Soluble C5b-9 detection: insight into complement activation and precise protection of every immune response
What traces will be left behind when complement is activated?
In the final stage of complement activation, C5b, C6, C7, C8 and multiple C9s assemble into the membrane attack complex (MAC), which aggregates to form pores on the cell membrane. When it is free in body fluids, it is regulated by S protein and clusterin, only binds one to three C9, and no longer forms pores - this is soluble C5b-9 (sC5b-9). As a key biomarker of complement terminal activation, sC5b-9 is closely related to the occurrence of various complement-involved diseases such as thrombotic microangiopathy and immune nephropathy.
Detection challenges: epitope selection
The complement components that make up C5b-9 (such as C5b, C6, C7) exist in free form in large amounts in body fluids. If the detection antibody recognizes the universal epitope of these components, the free monomers will be detected together, causing serious interference and false positives. Therefore, one must choose an epitope that is present only on the intact complex.
Core antibody AD99: only recognizes "neo-epitope"
AlloDx's core antibody AD99 does not recognize the monomeric complement protein itself, but only recognizes a special conformational nascent epitope - this epitope only exists on polymerized C9 and is formed by two adjacent C9 subunits and does not exist on monomeric C9.
The discovery of AD99 originated from a rigorous hybridoma screening system: mice were immunized with polymeric C9, splenic B lymphocytes were fused with myeloma cells, and thousands of hybridoma clones were obtained. Then, thousands of hybridoma clones were obtained. Then, only clones that "bind polymerized C9 but did not bind monomeric C9" were retained, and AD99 was finally targeted.
Double-antibody sandwich ELISA: dual recognition, only complete complexes are detected
AD99-based double-antibody sandwich ELISA: the capture antibody AD99 coated on the reaction plate specifically binds to the polymerized C9 neo-epitope, and the detection antibody binds to the C5b domain - both hit at the same time, and only the complete sC5b-9 complex will be detected.
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Linear range 15.6–250 ng/mL, covering the lower limit of the normal population
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The intra-batch and inter-batch variation coefficients are both less than 20%
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It is recommended to collect plasma samples for testing: comparison shows that the concentration of sC5b-9 in serum is significantly increased. Non-EDTA chelation sampling will continue to activate complement in vitro, directly affecting the accuracy of the results.
Products and testing services
The soluble C5b-9 detection kit uses AD99-specific monoclonal antibody. Each kit can detect 96 samples and is stored in the dark at 2–8°C.
AlloDx also provides professional sC5b-9 testing services. Standardized sampling is the first step for accurate testing:
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Use a purple-tipped tube (K2 EDTA) to collect 3 mL of peripheral blood. Immediately after blood collection, place it in a refrigerator or on ice, let it sit for more than 1 hour, and transport it to the AlloDx laboratory under refrigeration throughout the process.
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Samples after plasma exchange must be collected at least 48 hours apart
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If you need to separate plasma first, centrifugation must be completed within 4 hours (4°C, 1000–2000 G, 10 minutes), aliquot and freeze immediately at -80°C, and transport on dry ice
You only need to standardize sampling and cold chain delivery, and AlloDx will do the rest.