Silicon Investor (SI) -- The First Internet Community

STOCKTALK

We've detected that you're using an ad content blocking browser plug-in or feature. Ads provide a critical source of revenue to the continued operation of Silicon Investor. We ask that you disable ad blocking while on Silicon Investor in the best interests of our community. If you are not using an ad blocker but are still receiving this message, make sure your browser's tracking protection is set to the 'standard' level.

Biotech / Medical : momo-T/FIF -- Ignore unavailable to you. Want to Upgrade?

To: tuck who wrote (2934)	8/10/2005 7:13:23 PM
From: scaram(o)uche	Read Replies (1) \| Respond to of 12215

Need two antibodies. Need one that's isoform-specific, and that is most often a piece of cake. But you'd also need something that could be negative in the assay, next to any isoform-positive result. That antibody would need to recognize an epitope that is dependent upon the N-terminus. Polyclonal work can tell you if that's possible, pretty quickly, if available literature doesn't. Yeah, I think I could do it. Vox Sang. 1984;47(6):412-20. Detection of IgG-associated determinants in reduced and alkylated preparations of human IgG3 by monoclonal antibodies. Brown AM, Dumas ML, Reimer CB, Louie RE, Harmon RC. Using classical typing antisera, previous experiments have failed to demonstrate IgG3 in partially reduced and alkylated preparations of human IgG intended for intravenous application (IGIV). To establish that IgG3 is actually present in such preparations, we designed an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies as solid-phase reagents and protein A-purified IgG3 as antigen. Three different samples of reduced and alkylated antigen were used: (1) IgG3 isolated from a ready-for-infusion IGIV; (2) IgG3 which was purified from an intramuscular (Cohn fraction II) IgG solution before being subjected to a mild reduction and alkylation procedure, and (3) completely reduced and alkylated IgG3. The reduction and alkylation procedure did not affect the solubility of IgG3, indicating that IGIV prepared in this manner should contain normal quantities of IgG3. In the ELISA, solid-phase monoclonals which were cross-reactive with multiple IgG subclasses clearly reacted with reduced and alkylated IgG3. Furthermore, there was no substantial difference between the quantities of modified and native antigen required for 50% maximal ELISA signal. In contrast, solid-phase monoclonals with IgG3-restricted specificity did not recognize reduced and alkylated material. These results indicate that IGIV prepared by reduction and alkylation has a normal IgG3 content and confirm that some IgG3-specific determinants are altered by the modification procedure. For those who read this and ask....... "uh....... why?"?? Marketing wanted the study. Long story. :-)

To: tuck who wrote (2934)	8/12/2005 12:39:03 PM
From: former_pgs	Read Replies (1) \| Respond to of 12215

OT: I realize that you wanted an ELISA, but... It seems that focusing the protein by 2D SDS-PAGE or resolving it by 1D SDS-PAGE (you can fiddle with the gel concentrations to resolve) followed by Western blotting with an antibody against a common part of the protein (that is not cleaved off) would be more powerful than the ELISA. The detection limit / sensitivity would be basically the same, but the above two methods would be more powerful in that you would get some absolute numbers regarding the % of the protein cleaved. ELISA won't give you such numbers unless you know the affinity of the antibodies that you're using. Just in case you are not married to the ELISA technique... the above still qualifies as an immunoassay of sorts ;-)

To: tuck who wrote (2934)	8/25/2005 10:44:56 PM
From: scaram(o)uche	Read Replies (2) \| Respond to of 12215

Your question brought out fun commentary. Thanks. Polymorphism within the MHC is extreme. I was lucky as heck. For decades, the lab of George Snell and numerous other labs which recognized the brilliance in his approach had focused on dissecting the MHC with polyclonal antisera. It was a rush to isolate antibodies which recognized an epitope that Snell et al. (in the very large sense, as he cast a long shadow........ a frigging gentleman and unassuming guy, I might add) had characterized with a titer of 1:20 or sumthin (and they busted their butt to get that!), and find that you could ten-fold dilute seven times before starting your assay. It was a bigger rush to discover that one out of two antibodies found something that a score of the best had missed for years. Antibodies are fun. Wholesome, good for the entire family. I'm moving, folks. Gonna be spotty in the next week. Small-cap, big-science....... great summer. Phenomenal summer. Heaven. Best, all! Rick