Advances in biotechnology mean that antibody and protein sequencing services are available to offer monoclonal antibody sequencing and monoclonal antibody production. Such services facilitate recombinant protein expression for the protein manufacturing process in antibody engineering, function optimization, and database banking for therapeutic applications.
Antibody protein sequencing services may offer different packages, but they apply similar principles as explained below.
The History Of Antibody Sequencing
According to one historical account, the first chronological reference to antibodies was in a landmark publication published in 1890 by two physiologists, Emil Von Behring and Kitasato Shibasaburō. The publication proposed antibodies as significant immunity bodies by demonstrating how serum from immunized mammals caused an unimmunized mammal to develop immunity.
Von Behring and Shibasaburō reported developing antitoxins, equivalent to today’s antibodies, for two killer diseases, diphtheria and tetanus. Their publication laid the foundation for monoclonal antibody sequencing and other protein sequencing services.
The discovery of antibodies and their role in immunity set the tone for antibody repertoire studies focusing on understanding the human diversity repertoire. According to one literature review, recombination and somatic hypermutation (SHM) are the primary mechanisms facilitating human antibody repertoire diversification. The two mechanisms diversify possible light-chain and heavy-chain combinations, defining antibody affinity levels.
Antibody repertoire studies go hand-in-hand with antibody evolution studies. These studies trace clonal lineage to the same-descent B-cells that may differ at the sequence level, promoting clonal diversity.
Advances in antibody studies contributed to the generation of the first monoclonal antibody (mAb) in 1975. Monoclonal antibodies are artificial proteins cloned to act like the natural antibodies released by the immune system’s B-cells, but have a higher antibody specificity.
Following the developments in antibody sequencing services, the first monoclonal antibody became fully licensed in 1986. One statistical report shows that 53 monoclonal antibody medications have been available in the US and Europe since the initial monoclonal antibody production. Moreover, monoclonal antibody production is ubiquitous, with the therapeutic monoclonal antibody industry being worth $125 billion as of 2020.
Antibody specificity is a vital characteristic in monoclonal antibody sequencing, meaning the antibody production steps that antibody production services use must preserve target affinity. Besides monoclonal antibody sequencing, polyclonal antibody production is emerging as a vital niche in antibody protein sequencing services.
Each antibody type utilizes antibody specificity in different ways. You can learn more about polyclonal antibody VS monoclonal later in this post. However, now that you understand the concept behind antibody sequencing services and protein expression and purification services, keep reading to learn the advantages of such services.
Advantages Of Antibody Sequencing Services
Antibody sequencing services are helpful for recombinant antibody production. Below are the primary advantages of an antibody sequencing service to antibody reagent users.
An Antibody Production Service Facilitates Diversity
Antibodies play a vital role in the medical field by facilitating research and practical applications in preventative medicine and therapeutics. However, antibodies' role in therapeutics and immunology stems from antibody repertoire studies, exploring antibody diversity and binding affinity via antibody sequencing.
The Service Provider Can Proceed Without Producer Cells
The producer cell or hybridoma is the primary structure that antibody sequencing services use to originate an antibody culture in recombinant antibody production. Hybridoma generation entails fusing plasma cells from an immunized host cell with a cell line to form an immortal, antibody-producing cell. The immortal hybridoma constantly facilitates monoclonal and polyclonal antibody production.
Ideally, cryopreservation using liquid nitrogen is the primary method that an antibody production service uses to store a hybridoma cell. However, while hybridomas are immortal and stable in storage, they have a low antibody diversity and are prone to contamination, affecting macrophage screening. Macrophage screening technology is a crucial recombinant protein expression stage that guides essential processes during custom gene synthesis and custom protein expression.
However, biotechnology advances eliminate hybridoma-related inconveniences, and any antibody sequencing service can use synthetic techniques like the solid-phase peptide synthesis protocol to produce antibodies.
According to one expert review, synthetic peptides featuring 10-20 amino acids can facilitate monoclonal and polyclonal antibody production. Such peptides can produce antibodies with titers higher than 20000, and ELISA kit manufacturers make kits that can verify such titers via antibody specificity and affinity.
Accuracy
Protein expression and purification service providers use various mass spectrometry techniques to test the antibody sequencing accuracy levels. The advanced mass spectrometry techniques use sequence signatures to identify antibodies within the same class after monoclonal and polyclonal antibody production processes.
Moreover, one literature review posits that an antibody sequencing service can optimize signature sequences to identify specific antibody classes. The optimization aspect helps improve biotechnological services relying on antibody sequencing, including recombinant protein expression, custom gene synthesis, and custom DNA synthesis. Most custom protein production activities involving antibody sequencing have a success rate exceeding 90%.
Patent Application
Antibody patents focus on the antibody’s structure rather than its functionality. The antibody sequencing service facilitates custom antibody production because identifying antibody heavy-chain and light-chain antibody combinations that improve specificity and affinity helps generate custom gene sequences.
Custom gene sequences facilitate custom protein expression and custom protein synthesis to generate antibodies with unique structures. The protein expression and purification service that yields a de novo antibody sequence can apply for a patent on the antibody’s structure. Besides an antibody sequencing service, research bodies and individuals dealing in antibody reagents can apply for the patents.
Cost-effectiveness
Historically, custom antibodies were expensive to produce using traditional biological methods. The advent and ubiquity of modern antibody sequencing technology have significantly driven down costs. Sequencing allows for efficient, large-scale recombinant production, offering high consistency and speed, which has made custom antibodies substantially more affordable and accessible today.
Moreover, high-throughput monoclonal and polyclonal antibody production is possible, facilitating mass recombinant antibody production. However, various factors like antibody production steps influence the custom antibody cost among custom protein expression service providers.
Comprehensive Reports
Antibody production services include an analysis stage similar to protein characterization, one of the antibody production steps in custom protein production. The report details the sequence for native and de novo antibodies, including heavy and light chain sequences that enhance specificity and affinity in recombinant antibody production.
Such reports help in creating antibody libraries for antibody production services. Moreover, the detailed reports are helpful for custom protein expression and custom protein synthesis in recombinant antibody production.
Antibody Sequencing Protocol/Workflow: Antibody Sequencing Steps
The antibody sequencing workflow or antibody production steps fall into either classical or contemporary protocols. Both protocols determine immunoglobulin heavy and light chains and complementary regions expressed via B-cells to monitor full-length antibody repertoires. However, while classical and contemporary protocols share similar end goals, they have different steps, as highlighted below.
Classical Hybridoma Sequencing
Hybridoma sequencing protocols proceed as follows:
mRNA Isolation From Hybridoma
The first step in classical hybridoma sequencing is isolating an mRNA strand from the DNA content in the hybridoma cell. Isolation entails using filtration techniques to detect a specific immunoglobulin mRNA.
cDNA Synthesis
Once you have the isolated mRNA, proceed by generating a cDNA from the mRNA, as with typical custom protein production. The isolated mRNA serves as a template for the cRNA, and reverse transcription is necessary to transfer the sequence code from the mRNA to the cDNA. Moreover, hexamer molecules introduced into the sample at this stage help generate an antibody library to aid the sequencing.
Variable Region PCR (Polymerase Chain Reaction) Amplification
PCR chains serve the primary purpose of detecting genetic material from organic matter. The chain reaction serves as an amplification technique, highlighting target parts of the cDNA cloned from the hybridoma featuring sequences.
Ideally, the variable regions holding heavy-chain (VR) and light-chain (VL) sequences have the highest genetic matter concentration. The areas require multiple amplification steps using primers to highlight the variable parts.
Gene Cloning Into A Vector
PCR signal amplification helps assemble the revealed genetic sequences or library into contigs. Contigs are overlapping clone sequences that map out the genome sequences established during the antibody sequencing process. The contigs help decipher the active regions of the antibody along the VR and VL regions to generate a diverse repertoire.
Hybridoma sequencing utilizes classic techniques like E. coli protein expression to generate the genetic sequences for specific antibodies. Moreover, ELISA kit manufacturers produce kits to support this library creation step for custom protein synthesis. Hybridoma antibody sequencing is standard for polyclonal antibodies vs. monoclonal ones.
However, as stated, hybridoma antibody sequencing has multiple challenges similar to the classic heterologous protein manufacturing process and E coli protein expression. For instance, the hybridoma in question may become tainted or degraded. Consequently, antibody sequencing services are shifting towards contemporary techniques, as explained below.
Contemporary Antibody Protein Sequencing Protocol
The protein sequencing protocol helps overcome the challenges associated with classical hybridoma sequencing. Below is a step-by-step breakdown of the contemporary antibody protein manufacturing procedure.
Enzymatic Digestion Of The Antibody
Enzyme fragmentation is the first necessary step in contemporary or cell-free antibody sequencing. Fragmentation is similar to the protein manufacturing process using modern techniques like solid-phase peptide synthesis protocols.
It entails using proteolytic enzymes to reveal protein structure for study purposes. The proteolytic enzymes facilitate rapid fragmentation.
Liquid Chromatography-Mass Spectrometry (LC-MS/MS)
Unlike the classical hybridoma technique, which uses simple analytical methods, contemporary protein sequencing does not rely on an ELISA kit for complementary ELISA testing.
Instead, it uses advanced techniques, combining liquid chromatography and mass spectrometry to detect genetic molecules within the antibody. The custom protein production service provider utilizes the LC-MS/MS technique on each antibody molecule.
De Novo Peptide Sequencing
Classical hybridoma peptide sequencing means you can only work with known antibody sequences. However, de novo peptide sequencing allows you to determine the genetic sequence of an unknown antibody in a sample.
While an ELISA kit can detect the presence of an antibody in a sample, it cannot detect the antibody’s structure. You can learn more about de novo peptide sequencing and what polyclonal peptides are in the segment below on polyclonal antibody vs monoclonal de novo sequencing.
Overlapping Peptides Assembly
The antibody’s structure features peptides and amino acids as building blocks. Therefore, supramolecular peptide assembly is necessary.
Overlapping peptide assembly using codons helps generate an overlapping peptide library. Peptide libraries are essential in creating epitope mapping to show segments of the antibody peptide containing essential amino acids because the critical amino acids influence an antibody’s functionality.
Antibody Sequencing Service Requirements and Deliverables
Although the antibody sequencing techniques above vary in advancement, both apply to mass and custom antibody production, depending on the project. Moreover, the methods used influence high throughput and custom antibody costs.
Different antibody sequencing services have diverse requirements and deliverables when processing an antibody sequencing order. Below are some pointers on requirements and deliverables to consider when shopping for a peptide sequencing service company.
Requirements
The antibody sequencing service provider requests how to ship your hybridoma or other antibody samples, e.g., freezing and other culture cell conditions. They also advise on acceptable purity levels for the hybridoma and other samples. Third, they also highlight the minimum antibody sample size, especially for de novo protein sequencing; some accept sample sizes as small as 0.1mg. Depending on the time it takes to complete sequencing, you should order the service in advance. Some services take two to three weeks, while others take several months.
Therefore, reaching out to a service provider beforehand for a detailed quotation is prudent.
Deliverables
The antibody sequencing service provider should deliver the following.
- The mapped antibody sequence within the agreed-upon duration.
- 100% sequence coverage and at least 90% working sequence.
- A synthetic peptide sequence that is equal or perform better in specificity and affinity.
- Specify the custom antibody cost and the parameters for custom protein synthesis and custom protein production.
- They should highlight the media available for antibody protein sequencing, e.g., the solid-phase peptide synthesis protocol.
- After a thorough analysis, they should issue a comprehensive report on the peptide sequence. The study should cover everything, including expression mechanisms like the solid phase peptide synthesis protocol and E coli protein expression.
Polyclonal Antibody VS Monoclonal Antibody
Antibody sequencing services provide sequencing options for monoclonal antibodies in mass and custom antibody production. However, what are polyclonal antibodies, and how do they differ from monoclonal antibodies?
The primary difference when considering polyclonal and monoclonal antibodies is that while monoclonal antibodies feature identical immune cells, polyclonal antibodies feature diverse immune cells. Second, polyclonal antibody production begins with custom peptide synthesis, while antigen preparation is the first step in monoclonal antibody production. Regarding antibody sequencing, monoclonal antibody sequencing favors classical hybridoma sequencing, while polyclonal antibody sequencing favors de novo sequencing.
Conclusion:
Antibody sequencing is vital in drug discovery and therapeutics development, helping improve human quality of life. However, not all antibody sequencing services are equal, and learning how they function is vital to advancing therapeutics.