There are paper where is written that "a protein is less express in vitro compare to in intact tissue", such as caveolins. The epitope is recognized by antibodies by its linear sequence of amino acids, or primary structure. Most antibodies recognize a conformational epitope that has a specific three-dimensional shape and its protein structure.
Could it be that some epitopes lose their conformational structure in vitro and in turn leading to an reduction of antibody affinity? Could it be an explanation why WB or IHC detect less proteins in vitro compare to in vivo?
Dear Mario,
I do agree with you that epitope loses its conformational structure in vitro system leading to a less affinity against the antibody. Support to this notion a study entitled " Reducing Epitope Spread during Affinity Maturation of an Anti-Ganglioside GD2 Antibody" which was published by Cheung's group in the journal of immunology:
Reducing Epitope Spread during Affinity Maturation of an
Anti-Ganglioside GD2 Antibody1
Jian Hu,* Xiaodong Huang,* Chang-Chun Ling,† David R. Bundle,‡ and Nai-Kong V. Cheung2
*
Ab affinity maturation in vivo is always accompanied by negative selection to maintain Ag specificity. In contrast, in vitro affinity maturation can lead to epitope spread, resulting in loss of specificity. Anti-ganglioside-GD2 mAbs are clinically
effective against neuroblastoma; pain and neuropathy are major side effects. We used structural relatives of GD2 to define epitope spread during in vitro affinity maturation of an anti-GD2 single-chain variable fragment (scFv) called 5F11-scFv.
Clonal dominance identified by polyclonal sequencing was confirmed by analyzing individual clones. Affinity-matured mutations were introduced into scFv-streptavidin for functional studies. Without a negative selector, 19-fold affinity improvement
(clone Q, where Q is the symbol for glutamine) was associated with strong cross-reactivity with GM2 and GD1b and moderate cross-reactivity with GD3, resulting in positive immunohistochemical staining of all 13 non-neural normal human
tissues, in contrast to none of 13 tissues with parental clone P. With GM2 as a negative selector, clone Y (where Y is the symbol for tyrosine) was generated with only weak cross-reactivity with GD1b, adrenal and thyroid glands, and no staining
of other non-neural normal tissues. Even though there was only a 3-fold affinity improvement, clone Y showed significantly higher tumor uptake over parental clone P (134%, p 0.04), whereas clone Q was inferior (54% of clone P; p 0.05) as
confirmed by tumor-to-normal tissue ratios across 16 organs (41% of clone P; p < 0.0001). Using the less efficient negative selector GD3, a clone mixture (Q, V, and Y, where V is the symbol for valine) emerged. We conclude that epitope spread
during affinity maturation can be reduced by negative selection. Furthermore, efficiency of the negative selector depends on its cross-reactive affinity with the matured scFv. The Journal of Immunology, 2009, 183: 5748 –5755.
For more details, please see attached file.
Hoping this will be helpful,
Rafik
Dear Rafik,
many thanks for your answer.
So my next question is : If the epitope loses its conformational structure in vitro system leading to a less affinity against the antibody, could it affect the protein at function level?
Thank you once again
Best Regards
Mario
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