Antibodies (immunoglobulins) are Y-shaped proteins secreted by B cells, which specifically recognize pathogen epitopes (such as viral spike proteins) through Fab, and use crystallizable fragments (Fc mediates immune effects such as neutralization: blocking pathogens from invading host cells (such as anti-SARS-CoV-2 antibodies blocking the binding of virus RBD with ACE2 receptors); Conditioning phagocytosis: Fc segment binds to Fcγ receptor on macrophage surface to promote pathogen clearance; Complement activation: triggering the membrane attack complex (MAC) to dissolve the pathogen through the classical way.
Antibody sequencing is the process of obtaining antibody gene sequence, which is very important for the research and application of antibodies. Through antibody sequencing, the complete amino acid sequence of antibody can be determined, and its three-dimensional structure and antigen binding site can be inferred. It is helpful to optimize antibodies, such as enhancing affinity, improving stability and reducing immunogenicity. It can help researchers to find new antibodies, especially when there is no ready-made antibody library, and obtain the target antibodies by methods such as de novo sequencing or gene bank screening.
The following is a detailed experimental flow of antibody sequencing:
1. Sample preparation
1.1 Collect antibody samples
- Source: There are many ways to source antibody samples. Common ones are:
- Immunized animals: for example, mice, rabbits or monkeys are immunized, and B cells in peripheral blood, spleen or lymph nodes are collected.
- Monoclonal antibody: Monoclonal antibody screened from antibody library or recombinant antibody library.
- Cell culture solution: antibody obtained by expression system culture such as transgenic cells or CHO cells.
- Concentration requirements: In sequencing experiments, the concentration of antibody samples needs to be high enough to ensure that the number of antibody molecules extracted is enough for subsequent experiments. The common concentration range is 1-10 mg/mL. If the sample concentration is low, concentration techniques can be used, such as dialysis or ultrafiltration.
Antibody-specific workflow of PCs in convalescent patients with COVID-19's disease (Ehling RA et al., 2021).
1.2 Purified antibody
- Methods: Affinity chromatography is usually used for purification, and Protein A/G affinity chromatography is the most commonly used method.
- Affinity chromatography of Protein A: Protein A binds to the Fc segment of the antibody, which can separate the antibody from the mixed sample. It is especially effective for IgG type antibodies.
- Ion exchange chromatography: It can be separated by adjusting the pH or salt concentration of the solution and using the charge difference between antibodies and other impurities.
- Analysis after purification: The purified antibody can be analyzed by SDS-PAGE to confirm its molecular weight and purity. Western Blot can also be used to confirm the specificity of antibody.
2. Cloning of antibody gene
2.1 Extraction of antibody coding genes
The key to the process of extracting antibody coding genes is to extract mRNA containing antibody gene information, and then convert the mRNA into cDNA by reverse transcription technology.
- MRNA extraction: by extracting total RNA from B cells or blood of immunized animals (such as mice and rabbits). High quality RNA can be obtained by using commercial RNA extraction kit (such as TRIzol).
- Reverse transcription into cDNA: RNA is converted into cDNA by reverse transcriptase, and efficient reverse transcriptase (such as SuperScript II or III) is usually selected for operation. Specific primers are added during reverse transcription to ensure the transcription of heavy chain and light chain.
2.2 reverse transcription and PCR amplification
- PCR amplification: The heavy chain (VH) and light chain (VL) sequences of antibody genes were obtained by PCR amplification of cDNA. Commonly used PCR primers include:
- VH primer: The family primer of V region is often used to amplify the variable region of antibody heavy chain.
- VL primer: the variable region of antibody light chain is amplified, and the primers for κ chain and λ chain are different.
- Conventional PCR conditions: the reaction system usually contains Taq enzyme, dNTP, primers, template DNA, etc., and the PCR cycle conditions are adjusted according to the target sequence.
2.3 product verification
- Agarose gel electrophoresis: PCR amplification products were analyzed by agarose gel electrophoresis to detect whether specific antibody gene fragments were produced. If the product is correct, you will see bands in the gel that meet the expected size.
- Sequencing verification: The PCR product was cloned and sequenced to further verify whether the amplification was successful and the sequence was accurate.
3. Cloning of heavy chain and light chain genes
3.1 Cloning into a vector
The antibody heavy chain and light chain genes amplified by PCR were inserted into the vector respectively. Commonly used vectors include pUC series or pCDNA3.1, etc. These vectors provide promoters, terminators, marker genes and other elements that are convenient for expression.
- Vector selection: For gene cloning of antibodies, vectors with selectable markers (such as antibiotic resistance genes) are often selected, and successfully transformed bacteria can be screened.
- Enzyme digestion connection: Restriction endonucleases (such as EcoRI, XhoI, etc.) are usually used to endonuclease the vector and PCR products to generate complementary sticky ends, and the two are connected by DNA ligase.
3.2 Transformation and Screening
The clones containing heavy chain and light chain genes were transformed into Escherichia coli. Antibiotic selective medium was used to screen positive clones.
- Transformation method: DNA was introduced into cells by thermal shock or electric shock.
- Screening: screening with appropriate antibiotics to ensure that only strains containing inserted fragments can grow.
3.3 Identification of clones
- Agarose gel electrophoresis: PCR verification was carried out to ensure that the inserted gene fragment was consistent with the target antibody gene.
- Sequencing verification: sequencing the forward and reverse primers respectively to verify the correctness of the antibody gene.
4. Expression and purification of antibody
4.1 Expression of Heavy Chain and Light Chain
The heavy chain and light chain genes were transfected into host cells respectively. Commonly used host cells are CHO (China hamster ovary) cells and HEK293 cells, which can express antibodies efficiently.
- Transfection: Transfection with plasmid or virus vector. Common transfection methods include liposome-mediated transfection and electrotransformation.
- Screening and cloning: screening positive clones by antibiotic screening or fluorescent labeling to obtain cell lines stably expressing antibodies.
4.2 Purified antibody
- Affinity chromatography: Protein A/G affinity chromatography column was used for antibody purification. By binding to the Fc segment of the antibody, the antibody is specifically captured.
- Impurities removal: In the purification process, dialysis, gel filtration and other methods are usually needed to remove impurities from cell lysate.
4.3 Quality Control
- SDS-PAGE: The purity and size of antibody molecules were confirmed by SDS-PAGE electrophoresis.
- Western Blot: to detect the specificity and function of antibody.
5. Antibody gene sequencing
5.1 DNA extraction
Genomic DNA was extracted from transfected host cells. Commonly used DNA extraction methods include phenol-chloroform method, column kit method and so on.
- Cell collection: the transfected cells were collected by centrifugation and other methods.
- DNA extraction: use appropriate kits to extract genomic DNA, or use traditional methods.
5.2 PCR amplification of antibody gene
- Amplification of heavy chain and light chain genes: PCR amplification was carried out using specific primers for VH and VL regions.
- PCR conditions: Set appropriate annealing temperature and amplification cycle times to ensure the accuracy of PCR products.
5.3 Connecting to Sequencing Vector
PCR products are inserted into sequencing vectors, usually using linearized plasmid vectors.
- Enzymatic ligation: the vector and PCR products are cut by restriction enzymes to generate sticky ends, and they are connected by ligase.
5.4 sequencing analysis
The heavy chain and light chain genes were sequenced and analyzed by Sanger sequencing or high-throughput sequencing technique.
- Sanger sequencing: It is usually used to obtain high-quality and long-read sequence data.
- High-throughput sequencing: A large number of antibody samples were sequenced at the same time to obtain more extensive genetic data.
5.5 Data analysis
Through the comparison and analysis of gene sequences by professional software (such as IgBLAST, IMGT), the structure and diversity of antibodies were inferred.
6. Antibody function verification
6.1 Antibody Affinity Determination
- ELISA: The antigen was immobilized on the plate to detect the ability of antibody to bind to antigen. The affinity of antibody was quantified by chromatographic change reaction.
- Flow cytometry: the binding strength was detected on cells by labeling antigens and antibodies.
6.2 Neutralization activity detection
- Virus neutralization test: the neutralization effect of antibody is evaluated by neutralizing the activity of virus in cell culture.
7. Data collation and archiving
The sequencing data, antibody sequence and functional analysis data are sorted out and uploaded to the database for subsequent use.
- Database upload: public databases such as GenBank and IMGT store antibody sequences and share them with researchers.
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Reference
- Meyer L, López T, Espinosa R, Arias CF, Vollmers C, DuBois RM. "A simplified workflow for monoclonal antibody sequencing." PLoS One. 2019;14(6):e0218717. doi: 10.1371/journal.pone.0218717
