In today’s modern world, biotechnology has gained significant ground in the commercial market. Gone are the days when a simple antibody test would take forever. However, if you’re wondering which antibodies we’re referring to, you might be surprised.
Even though antibodies are important proteins that the immune system produces to protect the body from harmful pathogens, they attach themselves chemically to the substances the body identifies as foreign, like viruses, bacteria, and other foreign matter in the blood.
On the other hand, industrial antibody-based therapeutics have become quite popular in the health industry due to their wide range of applications. As a result, this has increased the demand for the accurate sequencing of these proteins.
Antibody protein sequencing is an efficient and reliable way of assessing a protein’s amino acid sequence for identification and characterization. Contemporary biochemistry and biology depend heavily on commercial antibodies. Notably, the United States spends approximately $2 billion annually on these antibodies. However, most of them seem ineffective and could accelerate the “reproducibility crisis” that biological sciences are experiencing.
This post will provide an overview of antibody protein sequencing services, including the basics of antibody sequencing, its growth, the techniques used to sequence antibodies, alignment tools, and more. Please continue reading to discover more about these sequencing services.
What is Antibody Sequencing?
As the introduction mentions, antibodies are fundamental components of one’s immune system, fighting sickness and infection and keeping the body safe. Antibody protein sequencing involves identifying the order of amino acids in an unidentified antibody without needing a hybridoma cell line or mRNA.
Many researchers and scientific organizations rely on these services, including antibody engineering, function optimization, database banking, obtaining new antibody clones, and more. These disciplines evaluate the amino acid makeup to understand their sequencing – whether monoclonal or polyclonal, from a humanized, recombinant, or hybridoma.
Mass spectrometry has been the preferred method for sequencing proteins in recent years, and it supports amino acid sequencing from start to end. It provides better accuracy and facilitates sequencing even if you don’t have the DNA sequence. Conjugated or contaminated antibodies can also be sequenced, and the results from this technique are highly precise.
On the other hand, de novo antibody sequencing evaluates the mass difference between fragment ions to identify the residue mass in a peptide sequence. It is theoretically feasible to obtain the peptide sequence since most amino acids have a unique mass, except for isoleucine and leucine. However, numerous other techniques exist for identifying the protein sequence in unknown antibodies. But first, let’s discuss antibody sequence alignment tools.
Antibody Sequence Alignment Tools
Did you know antibody sequences are crucial in therapeutic development because they help in antibody engineering and protein characterization? Conducting sequence alignment/analysis offers users of antibody reagents important information that helps identify cross-reactivity and binding, both of which are key prerequisites for rational experiment design. Several antibody databases are available online, storing different data types, such as usage statistics, suppliers, and publication citations. These antibody databases are often employed to support reproducibility.
Employing an antibody sequence alignment tool helps decipher a particular antibody that has not been previously sequenced. However, when researchers know the sequence, they can use other antibody sequence databases to analyze it.
Some of the top antibody sequence databases comprise:
1. International ImMunoGeneTics (IMGT)
The International ImMunoGeneTics data system, or IMGT, is an integrated knowledge resource that many organizations use as a global reference. This information system specializes in immunoglobulins (Ig), Major Histocompatibility Complex (MHC), and T cell receptors (TCRs) molecules found in humans and most vertebrates.
The database consists of archives detailing antibody sequencing, structure, and genome. For example, IMGT/mAb-DB, a monoclonal antibody database, is widely used in clinical setups and fusion proteins in immune-based research.
2. abYsis
This online database for antibody research provides a central storehouse for structural and sequence information regarding antibodies. You can apply the abYsis database in three ways:
- To perform a database search to find more information about sequences, like thorough annotations, post-translational modification sites, locations of odd residues, and more
- To assess constant patterns in the database sequences
- To input sequences for further examination
3. UniProt
This database is one of the highest-quality, readily available, and most extensive functional and protein sequence information databases. Since UniProt is not primarily focused on antibodies, it only has a limited number of non-germline antibody sequences and a representative sample of germline antibody sequences.
4. SabDab
Also known as the Structural Antibody Database, SabDab is an online database comprising all the antibody structures available in the PDB. These structures are standardized and annotated. Users employ these traits and structural factors, such as variable domain orientation and complementarity-determining region (CDR) loop conformation, to narrow their search for the proper structure.
5. ABCD database
Established in 2020, this database is an assortment of sequenced antibodies comprising protein structures, cross-links of regulated chemical databases, and curated data on various antibodies and their antigens. Every antibody is assigned a unique ID number for scholarly publications to enhance experimental reproducibility.
Polyclonal Vs. Monoclonal Antibodies
The terms "polyclonal" and "monoclonal" refer to how the immune system makes antibodies. The B-cell lymphocyte differentiates into memory B-cells and plasma cells in response to a foreign antigen binding to its surface antigen receptor. This produces antibodies that are unique to the triggering antigenic epitope.
Since antigens differ in size, from small molecules to massive recombinant proteins, a single antigen could include many epitopes. Since numerous B-cells can detect the same antigen, the immune response comprises antibodies generated by different B-cells that target different epitopes on the same antigen. This is known as a polyclonal response. In simple terms, a polyclonal antibody is a collection of antibodies produced by several B-cell clones in response to the same antigen.
Assume that just one B-cell responds to a specific antigen. Because this cell detects just one epitope, it will grow, divide, and generate antibodies specific to that epitope. This is known as a monoclonal reaction. A monoclonal antibody is a set of antibodies generated by a single B-cell clone specific to a particular antigenic epitope.
Between monoclonal and polyclonal antibodies, the former has proven highly reliable in research, diagnostic, and biotherapeutic production. Monoclonal antibodies possess high specificity, as they can only attach themselves to a single epitope of an antigen. This is why they are gaining massive popularity, and their use continues to grow. In contrast, polyclonal antibodies, which are less consistent and stable, can attach themselves to various epitopes; hence, they are more likely to experience cross-reactions.
Techniques of Antibody Protein Sequencing
There are numerous methods used for protein sequencing. Traditional Sanger sequencing, suitable for small-scale applications, was the pioneer in identifying an antibody’s nucleic acids. Large-scale techniques, known as next-generation sequencing, are more modern and effective strategies. Modern antibody sequencing includes Edman degradation, mass spectrometry, de novo sequencing, hybridoma sequencing, etc. Let’s discuss these techniques in more detail.
1. Edman Degradation
Edman degradation is a chemical method that involves breaking down the amino acid sequence of a protein one amino acid at a time. This technique depends on removing one amino acid and its subsequent identification, then moving on to the next. This method has been used for decades and is still considered reliable for sequencing proteins. However, scaling up for large-scale sequencing projects is time-consuming and challenging.
2. Mass Spectrometry
Mass spectrometry is a potent analytical technique that determines the mass-to-charge ratio of individual molecules. In protein sequencing, mass spectrometry fragments the protein into smaller peptides and analyzes them to determine their sequence. This method is highly sensitive and can sequence proteins with high accuracy.
3. Hybridoma Sequencing
Hybridoma sequencing entails sequencing the cDNA in the hybridoma cell line’s variable light (VL) and variable heavy (VH) domains. This technique is one of the most economical protein sequencing techniques, as long as the cell line that produces the required antibody is easily accessible.
4. Next-generation Sequencing
Next-generation sequencing (NGS) is a high-throughput method that has revolutionized many areas of biology. In NGS, millions of DNA or RNA sequences are generated in parallel, leading to fast, accurate sequencing of a large number of proteins. This method is relatively new in the biotech industry and requires further optimization for protein sequencing.
5. De Novo Peptide Sequencing
De novo peptide sequencing is a technique that determines the amino acid sequence in a particular antibody. It is often used instead of sequencing nucleic acids. The protein is broken down into several pieces, and then mass spectrometry is employed to determine the mass-to-charge ratio of every piece. The researcher uses bioinformatics software to identify overlapping sequences to reconstruct the fragments into one complete sequence.
Some significant advantages of de novo sequencing include:
- End-to-end sequencing: If you have proprietary software for de novo protein sequencing and high-quality mass spectrometry in optimal settings, you can sequence the whole protein from the C-terminus to the N-terminus without leaving any amino acid out.
- Numerous applications: Besides human antibodies, mass spectrometry can sequence any antibody regardless of its mass, including IgA, IgY, IgM, IgG, scFv, Fab, etc., from any animal. It can also sequence antibodies that are conjugated, contaminated, or possess two unique light chains.
- Better accuracy: Powerful signal peak patterns from various mass spectrometry readings allow this technique to identify every amino acid in the sequence precisely.
- No need for the producer cell: Even when the hybridoma that produced the initial custom antibody is not accessible, you can still recreate the antibody from as little as 0.2g of antibody protein via recombination.
6. Intact Mass Analysis and Peptide Mapping
Although these tools can’t replace de novo or mass spectrometry antibody sequencing, they can accurately verify an unidentified antibody’s protein sequences. Intact mass analysis is deemed successful if the protein’s molecular weight is similar to the results observed experimentally.
Peptide mapping helps you determine if the precursor peptides are what you initially thought. These techniques are insufficient for independent sequencing because of their inability to determine specific amino acids.
Applications of Antibody Protein Sequencing Services
Antibody protein sequencing services have many applications in the healthcare industry and biomedical research:
Analyzing antibody concentration
You can estimate the quantity of antibodies using a general protein assay or a technique specific to the immunoglobulins and species, like specialized micro-agglutination assay kits. This process involves linking the titer to the effective potency of the antibody sample. To obtain the titer measurement, researchers use ELISA kits to determine the appropriate dilution of the antibody sample for detection in a particular experiment.
Quality control of monoclonal antibodies
Monoclonal antibodies (mAbs) are an essential class of therapeutic proteins that treat various diseases, including cancer and autoimmune disorders. Accurate sequencing of mAbs is essential, as it plays a substantial role in their safety, efficacy, and production consistency. Antibody protein sequencing can verify the identity and purity of mAbs and detect any potential impurities or modifications.
Identification of unknown antibodies
Antibody protein sequencing helps identify unknown antibodies to determine their specificity and affinity for their target antigens. Researchers use this information to develop new diagnostic tests or therapeutic agents.
Characterization of immune responses
Antibody protein sequencing studies the body’s immune response to various pathogens or vaccines. Sequencing the antibodies the immune system produces helps researchers gain insights into the nature and duration of the immune response.
Factors to Consider When Choosing an Antibody Protein Sequencing Service
- Experience and expertise: It is crucial to choose a service provider with extensive antibody protein sequencing experience who employs a team of highly skilled scientists. An experienced team can ensure that the sequencing results are accurate and reliable.
- Quality and accuracy: You should select a provider using high-quality reagents and equipment with a proven track record of delivering accurate results.
- Turnaround time and scalability: It’s advisable to assess the provider’s turnaround time, especially if you have time-sensitive projects. You should also consider whether the provider can handle large-scale projects if necessary.
The Bottom Line
What do you need to know about antibody protein sequencing services? We hope you have received all the information you need to choose an exemplary service for your research. Antibody sequencing and custom protein synthesis face numerous challenges, including inadequate user awareness, poor supplier performance, and poor characterization. These challenges can cause substantial personal and financial expenses. It is best to assess your research needs and select the best antibody sequencing service provider available.