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IgM hybridoma protocol


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5 replies to this topic

#1 TonyL

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Posted 26 January 2009 - 04:21 PM

Hello all,

We are looking to generate an IgM antibody against our protein of interest. Would anyone have any information on a hybridoma protocol which specifically targets IgM antibodies? I read that they appear much earlier and therefore screening for them happens much sooner in the immunization stage and does not require additional booster steps. Any information would be much appreciated!

Cheers,
Tony

#2 tonix37

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Posted 27 January 2009 - 12:53 AM

Hello all,

We are looking to generate an IgM antibody against our protein of interest. Would anyone have any information on a hybridoma protocol which specifically targets IgM antibodies? I read that they appear much earlier and therefore screening for them happens much sooner in the immunization stage and does not require additional booster steps. Any information would be much appreciated!

Cheers,
Tony


Hi Tony,

Why do you would obtain an IgM hybridoma??
The IgG monoclonal antibodies will have, always, more affinity, more specificity, and more antibody concentration than an IgM hybridoma secreting cell.

If you are working with an hapten, or with a low immunogenic antigen, you can obtain an IgM monoclonal antibody sometimes, but if you can immunize sometimes the antibody switching (IgM-->IgG) and somatic hypermutation will give you an better antibody for your purpose.

bye,

#3 TonyL

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Posted 27 January 2009 - 01:36 PM

We have found that IgM works best with our application but unfortunately there are no vendors which sell IgM to that protein. So we wanted to look into making our own.

#4 klinmed

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Posted 08 February 2009 - 05:46 AM

To obtain IgM antibodies you have to minimize class switching. Short immunization protocols and avoiding use of complete Freunds adjuvant will certainly help. If you are using cells as immunogens this should be no problem, with soluble proteins would try alum adjuvant.

Of course, screening should be undertaken using a specific anti-IgM secondary antibody detection reagent.

One likely problem will be that your selected reagents will probably have low avidity, but maybe ok for your purposes.

Hope this helps

#5 TonyL

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Posted 12 February 2009 - 09:55 AM

Thank you for the information klinmed. We are planning for a 7-day immunization cycle, with the fusion being done after the third week. I understand that the fusion rate (# hybridomas obtained after fusion) is very low (5-10?) with this approach though.

#6 klinmed

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Posted 13 February 2009 - 07:33 AM

Thank you for the information klinmed. We are planning for a 7-day immunization cycle, with the fusion being done after the third week. I understand that the fusion rate (# hybridomas obtained after fusion) is very low (5-10?) with this approach though.


Of MAJOR importance in obtaining a good number of hybridomas is the quality of your FCS. We always pre-screen batches by cloning our myeloma cells (NS0) by limiting dilution WITHOUT feeder cells (1 cell/well). We only use FCS that gives >20% cloning efficiency (more than 20 colonies per 96 well plate).

When we fuse 5E7 splenocytes with 2.5E7 myeloma cells we invariably get more than 900 wells containing colonies.

As for the immunization, I would suggest a priming dose (20-100 ug; sc) in complete Freundīs then boosting one month later with 20-100 ug ip at day -4, -3, -2, -1 prior to fusion. With such a short immunization scheme even with Freunds adjuvant you should get many IgMs.

We develop highly sensitive immunoassays for clinical use and therefore need very high affinities. Our immunizations schemes usually take 6 - 8 months! and we still get loads of IgMs (which we donīt want).

Hope this helps and good luck with the fusion.

Edited by klinmed, 13 February 2009 - 07:54 AM.





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