1. What is Hybridoma Sequencing

Hybridoma cell antibody gene sequencing refers to the use of professional degenerate primer design (degenerate primer) and sequencing schemes, while optimizing economic costs and time costs, to provide customers with fast and accurate antibody variable region and full-length gene sequencing services.

Figure 1: Hybridoma sequencing

2. Purpose of  Hybridoma Sequencing

During the storage process, hybridoma cells may lose antibody genes, poor cell status may affect antibody production, and even cell death. In order to preserve excellent antibodies for a long time and produce them stably, the antibody genes in hybridoma cells can be extracted and sequenced, and the nucleotide sequence can be obtained and stored in the form of DNA for later exogenous expression and production.

3. Application of  Hybridoma Sequencing

The hybridoma cell monoclonal antibody gene sequence obtained by hybridoma cell antibody sequencing is one of the prerequisites for publishing articles or patents and recombinant expression of engineering antibodies, and is also the basis for antibody humanization work.

4. TekBiotech can provide customers with high-quality hybridoma sequencing services

Standard cycle monoclonal antibody sequencing services: antibody variable region VH and VL sequencing, antibody VH, VL and leader region sequencing, full antibody VH, VL, leader region and constant region sequencing, 3-4 weeks to complete the sequencing service.

Rapid monoclonal antibody sequencing service: antibody sequence quickly identified from the target cell line, sequencing results can be obtained within 10 days.

5. Introduction to the Full Process of Hybridoma Sequencing Services Provided by TekBiotech to Customers

TekBiotech provides customers with hybridoma sequencing services, the process includes: RNA extraction, cDNA cloning, T vector construction, antibody gene sequencing, bioinformatics analysis, project report.

Figure 2: Hybridoma sequencing process

5.1 Hybridoma Production and total RNA Extraction

Total RNA was extracted from hybridoma cells using TRIzol reagent according to the manufacturer's instructions.

5.2 Reverse Transcription to Synthesize Antibody Variable Region cDNA

5.2.1 Reverse transcription

Use a reverse transcriptase kit, in addition to RNA samples (hybridoma RNA samples from mouse antibodies or RNA samples from chimeric antibodies), primers, 10 mM deoxynucleotide triphosphate mixture (dNTPs), H2O, and 80U/μL RNAse inhibitor. Note: Due to its RNA content, the template switching oligonucleotides are aliquoted and stored at –80°C.

Perform reverse transcription according to the following protocol: Keep all reactions on ice during the operation. For each RNA sample, set up three cDNA synthesis reactions: one for the kappa chain, one for the lambda chain, and one for the heavy chain. Ideally, only one light chain will be amplified for each antibody.

Figure 3: Protein sequence comparison of 3H4 kappa and 3H4 lambda variable regions

5.2.1.1 In a PCR tube, prepare Mix 1: 2μL 50ng/μL RNA, 1μL 10μM antibody chain-based reverse RT primer (e.g. mIGKRT, mIGLRT, or mIGHGRT for mouse antibodies), and 1μL 10mM dNTPs. For one RNA sample, three tubes of Mix 1 are required, each containing a different reverse primer.

5.2.1.2 In a 0.5mL Eppendorf tube, prepare Mix 2: 1.95μL H2O, 2μL 5x SMARTScribe buffer, 1μL 20mM DTT, and 0.3μL 100μM template switching oligonucleotide. The volume provided for Mix 2 is for one cDNA synthesis reaction, so scale up as needed, i.e., prepare three times the volume of Mix 2 per hybridoma RNA sample. One master mix of Mix 2 is prepared for all reactions.

5.2.1.3 Denature RNA secondary structure by incubating tubes containing Mix 1 at 72°C for 3 minutes in a thermal cycler.

5.2.1.4 During denaturation of Mix 1, add the following to Mix 2: 0.25 μL 80U/μL RNAse inhibitor and 0.5 μL 100U/μL SMARTScribe reverse transcriptase per cDNA synthesis reaction.

5.2.1.5 Add 6 μL of Mix 2 to each tube of denatured Mix 1.

5.2.1.6 Incubate the combined mixes at 42°C for 60 minutes in a thermal cycler and terminate the reaction by incubating at 70°C for 5 minutes. The reactions are maintained at 4°C. PCR amplification is performed immediately after reverse transcription. No cDNA purification step is required.

5.2.2 PCR amplification of antibody variable regions

5.2.2.1 Set up PCR reactions for each cDNA synthesis: 10 μL 5x PCR buffer, 1 μL 10 mM dNTPs, 3 μL cDNA synthesized from RT reaction, 2.5 μL 10 μM universal forward primer ISPCR, 2.5 μL 10 μM reverse PCR primer on the antibody chain (e.g. mIGKPCR, mIGLPCR, or mIGHGPCR for mouse antibodies), 30.5 μL H2O, and 0.5 μL 2U/μL Phusion polymerase (or other high-fidelity polymerase).

5.2.2.2 Step-down/step-down PCR was performed according to the following thermocycler conditions: 98°C for 30 seconds; 10 cycles of 98°C for 15 seconds, 63–57.5°C for 30 seconds (reducing the temperature by 0.5°C per cycle), and 72°C for 30 seconds; 15 cycles of 98°C for 15 seconds, 56°C for 30 seconds, and 72°C for 30 seconds; followed by 72°C for 7 minutes; and hold at 4°C.

5.2.2.3 5 μL of each RT-PCR reaction was run on a 1% agarose gel in TAE buffer at 90V. The amplified mouse antibody product appeared between 550 and 600 base pairs. The amplified human antibody product appeared between 750 and 850 base pairs. Quick Load Purple 2-Log DNA Ladder (NEB, N0550S) was used as a standard.

Figure 4: PCR amplification of antibody variable region

5.3 T Vector Construction

PCR cleanup of RT-PCR reactions was performed using Macherey-Nagel's PCR cleanup and gel extraction kit. According to the blunt end cloning kit manual, 2 μL of each PCR cleaned product was blunt-ended and cloned into the pCR-Blunt-II-TOPO vector. Next, 3 μL of each TOPO cloning reaction was transformed into chemically competent E.Coli. 100 μL of each transformation was spread on an LB plate containing 50 μg/mL kanamycin and incubated overnight at 37°C.

5.4 Antibody Gene Sequencing

After obtaining colonies, 5-10 colonies per antibody chain were inoculated in 5 mL LB/kanamycin medium and shaken at 250 rpm overnight at 37°C. These cultures were miniprepared using Macherey-Nagel's miniprep kit, and the resulting plasmid DNA was Sanger sequenced by Sequetech Corporation using the M13 forward primer.

5.5 Bioinformatics Analysis

The sequencing data was analyzed using a custom Python program available on GitHub to remove non-functional DNA.

5.6 Project Report

The resulting antibody sequence was delivered.

The acquisition of hybridoma cell monoclonal antibody gene sequences is the basis for the preparation of recombinant antibodies and the development of antibody drugs. TekBiotech can accurately and skillfully sequence hybridoma cells from multiple species, including mouse hybridoma cells, rat hybridoma cells, rabbit hybridoma cells, and recombinant monoclonal antibodies of different species (obtained from monoclonal antibody genes constructed by phage technology). Strict quality control is used to ensure high accuracy.