antibody against denatured protein? - basic questions (Apr/26/2007 )

Hello Experts,

We are trying to raise antibodies in Pig against a denatured protein.

My question is how should I prepare the protein sample, such that protein stays denatured and is ready to inject into the animal??

- Run it on SDS-PAGE gel and cut out the band??
- Add urea/Guanidinium chloride and inject?? (this seems unlikely)
- Heat denature and then inject??

How does the protein stay denatured even upon injection (as the denaturing agent will get diluted in the body fluid of the animal)??

Does SDS/acrylamide have any toxicity issues in pig??

How does aggregation of the protein affect this process??

any suggestions/protocols will be greatly appreciated.

thanks.

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hi,

it would be maybe more appropriate to immunize your pig with some peptides derived from the linear sequence of the protein of interest
I am not sure that SDS-decorated protein will be still immunogen
And what about the toxicity of urea or guanidinium into the circulation?

Seb

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Hello Brami!


My question is how should I prepare the protein sample, such that protein stays denatured and is ready to inject into the animal??

- Run it on SDS-PAGE gel and cut out the band??
- Add urea/Guanidinium chloride and inject?? (this seems unlikely)
- Heat denature and then inject??


Some notes for denaturated proteins used for immunization
Because protein purification frequently denatures molecules and synthetic peptides usually do not achieve native conformations, immunization with synthetic peptides and gel-purified proteins generally has resulted in the production of MAb that recognize the antigen in its denatured form. While such MAb may be useful for immunoprecipitation and immunoblot studies, often they are not useful for flow cytometry analysis of cell-surface antigens (UNITS 5.3  5.4) or functional assays that require binding of the antigen in its native conformation

So you should keep it in mind!

HERE PROTOCOL FOR PREPARATION IMMUNOGEN CUTTED FROM PAAG
Note: this suitable for little lab animals but for pigs you need big amount of antigen so before using this procedure think about preparative phorez. But I think this method of AG preparation is not suitable for pigs ( protein amount)
So follow Tryptofan suggestions as one of the possible ways to prepare antigen

IMMUNIZATION TO PRODUCE POLYCLONAL ANTIBODIES USING FREUNDS ADJUVANT


Rabbit, rat, mouse, or hamster of appropriate strain
Complete Freunds adjuvant (CFA; Sigma)
1 to 2 mg/ml purified protein antigen in PBS
Incomplete Freunds adjuvant (IFA; Sigma)

50-ml disposable polypropylene centrifuge tubes
3-ml glass syringes with 19-, 21-, and 22-G needles
Double-ended locking hub connector (Luer-Lok, Becton Dickinson) or plastic 3-way stopcock


CAUTION: CFA is an extremely potent inflammatory agent, particularly if introduced intradermally or into the eyes and may cause profound sloughing of skin or loss of sight. Self-injection can cause a positive TB test and lead to a granulomatous reaction. Use gloves and protective eyewear when handling CFA.

1. Bleed the animal prior to immunization and collect blood sample in a 50-ml centrifuge tube. Prepare serum from blood and assay and store (see Support Protocol).

This preimmune bleed is critical as a control to ensure that the antibody activity detected in later bleeds is due to the immunization.

2. Shake CFA to disperse insoluble Mycobacterium tuberculosis bacilli. Add 2 ml CFA to 2 ml of 0.25 to 0.5 mg/ml purified protein antigen in PBS at 4C.

These volumes produce immunogen sufficient to immunize 4 rabbits or up to 80 mice. Do not use Tris-based buffers for generating the emulsion.

An effective and simple method for preparing purified protein antigen is by preparative SDS-PAGE
If a standard-size 1.5-mm slab gel is used with a large-toothed comb, as much as 2 mg of a homogeneous protein can be loaded across the entire gel. Following electrophoresis, an edge can be sliced off with a razor blade, fixed and stained, and used to identify the region containing the protein band The gel slice containing the protein may then be directly added to several milliliters of PBS and emulsified as described below with an equal volume of CFA. The acrylamide serves as an additional component for the protein depot provided by the adjuvant.

3. Draw up the CFA/antigen mixture into a 3-ml glass syringe with a 19-G needle. Remove needle, expel as much air as possible, and attach syringe to the double-ended locking hub connector or the plastic 3-way stopcock. Attach an empty 3-ml glass syringe at the other end and force the mixture back and forth from one syringe to the other repeatedly. When the mixture is homogeneous and white, disconnect the connector or stopcock, attach a 21-G needle, and test whether the emulsion is stable by extruding a small drop onto the surface of 50 ml cold water in a 100-ml beaker. A good oil-in-water emulsion should hold together as a droplet on the surface of the water. If the drop disperses, mix the antigen using the hub- connected syringes until it forms an emulsion.

Heat will be generated by this procedure. Chill on a bed of ice from time to time to keep the mixture as close to 4C as possible.

4. Transfer all of the adjuvant-antigen emulsion to one syringe and remove the connector or stopcock. Attach a 22-G needle to the syringe and remove air bubbles.

5. Restrain the animal and inject the adjuvant/antigen emulsion into multiple intramuscular (i.m.), intradermal (i.d.), or subcutaneous (s.c.) sites.

Discard the unused immunogen. For extremely valuable antigens, the emulsion may be stored at 4C for several weeks and reemmulsified before use. denaturation of protein antigens may take place under these conditions.



I saw in literature about usage of 8M urea injection for immunization for little lab animals. But can’t find it now.



How does the protein stay denatured even upon injection (as the denaturing agent will get diluted in the body fluid of the animal)??

It is not correct suggestion that under dilution proteins renaturate correctly. In most cases it is not so. Refolding ( to native active state) is a complex enough sophisticated process and unique for certain protein


Does SDS/acrylamide have any toxicity issues in pig??

It is depends on conc. But I think that not so because you use for immunization polymerized acrylamide . IT IS ONLY foreign matrix for organism and organism remove without serious problems I think.


How does aggregation of the protein affect this process??

Aggregation will increase immunogenicity of your antigen. From the other side I think that it may have negative result, masking your epitope of interest

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