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Rabbit IgG purification


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#1 thekid

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Posted 04 August 2009 - 01:05 AM

Hello all,

We raised antibodies to a virus in rabbits and bleed them once the titers are high ( after 3 dose of immunization). We used the Pierce 89978 NAB tm Protein A plus spin kit, 1 ml to purify the IgG from the rabbit serum.

Intial volume of the serum was 39ml. According to protocol we had to add an Equal volume of PBS which bought the volume to 78ml. The elution was done in 1 ml volume of elution buffer and we had at the end nearly 65ml of elute which had IgG ( this was determined by nanodrop). We decided to concentrate the IgG from all the elutes and used a spin cut off column from Vivaspin. The MWCO was 30KDa. But at the end of the concentration also we had nearly 40ml of elute which had 3.52mg/ml of protein ( by nanodrop). This essentially means that we have 140.8mg/40ml.

Is there any way to concentrate this protein into say 5 ml of volume so that the concentration would increase.

The antibody once purified will be used to coat ELISA plates, to capture Virus lysate for which we are detecting antibodies in human serum samples.

Please let me know if there is any way of concentrating the protein without lossing the activity of the protein.


thekid

#2 Roo

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Posted 04 August 2009 - 08:21 AM

But at the end of the concentration also we had nearly 40ml of elute which had 3.52mg/ml of protein ( by nanodrop). This essentially means that we have 140.8mg/40ml

Is there any way to concentrate this protein into say 5 ml of volume so that the concentration would increase.



It seems to me that 3.52 mg/ml is already a good concentration. If you try to get 140 mg of protein into 5 ml that would put your concentration at 28 mg/ml. I have never seen such a high concentration of antibody. My guess is that it would precipitate long before you reached your 5 ml volume.

As far as concentrating antibodies goes, I have used various methods, but I usually use an Amicon Ultra Concentrator following the manufacturer's directions. Just be careful that you don't spin the volume so low that everything precipitates. Occasionally in working with my antibodies, I lose some activity of my antibody. Usually it's because of the harsh conditions (low pH) used to elute the antibodies from purification columns and not from the concentrator. From my experience, you are more likely to lose antibody to precipitation than you are to inactivation during concentration.

Another thought...
I know you used Protein A to purify the IgG from the rabbit polyclonal serum. In many assays this works fine. I'm not familiar with the Pierce kit, but keep in mind that when you use Protein A to purify serum you are purifying total IgG in the serum meaning ALL IgGs not just the IgG specific to your antigen. According to the Antibodies, A Laboratory Manual by Harlow and Lane, polyclonal serum usually contains 10 mg/ml antibody, but only 1 mg/ml at best (10% max) is specific to your antigen. Chances are, the antibody you already have will work for many assays, but you might try immunoaffinity purification of a small volume. This will allow you to purify the antibody specific to your antigen, then your final antibody concentration won't necessarily need to be as high since 100% of it should be specific to your antigen.

#3 thekid

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Posted 04 August 2009 - 10:21 AM

Hi Roo,

Thank you for the suggestions. Would like to know what you meant by saying "Amicon Ultra Concentrator following the manufacturer's directions. Just be careful that you don't spin the volume so low that everything precipitates. " Could you be more specific about this, as I am also using a similar device to concentrate the IgG.

I was told that usually there will be around 10mg/ml of IgG from Rabbit serum, hence comparing to that the 120mg/40ml that I have is less is what I thought. Am I wrong?

Also I have read up that you can freezze dry the IgG and then reconstitute, can that be used here as a way to concentrate the IgG, it is currently in PBS, I can freeze dry the IgG and then realiquot in a small volume of PBS hence reducing the volume but overall increasing the concentration per ml.

Please let me know what you would feel is the best way to proceed from the 40ml that I have?

thekid

#4 Roo

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Posted 05 August 2009 - 02:03 PM

Thank you for the suggestions. Would like to know what you meant by saying "Amicon Ultra Concentrator following the manufacturer's directions. Just be careful that you don't spin the volume so low that everything precipitates. " Could you be more specific about this, as I am also using a similar device to concentrate the IgG

.

Amicon Ultra Concentrators are made by Millipore (We buy through Fisher Scientific). There are different Mol Weight Cut Offs (MWCO) and sizes (one for 15 ml or less and one for 4 ml or less). You add your solution to be concentrated to the column. As it spins, the liquid spins through thus gradually decreasing the volume. If you spin it dry or close to dry your protein and/or antibody will probably precipitate since it is concentrating at the volume decreases. Depending on the starting volume, it may precipitate earlier because it's already at a concentration that is close to precipitating. I usually have more volume than I can fit in the column so I stop it every 10 - 20 min to see how fast the buffer is flowing through. Once I have an idea of the flow through rate I estimate the volume needed and the time needed to spin. There is a booklet that comes with these so it would be best to read it and/or any booklet that comes with desalting/concentrator columns.

I was told that usually there will be around 10mg/ml of IgG from Rabbit serum, hence comparing to that the 120mg/40ml that I have is less is what I thought. Am I wrong?


In the lit, it usually states that there is around 10 mg/ml of IgG in the serum. I assume you are expecting to get 400 mg of IgG since you purified 40 ml. I never get 10 mg/ml, but then again I don't do this all the time so I may not be the expert you are looking for. I looked back at an old purification and saw that I purified about 22 mg from 35 ml in one rabbit and only 14 mg from 35 ml in another rabbit. You can try repurifying the flow through in case you maxed out the binding in the protein A, but it seems to me that 120 mg of antibody is a life time supply so I'm not sure if you really need more or just want to get the maximum based on the calculations. I'm usually pretty happy with any concentration over 2 mg/ml because we dilute it out for most applications.

Also I have read up that you can freezze dry the IgG and then reconstitute, can that be used here as a way to concentrate the IgG, it is currently in PBS, I can freeze dry the IgG and then realiquot in a small volume of PBS hence reducing the volume but overall increasing the concentration per ml.


I have purchased freeze dried antibodies and reconstituted them as needed, but I have never freeze dried one myself. You might check Current Protocols or other resources regarding the protocol for this. If I were in your shoes, I think I would try some test assays to make sure the antibody is working as expected. If so, aliquot and freeze it. If you really want a higher concentration, I think I would start with only part of it in case you have problems and/or precipitation.

I hope this helps. Like I said, I'm not the expert on this. I am constantly searching for more lit and reference material (The Harlow and Lane book mentioned in my last e-mail is a great resource as well as Current Protocols Online. Check to see if your institution has access to it). Most of the time though, it just trial and error. It's best to read up so you kind of know what to expect, but different antibodies behave differently as do the reagents used to purify them.

Best of luck to you.

#5 Roo

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Posted 05 August 2009 - 02:17 PM

I looked back at an old purification and saw that I purified about 22 mg from 35 ml in one rabbit and only 14 mg from 35 ml in another rabbit.



Oops, I just realized that the purification I was looking at was an immunoaffinity purification meaning that I purified it over resin previously conjugated with my antigen. This means that my 22 mg or 14 ml from 35 ml is specific antibody. If you assume that it is roughly 10% of the total IgG present in the serum then I would have purified 220 mg and 140 mg from 35 ml. It sounds like your purification is on the lower end of my range. NOTE: I usually pass the flow through over my columns multiple times meaning I bind, elute, equilibrate, re-bind flow through, elute, equilibrate, etc. I find that my column doesn't bind a lot. This could be because of amount of resin/antigen present compared to the antibody being passed over, but I haven't really tested that.

#6 mdfenko

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Posted 06 August 2009 - 11:05 AM

Also I have read up that you can freezze dry the IgG and then reconstitute, can that be used here as a way to concentrate the IgG, it is currently in PBS, I can freeze dry the IgG and then realiquot in a small volume of PBS hence reducing the volume but overall increasing the concentration per ml.

if you freeze-dry the igg in pbs then when you reconstitute in a lower volume your pbs concentration will also be higher. then, using pbs to reconstitute will increase the pbs concentration even more.

if you want to avoid co-concentrating the buffer then you have to exhaustively dialyze the igg against water. hopefully the igg will be stable during dialysis and won't bind to the dialysis bag.
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