For over 50 years, Cedarlane has been producing complement, and is now a worldwide leader in supplying the reagent. Complement products are one of VH Bio’s best-selling Immunology products and it’s not surprising why!

In this blog piece, we are going to take a look at why complement is so popular and how it has been applied in the scientific world. 

What is Complement Serum? 

Firstly, well, what is it? The complement reagent contains several proteins naturally occurring in serum. Complement is a collection of proteins which are activated by the presence of pathogens. This in turn activates a cascade of reactions leading to the formation of the membrane attack complex. This complex then destroys the pathogen through cell membrane disruption. The complement system is a major part in the first line of defence in innate immunity.  

There are three complement activation pathways in the presence of a pathogen. Simply, these are the classical pathway which is activated by antibody-antigen interactions, the alternative pathway which is activated through microbial surface structure interactions with complement proteins, and the lectin pathway which is activated through lectins binding to the microbial surface. All complement activation pathways lead to the complement protein cascade activation and subsequent membrane attack complex formation [1].  

How is Complement Serum used in the laboratory? 

Complement is a mixture of proteins with immunological properties, but how can they be harnessed in the laboratory? This can be done through a serum bactericidal activity (SBA) assay. This assay can effectively measure complement-mediated killing via the classical pathway (antigen-antibody interactions). 

An SBA assay is a gold standard test for measuring natural and vaccine-induced immunity against Neisseria meningitidis (Nm). Nm is a gram-negative bacterium which can cause outbreaks of invasive meningococcal disease (IMD) and complement is a key factor in protection from it [2]. The aim of this assay is to assess immunogenicity by assessing the induction of bactericidal activity. 

Complement is the most critical reagent within this assay as you do not want the presence of exogenous activity, which can mask the true activity of your patient sera. To prevent this, our customers assess internally for intrinsic activity and performance of each batch and lot of complement used.  The intrinsic activity test looks to see if a reaction occurs between the bacteria and complement, in the absence of patient serum. No bacterial death should occur here as the complement should only be activated in the presence of antibodies from patient serum. Patient serum is also heat inactivated to disrupt the complement proteins present which further prevents any exogenous activity. Performance is then assessed by mixing the complement reagent with the bacteria and serum from an immune patient. This should result in the efficient killing of bacteria via the complement pathway. Once the complement has passed for each serogroup, it can be used for the rabbit SBA (rSBA) assay. The result of this assay is expressed as an SBA titre where 50% killing occurs after 60 minutes compared to the control bacterial colony forming units. The control contains bacteria but only heat inactivated patient serum and baby rabbit complement and so no bacterial killing will be happening here. The higher the resulting SBA titre, the more effective the complement assay has been at killing the bacteria. A high titre would indicate that the patient’s immunity either pre or post-vaccination, is at a protective level. 

Complement species can be key here too. Baby rabbit complement has been validated to be superior in SBA assays for Nm, and is appropriate for serogroups A,C, W, Y and X. Serogroup B however, is measured more effectively using human complement as rabbit complement results in intrinsic activity and bacterial death without the presence of human antibodies. 

If you would like to know more about the rSBA test performed in Manchester, please see their publication linked here: [3] https://link.springer.com/book/10.1007/978-1-4939-9202-7#:~:text=About%20this%20book,in%20vaccine%20development%20and%20surveillance. 

How Our Customers Use Complement Serum 

One of our customers, the Manchester vaccine evaluation unit, specialises in serological determination of immune responses to Nm following vaccination or disease. They work with clinicians and researchers on trials and projects and provide their services to perform the rSBA assay. Here, they use the tissue culture grade baby rabbit complement from Cedarlane. 

They have used the rSBA assay to look at meningococcal vaccine evaluation and response. Nm is usually a harmless coloniser of the human nasopharynx, yet IMD can affect around 1.2 million people worldwide resulting in an estimated 335 thousand deaths and many survivors with life-changing consequences. This is due to the development of rapidly evolving meningitis and sepsis. The highest rate of disease occurs in infants less than a year old with another peak between the ages of 15 and 25 years old. In Africa, there is an area described as the meningitis belt. This is an area spanning east to west, from Senegal to Ethiopia and containing 26 countries. This region experiences annual seasonal outbreaks and recurring epidemics every 5 to 12 years. Prevention of Nm and IMD is mostly provided through vaccination [4,5]. 

There are 12 serotypes of Nm based on their polysaccharide capsule structure yet 6 (A, B, C, W, X & Y) are known to cause the most cases of invasive disease with varying regional distribution. The Manchester vaccine evaluation unit has worked on studies focusing on the use of newly developed vaccines to help curb the outbreaks of Nm within the meningitis belt. At the time, serotype X-associated infection was rising in incidence although there were no licenced vaccines available that protected against it. The Manchester vaccine evaluation unit has worked on multiple publications, here are three recent ones which demonstrate how the rSBA assay has been used within clinical trials. 

In a clinical trial published in 2022 [6], Katz et al, aimed to assess the natural immunity to serotype X present within a community-based in the meningitis belt prior to the introduction of a pentavalent vaccine NmCV-5 which covers serotypes A, C, W, X and Y. At the time, only quadrivalent vaccines containing A, C, W and Y were licenced and in use. This study also aimed to validate a seroprevalence method to assess the natural immunity present in participant serum samples. Here, the rSBA assay was validated to be used in future phase 3 trials which would aim to assess immunogenicity of vaccines. This study came to two conclusions. Firstly, natural immunity to serogroup X Nm was present in 52.3% of participants, mainly in 5-14 year olds (73.9%) with the lowest seen in < 1-year-old infants (0%). Secondly, the rSBA assay was validated for many parameters for analysis of seroprevalence such as specificity and precision. This demonstrated the suitability of the rSBA assay for testing for serogroup X complement-based immunity for future clinical trials based on serotype X vaccination. The presence of participants with no immunity to serogroup X highlights a space where vaccination may help reduce outbreaks in the meningitis belt. 

This then prompted the next study [7] where Tapia et al, conducted a phase 2 clinical trial comparing quadrivalent conjugate vaccine which targets 4 different serotypes A, C, W & Y with the newly developed pentavalent vaccine NmCV-5 which could target serotypes A, C, W, Y and X. The rSBA assay was used here to look at how effective the vaccine was in 375 12–16-month-old children at inducing patient immunity at different stages after vaccination. Participants were assigned one of two NmCV-5 vaccines, or the quadrivalent vaccine MenACWY-D, administered in two doses 12 weeks apart. It was concluded that one dose of either NmCV-5 vaccine gave an immune response which excelled that of the two doses of MenACWY-D. Three months after dose one, the percentage of participants who had either of the NmCV-5 vaccines had an SBA value of at least 128 was 91% for all 5 serogroups, compared to only 36% which was only seen for serogroup C in participants who had the MenACWY-D as well as excluding serogroup X. This study hence shows that the NmCV-5 vaccine has the potential to affect outbreaks of all 5 serotypes. This study allows the NmCV-5 vaccine to move into phase 3 clinical trials (NCT03964012 and NCT04358731). This is significant as a single dose is an important consideration due to the cost and issues involved in patients returning for their second dose. This was an added benefit to containing the serotype X.  

The Manchester vaccine evaluation unit has also been involved in studies comparing vaccines to allow high-risk countries to stockpile alternative vaccines when the more widely used ones are in short supply. As seen above, pentavalent vaccines were being tested at this time but were not accessible for countries within the meningitis belt. Sow et al, [8] used the previously validated rSBA assay in a Phase 4 study which looked to compare the safety and immunogenicity of a quadrivalent Nm vaccine MPV-4 to a more commonly used quadrivalent vaccine MC4-4 in healthy 2 – 10-year-old participants. Participants were assessed at 30 days and 6 months post immunisation for immunogenicity for all 4 serogroups and safety outcomes, respectively. The vaccines were concluded to have similar properties for both study areas. These types of studies allow countries to prepare for potential outbreaks while reducing the chances of vaccine shortages until pentavalent vaccines become more accessible. 

These studies demonstrate that the work that the Manchester vaccine unit performs using the complement reagent from Cedarlane contributes to the building of knowledge around Nm vaccination, to improve the immunity of the populations affected the most. 

Complement Options Available from VH Bio 

Now you know how complement has been utilised by one of our customers, what would work for you and your work? Special processing techniques are used to yield complement with high activity and low background cytotoxicity which is ideal for effective use within customer assays. VH Bio distributes on behalf of Cedarlane in the UK and Ireland, and we can offer complement serum from several species and in multiple formats to suit a variety of applications. You can either purchase bulk quantities, reserve lots while you evaluate a sample bottle or purchase custom formats and sizes. Please see our product list below: 

  • Baby rabbit complement – tissue culture grade (frozen) 
  • Baby rabbit complement – Lyophilized 
  • Rabbit Complement MA (Lyophilized) for use with Human Lymphocytes 
  • Low-Tox®-M Rabbit Complement for use with mouse cells 
  • Low-Tox®-H Rabbit Complement for use with human cells 
  • Low-Tox®-R Rabbit Complement for use with rat cells 
  • Llama complement serum 
  • Rat complement serum 
  • Mouse complement serum 

 

Contact VH Bio today to discuss your requirements, request a sample, or secure your lot reservation:

  

References 

 

  1. Merle, N., Church, S., Fremeaux-Bacchi, V., Roumenina, L. (2015) Complement System Part I – Molecular Mechanisms of Activation and Regulation. Frontiers in Immunology. 6(262) DOI: 10.3389/fimmu.2015.00262. eCollection 2015. 
  2. Lewis, L & Ram, S. (2013) Meningococcal disease and the complement system. Virulence. 5(1) DOI: 10.4161/viru.26515 
  3. Lucidarme J, Louth J, Townsend-Payne K, Borrow R. Meningococcal Serogroup A, B, C, W, X, and Y Serum Bactericidal Antibody Assays. Methods Mol Biol 2019;1969:169–79 https://link.springer.com/book/10.1007/978-1-4939-9202-7#:~:text=About%20this%20book,in%20vaccine%20development%20and%20surveillance 
  4. Meningococcal Disease in Other Countries. US Centers for Disease Control & Prevention. 2024. (https://www.cdc.gov/meningococcal/php/global/index.html#:~:text=The%20meningitis%20belt%20of%20sub,Africa%20aren’t%20fully%20understood.) 
  5. Meningococcal Meningitis. World Health Organisation, 2024(https://www.afro.who.int/health-topics/meningococcal-meningitis) 
  6. Katz, S., Townsend-Payne, K., Louth, J., Lee-Jones, L., Trotter, C., Dan Dano, I., Borrow, R., MenAfriCar Consortium. (2022) Validation and use of a serum bactericidal antibody assay for Nesseria meningitidis serogroup X in a seroprevalence study in Niger, West Africa. Vaccine. 40 6042-47. DOI:10.1016/j.vaccine.2022.08.013 
  7. Tapia, M., Sow, S., Naficy, A., Diallo, F., Haidara, F., Chaudhari, A., Martellet, L., Traore, A., Townsend-Payne, K., Borrow, R., Hosken, N., Smolenov, I., Pisal, S., LaForce, M., Dhere, R., Kapse, D., Tang, Y., Alderson, M., Kulkarni, P. (2023) Meningococcal Serogroup ACWYX Conjugate Vaccine in Malian Toddlers. The New England Journal of Medicine. 384. 2115-23. DOI:10.1056/NEJMoa2013615 
  8. Sow, S., Tapia, M., Haidara, F., Diallo, F., Traore, Y., Traoré, A., Kodio, M., Borrow, R., Townsend-Payne, K., Yuan, L., Yang, S., Shi, L., Chen, J., Fang, G., Lin, J., Hu, R., Viviani, S., Huang, Z. (2023) Safety and immunogenicity of quadrivalent meningococcal polysaccharide vaccine (MPV ACYW135) compared with quadrivalent meningococcal conjugate vaccine (Menactra®) in Malian children. Human Vaccines & Immunotherapeutics. 19(2) DOI:10.1080/21645515.2023.2230829 

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