polyclonal Ab against the variable region

[pery]

Hi all,

I need help please.

I want to produce polyclonal Ab against the part of the variable region (CDR1 mostly) of human monoclonal Ab (IgG).

In my previous trials I've injected mAb into the rabbit and cleaned the pAb on G-sepharose column. Eventually I’ve got the non specific pAb population i.e. it recognizes many common IgG determinants.

I have very little quantity of the mAb so I cannot slice it in order to rid of the common parts (like Fc for example).

Please advice if there are ways to produce determinant specific anti-Ab antibodies (poly or mono).

Thanks in advance.

Pery

[scicop]

You're using a big protein in an attempt to get a fragment specific antibody. The nature of polyclonals is that they will recognize multiple sites of your antigen. If you're using an entire mAB to create pAB specific to a region of the mAB, you're bound to get alot of not specific pABs!!

 

You would need to produce that CDR1 region (recombinantly) and inject that into the rabbit to get a specific pAB (or mAB) I think you're wasting your time injecting the entire mAB, you're just gonna get pAB that recognize the entire multiple sites on your mAB fragment.

 

Also, how are you screening for CDR1 selective binding, I assume you're have some form of CDR1 protein to do the screen right? Remember, your antibody is only as good as the screen you develop for it! If you are using a CDR1 fragment in your screen that maybe you can use that as an antigen. REALLY think about your screen if you have to alternative but to use your mAB as an antigen. If you're using the entire mAB in your screen then you're fighting a losing battle. I did my screens using ELISA, although i've know others that use western blot, I found ELISA more quantative. Whatever you use for screen, just make sure it will give you specific results.

 

There are other tips I can give..but I think you're wasting you're time if you're not using CDR1 fragment an antigen or a screening tool.

 

I've personally made monoclonal antibodies (using a subtractive hybridization method), and I can say its VERY labor intensive (it took amost 8 months to produce), so I would avoid that route (this is why your screen is essential!!)

 

The other question I have is why do you need an antibody to the variable region, in other words what is the nature of the experiment you intend to do? They can be other ways to get at the same question.

[pery]

Dear Scicop,

Thank you very much for the fast reply.

You are absolutely right re the polyclonal population.

As I have mentioned before, my problem is that I have a very small quantity of the mAb so I am not capable to produce it's CDR1.

I have done the screening (by ELISA) with whole mAb in addition to the screening against normal human IgG as a control group. I hoped that may be control group will provide a weaker signal. But unfortunately I get a higher signal even than mab that I have immunized with.

Regarding your question:

I have this mAb which is human and bearing common 16/6 idiotype.

I need to develop an ELISA method for screening of human antibodies in serum for presence or absence of antibodies bearing this common 16/6 Id.

Therefore I need pAb or mAb that can recognize this idiotype.

I would use an anti-idiotyping network but it works only than the antibodies injected within the same species, so it is not particle in my case (we are talking about humans).

I appreciate a lot your professional opinion and will glad to here if you have any ideas regarding my problem.

Thanks in advance,

Pery.

[scicop]

Dear Scicop' date='

Thank you very much for the fast reply.

You are absolutely right re the polyclonal population.

As I have mentioned before, my problem is that I have a very small quantity of the mAb so I am not capable to produce it's CDR1.

I have done the screening (by ELISA) with whole mAb in addition to the screening against normal human IgG as a control group. I hoped that may be control group will provide a weaker signal. But unfortunately I get a higher signal even than mab that I have immunized with.

Regarding your question:

I have this mAb which is human and bearing common 16/6 idiotype.

I need to develop an ELISA method for screening of human antibodies in serum for presence or absence of antibodies bearing this common 16/6 Id.

Therefore I need pAb or mAb that can recognize this idiotype.

I would use an anti-idiotyping network but it works only than the antibodies injected within the same species, so it is not particle in my case (we are talking about humans).

I appreciate a lot your professional opinion and will glad to here if you have any ideas regarding my problem.

Thanks in advance,

Pery.[/quote']

 

Ok, well, I'd stop where you are. I think you need to go with trying to produce a monoclonal antibody, I feel you're wasting your time going down the pAb route. That being said you need a new batch of your bait mAb and ALOT of it. You will use alot of it in screening, because when you screen for monoclonals, you will screen ALOT of spleenocyte colonies. Each well you screen will represent a specific colony, and usually it is done in triplecate.

 

Before you start any monoclonal ab production you need to contact an expert. I have done it before, but I am limited with my ability to help you on-line. You basically need some to hold your hand (NOT KIDDING) when you start in. There are tricky parts to mAb production including immunizing, fusion, spleen cell selection, colony selection and growth, and screening. There is also a level of organization skills that will be necessary to develop and/or possess. Usually you can find such expertise in your institutions tissue culture facility. They will have the resources and equipment to help you.

 

Also, monoclonal Ab production is very time consuming and very expensive (consider your salary as well as the cost for tissue culture material and screening reageants..you will use LOTS of the stuff). You may wish to consider asking your PI to outsource the job. It may not be cheaper in the long run, but it will save time and you'll have an endless supply once you have the cell line. Also, you can use your time more wisely with other projects. Also, the risks will be higher if you have a novice try to make a mAB and you're in a rush for data. I failed on my first attempt, set me back a couple months. Got it with my second, but with alot of HAND HOLDING. mAB production takes a few times to master and even then, you always learn new tricks.

 

If you want and you have the time to learn mAB production, it can be a very vaualbe skill to have, pharmaceutical and biotech companies will compete for you. So, its basically how much time and energy you're willing to invest.

 

Good luck!!

[pery]

Thanks a lot for the explanations!

I think I will outsource the mAb production.

Best regards,

Pery