impact antibody binding studies?

The subcellular localization of HLA-H exhibits remarkable tissue-specific variation, which significantly impacts experimental design for antibody-based detection and functional studies. Research has identified three distinct patterns of HLA-H distribution:

• Non-polarized plasma membrane distribution: In stratified squamous epithelial cells of the esophagus and submucosal leukocytes, HLA-H is distributed around the entire plasma membrane. This pattern allows for more accessible antibody binding in non-permeabilized cells .

• Polarized basolateral localization: In polarized epithelial cells of the stomach and colon, HLA-H expression is restricted to the basolateral plasma membrane. This polarized distribution necessitates careful consideration of antibody accessibility in experimental systems, potentially requiring cell permeabilization or basolateral access .

• Intracellular perinuclear localization: Uniquely in the small intestine (duodenum, jejunum, and ileum), HLA-H shows predominantly intracellular, perinuclear staining, limited to cells in deep crypts. This pattern requires cell permeabilization protocols for effective antibody binding in immunohistochemistry or flow cytometry applications .

These distinct localization patterns are especially relevant when designing experiments to study HLA-H in proximity to sites of iron absorption in the small intestine. Researchers must consider appropriate fixation and permeabilization techniques, accessibility of epitopes, and potential masking of binding sites due to protein-protein interactions depending on the specific tissue under investigation .

What affinity determination methods are most effective for HLA-H antibody characterization?

Comprehensive characterization of HLA-H antibody binding properties requires multiple complementary approaches to assess both affinity and avidity. Research comparing different methodologies has revealed important distinctions:

Solid-Phase Technologies:

• Surface Plasmon Resonance (SPR): Provides real-time, label-free detection of antibody-antigen interactions and can determine association and dissociation rates. Studies have shown that SPR tends to measure avidity rather than true affinity for HLA antibodies .

• Bio-layer Interferometry (BLI): Similar to SPR but using optical interference patterns, BLI also predominantly measures avidity for HLA antibodies. Research has demonstrated comparable high binding-strength measurements between SPR and BLI .

• Luminex Single Antigen Beads (SAB): Widely used in clinical settings, this flow cytometry-based method detects antibodies bound to HLA-coated microbeads. While effective for screening, SAB also measures avidity rather than true affinity .

Solution-Phase Technology:

• Flow-Induced Dispersion Analysis (FIDA): This newer in-solution approach has been shown to yield slightly lower binding-strength measurements compared to solid-phase methods, suggesting it more accurately measures true affinity rather than avidity .

The in-solution FIDA approach offers a significant advantage by simultaneously determining both antibody affinity and concentration in patient serum samples, making it particularly valuable for clinical applications . For comprehensive HLA-H antibody characterization, researchers should consider employing both solid-phase and solution-phase methodologies to obtain complete binding profiles.

How is HLA-H gene expression quantified in different tissues and cell types?

Accurate quantification of HLA-H expression is essential for understanding its biological roles and potential involvement in disease processes. Several methodologies have been validated for this purpose:

Real-Time PCR Analysis:

Real-Time PCR represents a primary approach for measuring HLA-H mRNA expression levels. Researchers have established specific primer/probe sets with verified efficiency (e.g., 2.05 in dilution assays) that demonstrate no cross-reactivity with genomic DNA or negative control cell lines like K562 . This technique has enabled quantitative comparison of HLA-H expression across different tissues, revealing that:

• HLA-H is expressed in Peripheral Blood Mononuclear Cells (PBMCs) at levels of 5.334 (4.291–6.758) relative units

• Human Bronchial Epithelial Cells (HBECs) show similar expression at 5.322 (4.268–8.410) relative units

• HLA-H transcriptional expression in HBECs is comparable to HLA-G but significantly lower than HLA-E (p < 0.0001)

RNA Sequencing:

Analysis of RNA-seq data from lymphoblastoid cell lines provides another approach to assess HLA-H expression patterns, enabling allele-specific expression analyses that have revealed hemizygous effects and allele-dependent expression levels, with HLA-H*02:07 showing notably higher mRNA expression compared to other alleles .

Flow Cytometry for Indirect Detection:

While direct detection of HLA-H protein faces technical challenges, researchers have employed indirect methods such as measuring HLA-E mobilization to the cell surface following HLA-H signal peptide treatment. This approach has been successfully applied to various cell types including T-lymphocytes, monocytes, B-lymphocytes, and primary epithelial cells .

These complementary methodologies provide a comprehensive toolkit for investigating HLA-H expression in various experimental and clinical contexts.

What is the evolutionary significance of HLA-H alleles and how does this impact antibody studies?

The evolutionary history of HLA-H alleles presents fascinating insights with significant implications for immunological research and antibody characterization:

Archaic Human Genetic Contribution:

Research has revealed that the HLA-H02:07 allele, which encodes a functional membrane-bound protein, is of Denisovan origin. This finding was established through in silico genotyping of the Denisovan genome and haplotypic association with the Denisovan-derived HLA-A11 allele . The presence of this archaic allele in modern European and Asian populations represents an important instance of adaptive introgression from archaic humans into modern human genomes .

Functional Implications:

The HLA-H*02:07 allele's ability to encode a complete membrane-bound protein capable of inhibiting human effector cells (including IL-2–activated PBMCs and the human IL-2–independent NK92-MI cell line) suggests that admixture with archaic humans introduced functionally significant immune regulatory elements into modern human populations . This challenges the traditional view of HLA-H as a non-functional pseudogene.

Research Design Considerations:

When developing or characterizing antibodies against HLA-H, researchers must consider:

• Allelic variation: Antibodies may have differential binding to various HLA-H alleles, particularly between archaic-derived alleles like H*02:07 and other variants.

• Expression level differences: Studies have shown that H*02:07 exhibits higher mRNA expression levels compared to other HLA-H alleles, potentially affecting antibody detection sensitivity .

• Population specificity: The distribution of functional HLA-H alleles varies across populations, necessitating consideration of demographic factors in experimental design and interpretation.

These evolutionary insights reinforce the importance of comprehensive validation when developing antibodies against HLA-H, ideally testing against multiple allelic variants to ensure broad applicability across genetically diverse populations and research contexts.

How do HLA-H antibodies compare with antibodies against other HLA molecules?

Comparing HLA-H antibodies with those targeting other HLA molecules reveals important distinctions in terms of binding characteristics, cross-reactivity patterns, and functional properties:

Epitope Recognition:

Unlike classical HLA class I antibodies that typically recognize epitopes formed by polymorphic amino acid residues on the outer surface of the molecule, antibodies against HLA-H often target specific regions that differentiate it from other HLA molecules. For example, researchers have strategically designed antibodies against the C-terminal peptide of HLA-H, which shares minimal sequence homology with other HLA molecules (only 4 amino acids with HLA-A2, 1 with HLA-G, and 2 with human neonatal Fc receptor) .

Cross-Reactivity Considerations:

Due to the sequence homology between HLA-H and other HLA molecules, cross-reactivity remains an important consideration when developing and characterizing HLA-H antibodies. Rigorous validation is required, typically involving:

• Peptide-specific blocking assays

• Testing against multiple HLA molecules

• Evaluation with cells expressing only specific HLA genes

• Demonstrating reactivity with HLA-H transfected cells but not vector-only controls

These methodological approaches help ensure the specificity of antibodies developed against HLA-H, critical for accurate research applications in studying its unique biological functions.

What is the relationship between HLA-H and iron metabolism disorders?

The connection between HLA-H and iron metabolism disorders, particularly hereditary hemochromatosis (HH), represents one of the most significant clinical applications for HLA-H research:

Genetic Basis:

Hereditary hemochromatosis is a common autosomal recessive disorder of iron metabolism characterized by excessive iron storage in the liver and other organs. Between 83-100% of HH patients have been found to be homozygous for a specific Cys-282 → Tyr mutation in the HLA-H gene . This mutation is believed to disrupt a critical disulfide bridge, compromising the function of the HLA-H protein .

Molecular Mechanisms:

The physiological role of HLA-H in iron metabolism appears to be related to its unique expression pattern in the gastrointestinal tract, particularly its intracellular, perinuclear localization in the crypts of the small intestine - anatomically proximal to the primary sites of iron absorption . This suggests that HLA-H may play a regulatory role in intestinal iron uptake, with mutations leading to dysregulated absorption and subsequent systemic iron overload.

Applications of HLA-H Antibodies:

Antibodies against HLA-H have proven instrumental in elucidating these relationships by:

• Mapping tissue expression patterns: Immunohistochemical studies using HLA-H antibodies have revealed the protein's variable distribution throughout the gastrointestinal tract, with particular significance in the deep crypts of the small intestine .

• Subcellular localization analysis: The distinct subcellular distribution patterns of HLA-H (plasma membrane in some tissues vs. intracellular/perinuclear in others) provide important clues about its potential mechanistic role in iron homeostasis .

• Mutational impact assessment: Comparing antibody binding patterns between wild-type and mutant HLA-H can help elucidate how specific mutations alter protein localization, stability, or function.

These findings establish HLA-H antibodies as critical research tools for investigating the pathophysiology of iron metabolism disorders and potentially developing targeted therapeutic interventions.

How does HLA-H interact with HLA-E and what methods are used to study this relationship?

The functional interaction between HLA-H and HLA-E represents a fascinating aspect of HLA biology with significant implications for immune regulation:

Molecular Interaction Mechanism:

HLA-H appears to function through dual pathways: directly as a transmembrane molecule and indirectly via its signal peptide, which can mobilize HLA-E to the cell surface . HLA-E regulates natural killer (NK) and cytotoxic T-lymphocyte cells through interaction with the inhibitory receptor CD94/NKG2 . While HLA-E is widely transcribed in most tissues, its surface expression requires signal peptides from various sources, including HLA class I molecules, HLA-G, and peptide ligands from stress proteins and viruses .

Research Methodologies:

Several experimental approaches have been successfully employed to investigate the HLA-H/HLA-E relationship:

• Signal Peptide Incubation Studies: Researchers have demonstrated that synthetic HLA-H signal peptides can mobilize HLA-E to the cell surface of multiple cell types, including T-lymphocytes, monocytes, B-lymphocytes, and primary epithelial cells . This approach typically involves:

  • Incubation of cells with synthetic HLA-H signal peptides at 37°C for varying time periods (1-16 hours)

  • Flow cytometric analysis using validated anti-HLA-E antibodies (such as Mouse IgG1 antibody clone 3D12-PE)

  • Appropriate isotype controls (e.g., Mouse IgG1 antibody clone 679.1Mc7-PE)

  • Analysis on flow cytometry platforms like FACSCalibur with specialized software (e.g., BD FACSDiva)

• Comparative Expression Analysis: Real-Time PCR analyses comparing HLA-H, HLA-G, and HLA-E expression levels in various tissues have shown that while HLA-H expression is comparable to HLA-G, both are significantly lower than HLA-E expression (p < 0.0001) .

These methodologies have established that HLA-H can influence immune regulation indirectly through HLA-E mobilization, suggesting a potential role in immune tolerance mechanisms that may be relevant in contexts ranging from pregnancy to transplantation.

What phage display techniques are used for developing HLA-H antibodies?

Phage display represents a powerful technique for developing novel antibodies against HLA-H with specific binding characteristics:

Methodology Overview:

The phage-Fab display approach involves several key steps:

• Antigen Preparation and Immobilization:

  • Coating maxisorp immunotubes with streptavidin (20 μg/mL) at 4°C overnight

  • Blocking with 4% skim milk in PBS

  • Incubating with biotinylated, refolded recombinant HLA (20 μg/mL) at room temperature

• Phage Library Preparation and Binding:

  • Preabsorbing PEG-precipitated human Fab phage library with streptavidin

  • Blocking with 2% skim milk in PBS

  • Incubating with antigen-coated immunotubes for 1.5 hours at room temperature

• Selection Process:

  • Washing with 4% skim milk in PBST, PBST alone, and PBS

  • Eluting bound phages with trypsin at 37°C

  • Infecting E. coli TG1 with eluted phages

• Amplification and Enrichment:

  • Infecting phage-containing E. coli with helper phage (M13KO7)

  • Culturing on selective media (2xYT agar with carbenicillin and kanamycin)

  • Harvesting and precipitating phages for subsequent panning rounds

  • Typically repeating the process for four panning cycles to enrich for specific binders

Expression System Considerations:

For successful production of recombinant HLA-H for phage display, researchers have employed prokaryotic expression systems:

• Transformation of E. coli BL21 with plasmids encoding human β2m and the extracellular portion of the HLA α chain

• Extraction of protein inclusion bodies

• In vitro refolding of peptide-HLA complexes by rapid dilution

• Biotinylation and purification through Fast Protein Liquid Chromatography and size exclusion chromatography

While these methods have been successfully applied to various HLA molecules, adaptation specifically for HLA-H requires careful consideration of its unique sequence characteristics and potential folding requirements.

What assays are used to evaluate HLA-H antibody specificity and cross-reactivity?

Ensuring high specificity and minimal cross-reactivity is critical when developing and characterizing HLA-H antibodies. Several complementary approaches are employed for comprehensive validation:

Western Blot Analysis with Peptide Competition:

This approach assesses antibody specificity by demonstrating that binding can be blocked by the specific peptide used for immunization:

• Tissue homogenates are separated by electrophoresis and blotted

• Parallel blots are probed with the antibody alone or antibody pre-incubated with the immunizing peptide

• Specific binding is indicated by the absence of bands in the peptide-blocked condition

Comparative Antibody Validation:

Testing whether antibodies raised against different regions of the same protein recognize identical bands:

• Researchers have demonstrated that a C-terminal HLA-H-specific IgG shows strong immunostaining of a 45-50 kDa protein that also reacts with an antibody raised against a different epitope (α3 loop in the extracellular domain)

Control Protein Testing:

Verifying lack of cross-reactivity with structurally related proteins:

• For example, confirming that the HLA-H C-terminal peptide antibody does not react with affinity-purified Fc receptor from human placenta, despite some sequence similarity

Transfection Studies:

One of the most definitive approaches involves comparing reactivity in transfected versus non-transfected cells:

• COS-7 cells transfected with HLA-H cDNA show strong antibody binding to a 45-50 kDa polypeptide

• No signal is observed in COS-7 cells transfected with vector only

• Similarly, no binding is seen with truncated HLA-H lacking the C-terminal amino acids

One Lambda Single Antigen Bead (OL-SAB) Assay:

For evaluating potential cross-reactivity across multiple HLA alleles:

• Antibody is diluted in negative control serum (typically to 1 μg/mL final concentration)

• Testing is performed using LABScreen® Single Antigen HLA Class I platforms

• Results are analyzed by specialized laboratories with international accreditation for conducting solid-phase anti-HLA antibody detection assays

These methodologies collectively provide a robust framework for ensuring the specificity of antibodies developed against HLA-H, critical for accurate research applications.

How are HLA-H antibodies used to study hereditary hemochromatosis pathophysiology?

HLA-H antibodies have proven instrumental in investigating the pathophysiology of hereditary hemochromatosis (HH), providing critical insights into the molecular mechanisms underlying this common genetic disorder:

Immunolocalization Studies:

Using antibodies against HLA-H, researchers have mapped the protein's distribution throughout the gastrointestinal tract in normal subjects, revealing crucial patterns relevant to iron absorption:

• Tissue-Specific Expression: HLA-H is expressed in epithelial cells throughout the alimentary canal from the esophagus to the rectum, as well as in subepithelial leukocytes .

• Subcellular Localization Patterns: Three distinct patterns were observed:

  • Non-polarized plasma membrane distribution in esophageal epithelium and leukocytes

  • Polarized basolateral membrane localization in stomach and colon

  • Unique intracellular, perinuclear localization in the small intestine, specifically in cells of the deep crypts

• Anatomical Correlation: The specific perinuclear localization in small intestinal crypts is particularly significant as it places HLA-H in proximity to the presumed sites of iron absorption .

Mutation Impact Assessment:

Antibodies enable comparative analyses between normal and mutant HLA-H proteins:

The Cys-282 → Tyr mutation found in 83-100% of HH patients disrupts a critical disulfide bridge, likely affecting protein folding and function . Antibody-based studies can reveal how this mutation impacts:

• Protein stability and expression levels

• Subcellular trafficking and localization

• Interactions with other proteins involved in iron metabolism

Mechanistic Insights:

The distinctive subcellular localization patterns revealed through antibody studies suggest that HLA-H may play different roles in different segments of the gastrointestinal tract:

• In the small intestine, its intracellular/perinuclear localization suggests potential involvement in iron sensing or trafficking pathways rather than direct membrane transport

• The basolateral localization in stomach and colon points to possible roles in intercellular signaling or transport

These antibody-enabled discoveries provide a foundation for understanding how mutations in HLA-H lead to the dysregulated iron absorption characteristic of hereditary hemochromatosis.

What are the challenges in producing monoclonal antibodies specific to HLA-H?

Developing highly specific monoclonal antibodies against HLA-H presents several significant challenges that researchers must address through careful experimental design:

Sequence Homology Considerations:

HLA-H shares substantial sequence similarity with other HLA molecules, creating specificity challenges:

• Researchers must carefully select target peptides that minimize shared sequence elements with other HLA proteins

• For example, strategic selection of the C-terminal region which has limited homology (only 4 amino acids in common with HLA-A2, 1 with HLA-G, and 2 with human neonatal Fc receptor)

Expression and Purification Hurdles:

Obtaining sufficient quantities of properly folded HLA-H protein for immunization or screening presents technical challenges:

• Expression in E. coli often results in inclusion bodies requiring refolding

• The complex tertiary structure of HLA molecules, including disulfide bonds, makes proper folding critical

• Association with β2-microglobulin may be necessary for native conformation

• Protocols typically involve protein extraction from inclusion bodies, in vitro refolding through rapid dilution, and purification via chromatography techniques

Validation Complexity:

Establishing true specificity requires multiple complementary approaches:

• Peptide competition assays to confirm epitope-specific binding

• Testing against multiple HLA molecules to assess cross-reactivity

• Validation in both recombinant systems and native tissues

• Confirming recognition of the correct molecular weight protein (45-50 kDa for HLA-H)

Allelic Variation Considerations:

Different HLA-H alleles may have distinct properties requiring separate validation:

• Some alleles like HLA-H02:07 and H02:14 encode complete membrane-bound proteins while others may not

• Expression levels vary between alleles, with H*02:07 showing notably higher mRNA expression

• The Denisovan origin of H*02:07 suggests potential structural or functional distinctions that may affect antibody recognition

Addressing these challenges requires rigorous methodology combining strategic epitope selection, careful immunization protocols, and comprehensive validation across multiple systems to ensure the development of truly HLA-H-specific monoclonal antibodies.

How does the HLA-H signal peptide mobilize HLA-E to the cell surface?

The interaction between the HLA-H signal peptide and HLA-E represents a fascinating example of cross-talk between different HLA molecules with important implications for immune regulation:

Molecular Mechanism:

Like other HLA class I molecules, HLA-H contains a signal peptide that is cleaved during protein processing. This signal peptide can subsequently:

• Be processed and loaded onto HLA-E molecules in the endoplasmic reticulum

• Stabilize HLA-E, facilitating its transport to the cell surface

• Present as part of the HLA-E complex to NK cells and T cells bearing the CD94/NKG2 receptor

Experimental Evidence:

Research has demonstrated that the HLA-H signal peptide can effectively mobilize HLA-E to the cell surface across multiple cell types:

• Cell Types Responsive to HLA-H Signal Peptide:

  • T-lymphocytes

  • Monocytes

  • B-lymphocytes

  • Primary epithelial cells (including Human Bronchial Epithelial Cells)

• Methodology:

  • Cells expressing known HLA-E genotypes are incubated with synthetic HLA-H signal peptides

  • Incubation periods typically range from 1-6 hours for lymphocytes and monocytes, and up to 16 hours for epithelial cells

  • Surface HLA-E expression is measured by flow cytometry using validated antibodies (Mouse IgG1 antibody clone 3D12-PE)

Functional Significance:

This mechanism has important implications for immune regulation:

• Indirect Immunomodulation: While direct HLA-H function remains under investigation, its ability to mobilize HLA-E provides an indirect pathway for influencing immune responses

• Inhibitory Signaling: Surface HLA-E engages the inhibitory CD94/NKG2A receptor on NK cells and some T cells, potentially dampening cytotoxic responses

• Tolerance Promotion: Similar to other non-classical HLA molecules (HLA-G and HLA-F), this pathway may contribute to immune tolerance in contexts such as pregnancy, transplantation, and tumor microenvironments

This mechanism illustrates how HLA-H may influence immune regulation beyond its direct membrane-bound form, highlighting the complex interplay between different components of the HLA system.

What technological advances are improving HLA-H antibody development and characterization?

Recent technological innovations have significantly enhanced the development, validation, and application of HLA-H antibodies for research and potential clinical applications:

Advanced Phage Display Platforms:

Modern phage display technologies enable the isolation of high-affinity antibodies against HLA-H through:

• Human Fab phage libraries with extensive diversity

• Multi-round panning against properly folded recombinant HLA-H

• Stringent washing protocols to select for high-affinity binders

• E. coli expression systems optimized for subsequent antibody production

Flow-Induced Dispersion Analysis (FIDA):

This novel in-solution approach offers significant advantages for antibody characterization:

• Measures true antibody affinity rather than avidity, unlike solid-phase methods

• Simultaneously determines both antibody concentration and binding strength

• Particularly valuable for patient sample evaluation

• Enables more accurate assessment of potential clinical significance

Comparative Multi-Platform Binding Assessment:

Comprehensive antibody characterization now commonly employs multiple complementary technologies:

• Surface plasmon resonance (SPR) for real-time binding kinetics

• Bio-layer interferometry (BLI) for label-free interaction analysis

• Luminex single antigen beads (SAB) for multiplex specificity testing

• FIDA for in-solution affinity determination

• These approaches collectively provide a more complete picture of antibody properties than any single method alone

Recombinant Expression Advances:

Improvements in recombinant protein production have enhanced antibody development:

• Optimized protocols for expressing HLA heavy chains and β2m in E. coli

• Refined refolding techniques for generating properly conformed HLA molecules

• Enhanced biotinylation strategies for downstream applications

• Advanced chromatography methods for high-purity protein isolation

These technological advances collectively facilitate the development of better-characterized, more specific antibodies against HLA-H, enhancing their utility as research tools and potential diagnostic or therapeutic agents in contexts ranging from iron metabolism disorders to transplantation medicine.