Introduction
Hybridoma cells are susceptible to contamination, gene loss, poor cell condition and even death during storage,
resulting in the permanent loss of important mAbs. Therefore, obtaining the gene sequences of monoclonal antibodies
from hybridoma cells is of great significance for the large-scale stable expression of monoclonal antibodies.
Compared with mass spectrometry sequencing, monoclonal antibody gene sequencing based on PCR amplification
technology can provide more accurate and reliable results, and can more accurately distinguish leucine/isoleucine of
amino acids that are difficult to distinguish in mass spectrometry identification.
Fig. 1.
Schematic of workflow for Selective PCR for Antibody Retrieval (SPAR). (Horns F, et al., 2020)
Our Services
Creative Proteomics has developed a PCR based antibody sequencing workflow combined with degenerate
primer design, which extracts and sequences the antibody genes from hybridoma cells to obtain the corresponding
nucleotide sequences, ensuring the stable expression and production of exogenous antibodies in the later stage. In
this method, professional degenerate primer design (degenerate primer) and sequencing solutions were applied to
provide customers with fast and accurate antibody variable region and full-length gene sequencing services. We
provide a one-stop service for the entire analytical steps of antibody sequencing. At the same time, we can also
provide corresponding customized services according to the specific needs of customers.
At the Creative Proteomics, we customize a complete set of antibody sequencing service processes for
your research.
Ⅰ. Extraction of total RNA from hybridoma cells.
Ⅱ. Reverse transcription of total RNA to cDNA.
Ⅲ. Design degenerate primers for the variable region sequence of the monoclonal antibody, and amplify the light chain
and heavy chain region DNA fragments of the variable region of the monoclonal antibody.
Ⅳ. Clone the DNA fragment into a plasmid vector dedicated to sequencing.
Ⅴ. Sequencing analysis of the DNA cloned antibody to obtain the DNA report of the monoclonal antibody.
Fig. 2. Workflow of the
monoclonal antibody sequencing service.
Advantages of Our Services
- Extremely Fast Experience
We provide you with efficient and fast analysis and delivery speed with PCR
results can be produced in 8 hours.
- Less Cell Consumption
Only 1~5 cells are needed, or well plate samples could be received.
- Independent Research and Development
Highly efficient enzymes and primers were developed in-house to ensure
a 100% success rate while delivering in just 5 days.
- Advanced Technology
Sequencing of antibodies derived from multiple common species, including human, mice,
rat, etc, and sequence analysis of the mutation-prone FR1 region of the antibody.
- High Accuracy
Multiple optimized workflows and custom PCR sequencing primer sets can analyze multiple DNA
clones to ensur accurate and high-quality sequencing analysis while you only need to provide enough hybridoma
cell lines to assist you in sequencing.
- One-Stop Service
Full-package service, expression verification service and custom service is also supported
with our extensive experience.
Creative Proteomics provides fast, reliable, and high-quality sequencing services for customers
around the world to advance your discoveries with a team of experts who can help you determine the best path
forward. Whether your ultimate goal is simple sequence or recombinantly expressed purified antibody, we have a
solution for you. Please contact us today to find out how we can advance
your next drug discovery project.
References
- Charlotte V, Amelia E, Spencer T C, et al. (2021) Sequencing, cloning, and antigen binding analysis of
monoclonal antibodies isolated from single mouse B cells. STAR Protocols. 2(2):100389.
