[Histonet] CD3 clean with RB monos
koellingr <@t> comcast.net
koellingr <@t> comcast.net
Sun Apr 29 12:06:47 CDT 2007
thanks for this tremendous reply. I hope many have copied it for you have provided a wealth of background information that goes to core of why IHC even works in the first place. I still have an uneasy feeling when I hear or read advertisements from companies regaling nano to pico molar potential improvements. My sense of theory is suddenly shocked until I can think this through. At that 25 square angstrom or so (or 3,000 cubic angstrom or so), whichever kind of figure you want to use, the 4 non-covalent forces of hydrogen bonding, electrostatic interactions, hydrophobic interactions and Van der Waals dispersion interactions, rule the day and determine strength between the binding "pocket" and its binding partner. But amino acids are amino acids whether from mouse or rabbit. The degrees of freedom of the carbons and/or nitrogen in the amino acid chain are set not by whether it is mouse or rabbit. All chemistry (of the atoms themselves) is the same for mouse or rabbit. So whi
le things might certainly be different between mouse and rabbit (accessory molecules or amino acid distributions or breaking of immunological tolerance or somatic hypermuation differences or anything like that) I just can't see how the binding sites, which affect the strength of the binding, can be up to 1,000 fold different.
But if there are immunological type chemists out there who can explain it to the Histonet, I'd be super happy to hear from you.
Thanks again Marvin,
-------------- Original message --------------
From: Marvin Hanna <mhanna <@t> histosearch.com>
> Hi Ray,
> Thanks for the additional input. I stand corrected. Herceptin is a
> HUMANIZED mouse monoclonal antibody. From Dr. Kimball's online
> Biology textbook, "The [Humanized] antibody combines only the amino
> acids responsible for making the antigen binding site (the
> hypervariable regions) of a mouse (or rat) antibody with the rest of
> a human antibody molecule thus replacing its own hypervariable
> regions." This helps reduce the problem of HAMA (human anti-mouse
> antibodies), which can cause damage to the kidneys and cause the
> therapeutic antibodies to be quickly eliminated from the human patient.
> I will also qualify my statement, "with up to a 10 times greater
> affinity" with "according to a number of IHC vendors of rabbit
> monoclonal antibodies". I have found at least 4 making claims of
> higher affinity. The Epitomics website claims that mouse monoclonals
> have affinities of "Nanomolar (~10-9 KD M)" and rabbit monoclonals
> have affinities of "Picomolar (10-12 KD M) possible", a thousand fold
> potential increase. I did not know that there are researchers who
> disagree with these statements and appreciate you pointing it out. I
> agree additional research should be done.
> According to a news release on the Epitomics website, they are
> providing humanized antibodies for therapeutic research. It is early
> to speculate that (possible) higher affinities of humanized rabbit
> monoclonals will provide a better therapeutic response in patients
> compared to humanized mouse monoclonals and will require many years
> of research and clinical trials to determine.
> I also agree I have used rabbit monoclonal antibodies in IHC that
> have not given better results, and sometimes worse results, than
> their mouse monoclonal counterparts.
> Best Regards,
> Marvin Hanna
> mhanna <@t> histosearch.com
> On Apr 23, 2007, at 4:32 PM, koellingr <@t> comcast.net wrote:
> > PS.
> > As far as I know Herceptin (trastuzumab) is a HUMANIZED monoclonal
> > antibody and not a mouse or rabbit monoclonal. While certainly
> > many things are possible, I'm skeptical of rabbit monoclonals, no
> > matter how great they are for IHC, going into human therapeutics in
> > light of the norm which is to humanize these reagents. Unless you
> > were to "humanize" the rabbit monoclonal itself similar to
> > humanizing mouse monoclonals.
> > Ray Koelling
> > Phenopath Laboratories
> > Seattle, WA
> > -------------- Original message ----------------------
> > From: koellingr <@t> comcast.net
> >> Rena,
> >> I agree that the epitomics website is great for learning about
> >> this technology
> >> and I further agree it could be of help as has been mentioned
> >> before. I am a
> >> bit skeptical of claims of a 10-fold better affinity than mouse
> >> monoclonals as a
> >> blanket statement.
> >> At a previous meeting a while back we asked about claims of higher
> >> affinity and
> >> couldn't get a sufficient (to our mind) response. To me that
> >> response would be
> >> in the form of a side to side comparison, using the same
> >> immunogen, and with a
> >> host mouse and rabbit side by side each producing monoclonals to
> >> same target to
> >> see which might be better. I believe the concept of a superior
> >> rabbit
> >> monoclonal technology in terms of being able to break
> >> immunological tolerance in
> >> the immunized systems, especially for some difficult targets, is
> >> probably right
> >> on.
> >> But simply breaking tolerance is not the same as producing higher
> >> affinity
> >> antibodies.
> >> For the blanket statement that rabbit monoclonals have higher
> >> affinity, I'd like
> >> to see data comparing them to their (identical) target in a mouse
> >> host and see
> >> data such as from Biacore, x-ray crystallography and that
> >> differences in somatic
> >> hypermutation are indeed making these higher affinity.
> >> That being said, I agree that the rabbit monoclonals have great
> >> use and even
> >> more potential. I've used several (many) that are far superior to
> >> their
> >> counter-part mouse monoclonals. However, I've used (and heard of)
> >> a few that
> >> weren't as good.
> >> So while I like rabbit monoclonals, use them, advocate for them
> >> and endorse the
> >> suggestion you try them, I'm not convinced that as a rule they
> >> have 10x higher
> >> affinity than do mouse monoclonals.
> >> Ray Koelling
> >> Phenopath Laboratories
> >> Seattle, WA
> >> -------------- Original message ----------------------
> >> From: "Mildred Fail"
> >>> We have had quite a problem with CD3s on bone marrow biopsies being
> >>> "messy" both with mouse monoclonals and rabbit polyclonals. Protein
> >>> block is used. Diluting the Ab out further lost some cells in the
> >>> lymph
> >>> node control. We tried a rabbit monoclonal. The staining is very
> >>> specific and intense. The slide is beautifully free of extraneous
> >>> staining. The higher dilution has not appeared to have effected the
> >>> number of cells stained. Question is why would the rabbit
> >>> monoclonal produce a cleaner slide?
> >>> Rena Fail
> >>> Rena Fail
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