Looking for sources of alpha-chymotrypsin, Trypsin and Pronase to help with our ID's of antibodies to high-prevalence antigens.  Any suggestions or personal experiences on where to source these enzymes from? We have freeze-dried papain and DTT already in-house.

Thanks!

Sigma-aldrich?

Preparation and standardization of enzymes is notoriously VERY difficult (making trypsin is a nightmare). Each batch of source material may have a very different activity level than the previous. Stability is also an issue, even when frozen.

You can probably get all of the enzymes you list from Sigma, but they will undoubtedly have 15 versions of each and it may be difficult to choose which flavor is most suitable.

I would stick with papain and DTT. They'll be most useful, most often. You'll get a lot of information from just those two. Only the very highest-level Reference Labs. should mess with alpha-chymotrypsin, trypsin and pronase.

I agree, my experience with trypsin is not good either.  It took us quite a few tries to get it correct - the stability was the biggest issue.  It took lots of time to figure that out

Thanks for your replies! 

Yeah I'm seeing that especially with trypsin.  Using Judd's Methods (and the BAEE units of activity per mg calculation) and trying to choose a trypsin on Sigma's website has already proven to be quite a headache

Edited September 15, 20177 yr by BBNC17

Curious as to the benefits vs. the time it takes to prepare, validate and store these enzyme stock solutions (alpha-chy, pronase and trypsin)? 

Currently at a reference lab that would likely send antibody to high-incidence antigen workups out as we don't have much access to rare antisera and cells at the moment.  However, before we send it out (or while it's being worked up), we would like to at least try a narrow down the classification of the antibody and also perform enzyme and/or DTT treatment on pheno-similar cells, or adsorb out the antibody, to investigate any underlying allo to common antigens.  This way we can at least provide the hospital with a preliminary report of the patient phenotype and a potential aby to high-incidence antigen and any ID'd underlying aby.  

Eventually, when we build up our rare antisera inventory, we'd like to perform these IDs in-house.  For now, do you think DTT and papain are sufficient enough since we are sending these workups out anyways?  Looking at a few enzyme/chemical reactions on high-prevalence antigen charts and, other than -Yta, trypsin doesn't seem to determine any of these abys.

 

It sounds like if at that point (after throwing everything at it including DTT and papain) you don't have the rare antisera/cells and reagents to identify the antibody then you are going to have to send it out anyways and the reference lab will probably end up repeating most of that testing in order to ID the reactivity on their end.  You are right, you have to consider that implementing trypsin is a big project with SOP revisions, may involve added costs, is known to have stability issues, and how often do you expect you will really need it and keep your staff competent?  Will it really have any added benefit to your testing?  

From personal experience in a smaller reference lab with limited rare reagents as well as in a larger IRL, I would say it sounds like you may want to wait on the trypsin until you have a better inventory of rare reagents to aid in completing antibody ID?