Albuminomics

SEB Test (Serum Enhanced Binding Test)

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SEB Test (Serum Enhanced Binding Test)

Definition

The Serum Enhanced Binding (SEB) test is a novel functional assay developed by Soli’s group (INSERM UMR1248 / CHU Limoges) for detecting HSA post-translational modifications in patient serum without requiring mass spectrometry. It measures whether specific metal ligands can bind to their corresponding sites on albumin — sites that are disrupted when HSA carries clinically relevant PTMs.

Primary paper: el-balkhi-2024-seb (Scientific Reports, 2024)

Principle

Logic

  1. Metal ligands (gold, copper, cadmium) bind to specific sites on HSA that depend on the structural integrity of the molecule
  2. If PTMs (cysteinylation, glycation, oxidation) have disrupted these sites → the metal cannot bind → more ligand remains free in solution
  3. Free fraction of each ligand is measured → elevated free fraction = positive signal = HSA PTM present

Ligands and their binding targets

LigandAlbumin binding sitePTMs disrupting this site
Gold (Au)—General PTM disruption; broad signal
Copper (Cu)N-terminal copper-binding site; also Cys34 regionCysteinylation, oxidation (HNA1/HNA2)
Cadmium (Cd)Specific metal-binding domainPTM combinations

Comparison to IMA/ACB test

  • The FDA-approved Ischemia Modified Albumin (IMA/ACB) test uses cobalt binding to the N-terminus of albumin to detect ischemia-modified albumin (reduced cobalt binding in ischemia)
  • SEB test is broader (3 ligands) and liver-disease-focused rather than cardiac

Procedure (general scheme)

  1. Patient serum collected (standard blood draw)
  2. Ex vivo spiking with specific metal ligand solution (Au, Cu, or Cd) at defined concentrations
  3. Incubation (room temperature or 37°C; defined time)
  4. Separation of protein-bound vs free ligand (ultrafiltration or similar)
  5. Measurement of free ligand fraction (by spectrometry/ICP-MS or colorimetric method)
  6. Interpretation: high free fraction = positive SEB test = HSA PTMs present

Performance — el-balkhi-2024-seb (Sci Rep 2024)

Human cohort

  • 45 cirrhotic patients vs 45 controls (+ independent 12 vs 12 confirmation set)
  • SEB ligands discriminated cirrhotic from control patients with individually high sensitivity/specificity
  • Combined threshold: 17/45 (37.8%) cirrhotic flagged ≥1 ligand above threshold vs 4/45 controls
  • First human proof-of-concept for the functional assay

Rat hepatotoxicity model

EndpointSEB testALT/AST
Detection of liver injuryDay 3Day 7
Sensitivity (early timepoint)PositiveNegative
Specificity (histology correlation)Confirmed D3 lesionsDetected D7 necrosis
  • Detection 4 days earlier than classical liver enzyme tests
  • Validated in two models: EtOH (chronic alcohol) and CCl₄ (direct hepatotoxin)

Advantages

  • No mass spectrometer required → clinically deployable in routine laboratories
  • Simple workflow: serum + ligand + measurement
  • Sensitive to early PTMs: detects functional binding site disruption before hepatocyte necrosis (and thus before enzyme release)
  • Potential multiplexing: 3 ligands can be tested simultaneously

Limitations and gaps

  • ⚠️ Human evidence is a single-centre cirrhosis-vs-control pilot (45 v 45 + 12 v 12); large multicentric validation ongoing via MALAHBAR (560 patients / 8 French CHUs, readout ~2027)
  • ⚠️ Early-detection/DILI timing advantage (D3 vs D7) shown in the rat model, not yet a human DILI cohort
  • Ligand panel (Au, Cu, Cd) requires optimization for human application — clinical free-fraction thresholds not yet standardized across centres
  • Mechanism of individual ligand sensitivity to specific PTMs not fully characterized (molecular dynamics simulations provide structural basis but not definitive mapping)
  • Does not identify which PTM is present — only that a binding site is disrupted
  • Comparison to IMA test not directly performed

Relation to mass spectrometry approach

The SEB test is positioned as the clinically scalable, equipment-light companion to the full LC-HR-MS isoform profiling approach (Top-down proteomics, el-balkhi-2025). Workflow concept:

Clinical suspicion of liver injury
↓
SEB test (routine lab; quick; no MS needed) → positive screening signal
↓ (if positive or complex case)
LC-HR-MS isoform profiling (specialized lab; full PTM landscape) → definitive characterization

Regulatory pathway

  • FDA-approved precedent: IMA/ACB test (cobalt-albumin binding); demonstrates regulatory feasibility for functional albumin binding assays as biomarkers
  • SEB test would require human clinical validation cohort → IVD regulatory submission (FDA/CE)

Development status

☑ Animal validation (done — rat model)
☑ Human pilot study (done — 45 cirrhotic vs 45 controls, Sci Rep 2024)
◧ Clinical validation (large multicentric cohort) — MALAHBAR ongoing (560 / 8 CHUs, readout ~2027)
☐ IVD regulatory submission
☐ Clinical approval

IP Status

The SEB test is protected by US20220018852 (US patent application, filed 2019, published 2022) and its European equivalent EP 3 884 280 B1 (granted). The patent covers the 5-ligand panel (Cu, Co, Au, Cd, T4/dansylsarcosine), the ICP-MS measurement method, and kits. See portfolio-overview for full patent portfolio context.

Key references

  • el-balkhi-2024-seb — primary paper describing SEB test development and validation in rat model
  • el-balkhi-2025 — ALBOM study; LC-HR-MS isoform approach that SEB test is designed to complement
  • US20220018852 — patent covering this method
  • HSA — protein whose binding sites the SEB test interrogates

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