Dear Huiwu,

Fresh mouse serum should be able to activate via Classical pathway with human IgM. It works on ELISA setting as an activation ligand, but I have no hands on experience on cell-based assays. IgM alone can support C1q mediated binding as it is pentameric.

Word of warning on mice complement tough. The C-system between females and males differs greatly, especially on the Terminal Pathway components (C5 through C9). Therefore female derived serum will likely be much slower or weaker in killing compared to male serum. Second, serum must be (should be) fresh, stored in -80C, unthawed and kept on melting ice during preparation for the assay. Repeated fz-thaw will degrade mouse C, -20°C storage results in steady degradation and incubation at RT or higher even during melting can result in degradation and assay to assay variation.

Assays like yours may consume high amounts of mouse serum, therefore you may need to have larger single batch of serum at hand. Instead of preparing serum from 30-100 animals yourself I would suggest getting it commercially. I have relied Innovative Research Complement preserved CD1 and C57bl/6 mouse serum in my work. They do not specify the sex of the "donors" with the off the shelf products, which can result in batch to batch variations. You can request a custom batch of males only at a decent(ish) price. I have been happy with batches up to 25ml, which I have thawed, aliquoted and stored at -80C up to a year after receiving.

For further information on assay setup you might want to look through these publications by Bart de Vries and Leendert Trouw: 

http://www.ncbi.nlm.nih.gov/pubmed/15781117

http://www.ncbi.nlm.nih.gov/pubmed/12589162

Kind Regards

Juha Kotimaa

Mice are the experimental tool of choice for the majority of immunologists and the study of their immune responses has yielded tremendous insight into the workings of the human immune system. There are significant differences. Here we outline known discrepancies in both innate and adaptive immunity, including: balance of leukocyte subsets, defensins, Toll receptors, inducible NO synthase, the NK inhibitory receptor families Ly49 and KIR, FcR, Ig subsets, the B cell (BLNK, Btk, and λ5) and T cell (ZAP70 and common γ-chain) signaling pathway components, Thy-1, γδ T cells, cytokines and cytokine receptors, Th1/Th2 differentiation, costimulatory molecule expression and function, Ag-presenting function of endothelial cells, and chemokine and chemokine receptor expression.

Hi Kalpesh, thank you for points. Do you know whether mouse complement could be used for human IgM dependent cell cytotoxicity assay?

Hi Huiwu. I think mouse complement should work. There are a number of in vivo mouse studies with rituximab, a human IgG1 antibody that depends on CDC to work, to suggest that human Ig can also activate mouse complement. But rabbit and guinea pig complement is a lot cheaper to buy though. Hope that helps.

Dear Huiwu,

Fresh mouse serum should be able to activate via Classical pathway with human IgM. It works on ELISA setting as an activation ligand, but I have no hands on experience on cell-based assays. IgM alone can support C1q mediated binding as it is pentameric.

Word of warning on mice complement tough. The C-system between females and males differs greatly, especially on the Terminal Pathway components (C5 through C9). Therefore female derived serum will likely be much slower or weaker in killing compared to male serum. Second, serum must be (should be) fresh, stored in -80C, unthawed and kept on melting ice during preparation for the assay. Repeated fz-thaw will degrade mouse C, -20°C storage results in steady degradation and incubation at RT or higher even during melting can result in degradation and assay to assay variation.

Assays like yours may consume high amounts of mouse serum, therefore you may need to have larger single batch of serum at hand. Instead of preparing serum from 30-100 animals yourself I would suggest getting it commercially. I have relied Innovative Research Complement preserved CD1 and C57bl/6 mouse serum in my work. They do not specify the sex of the "donors" with the off the shelf products, which can result in batch to batch variations. You can request a custom batch of males only at a decent(ish) price. I have been happy with batches up to 25ml, which I have thawed, aliquoted and stored at -80C up to a year after receiving.

For further information on assay setup you might want to look through these publications by Bart de Vries and Leendert Trouw: 

http://www.ncbi.nlm.nih.gov/pubmed/15781117

http://www.ncbi.nlm.nih.gov/pubmed/12589162

Kind Regards

Juha Kotimaa

Hi, everyone. Thank you so much. Your points are very helpful.

Hi, Huiwu,

Maybe you can find some useful information in these papers:

Julien Ratelade, A.S. Verkman. Inhibitor(s) of the classical complement pathway in mouse serum limit the utility of mice as experimental models of neuromyelitis optica. Mol. Immunol., 62(1):104-113, 2014.

Ira Bergman, Per H. Basse, Mamdouha A. Barmada, Judith A. Griffin, Nai-Kong V. Cheung. Comparison of in vitro antibody-targeted cytotoxicity using mouse,

rat and human effectors. Cancer Immunol. Immunother., 49:259-266, 2000.

Kind regards

Luciana

Hi Juha,

Is there any reliable quantitative ELISA based assay (based on C3 deposition) to assess the complement fixing capability of antigen specific antibodies? I have few chimeric (mouse Vh and Human Fc) antigen specific antibodies variants and I want to check them for their ability to fix mouse complement.

Thanks!

Pankaj

Dear Pankaj,

Reliable ELISA based systems are not that difficult to set up. I do not think there is commercially available mouse assays yet, and the only one I know of is focused on the level of C9 and is in development.

I think ELISA based system in general is not the right option for you. If I understood your question correctly, you want to test whether your mAb can initiate complement after binding to its target (membrane) protein? 

If that is the case the ELISA based system can be a hit-and-miss approach. It as recently published that the C1q mediated complement activation requires hexameric IgGs, and in most cases a cell membrane-like platform where your protein is to accomodate the hexamerisation. I know that sometimes depending on the experimental conditions you can coax the complement to bind adsorbed IgGs, but I would not read too much in to it based on the finding by Diebolder et al.:

http://www.sciencemag.org/content/343/6176/1260

Cell-based systems might be an interesting system for you to explore. You just need your target protein on the surface and then go with the traditional way of studying whether you get C3/C4 or C5b-9 deposition on the surface after exosure to your antibody and complement preserved serum. 

I hope this helps.

Kind Regards

Juha

Thanks a lot Juha! I agree Cell based assay makes sense in this case.

Best!

Pankaj

Be aware of that the amounts (activity)  of complement in mice is generally very low compared to guineapig, rabbits and humans. The immunobiological importance of complement in mice may be equally limited. See

Journal of Immunological Methods

Volume 125, Issues 1–2, 20 December 1989, Pages 147–158

Hans

"Mice are the experimental tool of choice for the majority of immunologists and the study of their immune responses has yielded tremendous insight into the workings of the human immune system." This sentence is from The Journal of Immunology

March 1, 2004

vol. 172 no. 5 2731-2738. Plagiarism is plagiarism, Kalpesh.