Quantification of C3b Deposition Service

Creative Biolabs' C3b deposition assay service provides high-fidelity, pathway-aware measurements of C3/C3b/iC3b deposition on cell surfaces. Whether you're profiling a monoclonal antibody's CDC potential, screening complement modulators, or de-risking biomaterials and nanoparticle formulations, we build the right assay around your question and deliver data you can act on.

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Why C3b and iC3b Deposition Matters

C3 is the hub of the complement cascade. Once activated, it converts to C3b, a potent opsonin that tags targets for clearance, amplifies the cascade, and drives downstream events (C5 convertase formation and, ultimately, MAC assembly). Regulated cleavage of C3b yields iC3b, C3c, and C3dg, each carrying distinct biological consequences for phagocytosis, immune cell signaling, and immunogenicity. Measuring where, when, and how strongly C3 fragments deposit answers pivotal questions.

Does a therapeutic antibody trigger classical-pathway opsonization on target cells?
Will a lipid nanoparticle, AAV capsid, exosome, or polymeric scaffold be rapidly opsonized in blood?
Are new small-molecule or biologic candidates effectively modulating complement?
How do pathway-specific conditions (classical, lectin, alternative) shape a product's complement footprint?

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What We Test

Antibody & Fc Variant Assessment

Compare C3b/iC3b deposition across clones, subclasses (IgG1-4), glycoforms (afucosylated, sialylated), or engineered Fc backbones.

CDC Readiness

Pair C1q binding and C3b deposition readouts with functional hemolysis (CH50/AH50) or target-cell lysis assays to contextualize CDC potential.

Nanoparticles & Gene Delivery Vectors

Assess opsonization on LNPs, liposomes, polymers, or viral vectors under human serum conditions to anticipate pharmacology and PK challenges.

Cell & Pathogen Models

Quantify complement coating of tumor cells, primary cells, bacteria, or yeast to inform opsonophagocytic killing or immune evasion studies.

Complement Inhibitor Screening

Rank molecules or biologics that block C3 convertase assembly, Factor B activity, Factor D activity, or C3b-mediated amplification.

Autoimmunity and Inflammation Research

Explore how immune complexes or altered self-surfaces drive pathway-specific deposition.

Assay Formats	Principle	Ideal for
Microplate-Based C3/C3b Deposition	Immobilize the target (antigen/complex/surface) on a plate; incubate with serum (native, heat-inactivated, pathway-biased); detect deposited C3/C3b/iC3b with well-validated monoclonal antibodies and HRP-based or fluorescence readouts.	Antibodies, antigens, immune complexes, recombinant proteins, or coated surfaces
Cell/Particle-Based C3b Opsonization by Flow Cytometry	Incubate targets with serum; label deposited fragments (C3b/iC3b/C3d) using fragment-specific antibodies; quantify by MFI and percent-positive.	Intact cells (adherent or suspension), RBC/platelets, bacteria/yeast, EVs, LNPs, AAV, polymeric beads

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Species & Matrices

Species

Human (pooled NHS or donor-specific), NHP, mouse, rat

Matrices

Serum (preferred), citrate plasma, customized buffer systems

Depleted Panels

C1q-depleted, C2-depleted, Factor B-depleted, C3-depleted sera

Our Typical Workflow

Creative Biolabs receives and processes client samples according to validated protocols, employing highly sensitive C3b assays for precise quantification. We provide clients with a comprehensive report detailing experimental procedures, results, and any relevant observations or interpretations.

Fig. 1 Workflow of ELISA-based C3b deposition assay.

Design Your Workflow

Sample Requirements

Serum

Fresh frozen, no EDTA
0.5-1.0 mL per condition
Provide donor info if relevant

Plasma

Citrate (with caveats)
1-2 mL
For specialized questions, not ideal for activation readouts

Antibodies/Proteins

≥0.2 mg/mL
50-100 µL
Provide isotype, subclass, Fc engineering notes

Cells/Particles

0.5-2×10⁶ cells/test
≥10⁹ particles/mL for nanos
As needed, ship on cold packs or in validated media

Every project is unique, and we provide extensive customization to maximize relevance.

Design Your Customization

Add-On Assays That Sharpen Interpretation

Assays. (Creative Biolabs Authorized)

Design Your Customization

Case Studies

Case 1

C3b deposition assay test in the normal human serum

Creative Biolabs conducted C3b deposition assays on client-provided samples. The following figure illustrates a representative C3b deposition assay. The results indicated that the sample inhibited C3b deposition in normal human serum. This assay was performed using seven serial dilutions, with each dilution tested in duplicate. Further customization of the experimental plan can be accommodated based on specific requirements.

C3b deposition assay. (Creative Biolabs Original)

Fig. 2 C3b deposition assay test in the normal human serum.

Case 2

C3b deposition assay in the presence of CRP and human Ig preparation

Increasing concentrations of CRP were coated onto the ELISA microplate and overlaid with normal human serum +/− recombinant C2 variants. C3b deposition was detected by anti-C3b antibody. Purified C3b directly coated on the plate was used as a standard.

C3b deposition was detected by anti-C3b antibody. (OA Literature)

Fig. 3 C3b deposition assay in the presence of CRP and human Ig preparation.^1,2

References

Urban, Aleksandra, et al. "Gain-of-function mutations R249C and S250C in complement C2 protein increase C3 deposition in the presence of C-reactive protein." Frontiers in Immunology 12 (2021): 724361. https://doi.org/10.3389/fimmu.2021.724361
Distributed under Open Access license CC BY 4.0, without modification.

What Our Clients Say

"We needed a decision-ready way to rank five IgG1 leads by complement engagement. Creative Biolabs gave us pathway-resolved C3b/iC3b data, clean controls, and a crisp story that aligned with our C1q and CDC follow-ups."

— Director of Antibody Engineering, Mid-size Biotech

"Their flow cytometry opzonization assay on primary cells was outstanding. Gating was transparent, controls were done right, and we received files with a ready-to-use template. That level of data integrity is why we came back."

— Associate Professor, Academic Immunology Lab

"As a complement inhibitor shop, we needed believable data. They separated classical vs. alternative components with controls and depleted/reconstituted sera."

— CEO, Complement Therapeutics Startup

"This was our second engagement. The study plan referenced our prior lots, used the same reference standards, and bridged seamlessly. Consistency across quarters is rare; they delivered."

— Operations Manager, Biotherapeutics Division

Frequently Asked Questions

How do you decide between plate-based C3b deposition and flow cytometry opsonization?

Plate formats are ideal for high-throughput ranking of purified antigens, immune complexes, and coated materials with tight CVs and straightforward normalization. Flow cytometry is best when biological context matters—intact cells, microbes, EVs, LNPs, or AAV—because you gain single-event resolution, subpopulation gating, and easier translation to ADCP/ADNP. Many programs start on plates for speed, then confirm on flow for biological relevance.

What sample types and species can you work with?

Serum is standard. We also support NHP, mouse, and rat with validated cross-reactive detection. For plasma, citrate can be used with caveats; EDTA plasma is unsuitable for activation readouts. We routinely test purified antibodies/proteins, intact cells, bacteria/yeast, EVs, LNPs, AAV, coated coupons, and microfluidic surfaces.

Can you correlate C3b deposition with CH50/AH50, CDC, or phagocytosis?

Absolutely. Many clients request a cascade-spanning panel: C1q (priming)-C3b/iC3b (opsonization)-sC5b-9 (terminal) plus a functional endpoint (CH50/AH50, CDC, or ADCP/ADNP).

Can you distinguish C3b from iC3b and C3d, and why does that matter?

Yes. We deploy fragment-specific antibodies and orthogonal checks to separate C3b, iC3b, and C3d. Mapping this trajectory clarifies convertase activity, Factor I-mediated regulation, and persistence of opsonization signals that can influence downstream immune readouts and biomaterial compatibility.

What do you deliver at closeout?

You will receive an interpretive report (methods, figures, conclusions), raw files, tidy processed tables, and a QC dossier (acceptance criteria performance, deviations, corrective actions, reagent lots). Others are available on request.

What is the typical timeline from kickoff to report?

The timelines are contingent on sample availability, species, and scope. If you're milestone-driven, we propose phased gates to de-risk timelines.

Do you support downstream functional assays if C3b deposition is positive?

Definitely. We commonly add CDC or CH50/AH50 to link opsonization to effect. We can include sC5b-9 to assess terminal pathway progression and recommend design tweaks that reduce unwanted complement activation.