C8B Antibody

What is C8B and what is its biological function?

C8B is one of the three subunits that comprise complement component 8 (C8) of the complement system. It is primarily expressed in liver tissue where it contributes to the formation of the C8 protein complex, a component necessary for the assembly of the membrane attack complex (MAC) . The MAC plays a crucial role in the immune response by creating pores in target cell membranes, leading to cell lysis. C8B functions as a constituent of this complex, enabling the immune system to eliminate pathogens effectively. Patients with C8B deficiency often show increased susceptibility to bacterial infections, highlighting its importance in immune defense mechanisms .

What types of C8B antibodies are available for research?

Various C8B antibodies are available for research applications, including both monoclonal and polyclonal variants. Monoclonal options include rabbit recombinant antibodies such as EPR23764-1, which offer high specificity . Polyclonal antibodies targeting different amino acid regions of the C8B protein are also available, with options targeting specific regions such as AA 55-350, AA 321-370, and AA 451-500 . Some antibodies come in conjugation-ready formats designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes, making them suitable for multiplex imaging applications and flow-based assays .

What are the validated applications for C8B antibodies?

C8B antibodies have been validated for multiple research applications:

How should researchers validate C8B antibody specificity?

Validating C8B antibody specificity requires a multi-faceted approach. First, researchers should use recommended positive controls such as HeLa, HepG2, and HCT116 cell lines . Epitope analysis through competitive binding assays can help classify antibodies into different specificity groups, as demonstrated in studies where antibodies to the C8 alpha-subunit were classified into four overlapping epitope groups . Immunoblotting after SDS-PAGE separation of C8 subunits provides a definitive method to confirm subunit specificity, as shown in research where six antibodies reacted with the alpha-subunit, two with the beta-subunit, and two with the gamma-subunit . Additionally, comparing reactivity patterns across species with varying homology (e.g., human, rat, pig, rabbit, and bovine) can further validate antibody performance, with predicted reactivity percentages ranging from 80% to 86% depending on the species .

What is the optimal protocol for immunoprecipitation of C8B?

For successful immunoprecipitation of C8B, the following protocol has been validated: use the antibody at a 1/30 dilution (approximately 2μg in 0.35 mg lysates) when working with tissue samples such as human kidney tissue lysate . After immunoprecipitation, confirm results by Western blot using the same antibody at 1/1000 dilution. For detection, VeriBlot for IP Detection Reagent (HRP) should be used at 1/5000 dilution . This approach effectively isolates C8B while minimizing background interference, allowing for the detection of protein interactions and modifications in subsequent analyses.

How do C8B antibodies contribute to studying complement pathway activation?

C8B antibodies serve as essential tools for investigating complement pathway activation, particularly in assessing the formation and function of the membrane attack complex. Researchers can use these antibodies to measure C8B levels in patient serum to identify potential complement deficiencies or abnormal activation patterns . In experimental settings, monoclonal anti-C8 immunoaffinity columns have been successfully employed to purify C8 from fresh human plasma and to prepare C8-depleted serum, enabling functional studies of complement activity . Salt elution techniques can isolate either alpha-gamma or beta subunits when C8 is bound to anti-beta or anti-alpha immunoaffinity columns, respectively, allowing for detailed analysis of subunit contributions to complement activation .

How can researchers investigate the impact of C8B genetic variations?

Investigating C8B genetic variations requires an integrated approach combining genetic, biochemical, and functional analyses:

• Genetic analysis: Identify variations through sequencing, focusing on known variants such as c.1625C>T (p.Thr542Ile)

• Protein expression and function: Use Western blotting with C8B-specific antibodies to quantify protein levels in patient serum compared to controls, using native purified C8 as a standard (loaded at 10, 25, 50, 75, and 100 ng)

• Functional complement assessment: Evaluate the impact on complement pathway activation using hemolytic assays to assess whether identified variants affect MAC formation and function

• Reconstitution experiments: Test whether adding purified C8 components can restore normal complement function in deficient samples

• Cellular impact assessment: Evaluate effects on immune cell function, including phagocytosis, reactive oxygen species generation, and receptor expression (e.g., C5aR1)

What experimental approaches can assess the functional consequences of C8B deficiency?

C8B deficiency can be studied using several complementary approaches. Researchers have employed comprehensive investigations of the innate immune system on both cellular and humoral levels to define functional consequences . One approach involves assessing the functionality of neutrophils and monocytes regarding phagocytosis and reactive oxygen species generation, as well as measuring the expression of complement receptors such as C5aR1 . Extensive complement analysis can reveal activation potential for both alternative and classical pathways in C8B-deficient samples. Reconstitution experiments, where purified C2 and C8 are added to patient serum, can help determine whether the dysfunction can be normalized . Interestingly, in some cases, the addition of C3 rather than C8 improved hemolytic activity, suggesting complex interactions within the complement cascade .

What is the optimal protocol for Western blotting detection of C8B?

For optimal Western blot detection of C8B, the following protocol has been validated:

• Sample preparation: Mix 1 μL serum with 2× Laemmli sample buffer and run under nonreducing conditions for 45 min at 200 V on a precast stain-free gel (4–15%)

• Controls: Include native purified C8 as a positive control loaded at 10, 25, 50, 75, and 100 ng for standard curve generation

• Transfer: Perform protein transfer to an Amersham hybond membrane using a 7-min blotting program specifically optimized for mixed-molecular-weight proteins

• Blocking: Block the membrane in 5% dry skim milk in TBST at room temperature for 1 hour

• Primary antibody: Incubate with a polyclonal rabbit antihuman C8B antibody (e.g., PA5-31290) at 1:2,500 dilution overnight at 4°C under rolling conditions

• Washing: Perform three 15-minute washing steps in TBST

• Secondary antibody: Incubate with goat anti-rabbit HRP-linked antibody at 1:4,000 dilution for 1 hour

• Development: After washing twice in TBST for 15 min and once in TBS for 10 min, develop the membrane in clarity Western ECL solution for 40 seconds before capturing a chemiluminescence image

• Analysis: Conduct densitometric analysis using appropriate image analysis software

How can researchers differentiate between C8B and other complement components?

Differentiating between C8B and other complement components requires careful antibody selection and experimental design. Epitope analysis through competitive binding assays helps identify antibodies with high specificity for C8B . Research has shown that antibodies to the beta- and gamma-subunits, while binding to a single antigenic site on each, may cross-react with antigenic sites on the alpha-subunit . To ensure specificity, researchers should use antibodies validated for the beta-subunit and include appropriate controls, such as recombinant C8B protein or C8B-knockout samples. Additionally, immunoprecipitation followed by mass spectrometry can confirm antibody specificity by identifying the exact proteins being pulled down. For functional studies, salt elution techniques can be employed to purify either alpha-gamma or beta subunits when C8 is bound to anti-beta or anti-alpha immunoaffinity columns, respectively .

What considerations are important when using C8B antibodies for immunohistochemistry?

When using C8B antibodies for immunohistochemistry (IHC), several considerations are crucial for obtaining reliable results:

• Tissue selection: Since C8B is primarily expressed in liver tissue, this represents an appropriate positive control for IHC validation

• Antibody selection: Choose antibodies specifically validated for IHC applications, such as EPR23764-1 for IHC-P (paraffin-embedded tissues)

• Controls: Include both positive controls (liver tissue) and negative controls (tissues known not to express C8B or primary antibody omission controls)

• Antigen retrieval: Optimize antigen retrieval methods, as complement proteins may require specific conditions to expose epitopes after fixation

• Signal amplification: Consider signal amplification systems for detecting low-abundance complement proteins

• Cross-reactivity: Be aware of potential cross-reactivity with other complement components, particularly C8 alpha and gamma subunits

• Counterstaining: Use appropriate counterstains to visualize tissue architecture while maintaining the visibility of the C8B signal

• Validation: Confirm IHC findings with alternative techniques such as in situ hybridization or Western blotting

How might single-cell analysis techniques advance our understanding of C8B function?

Single-cell analysis techniques represent a promising frontier for understanding C8B function at unprecedented resolution. These approaches could help map C8B expression patterns across different cell types within the liver and other tissues, potentially uncovering previously unknown sites of complement component synthesis beyond the primary hepatic source . Single-cell RNA sequencing could reveal how C8B expression changes during immune responses or in disease states, while single-cell proteomics might identify cell-specific post-translational modifications affecting C8B function. Additionally, spatial transcriptomics could map the distribution of C8B-expressing cells within tissue microenvironments, providing insights into local complement activation patterns and their relationship to disease processes.

How can systems biology approaches integrate C8B data with other complement components?

Systems biology approaches offer powerful frameworks for integrating C8B data with information about other complement components. Network analysis could map protein-protein interactions between C8B and other MAC components, revealing how genetic variations in C8B might affect the entire complement cascade . Mathematical modeling of complement activation could predict how alterations in C8B levels or function impact MAC formation kinetics and efficiency. Multi-omics integration could correlate C8B genetic variations, expression levels, and functional outcomes with broader immune system parameters, helping to explain why some C8B variants lead to clinical manifestations while others remain asymptomatic. These approaches would be particularly valuable for understanding combined heterozygous genetic variations in complement components, such as those described in C2 and C8B, which cannot be fully explained by studying each component in isolation .