How Do You Obtain the Sequence of an Antibody?

Introduction

Antibody sequences are critical for antibody engineering and protein characterization in therapeutic development. For antibody reagent users, knowing the sequences allows them to perform sequence analysis/alignment to identify binding and cross-reactivity so they can conduct rational experimental design.

There are a number of online antibody databases storing different types of information such as suppliers, usage data, and publication references. These types of antibody databases are often used for reproducibility purposes. However, other databases containing sequences from immunoglobulin genes or sequenced antibody proteins offer much more insight for the purposes of antibody engineering and characterization.

Sometimes, some genomes have not been sequenced, and this requires the use of de novo protein sequencing to elucidate the sequence. However, when the sequence is available, there are numerous antibody sequence databases that can be used by researchers for the purpose of sequence search.

Antibody Sequence Databases

UniProt 

UniProt is a comprehensive, high-quality, and freely accessible resource of protein sequence and functional information. UniProt does not specifically focus on antibodies; as such, it only includes a representative set of germline antibody sequences and a limited number of non-germline antibody sequences. 

IMGT 

The international ImMunoGeneTics information system (IMGT) is a global, high-quality integrated knowledge resource specializing in immunoglobulins (Ig), T cell Receptors (TcR), and Major Histocompatibility Complex (MHC) molecules of human and other vertebrates. IMGT comprises seven databases encompassing antibody sequence, genome, and structure. For example, IMGT/mAb-DB, the monoclonal antibody (mAb) database of IMGT, provides an expert resource on mAbs with clinical indications, and on fusion proteins for immune applications (FPIA).

abYsis

abYsis is a web-based antibody research system that includes an integrated database of antibody sequence and structure data. abYsis can be used in three main ways: 

1. to search the database to find information about sequences (and structures) including detailed annotations, identification of unusual residues, post-translational modification sites, etc.,
2. to analyze trends across sequences in the database, and 
3. to enter one’s own sequences for analysis. 

The ABCD (for AntiBodies Chemically Defined) database

Launched in 2020, the ABCD Database is a repository of sequenced antibodies, integrating curated information about the antibody and its antigen with cross-links to standardized databases of chemical and protein entities. Each antibody is assigned a unique ID number that can be used in academic publications to increase the reproducibility of experiments. Check out their tutorial to learn How to obtain an antibody sequence, Step-by-step.

SAbDab

Structural Antibody Database (SAbDab) is a database containing all the antibody structures available in the PDB, annotated and presented in a consistent fashion. The user can select structures, according to these attributes as well as structural properties such as complementarity determining region loop conformation and variable domain orientation.

Thera-SAbDab 

Therapeutic Structural Antibody Database (Thera-SAbDab) tracks all antibody- and nanobody-related therapeutics recognized by the World Health Organisation (WHO), and identifies any corresponding structures in the SAbDab with near-exact or exact variable domain sequence matches.

AHo’s Amazing Atlas of Antibody Anatomy

AAAAA is a tool for antibody structural analysis, modeling, and engineering. All sequences of variable domain germline and rearranged sequences in AAAAA are numbered in an identical way. AAAAA is quite useful for users to check antibody sequences for plausibility to find out about unusual residues, sequencing mistakes, frameshifts, insertions, and deletions.

Observed Antibody Space database

OAS is a project to collect and annotate immune repertoires for use in large-scale analysis. It currently contains over one billion sequences, from more than 79 different studies. These repertoires cover diverse immune states, organisms (primarily human and mouse), and individuals. OAS now contains both unpaired and paired antibody sequences.

cAb-Rep

cAb-Rep is a database of curated antibody repertoires. It currently includes 306 B cell repertoires collected from 121 human individuals, including healthy, vaccinated, and autoimmune disease donors. The database has a total of 267.9 and 72.9 million curated full-length V(D)J heavy and light chain transcripts respectively, which were generated using Illumina sequencing technology in previous studies.

The Lack of Antibody Sequence Information is a Common Problem

Due to proprietary ownership and the lack of fully sequenced genomes, the sequences of many antibodies are not available to the public. Researchers may use these databases to find sequences of closely related antibodies, as well as rely on bioinformatics tools to make predictions for the antibody of interest; however, this is not an ideal way to conduct confident and rational research. 

With immune systems evolving constantly, global antibody discovery efforts are continuously producing novel antibodies that are not listed in any database. As a result, these online databases may not offer much value in acquiring sequence information when working with newly discovered or uncharacterized antibodies.

Why is it Difficult to Obtain the Sequences of Antibodies?

Various techniques can be used to determine and confirm the amino acid sequence of an antibody. Traditionally, researchers go for hybridoma sequencing if they have the cell line for a mAb. However, sequencing with hybridomas suffers from certain drawbacks, which can cause inaccurate results. To make things worse, researchers do not always have available, viable hybridomas. B cell sequencing, including single-cell approaches, can give nucleotide sequence information (blueprints for thousands of possible antibodies), but may not be able to access the sequence of the full circulating antibody protein repertoire.

How is a Protein Sequence Determined at Rapid Novor?

With our proprietary REmAb® de novo protein sequencing platform, we are able to determine the primary amino acid sequences of mAbs without the need for hybridoma cell lines or prior knowledge of DNA sequences. REmAb® directly analyzes the antibody protein (only 0.1 mg required) with 100% coverage and accuracy. Our team has successfully sequenced thousands of antibodies, including antibody reagents, therapeutics, and newly isolated antibodies, helping researchers decode their antibodies within two weeks. So, for researchers who cannot find sequences of their antibodies in online databases, REmAb® could be considered a straightforward solution with the above-mentioned benefits.

Can I Get Sequences from Polyclonal Antibodies?

Now you can. Rapid Novor recently became the world’s first to sequence antibodies directly from a polyclonal protein sample without the use of DNA or other nucleotide sequencing technologies. This means from now on researchers can expect to obtain amino acid sequences from a polyclonal antibody (pAb) mixture using Rapid Novor’s REpAb® antibody discovery platform.

Built on years of experience in mass spectrometry-based proteomics and bioinformatics, our team has made the breakthrough to accomplish what used to be impossible. Our polyclonal sequencing technology can derive the sequences of dominant mAbs directly from a pAb mixture. Since the majority of antibody reagents and all naturally produced antibodies are polyclonal, the applications of REpAb® are limitless. If you’re interested in learning more, contact us here.