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Human Immune Checkpoint 37-Plex Panel

Human Immune Checkpoint 37-Plex Panel

Q: Can this 37-plex be combined with other panels on the same plate?

Yes. If your project requires immune checkpoint profiling plus additional cytokine or chemokine panels, we can coordinate a multi-panel workflow on the same Luminex plate. This maximizes data from each sample.

Simultaneous quantification of 37 key immune checkpoint proteins in a single well using Luminex xMAP technology. Covers co-inhibitory checkpoints, co-stimulatory receptors, NK cell ligands, and soluble immune regulators for cancer immunotherapy and immune profiling research.

37 TargetsHuman25 μL SampleSub-pg/mL Sensitivity

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Immune checkpoint proteins are key regulators of T cell activation, exhaustion, and anti-tumor immunity. The balance between co-stimulatory and co-inhibitory signals determines whether an immune response is activated or suppressed. In the tumor microenvironment, cancer cells exploit checkpoint pathways — particularly the PD-1/PD-L1 axis and CTLA-4 — to evade immune destruction. Blocking these pathways with checkpoint inhibitors (e.g., anti-PD-1, anti-PD-L1, anti-CTLA-4 antibodies) has revolutionized cancer treatment.

Creative Proteomics offers the Human Immune Checkpoint 37-Plex Panel based on the Luminex xMAP platform for simultaneous quantification of 37 key immune checkpoint proteins in a single well. This panel provides comprehensive coverage of co-inhibitory checkpoints, co-stimulatory receptors, NK cell ligands, and soluble immune regulators — all from just 25 μL of serum or plasma.

The panel is organized into three modular sub-panels that can be used independently or combined for full 37-plex coverage. It is validated for serum, plasma, and cell culture supernatants, compatible with MAGPIX, Luminex 200, and FLEXMAP 3D systems.

Panel Specifications

TechnologyLuminex xMAP

Panel Size37-plex (3 sub-panels)

SpeciesHuman

Sample Volume25-50 μL

SensitivitySub-pg/mL

Dynamic Range4-5 logs

Assay Time~4 hours

Complete Analyte List (37 Immune Checkpoint Targets)

The Human Immune Checkpoint 37-Plex Panel is organized into three modular sub-panels that can be used independently or combined for full 37-plex coverage.

Panel 1: Co-Inhibitory & NK Cell Ligands (14-Plex)

Target	Alternative Name	Immune Function
CTLA-4	CD152	T cell co-inhibitory receptor; immune suppression
CD28	Tp44	T cell co-stimulatory receptor
CD80	B7-1	Co-stimulatory ligand for CD28/CTLA-4
CD96	TACTILE	NK cell adhesion and immune regulation
MICA	MHC class I chain-related A	NKG2D ligand; NK cell activation
MICB	MHC class I chain-related B	NKG2D ligand; immune surveillance
Nectin-2	CD112, PVRL2	Cell adhesion; NK and T cell modulation
PVR	CD155	NK cell receptor ligand; immune evasion
ULBP-1	RAET1I	NKG2D ligand; tumor immune recognition
ULBP-3	RAET1N	NKG2D ligand; innate immunity
ULBP-4	RAET1E	NKG2D ligand; stress-induced immunity
Arginase-1	ARG1	T cell suppression via arginine depletion
B7-H6	NCR3LG1	NKp30 ligand; tumor recognition
CD48	BCM1, SLAMF2	NK and T cell co-stimulation

Panel 2: PD-1/PD-L1 Axis & Co-Stimulatory Receptors (9-Plex)

Target	Alternative Name	Immune Function
PD-L1	CD274, B7-H1	Key immune checkpoint; immunotherapy target
PD-1	CD279	T cell exhaustion marker
PD-L2	CD273, B7-DC	Second ligand for PD-1
OX40	CD134, TNFRSF4	T cell co-stimulation; memory formation
4-1BB	CD137, TNFRSF9	T/NK cell activation; cancer immunotherapy target
CD47	IAP, MER6	"Don't eat me" signal; macrophage checkpoint
B7-H3	CD276	Co-inhibitory molecule; tumor immune evasion
GITR	CD357, TNFRSF18	Treg modulation; immunotherapy target
HVEM	CD270, TNFRSF14	Dual co-stimulatory/co-inhibitory signaling
IDO	IDO1	Tryptophan metabolism; T cell suppression
TIM-3	CD366, HAVCR2	T cell exhaustion; dual checkpoint target
VISTA	B7-H5, PD-1H	T cell suppression; emerging checkpoint
BTLA	CD272	Co-inhibitory receptor; binds HVEM
CD27	TNFRSF7	T/B cell co-stimulation; memory formation

Panel 3: Emerging & Soluble Immune Regulators (14-Plex)

Target	Alternative Name	Immune Function
CD73	NT5E	Adenosine production; immunosuppression
E-Cadherin	CDH1, CD324	Cell adhesion; EMT marker; immune exclusion
ICOS Ligand	B7-H2, CD275	Tfh and B cell co-stimulation
LAG-3	CD223	T cell exhaustion; dual checkpoint with PD-1
Perforin	PRF1	Cytotoxic granule; CTL/NK effector function
S100A8/A9	Calprotectin, MRP8/14	Inflammation; MDSC recruitment
Siglec-7	CD328	NK cell inhibitory receptor
Siglec-9	CD329	Immune inhibitory receptor; tumor evasion
TIMD-4	TIM4	Phosphatidylserine receptor; efferocytosis

Technical Specifications

Validated performance parameters for the Human Immune Checkpoint 37-Plex Panel.

Platform and Assay

PlatformLuminex xMAP (MAGPIX / Luminex 200 / FLEXMAP 3D)

Panel Size37-plex (3 modular sub-panels)

SpeciesHuman

Sample TypesSerum, EDTA/Heparin Plasma, CCS

Sample Volume25 μL (serum/plasma), 50 μL (CCS)

Assay Time~4 hours

Performance Metrics

SensitivitySub-pg/mL (varies by analyte)

Dynamic Range4-5 logs

Intra-Assay CV<10%

Inter-Assay CV<15%

Spike Recovery80-120%

Standard Curve5PL fit, R² >0.98

Luminex vs ELISA for Immune Checkpoint Analysis

Traditional ELISA requires a separate assay for each target. Our Luminex multiplex panel quantifies all 37 immune checkpoint proteins simultaneously in a single well.

Parameter	Luminex 37-Plex Panel	Traditional ELISA (37 assays)
Targets per Well	37	1
Wells Required	1	37
Sample Volume	25 μL	925 μL
Assay Time	~4 hours	~148 hours
Dynamic Range	4-5 logs	1-2 logs
Data Points per Sample	37	1
Re-dilution Required	Rarely needed	Often required

Traditional ELISA would require 37 separate assays to cover all targets in this panel, consuming nearly 1 mL of sample — impractical for precious clinical specimens. Our Luminex multiplex panel captures the complete immune checkpoint landscape from just 25 μL of serum, making it ideal for multi-timepoint immunotherapy monitoring studies where sample volume is limited.

Sample Requirements for Immune Checkpoint Luminex Assays

Proper sample collection and handling are essential for reliable immune checkpoint protein measurements.

Recommended Sample Types

Serum25 μL, no hemolysis, SST tubes

EDTA/Heparin Plasma25 μL, clear, no fibrin

Cell Culture Supernatant50 μL, centrifuge 10,000 rpm

Tissue/Cell Lysate50 μL, clarify by centrifugation

Handling Notes

Sample Storage-80°C, avoid freeze-thaw

ShippingDry ice or wet ice

ReplicatesDuplicate recommended

ImportantConsistent protocols across study

Soluble immune checkpoint proteins may be present at very low concentrations in healthy serum (typically low pg/mL range). Use consistent collection and storage protocols across all samples within a study to minimize pre-analytical variability. For longitudinal immunotherapy monitoring studies, collect samples at standardized time points relative to treatment cycles.

Which Sub-Panel to Choose?

The 37-Plex panel is organized into three modular sub-panels. Choose the configuration that matches your research focus, or combine all three for maximum coverage.

Co-Inhibitory & NK Cell Ligands

Covers NKG2D ligands (MICA, MICB, ULBP-1/3/4), NK cell receptors (PVR, Nectin-2, B7-H6, CD96, CD48), and co-inhibitory molecules (CTLA-4, CD28, CD80, Arginase-1). 25 μL per well.

Choose this for: NK cell-mediated immunity

PD-1/PD-L1 Axis & Co-Stimulatory Receptors

Complete PD-1/PD-L1 pathway coverage (PD-1, PD-L1, PD-L2) plus co-stimulatory receptors (OX40, 4-1BB, GITR, CD27), co-inhibitory checkpoints (TIM-3, VISTA, B7-H3, BTLA, HVEM), and metabolic regulators (IDO, CD47). 25 μL per well.

Choose this for: PD-1/PD-L1 axis research

Emerging & Soluble Immune Regulators

Covers emerging checkpoint targets (LAG-3, Siglec-7/9, TIMD-4), adenosine pathway (CD73), immune exclusion markers (E-Cadherin), and effector molecules (Perforin, S100A8/A9, ICOS Ligand). 25 μL per well.

Choose this for: emerging checkpoint targets

Maximum coverage for biomarker discovery? Combine all three sub-panels for the full 37-plex from a single 25 μL sample. Contact us to configure your panel

Immune Checkpoint Panel Research Applications

The Human Immune Checkpoint 37-Plex Panel supports research across immuno-oncology, autoimmune disease, and drug development.

Cancer Immunotherapy Monitoring

Quantify soluble PD-L1, PD-1, CTLA-4, TIM-3, and LAG-3 in serum from research participants receiving checkpoint inhibitors. Wang et al. (2022) demonstrated that sCD27 and sPD-L2 distinguish invasive from pre-invasive NSCLC (AUC=0.845). Published data shows elevated sPD-L1 in multiple cancer types versus healthy controls.

Liquid Biopsy Biomarker Discovery

Profile 37 soluble immune checkpoint proteins as potential blood-based biomarkers. A 2022 study found all five co-inhibitory checkpoints (CTLA-4, LAG-3, PD-1, PD-L1, TIM-3) significantly elevated in basal cell carcinoma patients versus controls.

Tumor Microenvironment Characterization

Analyze checkpoint protein expression in tumor cell lysates and conditioned media from cancer cell lines. Covers both established targets (PD-L1, CTLA-4) and emerging checkpoints (VISTA, B7-H3, Siglec-7/9).

Autoimmune Disease Monitoring

Track soluble checkpoint protein changes in rheumatoid arthritis, lupus, and inflammatory bowel disease. Checkpoint dysregulation is increasingly recognized as a driver of autoimmune pathology.

Infectious Disease & Sepsis

Evaluate immune exhaustion markers (PD-1, TIM-3, LAG-3) in chronic infections and sepsis. Soluble checkpoint proteins have been identified as prognostic markers in sepsis cohorts.

Preclinical Drug Development

Screen novel checkpoint-targeted therapeutics for on-target and off-target effects across 37 immune regulatory proteins in a single assay.

Deliverables and Quality Metrics

Every Luminex multiplex assay includes a comprehensive data package with full quality control documentation.

Data Package

Raw fluorescence intensities (.csv)
Calculated concentrations (pg/mL)
5PL standard curves for each analyte
Full QC report (.xlsx format)

Quality Control

Standard curve: 8-point, R² > 0.98
Intra-assay CV < 10%
Inter-assay CV < 15%
Spike recovery: 80-120%

Assay Performance

Duplicate sample measurements
Bridge sample for multi-plate studies
Method summary with lot numbers
Platform: Luminex xMAP

Case Study: Soluble Immune Checkpoints in NSCLC (Wang et al. 2022)

Published research using a Luminex multiplex bead-based assay to measure 14 soluble immune checkpoint-related proteins in plasma from patients at different stages of lung cancer progression.

Wang Q, et al. (2022) used a Luminex multiplex bead-based assay to measure 14 soluble immune checkpoint-related proteins (BTLA, LAG-3, GITR, IDO, PD-L2, PD-L1, PD-1, HVEM, TIM-3, CD28, CD27, CD80, CD137, CTLA-4) in plasma from 43 patients with pre-invasive adenocarcinoma in situ (AIS), 81 invasive adenocarcinoma patients (IAC: 50 early-stage + 31 late-stage), and 35 healthy controls. The goal was to determine whether soluble checkpoint proteins in blood could distinguish pre-invasive from invasive lung cancer.

Checkpoint	Healthy (n=35)	AIS (n=43)	IAC Early (n=50)	IAC Late (n=31)	P (AIS vs IAC)
sCD27	556.3	489.7	735.4	1,382.8	1.1E-06
sPD-L2	268.8	321.2	467.2	702.5	1.2E-06
sCD80	106.4	66.4	109.7	285.0	4.4E-05
sCD137	177.0	65.1	132.1	176.8	2.3E-05
sPD-L1	0.35	0.29	0.18	0.24	0.05 (NS)
sCTLA-4	7.81	5.19	5.91	9.63	0.14 (NS)
sTIM-3	131.3	29.0	39.5	67.8	0.33 (NS)
sHVEM	13.9	4.9	20.1	22.5	0.02

Median concentrations in pg/mL. NS = not significant.

Prediction Model Performance

Model	Variables	AUC	Sensitivity	Specificity
Model 1	Clinical only (age, sex, BMI, smoking)	0.779	81.4%	66.7%
Model 2	Clinical + CEA	0.806	75.0%	71.1%
Model 3	Clinical + sCD27 + sPD-L2	0.845	81.4%	75.3%

Key Findings

AUC = 0.845: Adding just two soluble checkpoint markers (sCD27 and sPD-L2) to clinical variables improved the AUC from 0.779 to 0.845, outperforming the established tumor marker CEA (AUC = 0.806).
Dynamic range matters: sCD27 ranges from ~400 to >1,700 pg/mL across groups. Luminex's 4-5 log dynamic range captures this spread without re-dilution, which is critical for studies comparing healthy vs. late-stage cancer levels.
Multiplex outperforms single analytes: The best model used sCD27 + sPD-L2 combined. A 37-plex panel enables systematic identification of the optimal biomarker combination for your research question.
Longitudinal design recommended: Inter-subject variability is substantial, especially for sCD27 (IQR spans 2-3x). Each research participant should serve as their own baseline in immunotherapy monitoring studies.
Optimal cutoffs: sCD27 > 453 pg/mL (OR = 2.9) and sPD-L2 > 287 pg/mL (OR = 4.23) for distinguishing invasive from pre-invasive NSCLC.

Source: Wang Q, et al. Soluble immune checkpoint-related proteins in blood are associated with invasion and progression in non-small cell lung cancer. Front Immunol. 2022;13:887916. DOI: 10.3389/fimmu.2022.887916

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Supporting Publications for Immune Checkpoint Multiplex Assays

Selected references utilizing Luminex multiplex assays for immune checkpoint protein profiling in research.

IMMUNO-ONCOLOGY

Wang Q, et al. (2022) Soluble immune checkpoint-related proteins in blood are associated with invasion and progression in non-small cell lung cancer. Front Immunol. 13:887916.

DOI: 10.3389/fimmu.2022.887916

DERMATOLOGY

Malinga NZ, et al. (2022) Systemic levels of the soluble co-inhibitory immune checkpoints, CTLA-4, LAG-3, PD-1/PD-L1 and TIM-3 are markedly increased in basal cell carcinoma. Transl Oncol. 19:101384.

PMID: 35255355

IMMUNOTHERAPY

Neuperger P, et al. (2023) Single-cell mass cytometric analysis of peripheral immunity and multiplex plasma marker profiling of NSCLC patients receiving PD-1 targeting immune checkpoint inhibitors. Front Immunol. 14:1243233.

DOI: 10.3389/fimmu.2023.1243233

Customization available: Select specific targets, expand plex capacity, or optimize for unusual sample matrices. Contact us for a custom quote

Frequently Asked Questions About Immune Checkpoint 37-Plex Panel

Common questions about our human immune checkpoint Luminex multiplex panel service.

Can I order individual sub-panels instead of the full 37-plex?

Yes. The panel consists of three modular sub-panels (14-plex co-inhibitory/NK ligands, 14-plex PD-1 axis and co-stimulatory receptors, 9-plex emerging immune regulators) that can be ordered independently or together. Choose the sub-panel(s) that match your specific research targets.

What concentration ranges should I expect for soluble checkpoints in serum?

Soluble checkpoint protein concentrations in healthy human serum are typically in the low pg/mL range. Published studies report that sPD-L1, sCTLA-4, sTIM-3, and sLAG-3 are detectable at pg/mL levels in healthy controls and significantly elevated in cancer patients. Blood levels of these markers have been shown to correlate with tumor tissue expression in several published studies.

Is a full 25 μL of serum sufficient to measure all 37 targets?

Yes. A single 25 μL serum/plasma sample is sufficient for the full 37-plex assay. The Luminex xMAP technology enables simultaneous quantification from minimal sample volume, making it ideal for precious clinical specimens.

Can this panel be used to monitor response to checkpoint inhibitor therapy?

Yes. Published studies have used similar multiplex checkpoint panels to track changes in soluble PD-L1, PD-1, CTLA-4, TIM-3, and LAG-3 levels during immunotherapy treatment. Within-subject longitudinal monitoring is recommended for tracking treatment response.

What is the difference between soluble and membrane-bound checkpoint proteins?

This panel measures soluble (shed/secreted) forms of checkpoint proteins in biological fluids. While membrane-bound forms require tissue biopsy or flow cytometry, soluble forms can be detected in blood samples. Soluble checkpoint levels have been shown to reflect immune activation status and have prognostic value in multiple cancer types.

Can this panel detect both PD-L1 and PD-1 in the same sample?

Yes. The panel includes both PD-L1 and PD-1, allowing simultaneous measurement of the ligand-receptor pair. This enables assessment of the PD-1/PD-L1 axis in a single assay well, which is valuable for understanding the balance of this key checkpoint pathway.

How are values below the lower limit of detection (LLOD) reported?

Concentrations below the LLOD are flagged as "LLOD" in the final report. For checkpoint proteins that may be near or below detection limits in healthy serum (sPD-L1 in healthy individuals is typically < 1 pg/mL), we can recommend high-sensitivity assay configurations. Contact us to discuss expected concentration ranges for your study population.

Can this 37-plex be combined with other panels on the same plate?

Yes. If your project requires immune checkpoint profiling plus additional cytokine or chemokine panels, we can coordinate a multi-panel workflow on the same Luminex plate. This maximizes data from each sample. Contact us with your full target list for a combined panel quote.

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