I am in need of some advice about a novel method my supervisor wants me to try to purify immunoglobulin that is specific for the bacterium Neisseria meningitidis. It's sort of affinity chromatography with a twist. He wants me to incubate whole bacteria with the antibody source (probably plasma)and then mix this with some sort of beads and pack a column with it. He then wants me to elute the antibody using a low pH buffer (probably glycine-HCl pH 2.7). I am worried that the bacteria will break up when mixed with the beads, or at the very least that they will not be uniformly dispersed throughout the column. The bacteria themselves are about 1 micron diameter.

Anybody got any advice on suitable matrix components or any other suggestions (other than to find myself another supervisor!!)?

Thanks,
Julia

Can you give us any more info on the beads? I assume that they are of a certain pore size that will capture the bacteria and hold on to them long enough for a wash of the column and elution of the specifically-bound antibodies. I don't think you need to worry about the bacteria breaking up, and you shouldn't worry about whether they are evenly dispersed.
We frequently do immunoaffinity purification of antibodies, but without your 'twist' (our antigen is usually directly coupled to the beads.
We use 50mM Tris pH7 as the equilibration buffer and 0.1M Citrate buffer pH3 as the elution buffer. The Glycine buffer should be fine, but if you are worried about the effect of the low pH on the bacteria, try the slightly higher pH first.
It is advisable to neutralise the eluted antibody as soon as possible (we use 1M Tris pH9 - 150µl is usually enough to lift the pH of 1ml elution to approx 6-7).
You should dilute the 'antibody source' (serum
usually better than plasma) in the equilibration buffer, incubate with the bacteria then add to the beads. The incubation with the beads should probably be stirred continuously. Once packed into the column, wash with equilibration buffer and elute your antibody. The antibody might come through with some of the bacteria, so it might be worthwhile using another stage to separate them (even a 0.2µm filter should do it).
There's probably more info than you need here - since you're at Imperial you probably know most of it already! Let's hope your supervisor doesn't read this site!!

Thanks for the quick reply David!

I don't actually have any beads at the moment - that was what I wanted advice on first. My supervisor just wanted the bacteria evenly dispersed, so we had started off thinking of beads without pores (perhaps silica particles?), but do you think particles with pores would be more sensible?

Why do you think it is unimportant to have the bacteria evenly distributed among the beads? My worry is that they will all migrate to the bottom of the column and then the efficiency of Ab-elution will be much less.
Do you think that the bugs won't break up, or is it that you think it shouldn't matter if they do?

My problems with the bugs breaking up are several-fold. Firstly bacteria contain all sorts of proteases which I could really do without releasing. Secondly, if I end up with a soup of lysed fragments it is going to block the flow through the column. Finally, the whole point of us doing this is to recover immunoglobulins that bind only to surface-exposed epitopes on intact bacteria. Throughout the setting up of the column there will be continual association and dissociation of antibody, so even if at the start the antibody is only bound to surface epitopes, if the bugs lyse during mixing with beads, there is opportunity for Ab to bind to non-external epitopes on fragments. I'll then end up isolating a mixture of antibodies to both internal and external proteins.

I guess I could get round this final point by washing the bacteria thoroughly before they get mixed with the beads, but part of the problem I was having was resuspending such large amounts of bacteria well enough to ensure thorough washing. The column approach was suggested in part to avoid this problem and allow easier washing and eluting.

So do you have any suggestions for a bead type?

Thanks for your help, and sorry if I sound a bit negative about it all - I just keep finding more and more questions!

Why use beads?

Once the bacteria have been incubated with antibody they start aggregating a lot and I guess my supervisor thinks that beads (if in excess) will hold them apart in a uniform suspension. This would then mean that the elution would be more efficient. But I reckon that as soon as a flow of liquid is applied, the bugs will filter through the beads and end up packed at the bottom. My knowledge of physics is limited, and I don't know whether beads would have to be the same density or the same size as the bugs to achieve a uniform suspension.

Any ideas?

Unless you have bacteria that have the tendency to attach to surfaces I would think that they may depart discreetly. Are you the first to try the isolation of these immunoglobulins? There are all kinds of materials offered for purification of immunoglobulins. Do a search in google....

I'm not sure why you need to use chromatography for this. If I was given the task of purifying the antibody specific for surface proteins I can think of two ways of doing so, neither of which use chromatography, and both of which are relatively quick to perform:
1. Incubate the serum with the bacteria. Spin them down (bacteria will fall to the bottom of the tube along with any antibodies bound to them). Wash the 'pellet' - I don't think it would be necessary to resuspend. Respin. Add a low pH buffer, e.g. pH3 (it is probably necessary to resuspend at this stage & perhaps incubate for a short time). Spin again. This time keep the supernatant - this should contain your antibody.
2. Incubate the serum & bacteria. Filter through a small pore size filter (0.2µm or less). Wash. Add low pH buffer and collect the antibody (neutralise ASAP).
There is the possibility that the spinning of the bacteria may cause lysis, so care will be needed (leave the brake off the 'fuge to reduce shearing forces, etc.).
Getting back to the chromatography. I don't see why it is important in this instance to have the bacteria uniformly distributed throughout the beads. The bacteria are going to move as soon as you apply a flow through the column. The idea would be that you get the antibody off before the bacteria come through. If the bacteria start to move as soon as a flow is applied, they are probably going to pass through the column during the wash stage, so the idea would be to use something that will hold back the large bacteria long enough to wash the non-specific antibodies away and then elute the specific antibodies off the bacteria.
To HWM, I know that you are one of the regular experts on this site. Do you know of a matrix that will do what I've just outlined? On the subject of materials for purifying antibodies, none that I am aware of are suitable in this instance. Let us know if you know of anything.

Reply to HWM-
I'm not sure I understood what you meant by "depart discreetly". Do you mean that the bacteria would pass through the column and end up at the bottom?

In answer to your other question about the immunoglobulins - we don't just want to purify all of the IgG (protein G would be fine if that was what we wanted to do). What we want is just the IgG that is specific for a particular species of bacteria (Neisseria meningitidis). And yes, I think we are the first to be trying this approach.

Reply to David-
The first approach you suggested is exactly what we tried. The amounts of bacteria we want to use are necessarily large to allow purification of a sensible amount of Ab (I had 5 pellets of 0.5ml volume each). I found that it was taking at least 5 minutes to resuspend the pellets in the low pH buffer, followed by 2-3 minutes to centrifuge them to allow recovery of the Ab-containing supernatant. Such large amounts of bugs are difficult to resuspend effectively under any circumstances, and with Ab in there as well they aggregate even more. I failed to recover any functional antibody (ie bactericidal Ab) and assumed it was because the Ab was in low pH buffer too long

Does anybody know how long immunoglobulins can survive in low pH buffers before falling apart?

I liked the second approach, but I don't think it will work. If I put a suspension of bacteria through a 0.2 micron filter, it very rapidly becomes blocked. The pressure that would be required to pass wash or elution buffer through would be larger than the filter could cope with, the result being bursting. Filters like that are generally designed for filtering relatively clear suspensions - unless there are some commercially available ones that can withstand higher pressures that you know of?

The bead and column approach that my supervisor wants me to try is mainly to avoid the difficulties with resuspension of large bacterial pellets, and to allow quick elution of Ab. But it seems to be associated with even more potential pitfalls!

So yes, maybe I shouldn't worry so much about the bacteria moving, as long as they are relatively held back so the Ab could be eluted off. If the non-specific Ab could be washed off before combining the bugs with the beads and packing the column then they would only have to stay held back for the elution step.

The question now is: does anyone have a matrix that would fit the bill???

Julia,
with 5 µM particles and 1 µM bacteria one could expect them to be washed out of the column if they didn´t get attached to the beads (agree with David here).
Our experience with mab would indicate that if they denature, or whatever, they do it practically instantaneously. Generally, the stability of proteins is extremely dependent on the protein at hand. Five min. might be a bit short for the chromatography, or the equilibrium which frees your Ab, also? Don,t you have some purified Ab which you can use to determine some of it´s characteristics?
How about resuspending the cells (after washing) with a friendly (to the bacteria) buffer, then shifting the equilibrium away from the bacteria with a immobilized protein G, etc. etc.


Dave,
sorry, my thoughts are "theoretical", but in the back of my head it appears that somebody immobilized cells on beads. Checking "Affinity Chromatography" by Mohr and Pommerening, Decker, 1985, I found only cases were a substace interacting (affinity) with cells were immobilized to sparate cells.

HWM, your thoughts might be theoretical but they reminded me that bacteria and other organisms can be immobilized onto beads.
Julie, do a search on 'immobilized bacteria beads' on Google and see the response!!
I can remember someone in my old lab trying a version of this using beads from Bangs Laboratories (www.bangslabs.com). The bacteria were coupled to the beads using EDC/NHS coupling chemistry. They were trying to get something to bind the bacteria (not antibodies), so I guess that the same principle applies here. If I remember correctly, there was a problem with aggregation but it was overcome. I've tried getting in touch with the chap that did this, but unfortunately he has moved on.