5-Step Process To Sequence Monoclonal Antibodies

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In recent times, the world of biosciences has evolved significantly, with several new techniques, procedures, and alterations coming into the fold. Speaking about antibody production and degeneration, a multitude of scholars have conducted studies on how antibodies operate and function.

Meanwhile, when you touch base with monoclonal antibodies, they are proteins that bind to a particular molecule. This makes them useful in various fields such as research, medicine, or diagnostics. Sequencing of monoclonal antibodies involves the identification of the correct sequence of amino acids that make up the protein.

The sequencing is done by isolating antibodies and breaking them into single amino acids. Afterward, monoclonal antibody sequencing involves using methodologies like mass spectrometry and chromatography. This is to identify the sequence of monoclonal antibodies.

Moving on, sequencing of the monoclonal antibodies is essential. This is because it helps in the understanding of the structure of the antibodies and their functions. Moreover, it is critical because it helps develop new therapeutic applications.

What are Monoclonal Antibodies

Monoclonal antibodies are also called moAbs. They are proteins that are manufactured in laboratories and act like proteins called antibodies. Their primary function is to seek out antigens and stick them to destroy them.

Laboratory-manufactured monoclonal antibodies help stimulate our immune system. Moreover, monoclonal simply refers to how antibodies created in the labs are clones. They are just the exact copies of one antibody.

If you want to identify amino acid sequences, you can seek antibody sequencing services. They can determine if the antibody’s amino acid sequence is polyclonal or monoclonal.

Let’s explore the 5 step process to sequence monoclonal antibodies.

  1. Generation of Monoclonal Antibodies

Producing the antibodies themselves is the first step of sequencing monoclonal antibodies. Proteins known as monoclonal antibodies can be modified to bind precisely to a target molecule. Monoclonal antibodies are usable in various applications, such as therapeutic treatment and diagnostic tests, among many others.

This generation of monoclonal antibodies involves the use of hybridoma technology. Hybridomas are made by fusing rat B cells, producing antibodies with a myeloma cell line. The rat B cells are then fused with the myeloma cell line to produce commemorated cells.

The commemorated cells generate a single type of antibody known as a monoclonal antibody. The produced monoclonal antibody is then screened and tested for its affinity and specificity to the specific antigen.

  1. Purifications of Monoclonal Antibodies

After generating monoclonal antibodies through hybridoma technology, it is time to purify them. Purification of monoclonal antibodies helps in making them pure and active. Moreover, another critical need for this purification step is to separate the monoclonal antibodies from contaminants and proteins present.

The contaminants are often found in the hybridoma culture supernatant. The pollutants include secreted proteins, serum components, and cell debris. This step involves the chromatography techniques and multiple stages.

Chromatography is a technique for separating the constituents of a mixture. This technique is based on the chemical and physical properties of the mixture’s components. Furthermore, the chromatography technique used to purify monoclonal antibodies depends on the antibody. It is also dependent on the pollutants present in the culture supernatant.

  1. Fragmentation of Monoclonal Antibodies

Monoclonal antibodies are huge proteins, making it difficult to analyze them directly. So, for a more straightforward analysis, it is crucial for this fragmentation of the monoclonal antibodies step. Smaller or more fragmented pieces of monoclonal antibodies can be readily studied without difficulties.

These antibodies can be fragmented by using enzymatic digestion with a protease. Trypsin is one of the proteases that can be used. This is because the trypsin protease can break peptide bonds on the carboxyl side of lysine and arginine byproducts. When you add trypsin to a monoclonal antibody solution, it cleaves the antibody into small peptides.

For the smaller peptides produced, they can be separated by chromatography. This chromatography technique can help separate the monoclonal antibody molecules based on size or charge. The separation is also based on their chemical and physical properties. Techniques like mass spectrometry can analyze the fragments that have been separated.

  1. Mass Spectrometry Analysis of Monoclonal Antibodies

Following the fragmentation of monoclonal antibodies, the generated peptide fragments can be analyzed using mass spectrometry. Mass spectrometry is a methodology that ionizes and separates molecules. This enables the identification and quantification of single peptide fragments.

Various mass spectrometry techniques can be leveraged for the sequencing of monoclonal antibodies. Some of the methods include:

  • Electrospray Ionization
  • Matrix-assisted laser ionization or desorption

Mass spectrometry analysis of monoclonal antibodies also includes some key steps such as:

  • Sample preparation

In this step, the peptide fragments are derived from the monoclonal antibodies. They are then purified or screened using liquid chromatography to remove pollutants that may be there.

  • Ionization

After that, the peptide fragments are ionized using MALDI or ESI. However, the ionization of the pieces using MALDI or ESI depends on the specific mass spectrometry technique employed.

  • Mass Analysis

After the ionization, the peptide fragments are separated. The separation is based on their mass-to-charge ratio with the leverage of a mass analyzer such as a quadrupole analyzer.

  • Data Interpretation

Just after the mass analysis, the resulting mass spectra are examined using the software. This will aid in identifying single peptide fragments and their amino acids sequentially.

  1. Data Analysis and Confirmation

This is the final step in the monoclonal antibody sequencing process. You must evaluate and verify the results of the mass spectrometry analysis data. This step also involves comparing the recognized peptide fragments to the monoclonal antibody’s reference sequence.

Moreover, it involves performing other experiments to substantiate the sequence. This step is crucial since it confirms the sequencing data’s accuracy and the resulting data sequence. Once you approve the monoclonal antibody sequencing, you can use them for various applications.

Take Away

Monoclonal antibody sequencing is essential for creating applications in diagnosis and therapy. Monoclonal antibodies are unique and compelling. However, their properties are defined by their amino acid structure.

Monoclonal antibody sequencing enables the identification of a unique monoclonal antibody sequence. Therefore, sequencing provides good insight into the monoclonal antibody structure, applications, and functions. 

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