Antibody Basics: Part 1 - What are monoclonal, polyclonal, and recombinant antibodies?

Welcome to Antibody Basics by Biointron, Part 1. Here we’ll give an introduction on monoclonal, polyclonal, and recombinant antibodies.

Antibodies are Y-shaped proteins produced by the immune system to identify and neutralize foreign objects like bacteria and viruses. They recognize a unique molecule of the foreign object, called an antigen, via the antibody's variable region. When an antibody binds to an antigen, it triggers other parts of the immune system to eliminate the antigen.

Immunoglobulin, IgG, is made up of four polypeptide chains, comprising two identical light chains and heavy chains. The heavy chains are linked to each other by disulfide bonds and, each heavy chain is also linked to a light chain by a disulfide bond. The antibody has two identical antigen-binding sites, giving it the ability to bind simultaneously to two structures.

Antibodies, are classified into five isotypes (IgG, IgA, IgD, IgE, and IgM) based on the constant domains in their H chains, which give each isotype their different characteristics. They can be further classified into subtypes.

Because of their role in the body’s immune system, antibodies are highly valuable in research and therapeutics. Antibodies are essential research tools, being involved with ELISA testing, Western Blots, Immunofluorescence, Flow Cytometry, Immunoprecipitation, and many more. As a therapy, antibodies can be used to treat numerous diseases by utilizing their specificity and affinity in targeting specific molecules. The most common antibody drug type is the monoclonal antibody. 

Polyclonal antibodies were first used for research in the early to mid-20th century, after the discovery of serum antibodies in the late 19th century. Subsequently, the development of monoclonal antibodies in 1975 became a major research breakthrough, as it allowed for the production of highly specific antibodies with known binding characteristics. 

Polyclonal antibodies

• Produced by different B lymphocytes and recognize different epitopes of a single antigen.

• They can be generated in large quantities and rapidly (within several months of initiating immunizations), and at a lower cost than mAbs.

• Heterogenous: any structural changes on one epitope are unlikely to significantly effect binding, and pAbs are more stable over a broad pH and salt concentration.

• Monoclonal Antibodies 

• Produced by identical B lymphocytes which are clones of a single parent cell.

• Can be produced as a constant and renewable resource once the desired hybridoma has been generated.

• Homogeneous: they have high specificity and affinity. They are particularly useful in analyzing changes in molecular conformation, protein-protein interactions, phosphorylation states, and identifying single members of protein families.

Polyclonal antibodies are produced by different B cell clones. Here is an overview of the steps:

1. Antigen Preparation: Depending on the application, the target antigen will be a purified protein, peptide, or protein mixture.

2. Immunization: Animals are immunized with the antigen of interest. Booster immunizations administered.

3. Serum Collection and Processing: Animals are allowed time to mount a robust immune response. Blood is collected to obtain serum samples when antibody titers are sufficiently high.

4. Purification: Purification techniques to concentrate and isolate the antibodies of interest.

5. Characterization: Purified pAbs are assessed for specificity and binding properties.

Monoclonal antibodies are produced by identical B cells which are clones from a single parent cell. Here is an overview of three methods:

• Hybridoma Technology: After animal immunization with a specific antigen of interest, spleen cells are harvested to obtain B cells. Hybridoma cell lines are created by fusing these antibody-producing B cells with immortalized myeloma cells. Monoclonal antibody-producing hybridoma cells are screened for the specific binding and activity against the target antigen. This step may take several rounds to identify the most suitable clones.

• Single B Cell Technology: After animal immunization with a specific antigen of interest, antibody-secreting cells are harvested from spleen and bone marrow. Different platforms exist for single B cell sorting including nanowell, nanovial, and microfluidics-based methods. A microfluidic-based method involves protein binding and/or cell-based binding to detect and sort positive micro-droplets. Single-cell sequencing is then performed.

• Phage Display Technology: By genetically engineering the bacteriophage's coat protein, a fragment sequence can be linked to the phage DNA, enabling the displayed antibody fragment to be encoded by the viral genome. These phage display libraries screen the phage particles to identify antibodies that bind to specific target antigens in a process called panning.

Recombinant antibodies are monoclonal antibodies produced in vitro. Unlike traditional antibodies made by the immune system, recombinant antibodies are generated from a synthetic gene to target specific molecules, such as those found on the surface of cancer cells. One of the main advantages of recombinant antibodies is their ability to be produced on a large scale, making them useful in the development of diagnostic tools and therapeutic drugs.

The steps to produce recombinant antibodies are as follows:

1. Design and Cloning: The antibody gene sequence is cloned into an expression vector.

2. Transfection: Host cells (e.g., mammalian cells) are transfected. Host cells will produce the recombinant antibody.

3. Cell Culture: Transfected cells are cultivated in a bioreactor or cell culture and monitored for cell growth and antibody expression.

4. Harvest and Purification: The cell culture supernatant or lysate containing rAbs are harvested for purification to isolate the desired antibody.

5. Characterization and Quality Control: Purified antibodies are analyzed to confirm identity, functionality, antibody specificity and binding properties.

Biointron focuses on antibody discovery, expression, and optimization. Our main feature is antibody expression, with gene sequence to purified antibodies in just 2 weeks! Biointron’s high-throughput platform enables the rapid expression of recombinant antibodies in HEK293/CHO cells. We guarantee the delivery of 100ug-100g of recombinant antibodies. No antibody = no cost! 

We also have a VHH antibody library generation service. Biointron is a well-recognized leader in this space. With our self-owned alpaca breeding farm, we offer a 1 alpaca for 1 project commitment, and we guarantee the delivery of 20+ unique binders, high diversity and large capacity. 

We also have a wide variety of other services, including bispecific antibodies, afucosylated antibodies, single B cell screening, hybridoma sequencing, antibody optimization, recombinant protein expression in the mammalian system, and CHO-K1 stable cell line development.