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How topurify and measure stability of mAb IgM


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11 replies to this topic

#1 Kristina

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Posted 02 December 2009 - 05:02 AM

Hi,

I have an IgM mAb that I would like to use in ELISA.

1. Can anywone recommend a (not so expensive) method for purification of mouse IgM?

2. I would like to check the stability after storage at +4C as well as -20C for a few weeks.
I will also make a stability test after storage at 37C fore 3 weeks, which should simulate longtime storage.

What stability-tests should I do after storage?
I will compare the activity in ELISA, and also look at a SDS-PAGE (reducing conditions?).
Is there anything else I should test.

Regards,

Kristina

#2 Kristina

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Posted 03 December 2009 - 05:04 AM

Thank you for your reply.
I will purify mAb from concentrated cell culture supernatant (CellLine flasks). The cells will be cultured in a serum-free media.

/K






What will you be purifying the IgM from (i.e., ascites, supernatant)?

Your ELISA and SDS-PAGE (you should do both reduced and non-reduced conditions) strategy may be sufficient. If you are able, you may want to test also by FACS analysis in addition to your assessment by ELISA.



#3 BioMiha

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Posted 08 December 2009 - 10:43 AM

Hi,

I have an IgM mAb that I would like to use in ELISA.

1. Can anywone recommend a (not so expensive) method for purification of mouse IgM?

2. I would like to check the stability after storage at +4C as well as -20C for a few weeks.
I will also make a stability test after storage at 37C fore 3 weeks, which should simulate longtime storage.

What stability-tests should I do after storage?
I will compare the activity in ELISA, and also look at a SDS-PAGE (reducing conditions?).
Is there anything else I should test.

Regards,

Kristina



Hi Kristina,

We purify our IgMs from cell culture supernatants using an FPLC affinity column and it seems to work quite well. The exact interaction I think is still not clear but is based on the tiophilic adsorption of IgMs that have multiple disulfide bonds. You could go the gel filtration route, but I'm thinking you will probably have too large a volume for that. Pierce also sells IgM purification kits that are mannan based. Haven't tried them, but in the past I have been very satisfied with Pierce's products.
Regarding stability tests, the first thing I would say is that the SDS-PAGE will not tell you anything. If your mAbs are not degraded and there is no reason that they would be, you will see two nice shiny bands for the heavy and light chains respectively and nothing else. Stability testing is carried out to see if they are still active after a certain period of time. Activity or lack thereof is because your mAbs unfold or aggregate or precipitate => things you can't see in SDS-PAGE. You can only see that in ELISA or a similar immunoaffinity assay. However, a good thing to do is check for aggregation by gel filtration (perhaps after multiple freeze thaw cycles). We also do high temperature testing (up to 55 deg. C) to check for thermal stability.
That's just my 2c.
Hope it helps,

Miha

#4 sgt4boston

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Posted 09 December 2009 - 09:56 AM

I agree with Miha. You have to do some type of activity test. If you could also run some type of quantitative test for total IgM that may help as well. If the hevy chain degrades you may get elevated or decreased results compared to a control.

#5 fatemeh

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Posted 03 March 2010 - 02:56 AM

Hi,

I have an IgM mAb that I would like to use in ELISA.

1. Can anywone recommend a (not so expensive) method for purification of mouse IgM?

2. I would like to check the stability after storage at +4C as well as -20C for a few weeks.
I will also make a stability test after storage at 37C fore 3 weeks, which should simulate longtime storage.

What stability-tests should I do after storage?
I will compare the activity in ELISA, and also look at a SDS-PAGE (reducing conditions?).
Is there anything else I should test.

Regards,

Kristina



Hi Kristina,

We purify our IgMs from cell culture supernatants using an FPLC affinity column and it seems to work quite well. The exact interaction I think is still not clear but is based on the tiophilic adsorption of IgMs that have multiple disulfide bonds. You could go the gel filtration route, but I'm thinking you will probably have too large a volume for that. Pierce also sells IgM purification kits that are mannan based. Haven't tried them, but in the past I have been very satisfied with Pierce's products.
Regarding stability tests, the first thing I would say is that the SDS-PAGE will not tell you anything. If your mAbs are not degraded and there is no reason that they would be, you will see two nice shiny bands for the heavy and light chains respectively and nothing else. Stability testing is carried out to see if they are still active after a certain period of time. Activity or lack thereof is because your mAbs unfold or aggregate or precipitate => things you can't see in SDS-PAGE. You can only see that in ELISA or a similar immunoaffinity assay. However, a good thing to do is check for aggregation by gel filtration (perhaps after multiple freeze thaw cycles). We also do high temperature testing (up to 55 deg. C) to check for thermal stability.
That's just my 2c.
Hope it helps,

Miha

Hi Miha

I just saw this message. I am using the same system(FPLC affinity) but I couldn't get purified antibody yet. May I ask your protocol? Have you made any modification in procedures that introduced in its manual? I will be very appreciated to get your response, thanks
fatemeh

#6 biorob

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Posted 08 March 2010 - 12:38 PM

Hi,

I have an IgM mAb that I would like to use in ELISA.

1. Can anywone recommend a (not so expensive) method for purification of mouse IgM?

2. I would like to check the stability after storage at +4C as well as -20C for a few weeks.
I will also make a stability test after storage at 37C fore 3 weeks, which should simulate longtime storage.

What stability-tests should I do after storage?
I will compare the activity in ELISA, and also look at a SDS-PAGE (reducing conditions?).
Is there anything else I should test.

Regards,

Kristina



Hi Kristina,

We purify our IgMs from cell culture supernatants using an FPLC affinity column and it seems to work quite well. The exact interaction I think is still not clear but is based on the tiophilic adsorption of IgMs that have multiple disulfide bonds. You could go the gel filtration route, but I'm thinking you will probably have too large a volume for that. Pierce also sells IgM purification kits that are mannan based. Haven't tried them, but in the past I have been very satisfied with Pierce's products.
Regarding stability tests, the first thing I would say is that the SDS-PAGE will not tell you anything. If your mAbs are not degraded and there is no reason that they would be, you will see two nice shiny bands for the heavy and light chains respectively and nothing else. Stability testing is carried out to see if they are still active after a certain period of time. Activity or lack thereof is because your mAbs unfold or aggregate or precipitate => things you can't see in SDS-PAGE. You can only see that in ELISA or a similar immunoaffinity assay. However, a good thing to do is check for aggregation by gel filtration (perhaps after multiple freeze thaw cycles). We also do high temperature testing (up to 55 deg. C) to check for thermal stability.
That's just my 2c.
Hope it helps,

Miha

Hi Miha

I just saw this message. I am using the same system(FPLC affinity) but I couldn't get purified antibody yet. May I ask your protocol? Have you made any modification in procedures that introduced in its manual? I will be very appreciated to get your response, thanks
fatemeh


Hi fatemeh,
does your sample bind at the column? If not, try to put more sulfate salt in your sample. If it binds to heavy (no elution peak), try to use isopropanol in your elution buffer. Up to 20% should be sufficient. Or try a CaCl-containing buffer instead of isoprop (but do not mix it with phosphate buffer! ;-))
biorob

#7 fatemeh

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Posted 08 March 2010 - 02:42 PM

Hi fatemeh,
does your sample bind at the column? If not, try to put more sulfate salt in your sample. If it binds to heavy (no elution peak), try to use isopropanol in your elution buffer. Up to 20% should be sufficient. Or try a CaCl-containing buffer instead of isoprop (but do not mix it with phosphate buffer! ;-))
biorob
[/quote]
Hello biorob

very appreciated! I can see very small peak in elution , sometimes almost nothing. I decrease the pH to 6.5 and the peak can be seen but still very small with low amount of protein( I measure the protein content instead the IgM every time) The method to measure IgM is very expensive . About CaCl may I know how many molar and should I add it to regeneration buffer too?
May I know how you measure recovery and yeild ? :P

Regards,
fatemeh

#8 fatemeh

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Posted 08 March 2010 - 02:45 PM

[/quote]
Hi Miha

I just saw this message. I am using the same system(FPLC affinity) but I couldn't get purified antibody yet. May I ask your protocol? Have you made any modification in procedures that introduced in its manual? I will be very appreciated to get your response, thanks
fatemeh
[/quote]

Hi fatemeh,
does your sample bind at the column? If not, try to put more sulfate salt in your sample. If it binds to heavy (no elution peak), try to use isopropanol in your elution buffer. Up to 20% should be sufficient. Or try a CaCl-containing buffer instead of isoprop (but do not mix it with phosphate buffer! ;-))
biorob
[/quote]

very appreciated! I can see very small peak in elution , sometimes almost nothing. I decrease the pH to 6.5 and the peak can be seen but still very small with low amount of protein( I measure the protein content instead the IgM every time) The method to measure IgM is very expensive . About CaCl may I know how many molar and should I add it to regeneration buffer too?
May I know how you measure recovery and yeild ?

Regards,
fatemeh

#9 biorob

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Posted 09 March 2010 - 11:46 AM

very appreciated! I can see very small peak in elution , sometimes almost nothing. I decrease the pH to 6.5 and the peak can be seen but still very small with low amount of protein( I measure the protein content instead the IgM every time) The method to measure IgM is very expensive . About CaCl may I know how many molar and should I add it to regeneration buffer too?
May I know how you measure recovery and yeild ? :lol:

Regards,
fatemeh
[/quote]

Hi fatemeh, do you use "IgM purification HP"-Resin (GE)? I used it long time ago, and it worked well, but I had to use a "special" elution buffer to elute the tight bound IgM. As I said I used Isopropano or CaCl. The idea behind this is to disturb the hydrophobic interaction between column and IgM (depends on the IgM). You can do this by using Isopropanol (i think it displaces the hydrophobe IgM) or by using a chaotropic salt (eg. CaCl, see "Hofmeister serie"). These salts increase the solubility of nonpolar molecules ("salting in") and increase the order in water; in effect, they weaken the hydrophobic effect.
I donīt remember the exact molarity, but i think 0.2M CaCl should be fine. I didnīt used this for regeneration, only for elution.
Recovery / Yield I measured by UV-280nm (Photometric) and by anti-IgM-ELISA (Supernatant vs. Flowthrough and Eluate). For the ELISA you coat the plate with an anti-IgM-antibody (or the specific antigen), then sample, then detection antibody...
If the ELISA is too expensive, you can measure the protein content and put the sample on the SDS-PAGE. If the sample is pure, everything you measured is IgM. If there are impurities you can quantify them by densitometry (some labs have such devices, or simply scan your gel and use some of the free software to quantify the bands).
I hope I could help you. For what do you want to use the IgM?
greetings, biorob

#10 fatemeh

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Posted 11 April 2010 - 04:08 AM

Hi Biorob
Thank you so much for fruitful post and sorry for late reply.Actually I didn't check my post for a long time. I use IgM affinity column that is a prepacked column from GE, but seems you used the resin that needs to be packed, didn't you?
Regarding to special elution buffer, Do you mean I should add cacl to elution buffer ( 20mM Sodium phospate pH=7.5)or just use the cacl as the only salt of elution buffer? may I know how your peaks looks like? When I use pH=7.5 , I got 3 peaks, 1 band for each segment, when I use pH=6 I get 2 peaks.

I appreciate your detail explain for IgM quantification. I use IgM quantification from bethyl, it needs about 7hours time, do u know any shorter method :wacko:

This purification is a part of my PhD thesis to characterize a monoclonal antibody. first I use ascitic fluid as a source of my experiments but latter I shift to supernatant avoiding the suffer to animal

Thanks again,
fatemeh

#11 MouseMab

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Posted 14 April 2010 - 06:18 AM

Hi Fatemeh,

Have you had any luck purifying your IgM MAb? I too am trying to purify an IgM and it is proving to be quite challenging! I am using the GE -HiTrap IgM purification column and am trying to purify from supernatant harvested from the CELLine CL-350 flask. I have concentrated ny harvest before loading on the column but seem to get a very low MAb yield. Any advice would be much appreciated.

#12 fatemeh

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Posted 02 June 2010 - 08:19 PM

Hi Fatemeh,

Have you had any luck purifying your IgM MAb? I too am trying to purify an IgM and it is proving to be quite challenging! I am using the GE -HiTrap IgM purification column and am trying to purify from supernatant harvested from the CELLine CL-350 flask. I have concentrated ny harvest before loading on the column but seem to get a very low MAb yield. Any advice would be much appreciated.



Hi MouseMab

Sorry for very late reply, I hope you could get very good results by now. I should say I couldn't yet. I use the same column as yours but still no desired results. May I know what protocol you follow? My protocol is explained in my previous posts.

Regarding concentration, have u checked your IgM concentration after concentrating? If you are using concentrating filters, sometimes it adsorbed the protein you should be careful in choosing the concentrator avoiding the low yeild of concentrated antibody. Ammonium precipitation may be better choice if it doesn't affect IgM activity.

Good Luck,
fatemeh




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