Reagents
Reagents
Creative Biolabs is proud to offer a wide range of reagents dedicated to advancing complement
system research. In research, understanding the activation, regulation, and inhibition of this
system requires specific and high-quality reagents. We guide researchers in choosing the most
appropriate reagents to achieve robust and reliable results in complement assays.
Product List Overview Solutions Erythrocytes Optimizing Assays Reagent Guide FAQs
Product List
Reagents for Complement Research
The main categories of reagents that we offer include:
Buffers and Solutions
Buffers are critical for controlling the experimental conditions necessary to observe complement
activity. Key solutions include:
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Verona Buffered Saline (VBS): Ideal for classical and lectin pathway assays.
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Gelatin Veronal Buffer (GVB): A common buffer used for stabilizing complement components.
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EDTA-containing Buffers: Used to chelate calcium and magnesium ions, effectively inhibiting
complement activity in experiments where blocking activation is required.
Erythrocytes
Sheep, rabbit, and other erythrocytes are essential in hemolytic assays, which measure the activity
of
complement pathways. RBCs act as target cells in classical and alternative pathway assays.
Specifically:
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Sheep erythrocytes are commonly used as targets in classical complement pathway hemolysis
assays.
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Rabbit erythrocytes are often applied in the alternative pathway, as they activate the
complement
system more efficiently through this pathway.
Each reagent serves a specialized function, enabling researchers to dissect the multifaceted roles
of
the complement system in immunology. If you need other related research tools, you can click below
for
details.
Products You May be Interested In
Buffer Solutions for Complement Assays
The use of proper buffers in complement assays is
critical for maintaining optimal conditions for
complement activation and function. The complement system is highly sensitive to changes in pH,
ionic strength, and divalent cation concentration. The preparation of buffer solutions for
complement assays requires precision and an understanding of the roles played by various buffer
components. The selection of a buffer system is tailored to the specific complement pathway being
studied and the biological sample in use.
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Buffer
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Introduction
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Function
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EDTA
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A chelating agent
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EDTA is one of the most commonly used reagents in complement assays due to its ability
to chelate divalent cations like Ca²⁺ and Mg²⁺, both of which are essential for
complement activation. By chelating calcium and magnesium, EDTA inhibits the classical
and lectin pathways, as both pathways require these ions for activation.
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VBS
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A specialized buffer frequently employed in complement assays, particularly for the
classical pathway
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VBS is often supplemented with Ca²⁺ and Mg²⁺, which are essential for the proper
activation of the classical and lectin pathways. This ensures the physiological
relevance of the assay environment, allowing for reliable data on complement activation
under near-natural conditions.
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Mg-EGTA Buffer
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A chelating agent that binds Ca²⁺ but not Mg²⁺
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This buffer is designed to chelate calcium ions while leaving magnesium ions available,
thus allowing the selective activation of the alternative pathway.
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Erythrocytes in Complement Pathway Studies
Among the various techniques to study complement
activity, hemolysis assays stand out as a robust,
sensitive method to investigate the efficacy of the complement system. In hemolysis assays,
erythrocytes are often used as targets for lysis, providing an easily measurable endpoint for
complement activation.
The classical complement pathway is primarily activated by immune complexes—specifically,
antibodies bound to antigens on the surface of a target cell. In hemolysis assays, sheep
erythrocytes are coated with antibodies (usually IgM or IgG), which leads to the
activation
of C1, the first complement component in the classical pathway.
Why sheep erythrocytes?
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They possess high antigenicity, allowing for effective sensitization by antibodies.
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Their surface properties allow the classical pathway to proceed efficiently, leading to
MAC formation and subsequent lysis.
Unlike the classical pathway, the alternative pathway does not require antibodies for
activation. Instead, it is activated spontaneously on foreign surfaces in a process that
involves the direct binding of complement protein C3b to the surface of the target cell. In
hemolysis assays for the alternative pathway, rabbit
erythrocytes are commonly used as the target
cells because they can naturally activate the alternative pathway due to their surface
characteristics.
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They have low sialic acid content, which makes them susceptible to spontaneous
complement deposition.
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They are efficient in triggering the C3b amplification loop, a hallmark of alternative
pathway activation.
Get a free brochure
Creative Biolabs developed a comprehensive hemolysis assay platform to
support our clients’ complement therapy research. We offer all kinds of
hemolysis assays to best suit your program requirements.
Get the brochure
Complement Function Analysis Protocols
We briefly summarize the complement-related assay protocols. Find
complement-related assay protocols by clicking the following items.
Get the protocols
Optimizing Complement Pathway Assays
Achieving reliable and reproducible results in complement pathway assays requires careful
optimization of experimental conditions in addition to quality reagents.
Serum Source
Freshly prepared or rapidly frozen serum is preferred, as complement components are
highly sensitive to degradation and inactivation.
Buffer Composition
Use buffers with physiological ionic strength and pH to maintain complement protein
stability. Ensure the presence of necessary divalent cations.
Avoid Contamination
Endotoxin or microbial contamination in samples can artificially activate the complement
system, leading to erroneous results.
Temperature Control
Complement activity is temperature-sensitive, with assays typically conducted at 37°C to
reflect physiological conditions.
Incubation Time
Time of incubation should be optimized to ensure maximal pathway activation without
plateauing. Pilot experiments to determine the optimal incubation time for your assay
setup are recommended.
Control Setup
Include positive controls, negative controls, inhibition controls.
Choosing the Right Reagent
When conducting complement research, selecting high-quality, consistent reagents is paramount.
Variability in reagent purity or batch consistency can lead to unreliable results, causing
delays in experimental timelines and wasting resources. At Creative Biolabs, we offer an
extensive catalog of complement reagents that have been thoroughly tested for performance,
consistency, and reproducibility.
Rigorous QC testing that ensure reagents meet performance standards
High-purity reagents that reduce the risk of non-specific reactions
Reagents from consistent batches that ensures reproducibility across different
experimental runs
How should complement buffers be prepared for optimal activity?
Complement buffers should be prepared fresh or stored in aliquots at -20°C to avoid
degradation. Commonly used buffers, such as PBS or HEPES, should be free from divalent
cations like calcium and magnesium unless specifically required. Always use cold buffers to
prevent complement activation prior to the experiment.
How do I prepare sheep erythrocytes for complement assays?
Sheep erythrocytes need to be washed multiple times in a cold isotonic buffer (e.g., PBS or
saline) to remove any contaminants. After washing, resuspend the cells at the appropriate
concentration, typically in veronal buffer, before use in hemolytic complement assays.
What buffers are recommended for complement activation assays?
For complement activation assays, buffers like GVB (gelatin veronal buffer) and veronal
buffer (Barbital-buffered saline) are recommended as they support optimal complement
activity. Ensure the buffer contains the right concentrations of calcium and magnesium ions,
as these are essential for complement pathway activation.
What should I do if my erythrocytes are not lysing in the complement assay?
Several factors could cause this issue, such as insufficient complement protein
concentration, improper buffer preparation, or expired erythrocytes. Double-check the
concentration of reagents, the freshness of erythrocytes, and the preparation steps to
troubleshoot.
Can I freeze erythrocytes for future use in complement assays?
Freshly prepared erythrocytes generally give the best results in complement assays. Freezing
erythrocytes can cause cell damage and may affect their lysis in hemolytic assays. If you
must store erythrocytes, keep them at 4°C for short-term use, but avoid freezing unless the
protocol specifically allows it.
Form Lyophilized
Storage Short-term storage at 4°C and long-term storage at -20°C.
Form Liquid
Storage Store at 4°C and avoid freezing.
Form Liquid
Storage Store at 4°C and avoid freezing.
Form Liquid
Storage Store at 4°C and avoid freezing.
Form Liquid
Storage Store at 4°C and avoid freezing.
Form Liquid
Storage Storage at -70°C
Form Liquid
Storage Storage at -70°C
Form Liquid
Storage Store at 4°C and avoid freezing.
Form Liquid
Storage Store at 4°C and avoid freezing.
Form Liquid
Storage Store at 4°C and avoid freezing.
Form Frozen suspension
Storage Storage at -70°C.
For Research Use Only.
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