Mechanism of Antibody Sequencing

Antibodies are essential defense mechanisms of the immune system, characterized by their high specificity and diversity. Understanding the sequence information of antibodies is crucial for biomedical research and clinical applications. Antibody sequencing, which deciphers the amino acid sequences of antibodies, is a pivotal technology.

 

Structure and Function of Antibodies

Antibodies, or immunoglobulins (Ig), are proteins produced by B cells that recognize and neutralize antigens. A typical antibody comprises two heavy chains and two light chains, forming a "Y"-shaped structure. Each chain includes a variable region and a constant region, with the variable region responsible for binding specific antigens.

 

Importance of Antibody Sequencing

Antibody sequencing reveals the amino acid sequences of antibodies, providing detailed insights into their structure and function. This is vital for developing new therapeutic antibodies, studying antibody-antigen interactions, and monitoring antibody changes within the body.

 

Mechanism of Antibody Sequencing

1. Sample Preparation

The initial step in antibody sequencing is sample preparation, involving the extraction of antibodies from biological samples, typically using protein A/G affinity chromatography for purification. The extracted and purified antibody samples must have sufficient purity and concentration to ensure the accuracy of subsequent sequencing processes.

 

2. Antibody Degradation and Peptide Generation

Purified antibody samples are usually degraded into smaller peptides using chemical or enzymatic methods to facilitate mass analysis. Common enzymatic methods include trypsin digestion, which specifically cleaves peptide bonds in antibody molecules, generating a series of peptides.

 

3. Mass Spectrometry Analysis

Mass spectrometry (MS) is the core technology in antibody sequencing. The mass spectrometer ionizes peptides, converting them into charged ions, and then separates and detects these ions based on their mass-to-charge ratio (m/z). Common mass spectrometry techniques include tandem mass spectrometry (MS/MS), which performs secondary fragmentation analysis on peptides to obtain more detailed structural information.

 

4. Data Analysis and Sequence Assembly

The raw data generated by mass spectrometry requires complex computational analysis to assemble the complete amino acid sequence of the antibody. Common software tools such as Mascot and PEAKS compare mass spectrometry data with known protein databases to identify peptide sequences. By assembling these peptides, the complete antibody sequence is ultimately obtained.

 

5. Verification and Optimization

The obtained antibody sequence usually requires verification and optimization, including comparison with known antibody sequences to confirm accuracy. Further experiments and analysis optimize the sequence information to ensure its reliability and effectiveness in practical applications.

 

Antibody sequencing technology through systematic sample preparation, antibody degradation, mass spectrometry analysis, data analysis, and sequence assembly, the complete amino acid sequence of antibodies is obtained. MtoZ Biolabs provides integrate antibody sequencing service.