一、Introduction

Monoclonal antibodies see wide use in lab detection, diagnostic kits and antibody drug development. Hybridoma cell lines may degrade or get lost over time. Without cell stocks, researchers cannot produce matching antibodies anymore. Hybridoma sequencing solves this problem by extracting antibody gene sequences from cells, allowing gene-level preservation and recombinant antibody reconstruction.

If only purified antibody protein remains and no cell material exists, operators adopt N-terminal sequencing to obtain partial N-terminal amino acid data. For full sequences covering variable and constant regions, full-length sequencing is required.

The three methods differ greatly in required samples, technical logics and final outputs. Improper selection leads to incomplete sequences, missing CDR segments or mixed false antibody transcripts. These flaws directly cause failure of subsequent recombinant antibody expression.

The antibody sequencing platform of KMD-Bioscience provides hybridoma sequencing, N-terminal sequencing and full-length sequencing services. Technicians match suitable sequencing pipelines based on submitted materials (hybridoma cells, RNA or pure antibody protein) and deliver complete sequences ready for vector construction.

二、Principles and Standard Workflows of Three Antibody Sequencing Technologies

1. Hybridoma Sequencing

Hybridoma sequencing takes hybridoma cells or cell-derived RNA as raw materials. It stands as the most common way to obtain monoclonal antibody gene sequences.

Standard workflow:

①Recover and culture frozen hybridoma cells

②Extract total cellular RNA

③Reverse transcribe RNA into cDNA

④Amplify heavy and light chain genes via 5'-RACE or degenerate primer RT-PCR

⑤Clone amplified fragments into vectors

⑥Run Sanger sequencing to read variable domains, or extend amplification to gain full-length coding sequences ^[1,2]

Core challenge of this method: Hybridoma cells are fused cell lines. They produce abnormal false immunoglobulin transcripts alongside target mRNA. Bioinformatic comparison and follow-up recombinant expression verification are needed to screen out authentic heavy-light chain pairs that secrete functional antibodies.

Limitation: It only works with live cells or RNA extracts. Pure antibody protein cannot support this sequencing method.

Output advantage: Variable domain sequences directly support construction of scFv, Fab and full-length IgG expression vectors.

2. N-terminal Sequencing

N-terminal sequencing operates at protein level. It requires no cells or nucleic acids, only purified antibody samples.

Traditional workflows adopt Edman degradation. Modern platforms rely on mass spectrometry to decode the first several amino acids on heavy and light chain N-termini.

Main uses: Confirm signal peptide cleavage sites, identify N-terminal residues, cross-check if translated protein matches gene sequences from hybridoma sequencing.

Major drawbacks: It only captures partial N-terminal fragments. Complete CDRs and full variable regions cannot be acquired. Single N-terminal data cannot rebuild full functional antibodies.

When only trace pure antibody is left without hybridoma cells, N-terminal sequencing cannot supply full gene information. It must be combined with mass spectrometry denovo full-length sequencing to retrieve complete sequences.^[3]

3. Full-Length Sequencing

Two separate full-length sequencing pipelines are available: gene-based full sequencing and protein denovo full sequencing.

①Gene-based full sequencing

Built on hybridoma sequencing. Amplify complete heavy and light chains carrying signal peptides, variable domains and constant regions. Deliver full IgG nucleic acid sequences. It fits subtype switching and Fc domain modification research.

②Protein denovo full sequencing

Directly analyze pure antibody via mass spectrometry without hybridoma materials. Decode complete amino acid sequences. It rescues antibody resources when cell lines are lost ^[4].

Technical barriers: Protein denovo sequencing relies heavily on sample purity and antibody subtype. Some sequence segments show insufficient coverage. Gene-based full sequencing demands high-quality hybridoma RNA, and amplification difficulty exceeds simple variable domain amplification.

三、Applicable Scenario Comparison of Three Sequencing Tools

1.Hybridoma Sequencing

Required sample: Live hybridoma cells or cellular RNA

Strength: High accuracy, controlled cost, fast access to VH/VL variable sequences

Suitable for: Cell line preservation and recombinant antibody production

Weakness: Cannot run on purified antibody protein samples

2.N-terminal Sequencing

Required sample: Purified antibody protein

Output: Partial N-terminal amino acid residues

Suitable for N-terminal validation and signal peptide confirmation

Weakness: Cannot independently rebuild full antibodies; only used as auxiliary verification

3.Full-Length Sequencing

Two variants:

Gene-based: Needs hybridoma materials, outputs complete heavy/light nucleic acid sequences with constant domains

Protein denovo: Only requires pure antibody, rescues antibodies after cell loss

Weakness: Higher overall cost; protein denovo sequencing carries certain failure risks

四、Antibody Sequencing Services from KMD-Bioscience

1. Hybridoma Sequencing

Accept frozen hybridoma cell pellets or cellular RNA. Complete cell recovery, titer recheck, RNA extraction and RT-PCR amplification. Clone fragments for Sanger sequencing. Annotate V(D)J and CDR regions, distinguish true and false transcripts. Variable sequences are delivered; gene-level full-length extension is optional.

2. N-terminal Sequencing

Process purified antibody samples. Use mass spectrometry to resolve heavy and light chain N-terminal amino acids. Support signal peptide identification and sequence cross-verification.

3. Full-Length Sequencing

Two optional pipelines:

Gene full-length sequencing based on hybridoma samples to acquire complete heavy/light nucleic sequences including constant domains

MS denovo protein sequencing for clients who lost cell stocks and only retain pure antibody protein

4. Downstream Supporting Services

After sequence delivery, provide antibody vector construction, recombinant expression and purification, plus antibody humanization. Deliver raw sequencing files, formal sequence reports and finished recombinant plasmids or purified antibody products.

FAQs

Q：My hybridoma cell line is lost, and only purified monoclonal antibody protein is left. Can I run hybridoma sequencing directly? What sequencing scheme should I choose？

A: Hybridoma sequencing cannot be carried out in this case. This method relies on live hybridoma cells or cellular RNA to extract antibody mRNA. No cell material means no mRNA can be isolated.

The only available input material is purified antibody protein. Mass spectrometry denovo full-length sequencing is the primary choice. N-terminal sequencing can be added as auxiliary cross-check for N-terminal amino acid data.

After obtaining full amino acid sequences from denovo analysis, codon optimization is performed and target antibody genes are synthesized. New expression vectors are built to realize in vitro recombinant antibody reconstruction.

Note that protein denovo sequencing requires high-purity antibody samples. Excess impurities reduce sequencing accuracy significantly.

References

[1] Meyer L, López T, Espinosa R, Arias CF, Vollmers C, et al. (2019) A simplified workflow for monoclonal antibody sequencing. PLOS ONE 14(6): e0218717. https://doi.org/10.1371/journal.pone.0218717

[2] Subas Satish HP, Zeglinski K, Uren RT, et al. NAb-seq: an accurate, rapid, and cost-effective method for antibody long-read sequencing in hybridoma cell lines and single B cells. MAbs. 2022;14(1):2106621. doi:10.1080/19420862.2022.2106621

[3] de Graaf, S. C., Hoek, M., Tamara, S., & Heck, A. J. R. (2022). A perspective toward mass spectrometry-based denovo sequencing of endogenous antibodies. mAbs, 14(1). https://doi.org/10.1080/19420862.2022.2079449

[4] Resemann A, Jabs W, Wiechmann A, et al. Full validation of therapeutic antibody sequences by middle-up mass measurements and middle-down protein sequencing. MAbs. 2016;8(2):318-330. doi:10.1080/19420862.2015.1128607