C3b Rat
Description
Introduction to C3b Rat
C3b Rat is a 190 kDa glycoprotein generated when rat C3 undergoes cleavage by enzymes such as trypsin or cobra venom factor (CVF) . This cleavage releases the anaphylatoxin C3a and activates C3b, which retains a reactive thioester bond. This bond enables C3b to covalently attach to hydroxyl or amino groups on microbial surfaces, initiating opsonization and complement activation .
Structure and Biochemical Properties
C3b Rat consists of two chains:
-
α’ chain: ~110 kDa, containing the reactive thioester and binding sites for complement receptors.
Key biochemical features:
-
Cleavage Kinetics: Rat C3b cleavage is slower than human C3b, with residual α’ chains observed after prolonged incubation .
-
Thioester Reactivity: The nascent C3b’s thioester forms covalent ester or amide bonds with target surfaces, enabling opsonization .
-
Degradation: Surface-bound C3b is cleaved by factor I and cofactors (e.g., factor H) into iC3b and C3d, which interact with receptors on immune cells .
Functional Roles in Immune Response
C3b Rat participates in three primary immune mechanisms:
Monoclonal Antibodies
-
mAb 2B10B9B2: Binds to C3b and C3, enabling detection in immunoassays. Useful for studying C3 activation in rat models .
Humanized Rat Models
-
C3 Humanized Rats: Engineered to express human C3 instead of rat C3, these models test C3 inhibitors (e.g., compstatin) for therapeutic potential. Human C3 restores complement activity in C3-deficient rats, with hemolysis inhibited by compstatin .
Antibody Response Studies
-
HEL-C3b Complexes: Covalent binding of C3b to hen egg lysozyme (HEL) enhances anti-HEL IgG production in mice, demonstrating C3b’s adjuvant role .
Comparative Analysis with Human C3b
Enhanced Antibody Responses
-
HEL-C3b Complexes: Mice immunized with covalent HEL-C3b complexes showed elevated anti-HEL IgG levels compared to free HEL or C3b alone .
-
Mechanism: C3b-Ag complexes are processed by antigen-presenting cells (APCs), stimulating T-cell help and B-cell activation .
Tissue Repair and Apoptosis
Product Specs
C3b, a key component of the complement system, is generated when native C3 is cleaved by the alternative pathway C3 convertase. This process releases C3a and leaves C3b, which is vital for all three complement pathways. These pathways use C3 convertases to cleave C3, producing C3a and activating C3b. Surface-bound C3b is crucial for activating C5, leading to the formation of C5b-9 complexes that lyse target cells. Additionally, C3b, along with its breakdown products iC3b and C3d, are recognized by receptors on immune cells, facilitating antigen presentation and stimulating the immune response.
Rat Complement C3b is a protein with a molecular weight of 175kDa, produced from Rat plasma.
The product is a sterile, filtered solution.
The C3b solution is formulated in phosphate buffered saline at a pH of 7.2.
Rat C3b remains stable for 2-4 weeks when stored at 4°C. For prolonged storage, freezing below -20°C is recommended. Adding a carrier protein (0.1% HSA or BSA) can further enhance stability during long-term storage. To maintain product integrity, avoid repeated freeze-thaw cycles.
The purity of Rat C3b is greater than 95%, as determined by SDS-PAGE analysis.
Complement C3, C3 and PZP-like alpha-2-macroglobulin domain-containing protein 1, C3, CPAMD1.
Rat Plasma.
Q&A
What experimental methods are most reliable for detecting C3b deposition in rat tissues?
C3b deposition is typically assessed via immunohistochemistry (IHC) or immunofluorescence (IF) using monoclonal antibodies targeting rat-specific C3b epitopes. In studies of glomerular injury, IHC with anti-C3b antibodies (e.g., clone 3/26 for murine models, adapted for rats) has been validated for specificity . For quantitative analysis, ELISA with anti-C3b IgG (e.g., homemade assays using purified rat C3b) provides semiquantitative data on circulating C3b levels. A critical validation step involves comparing results with negative controls (e.g., C3-deficient rats) to confirm antibody specificity .
Table 1: Comparison of C3b Detection Methods
| Method | Sensitivity | Tissue Compatibility | Key Limitations |
|---|---|---|---|
| IHC/IF | High | Kidney, liver, spleen | Requires fresh-frozen sections |
| ELISA | Moderate | Plasma, serum | Cross-reactivity with C3 fragments |
| SPR Analysis | High | In vitro systems | Limited to purified protein studies |
How are C3b-deficient rat models generated, and what are their primary applications?
C3b-deficient rats are created via CRISPR-Cas9 targeting of the C3 gene, focusing on exons critical for C3b generation (e.g., exon 10, which encodes the thioester bond domain). Phenotypic validation includes:
-
Plasma C3 quantification: Levels should be undetectable via ELISA .
-
Functional assays: Impaired alternative pathway (AP) activation, measured by reduced Bb and sC5b9 in serum after zymosan stimulation .
These models are used to study diseases like C3 glomerulopathy (C3G) and ischemia-reperfusion injury, where C3b deposition drives pathology .
What biomarkers confirm AP activation in C3b rat models?
Key biomarkers include:
-
Bb fragment: Elevated levels indicate AP convertase activity. In anti-FB antibody-positive rats, Bb levels exceed 3,500 ng/mL (vs. <1,275 ng/mL in controls) .
-
sC5b9: A terminal pathway marker; levels >420 ng/mL suggest uncontrolled AP activation .
-
C3a: Elevated in fluid-phase activation (e.g., >1,200 ng/mL in nephrotic syndrome models) .
How can researchers resolve contradictions between in vitro and in vivo C3b activity data?
Discrepancies often arise from differences in complement regulator expression (e.g., factor H, CR1) across experimental systems. For example:
-
In vitro: Purified anti-C3b IgG stabilizes C3 convertase (C3bBb) in surface plasmon resonance (SPR) assays .
-
In vivo: The same IgG may show reduced activity due to endogenous regulators.
Methodological solutions:
What experimental design strategies minimize confounding factors in C3b-targeted studies?
-
Temporal profiling: Collect plasma at multiple timepoints (e.g., pre-injury, 24h, 48h) to distinguish acute vs. chronic AP activation .
-
Cohort stratification: In C3G models, segregate rats by autoantibody status (anti-C3b+ vs. anti-FB+) to isolate mechanistic pathways .
-
Dual staining: Pair C3b IHC with markers like fibrin(ogen) to differentiate thrombotic microangiopathy from pure complementopathy .
Table 2: Biomarker Profiles in C3b-Related Injury Models
How do anti-C3b autoantibodies influence C3 convertase stability in rat models?
-
Coexisting autoantibodies: Anti-FB IgG synergistically increases C5 convertase activity, worsening glomerular injury .
-
Regulator competition: Anti-C3b IgG reduces CR1 binding to C3b by 60%, impairing convertase dissociation .
Methodological recommendation: Use IgG depletion assays (e.g., protein A/G columns) to confirm antibody specificity in functional studies .
What mechanisms link C3b deficiency to impaired tissue repair in rat models?
In APAP-induced liver injury, C3-deficient rats exhibit:
Product Science Overview
Complement C3 is a large protein that, upon activation, is cleaved into two fragments: C3a and C3b. The cleavage occurs through the action of C3 convertases, which are enzyme complexes formed during the activation of the complement pathways . The C3b fragment contains a reactive thioester bond that allows it to covalently attach to the surface of pathogens or other target cells. This process is known as opsonization, which tags the target for destruction by immune cells .
C3b plays a multifaceted role in the immune system:
- Opsonization: By binding to the surface of pathogens, C3b enhances their recognition and uptake by phagocytic cells such as macrophages and neutrophils .
- Formation of C5 Convertase: Surface-bound C3b participates in the formation of C5 convertase, another enzyme complex that cleaves C5 into C5a and C5b. This step is crucial for the formation of the membrane attack complex (MAC), which can lyse target cells .
- Immune Complex Clearance: C3b binds to immune complexes (antigen-antibody complexes) and facilitates their clearance from the circulation by binding to complement receptors on erythrocytes .
The activity of C3b is tightly regulated to prevent damage to host tissues. Regulatory proteins such as Factor H and Factor I inactivate C3b by cleaving it into inactive fragments (iC3b, C3c, and C3d) . These fragments can still bind to complement receptors and play roles in immune regulation, but they do not form C5 convertase or participate in the formation of the MAC .
Research on complement C3b has significant clinical implications. Dysregulation of the complement system, including excessive activation or insufficient regulation of C3b, is associated with various diseases such as autoimmune disorders, infections, and inflammatory conditions . Understanding the mechanisms of C3b activation and regulation can lead to the development of therapeutic interventions targeting the complement system.
