HLA-G Antibody, Biotin conjugated

What is HLA-G and why is it important in immunological research?

HLA-G is a non-classical major histocompatibility class Ib molecule involved in immune regulatory processes, particularly at the maternal-fetal interface. Unlike classical HLA class I molecules, HLA-G has restricted tissue expression, low polymorphism, and exists in seven isoforms (HLA-G1 to -G7) .

Its importance in research stems from its role as an immune checkpoint protein (ICP) that is neoexpressed in tumor cells as an immune evasion mechanism. HLA-G inhibits:

• Allogeneic proliferation of T cells

• Natural killer cell cytotoxicity

• Antigen-specific T-cell cytotoxicity

These immune-inhibitory properties identify HLA-G as a mediator of immune tolerance with specific relevance at immune-privileged sites such as trophoblast or thymus. Its immune modulatory function is mediated via three inhibitory receptors: ILT2 (CD85j), ILT4 (CD85d), and KIR2DL4 (CD158d) .

What are the main applications for biotin-conjugated HLA-G antibodies in research?

Biotin-conjugated HLA-G antibodies are versatile tools in immunological research with several validated applications:

The biotin conjugation enables signal amplification through streptavidin-reporter systems, enhancing detection sensitivity in these applications .

How do different HLA-G antibody clones differ in their specificity and applications?

Different HLA-G antibody clones recognize distinct epitopes and isoforms, making clone selection critical for experimental design:

When designing experiments, researchers should select the clone based on which HLA-G isoform they aim to study and the specific application required .

What are the structural differences between HLA-G isoforms and how do they affect antibody selection?

HLA-G exists in seven isoforms with distinct structural characteristics that influence antibody selection:

• HLA-G1: Full-length isoform with structure similar to classical HLA class I molecules - a heavy chain (39 kDa) noncovalently associated with β2-microglobulin and a nonameric peptide

• HLA-G2: Lacks exon 3 (α2 domain), resulting in a junction between exons 2 and 4

• HLA-G3: Lacks exons 3 and 4

• HLA-G4: Lacks exon 4

• HLA-G5: Soluble isoform containing intron 4, recognized by antibodies targeting soluble-specific epitopes

• HLA-G6: Soluble isoform, recognized by antibodies like 5A6G7 that target C-terminal sequences unique to soluble forms

These structural variations necessitate careful antibody selection:

• For detecting all isoforms: Use pan-HLA-G antibodies like 4H84

• For distinguishing membrane-bound vs. soluble forms: Use isoform-specific antibodies like 5A6G7

• For detecting native conformation: Use antibodies like MEM-G/9

Researchers must consider whether they need to detect specific isoforms or all HLA-G variants when selecting antibodies .

What are the recommended storage and handling procedures for biotin-conjugated HLA-G antibodies?

Proper storage and handling are critical for maintaining antibody functionality and experimental reproducibility:

Additional handling precautions:

• Minimize freeze-thaw cycles which can lead to antibody degradation

• Centrifuge vial before opening to recover entire volume

• For optimal performance, follow the reconstitution protocol provided in the Certificate of Analysis

How can researchers validate the specificity of HLA-G antibodies for their experimental systems?

Validating antibody specificity is critical for reliable results. A comprehensive validation approach includes:

• Positive and negative controls:

  • Positive: JEG-3 choriocarcinoma cell line (naturally expresses HLA-G)

  • Negative: K562 cells (lack HLA expression)

• RNA-level validation:

  • RT-PCR targeting different HLA-G exons

  • QRT-PCR using HLA-G-specific primers and probes

  • Example from research: "We observed that effectively the genome was edited in all cases. Different InDels occurred with each sgRNA, with an accumulative genome modification rate of 63%, 88%, 79% and 71% for 1A, 1B, 2A and 2B-sgRNAs, respectively"

• Protein-level validation:

  • Western blot analysis: Expected molecular weight for HLA-G is ~39 kDa

  • Flow cytometry with multiple antibody clones

  • Immunoprecipitation followed by mass spectrometry

• Functional validation:

  • Inhibition of NK cell cytotoxicity assays

  • Blocking experiments with W6/32 mAb

  • T cell proliferation inhibition

• Genetic manipulation controls:

  • HLA-G transfectants vs. empty vector controls

  • CRISPR/Cas9-edited cell lines with disrupted HLA-G expression

These validation steps ensure that observed signals are specific to HLA-G and not due to cross-reactivity with other proteins .

What methodological approaches can be used to study HLA-G homodimerization and its functional consequences?

HLA-G can form homodimers that display unique functional properties. To study this phenomenon:

• Generation of homodimer-specific models:

  • Use cells transfected with mutant HLA-G (C42S) in which the cysteine required for homodimerization is mutated to serine

  • Compare with wildtype HLA-G (e.g., 4C4 cells) where approximately 90% of surface-expressed HLA-G exists as homodimers and 10% as monomers

• Detection and quantification methods:

  • Western blotting under non-reducing conditions to preserve disulfide bonds

  • Size exclusion chromatography to separate monomers and dimers

  • Native PAGE analysis

• Functional analysis protocols:

  • Cytokine secretion assays: "HLA-G homodimer, but not the monomer, induces secretion of the proinflammatory cytokines IL-6 and IL-8 and a small amount of TNFα (and probably also IL-1α, IL-1β, and IFNγ) from both decidual macrophages and NK cells"

  • Cell stimulation: "cells were stimulated by coincubation with three different cells for 5 h: 721.221 cells (a B lymphblastoid cell line that lacks expression of all MHC proteins) as control, the same cells transfected with a mutant HLA-G (C42S) in which the cysteine required for homodimerization was mutated to serine, and finally with the unmutated HLA-G cDNA (4C4)"

  • mRNA analysis: "up-regulation of mRNA for proinflammatory cytokines (IL-1α, IL-1β, IL-6, IL-8, and TNFα) seen with the HLA-G homodimer"

• Receptor interaction studies:

  • Analysis of binding to inhibitory receptors (ILT2, ILT4, and KIR2DL4)

  • Compare binding affinities of monomers versus homodimers

This methodological approach provides insights into the functional differences between HLA-G monomers and homodimers in immune regulation .

How can researchers optimize flow cytometry protocols for detecting HLA-G using biotin-conjugated antibodies?

Optimizing flow cytometry for HLA-G detection requires addressing several technical considerations:

• Surface versus intracellular staining:

  • Surface staining: Direct approach for membrane-bound isoforms (HLA-G1, G2)

  • Intracellular staining: Required for detecting internal pools of HLA-G

  • For isoform-specific detection: "This antibody does not cross-react with the full-length HLA-G1 isoform and can be used to distinguish secreted HLA-G5 and HLA-G6 from shedded HLA-G1"

• Acid treatment protocol for enhanced detection:

  • Some protocols recommend acid treatment: "Intracellular staining or surface staining after acid treatment (Polakova K. et al. Molecular immunology 1993; 1223-30)"

  • This can expose epitopes that may be masked in native conformation

• Optimized antibody concentration:

  • For 5A6G7 clone: 1-4 μg/ml for intracellular staining

  • For MEM-G/9 clone: 1-5 μg/ml

  • For 4H84 clone: 2-5 μg/ml

• Secondary detection system optimization:

  • Use streptavidin conjugated to appropriate fluorochromes (PE, APC, FITC)

  • Example protocol outcome: "Flow cytometric analysis of HLA-G transfectants (red), compared with K562 cells (orange) and blank (blue), labeling HLA-G with ab239333 at 1/500 dilution, followed by streptavidin-PE. Surface staining."

• Controls:

  • Positive control: JEG-3 human choriocarcinoma cell line

  • Negative control: K562 parental cell line

  • Isotype control: Mouse IgG1 [MOPC-21] (Biotin)

• Gating strategy:

  • For T cell subsets: "using forward- versus side-scatter characteristics and a gate on CD3+ events"

  • For identifying HLA-G+ cells: Compare to isotype and negative controls

These optimizations enhance the sensitivity and specificity of HLA-G detection by flow cytometry .

What methods can be used to assess the functional effects of HLA-G in immune regulation experiments?

To assess HLA-G's immunoregulatory functions, researchers can employ these methodological approaches:

• NK cell cytotoxicity assays:

  • Experimental setup: "To analyze how the HLA-G1 and HLA-G2 molecules act on NK cell-mediated cytotoxicity, we used either PBMC or polyclonal NK cells (CD3−CD16+CD56+) isolated from 20 donors as effector cells"

  • Controls: "This inhibition was reversed when the transfectant cells that express HLA-G1 or HLA-G2 were incubated with the pan class I W6/32 mAb, indicating that inhibition of NK lysis was due to the presence of HLA-G on the K562 transfected target cells"

• T cell proliferation assays:

  • Protocol: "T-cell proliferation was measured after 3 days in culture and an additional 16-hour pulse with [3H] Tdr (18.5 kBq per well) using a liquid scintillation counter"

  • Controls: Compare HLA-G-positive and HLA-G-negative T cell subsets

• Allogeneic stimulation experiments:

  • Method: "For allogeneic stimulation, freshly isolated T cells (whole CD4 T cells, and CD4 HLA-Gpos/neg and CD8 HLA-Gpos/neg T cells; 1–3 × 105 per well in a 96-well plate; triplicates) were cultured in the presence of different numbers of irradiated DCs or in a mix lymphocyte reaction (MLR) using a mix of allogeneic PBMCs from 5 different healthy donors (donor mix)"

• Cytokine secretion analysis:

  • ELISA or cytometric bead arrays for protein detection

  • RT-qPCR for mRNA expression: "Quantitative analysis of gene expression was performed by reverse-transcription–polymerase chain reaction (RT-PCR) using the ABI prism 7000 Sequence Detection System"

  • Key cytokines to measure: "These data show that the HLA-G homodimer, but not the monomer, induces secretion of the proinflammatory cytokines IL-6 and IL-8 and a small amount of TNFα (and probably also IL-1α, IL-1β, and IFNγ) from both decidual macrophages and NK cells"

• Receptor binding and signaling studies:

  • Analysis of interaction with ILT2, ILT4, KIR2DL4 receptors

  • Downstream signaling pathway analysis

• In vivo tumor models:

  • As described: "CAR-NK strategy exploits the dual nature of HLA-G as both a tumor-associated neoantigen and an ICP to counteract tumor spread"

These methodological approaches provide comprehensive insights into HLA-G's functional effects on immune regulation .

What are the considerations for using biotin-conjugated HLA-G antibodies in immunohistochemistry applications?

When utilizing biotin-conjugated HLA-G antibodies for immunohistochemistry (IHC), researchers should consider these methodological aspects:

• Sample preparation protocols:

  • Paraffin-embedded tissues (IHC-P): Requires appropriate antigen retrieval methods

  • Frozen sections (IHC-F): Generally preserves epitopes better but may have poorer morphology

  • Fixation: "For fixation details see: Emadi et al., Biotech Histochem. 2022 Feb;97(2):136-142"

• Antibody clone selection based on application:

  • MEM-G/9: Suitable for IHC-F

  • 4H84: Works for both IHC-F and IHC-P

  • 5A6G7: Appropriate for IHC-P and IHC-Fr

• Optimal concentration determination:

  • MEM-G/9: Recommended dilution 5-10 μg/ml

  • Other clones: Titration experiments recommended to determine optimal concentration

• Detection system selection:

  • Streptavidin-HRP systems offer high sensitivity

  • Consider tyramide signal amplification for low expression tissues

  • Be cautious of endogenous biotin, especially in liver, kidney, and brain tissues

• Controls for validation:

  • Positive tissue control: Placental tissues (trophoblasts express HLA-G naturally)

  • Negative tissue control: Normal tissues with no expected HLA-G expression

  • Antibody controls: Isotype control and blocking peptide controls

• Dual staining considerations:

  • For co-localization studies with other markers

  • Careful selection of detection systems to avoid cross-reactivity

• Interpretation guidelines:

  • Membrane versus cytoplasmic staining pattern interpretation

  • Scoring systems for expression intensity and distribution

These considerations ensure reliable and reproducible IHC results when studying HLA-G expression in tissue samples .

How can researchers develop and validate HLA-G-targeting chimeric antigen receptor (CAR) constructs for immunotherapy research?

Developing HLA-G-targeting CAR constructs involves several advanced methodological steps:

• Antibody fragment selection and optimization:

  • Process: "The HuScL-2 Human Single-Chain Antibody Library (Creative Biolabs) was screened; after four rounds, 40 clones from the fourth eluate were selected for analysis in ELISA using monoclonal phages. The highest affinity clone was selected and its single-chain variable fragment (scFv) was used to build the anti-HLA-G CAR construct"

  • Design consideration: "We developed a novel CAR strategy using natural killer (NK) cell as effector cells, featuring enhanced cytolytic effect via DAP12-based intracellular signal amplification"

• CAR construct design and assembly:

  • Vector component: "by synthesizing DNA corresponding to the leader peptide sequence 5′-ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCC-3′"

  • Targeting domain: scFv against HLA-G as the targeting moiety

  • Signaling domain: Consider DAP12-based intracellular signal amplification for enhanced cytolytic effect

• Expression system optimization:

  • Lentiviral or retroviral transduction protocols for primary NK or T cells

  • Stable expression systems for in vitro testing

• Functional validation assays:

  • In vitro cytotoxicity: "HLA-G CAR shows antitumor activity both in vitro and in vivo in an HLA-G-expressing hematopoietic tumor model based on K562 and JEG-3 cells"

  • Target specificity: Test against HLA-G+ and HLA-G- cell lines

  • Cytokine release assessment

• Combination strategies development:

  • Chemotherapy synergy: "pretreatment with low-dose chemotherapy to induce overexpression of HLA-G increases the antitumor efficacy of HLA-G CAR-NK cells both in vitro and in vivo"

  • Mechanism investigation: "we also investigated how CAR converted inhibitory HLA-G to activating signal and explained the mechanism of chemotherapy induced cell surface HLA-G on tumor cells"

• In vivo model systems:

  • Animal model selection: "the construct is tested both in vitro and in vivo on four different solid tumor models"

  • Efficacy assessment: Tumor growth inhibition, survival analysis

  • Safety evaluation: Off-target effects assessment

This methodological framework provides a comprehensive approach to developing HLA-G-targeting CAR constructs for cancer immunotherapy research .

What advanced techniques can be used to study HLA-G peptide presentation and complexes with β2-microglobulin?

Studying HLA-G peptide presentation and β2-microglobulin (B2M) complexes requires sophisticated methodological approaches:

• Recombinant protein expression and purification:

  • Co-expression systems: "Biotinylated Human HLA-G&B2M&Peptide (RIIPRHLQL) Complex Protein is produced by co-expression of HLA and B2M loaded with RIIPRHLQL peptide"

  • Purification strategies: "This protein carries a polyhistidine tag at the C-terminus, followed by an Avi tag (Avitag™)"

  • Quality control: ">95% as determined by SDS-PAGE"

• Structural analysis of HLA-G/B2M/peptide complexes:

  • Size determination: "The protein has a calculated MW of 36.3 kDa and 13.9 kDa. The protein migrates as 40-42 kDa and 15 kDa under reducing (R) condition (SDS-PAGE) due to glycosylation"

  • Crystallography approaches for 3D structure determination

  • Cryo-EM for visualization of larger complexes

• Peptide binding and repertoire analysis:

  • Peptide elution and mass spectrometry: "In complex with B2M/beta-2 microglobulin binds a limited repertoire of nonamer self-peptides derived from intracellular proteins including histones and ribosomal proteins"

  • Binding affinity measurements

  • Competitive binding assays

• Functional interaction studies:

  • Receptor binding assays: "Immobilized Biotinylated Human HLA-G&B2M&Peptide (RIIPRHLQL) Complex Protein at 1 μg/mL (100 μL/well) on streptavidin precoated (0.5 μg/well) plate can bind Anti-HLA class I Antibody, Human IgG1 (W6/32) with a linear range of 0.2-8 ng/mL"

  • SPR or BLI for kinetic analysis of interactions

  • Cell-based functional assays

• Advanced imaging techniques:

  • Super-resolution microscopy for localization studies

  • FRET for protein-protein interaction analysis

  • Single-molecule tracking for dynamics studies

• Computational approaches:

  • Molecular dynamics simulations

  • Peptide-MHC binding prediction algorithms

  • Structure-function relationship modeling

These methodological approaches provide detailed insights into the structural and functional properties of HLA-G/B2M/peptide complexes, critical for understanding their immunoregulatory roles .

What are the advanced methodologies for CRISPR/Cas9-mediated HLA-G gene editing to study its function?

CRISPR/Cas9 gene editing of HLA-G provides powerful approaches to study its function:

These methodological approaches enable precise genetic manipulation of HLA-G to study its functional roles in various biological contexts .

How can researchers identify and characterize HLA-G-expressing T cell subpopulations for immunoregulatory studies?

Identifying and characterizing HLA-G-expressing T cells requires specialized methodological approaches:

• Flow cytometry-based identification protocols:

  • Surface marker combinations: "a subpopulation of CD4 and CD8 T cells in human peripheral blood expressing the immune tolerizing molecule HLA-G"

  • Phenotypic characterization: "HLA-G–expressing T cells are hypoproliferative, are CD25- and FOXP3-negative, and exhibit potent suppressive properties that are partially mediated by HLA-G"

  • Origin tracking: "HLA-G–positive (HLA-Gpos) T cells are found at low percentages among CD4 and CD8 single-positive thymocytes, suggesting a thymic origin"

• Isolation and purification techniques:

  • Cell sorting: Flow cytometry-based sorting of CD4 HLA-Gpos and HLA-Gneg T cells

  • Enrichment protocols: Magnetic-based isolation systems

  • Purity assessment: Post-isolation purity confirmation by flow cytometry

• Functional characterization assays:

  • Proliferation assessment: "[3H] Tdr (18.5 kBq per well) using a liquid scintillation counter"

  • Suppression assays: Co-culture with responder T cells

  • Stability evaluation: "For assessing stability of HLA-G expression over time, CD4 HLA-Gpos and HLA-Gneg T cells were cultured in standard culture medium... for a period of 3 days"

• Molecular characterization methods:

  • Gene expression profiling: "Quantitative analysis of gene expression was performed by reverse-transcription–polymerase chain reaction (RT-PCR) using the ABI prism 7000 Sequence Detection System"

  • Cytokine profile determination: "cDNA templates were amplified for IFN-γ, IL-10, TGF-β, and 18S"

  • Transcription factor analysis: "For Foxp3 and HLA-G, cDNAs were amplified using qPCR MasterMix Plus and a primer pair with probe kit (MGB Probe)"

• Clinical relevance assessment:

  • Tissue distribution studies: "The presence of HLA-Gpos T cells at sites of inflammation such as inflamed skeletal muscle in myositis or the cerebrospinal fluid of patients with acute neuroinflammatory disorders suggests an important function in modulating parenchymal inflammatory responses in vivo"

  • Disease correlation analysis

These methodological approaches enable comprehensive characterization of HLA-G-expressing T cell subpopulations and their immunoregulatory functions .

What are the latest methodological approaches for studying HLA-G in tumor immune evasion and developing targeted immunotherapies?

Advanced methodological approaches for studying HLA-G in tumor immune evasion include:

• Combined chemotherapy and immunotherapy strategies:

  • Approach: "This study aimed to use a combined approach involving application of low-dose chemotherapy to increase membranous expression of HLA-G by solid tumor cells and then targeting it with HLA-G CAR-NK cells"

  • Outcome assessment: "The results show that pretreatment with low-dose chemotherapy to induce overexpression of HLA-G increases the antitumor efficacy of HLA-G CAR-NK cells both in vitro and in vivo"

• Mechanism analysis of HLA-G-mediated immune suppression:

  • Signal conversion studies: "we also investigated how CAR converted inhibitory HLA-G to activating signal and explained the mechanism of chemotherapy induced cell surface HLA-G on tumor cells"

  • Receptor interaction analysis: "Peptide-bound HLA-G-B2M complex acts as a ligand for inhibitory/activating KIR2DL4, LILRB1 and LILRB2 receptors"

• Gene editing approaches for HLA-G targeting:

  • CRISPR/Cas9 methodology: "We used CRISPR/Cas9 gene editing to block the HLA-G expression in two tumor cell lines expressing HLA-G, including a renal cell carcinoma (RCC7) and a choriocarcinoma (JEG-3)"

  • Functional outcome assessment: "Most importantly, HLA-G− cells triggered a higher in vitro response of immune cells with respect to HLA-G+ wild type cells"

• CAR design optimization for targeting HLA-G:

  • Signal amplification: "We developed a novel CAR strategy using natural killer (NK) cell as effector cells, featuring enhanced cytolytic effect via DAP12-based intracellular signal amplification"

  • Targeting strategy: "A single-chain variable fragment (scFv) against HLA-G is designed as the targeting moiety, and the construct is tested both in vitro and in vivo on four different solid tumor models"

• Tumor microenvironment modulation studies:

  • Analysis of HLA-G's dual role: "Our novel CAR-NK strategy exploits the dual nature of HLA-G as both a tumor-associated neoantigen and an ICP to counteract tumor spread"

  • Combination therapy assessment: "Further ablation of tumors can be boosted when combined with administration of chemotherapeutic agents in clinical use"

• Translational research approaches:

  • Wide applicability evaluation: "The readiness of this novel strategy envisions a wide applicability in treating solid tumors"

  • Clinical trial design considerations for HLA-G-targeted therapies