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The Updated Technique in De Novo Antibody Sequencing

Antibody Sequencing has been a crucial technique in researching and developing various antibodies. There are many manufacturers who are dedicated to make this technique much better.

To differ themselves from the crowd, they have develop something unique, Creative Biolabs, for instance. Their antibody sequencing method is quite unusual.

De Novo  Sequencing of the CDR3 region

While the CDR3 of the light chain is mostly encoded by the germline sequences, the CDR3 of the heavy chain is usually not available in databases. It is encoded by the so called D-segments but these are modified by nucleases and terminal transferases. Typically, only 1-4 AA of a D-segment remain in the matured antibody. The rest of the D-segment is “artificial” and has to be sequenced de novo.

Creative Biolabs generates many overlapping peptides during the fragmentation process, enabling us to sequence very long stretches of unknown amino acids. The high quality of MS/MS spectra in combination with intelligent data mining, allows them to read the CDR3 like a book. The technique is so powerful that they were able to sequence a 20 kDa protein, which had no homologue in the database.

Isobaric amino acids

In contrast to other MS based methods, they can discriminate most isobaric amino acid combinations. E.g.:
• W can be distinguished from GE, AD and SV (by mass difference)
• R can be distinguished from GV (by mass difference) 
• Q can be distinguished from GA and K (by fragment spectra and mass difference) 
• N can be distinguished from GG (by derivatization and fragment spectra) 
• Leucine and isoleucine cannot be distinguished. However, most of these positions can be determined using the corresponding germline sequence (see figure 1). As antigen binding is mostly mediated by salt bridges and hydrogen bonds the impact of Leu/Ile is usually negligible. 

Sequencing of fluorochrome mABs, IgMs and other non-standard antibodies

 Their method is usually not affected by small ligands (FITC, Biotin, Alexa) coupled to antibodies. Larger protein ligands make sequencing slightly more difficult. However, this can be compensated by using a higher protein concentration, because sequencing quality is dependent on sample amount.

 IgMs can usually be sequenced like normal IgGs. Slightly more sample may be required. As the constant region is modified by several glycans, which cannot be cleaved by PNGase F, they can only guarantee complete coverage for the variable part of the antibody. Their method works best with mouse and rat antibodies. However, ~50 antibodies from rabbit hamster and lama have been sequenced successfully. Besides, many older hybridoma produce 2 light chains (one is a nonsense light chain). These mixtures can usually be sequenced, even when the nonsense chain is in twofold molar excess.

Learn more about unique de novo antibody sequencing.

What You Need to Know About scFv/Fab

Fragment antigen-binding (Fab fragment) is a region on an antibody that binds to antigens. It is composed of one constant and one variable domain of each of the heavy and the light chain. Single-chain variable fragment (scFv) is not actually a fragment of an antibody, but instead is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids.

Fab fragments differ from scFv's as they contain both variable domains and constant regions, thus are more complex in construction and ultimate expression. Creative Biolabs is an expert with many years of experiences in antibody production. It has established a solid platform for scFv and Fab constuction. The same heavy and light variable chains used for scFv construction can also be used in the construction of Fab.

There are three routes to have a scFv or Fab target a specific antigen in Creative Biolabs. The first approach is to generate a mouse hybridoma clone, then convert full IgG [or IgM] into a scFv or Fab; the second one is to create an immunized phage display scFv or Fab mouse library, then use the antigen to screen the library; the third method is to use the antigen to screen a premade scFv or Fab antibody phage display library [usually a human one] to get scFv or Fab clones directly. We are also able to convert chicken IgY into scFv or Fab fragments.

Learn more by visiting scFv/Fab Construction.

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