Please find the following rapid protocol

Preparation of reagents:

Clarification of the samples to be purified by filtration or centrifugation.

Dissolve saturated ammonium sulfate buffer: 750mg (NH4) 2SO4 in 1 liter H2O. Dissolve all salts by heating, then cool down to + 4 ° C. The supernatant is used as the saturated solution

Purification of IgM:

Mixing, in a centrifuge tube, 1ml of supernatant to 1ml of buffer saturated sulfate, quickly but gently mixed by inverting the tube.

let precipitate at 4 ° C for 30 minutes.

Spin at 2500rpm for 30 minutes to pellet the precipitate.

Resuspend the precipitate in 1 ml of 20 mM Tris buffer

Dialyze overnight against the 20 mM Tris buffer, a white precipitate appeared.

Spin at 2500rpm for 10 minutes to pellet the precipitate.

Resuspend the precipitate in 1 ml of PBS buffer to 150 mM.

Read at 280 nm, 1.35 ratio.

attention: IgM is stable in isotonic buffer, do not dilute in hypotonic buffer

Dr. Pin gave you an excelent protocol based in precipitation with ammonium sulfate. Other possibility could be purification by affinity chromatography. ConA-Sepharose is a good option for purifing IgM antibodies, using the specific affinity of the lectin for the oligosaccharide in the IgM structure. Also, immunoaffinity is other option, but it requires other step for purify only the IgM: saline precipitation as described previously by Dr. Pin or ConA immunoaffinity chromatography. This last option are more laborious, but yields specific IgM molecules only, without contamination of nonspecific IgM. Selection of the adequate method depends of the purity, economic considerations and amount of starting material.

Jean-Jacques Pin

thanks for your answers！but referring to literature , i found this antibody was purified by DEAE, do you have experience about DEAE ? and how to avoid the IgM precipitation?

we do have an additional purification protocol.

We practice HPLC purification on ion-exchange column, rather than DEAE QMA, and HyperD resin.

This protocol is used to separate all antibody isotypes in high density or ascites (ascites that we no longer practice for 10 years for ethical reasons).

It is coupled to the protocol of ammonium sulfate: After dialysis in 20 mM Tris pH 8.0, the antibody is injected on the column and elution is by gradient of 0-1 M NaCl.

Each isotype is released at a salt concentration which is specific to it, for example: IgG1 going out to 11% and IgM in 26%.

But we must pay attention to two Igs: IgG3 and IgM because they precipitate as above both.

To avoid this we can do dialysis against PBS, but then it will dilute the sample in 20 mM Tris, four times just before injection.

Final note: IgG3 and IgM are unstable after purification: Never dilute in water.  

To purify IgM (monoclonal and polyclonal) I use prepacked, ready to use,  affinity columns (HiTrap IgM purification HP, GE Healthcare, code 17-5110-01), according to manufacturer's instructions, with good results. These columns can be used with a syringe or with a liquid chromatography system. Contaninating IgG can be removed with protein A columns.

IgM is an euglobulin, that is, it precipitates at low ionic strenght. An easy method to get IgM more than 90% pure is to dilute ascytes 1/10 in deionized water, incubate 3 hours at 4ºC, centrifuge 15 minutes at 10000 rpm, discard the supernatant and resuspend the pellet in PBS. You can further purify IgM by gel filtration or DEAE chromatography.

We used the Pierce IgM purification kit in the past: https://www.lifetechnologies.com/order/catalog/product/44897

Alternative might be to allow the hybridome a class switch and take it from there.

Thanks all the friends for your help , I will try to purify it as your advises .

Chromatography on ConA-Sepharose and elution with alpha Methyl mannoside it the best solution in my experience.

The product might still contain some other proteins with mannose in their polysaccharide side chains. You can get rid of that by chromatography on a Superose 6 column.

You can use a ligand-affinity resin from LigaTrap Technologies. It really works for human IgM purification from hybridoma or 10%FBS cell culture media..

Binding capacity 5-10mg/mL resin..

It can recover up to 90% IgM from the total load with > 95% purity.

Finally, an affinity ligand for IgM purification developed by LigaTrap Technologies. These products can be purchased from ImmunoReagents.com

LT-143

LigaTrap Human IgM Purification Resin

LT-143Kit

LigaTrap Human IgM Purification Kit

Jean-Jacques Pin is the ammonium sulfate method effective for IgG isotype purification?

Dear Bouam.To answer your question: Ammonium sufate is active on all Igs isotypes. 42% of ammonium sulfate preferentially precipitates IgGs, IgA and IgE. 50% ammonium sulfate is required to precipitate the majority of IgMs. But this is only a pre-purification. To purify and separate each isotype of IgG, it is necessary to pass the precipitated antibodies on an ion exchange column to obtain isolated peaks of each isotype.

The separation sequence being, IgG1, IgG2, IgE, IgA1, IgG3, IgG4, IgA2 then the residual IgMs

Thank you so much dear J. Jaques Pin