Antibodies are sequenced from the beginning

online search
"
antibody
sequencing from the beginning", the search results will show a long list of resources and services. There are too many descriptions of the technology, and many of them are much the same. Small editor want to use this article to tell you why we need antibodies from the beginning sequencing and how to sequence antibodies from the beginning.
the classification of antibody sequencing from
antibody sequencing from the beginning is distinguished according to the type of antibody sample. We usually describe antibody
based on their"clone"
(clonality). Cloning refers to the number of different antibody sequences or genetic linelogies present in the sample.
: The sample contains antibodies with the same sequence. Monoclonality is a prerequisite for antibody sequencing from the beginning. In rare cases, a simple mixture of monoantials can be sequenced
protein
group, but this is not necessarily a guarantee of success.
: The sample contains antibodies with different sequences, usually with a large quantitative range. If the antibody
serum
is indicated as a multiclonal antibody.
: Samples contain a limited number of anti-system spectrals, defined by
's decision cluster complementary region
(
complementarity determination regions,
CDR
s
).
the above antibody classification, antibody sequencing from the head is divided into monoclonal antibody sequencing from the head, multiclonal antibody sequencing from the head and oligoclonal antibody sequencing from the head. This paper mainly focuses on monoclonal antibody sequencing from the beginning.
do I need antibodies to sequence from the beginning?
two common methods for restoring monoclonal anti-weight chains and light chain sequences. The most direct method
sequence transcripts or genetic material of B cells in the united States. This method is suitable for hybrid tumor,
phage
display,
yeast
bacteria display or single-cell screening, is the most cost-effective and reliable method of antibody sequencing. However, the source cells are not always available. If hybrid tumors are lost, genetic material may not be available. In this case, antibody sequencing can be used to restore the antibody sequence, for example using Edman degradation or
mass spectrometry
analysis techniques.
is based
The sequencing of Edman degradation
Edman degradation, also known as Edman sequencing, is a mature protein sequence determination technique that starts at the N-side and reads one
amino acid
at a time. The main advantage of Edman degradation sequencing is the low sample size requirement (usually less than 1 ug). The mass of Edman degradation sequencing decreases as the number of amino acid processing increases, so the sequencing is typically used to determine the first 30-50 amino acids and is rarely used for antibody full sequence determination.
antibody sequencing based on mass spectrometry
mass spectrometry has become the preferred method for antibody full sequence assay. In almost all mass spectrometry scenarios for antibody sequencing, antibodies are broken down into
15-20 amino acid-length peptides, which are then analyzed in a mass spectrometer. Ideally, each peptide produces a fragment map. When sequencing antibodies from the beginning, each fragment map needs to be parsed and a portion of the antibody sequence revealed. Typically, we use a variety of enzymes with different cutting sequences to break down antibodies, ensuring that each area of the antibody produces several overlapping peptides. As shown in Figure 1, by producing overlapping peptides, antibody sequencing techniques can recombine the antibody sequence in a manner similar to that of the bird gun sequencing assembly
gene
group.
analysis and antibody sequencing from the beginning
here, it is necessary to distinguish between peptide spectrometry and antibody sequencing from the beginning. The goal of peptide spectrometry is to confirm known sequences, and antibody head-sequencing is the process of obtaining previously unknown sequences from fragment maps or map collections. Peptide spectrometry tends to require fewer enzymatic and mass spectrometry runs than antibody sequencing from the beginning, which means it can be less expensive.
figure
1. Antibody sequencing process from
.
, antibody samples are broken down using a variety of enzymes. Each enzyme has a different cutting pattern (expressed in different colors) and each enzyme does not produce new peptides for the antibody sequence. Antibody sequencing from the beginning uses overlapping peptides produced from multiple enzyme decompositions to achieve complete coverage of the antibody and ensure that no sequence is missing.
so what are the advantages of monoclonal antibody sequencing? The monoclonal antibody sequencing technology of
100te Parker
is summarized as an example:
1, the 100% full sequence of antibody molecular light and heavy chain can be achieved;
2, combined with PAKS and other mass spectrometry analysis software, developed a targeted data processing algorithm, do not miss any valid data information
。