Phage Display Technology: Antigen Presentation for Nanobody Discovery

During the phage display screening process, the target molecule needs to be immobilized. This process is called antigen presentation. Antigen presentation can be categorized as direct presentation or indirect presentation.

1. Direct Presentation

Direct presentation is the simplest and most widely used method of antigen presentation. The antigen is directly immobilized on the solid surface via physical adsorption. During direct antigen immobilization, the antigen is passively adsorbed to the solid surface, resulting in random, non-directional antigen presentation. However, proteins bind to polystyrene solid supports via physical adsorption, primarily through the interaction between hydrophobic groups in the protein's molecular structure and those on the solid support's surface. Therefore, adsorption is more likely to occur in areas rich in hydrophobic amino acids. Furthermore, this process is influenced by factors such as the protein's molecular weight, isoelectric point, and concentration. Direct adsorption can easily block some epitopes. Larger proteins typically contain more hydrophobic groups than smaller proteins, making them more susceptible to adsorption to the solid support's surface.

Direct coating is achieved through physical adsorption, which mainly relies on hydrophobicity. The following problems may occur during this process: 

(1) the conformation of the antigen is changed, and the screened antibodies cannot bind to the natural conformation antigen, such as membrane surface antigens; 

(2) for antigens with small molecular weight (peptides and small molecules), the antigen epitope cannot be fully exposed, and the blocking proteins (BSA and OVA) bring steric hindrance, resulting in the inability to screen antibodies with suitable epitopes or the screened antibodies cannot cover all epitopes.

2. Indirect Presentation

To avoid the adverse effects of direct antigen immobilization, indirect immobilization can be attempted during phage display antibody screening. This involves immobilizing the target antigen using capture molecules on the surface of a solid support.

Several commonly used indirect immobilization methods include:

(1)  The strong covalent reaction between streptavidin and biotin is used to indirectly and stably attach the antigen to the surface of the solid phase carrier. There are two different biotinylation strategies: site-specific biotinylation and random biotinylation. Site-specific biotinylation can be achieved by using Avi Tag for in vivo/in vitro biotinylation. The ratio of antigen to biotin can be controlled by using site-specific biotinylation. However, the Avi Tag system cannot be used when recombinant expression of the target antigen is difficult or when the Avi Tag interferes with the potential epitope of the antigen. Random biotinylation is to react the purified antigen with the biotinylation reagent to achieve covalent connection between the antigen and biotin. Random biotinylation is faster and cheaper, but it cannot cause the antigen to be over-biotinylated. Over-biotinylated antigens may also mask their important epitopes, inactivate the antigen, or cause antigen aggregation.

Nanoselector|Streptavidin Magnetic beads (Code: 067-101-003) use a directional immobilization technology to covalently couple recombinant neutral streptavidin to the surface of 2.8um magnetic beads, which can efficiently bind to biotinylated antigens. This product utilizes superparamagnetic microspheres with uniform particle size and regular morphology, facilitating the convenient and rapid capture of target molecules and enabling magnetic separation. It can be used with automated equipment for high-throughput screening.

(2) Indirect immobilization of antigens using different peptide tags and labeled specific capture molecules. Such as his tag and Anit his tag systems, nickel media, and the recently emerged SpyTag and SpyCatcher systems. SpyTag and SpyCatcher systems are irreversible, specific, and highly stable under various conditions such as pH, temperature, and buffer.

AlpVHHs provides a series of pre-coated magnetic beads and ELISA plates for easy screening.

Pre-coated magnetic beads:

C-His tag Nanoselector Magnetic beads are nano-antibodies with nM affinity that are site-specifically coupled to the surface of 2.8um superparamagnetic magnetic beads, which can efficiently bind to C-his tag antigens.

SpyCatcher Nanoselector Magnetic beads are expressed and purified SpyCatcher that are site-specifically coupled to the surface of 2.8um superparamagnetic magnetic beads, thereby achieving directional immobilization of SpyTag antigens.

Pre-coated ELISA plates:

For phage libraries from different species, we use antibodies from different species to coat the ELISA plates, significantly reducing nonspecific background signals during screening. For example, immunizing alpacas with a human Fc-fused antigen will produce a large number of antibodies targeting human Fc. Therefore, we recommend using llama antibody plates for screening to effectively avoid screening antibodies that recognize human Fc.

AlpVHHs® specializes in developing and producing customized, high-performance alpaca nanobodies. We provide tailored single-domain antibody (VHH or sdAb) pre-discovery CRO services and innovative nanobody products as the next-generation RUO tools for life science research.

We possess industry-leading Phage Display and Yeast Surface Display platforms, as well as two dedicated animal farms housing over 800 Camelids (including alpacas, llamas, and camels), ensuring traceable and wellcharacterized animal sources for the development of high-quality recombinant nanobodies (VHHs).

Our product portfolio includes the Nanoselector series, tag nano-antibody, nano-secondary antibody, recombinant nano-secondary antibody, Smart Booster, and more. These are widely used in immunoprecipitation (IP), immunofluorescence (IF), super-resolution imaging, cell sorting, and antibody internalization assays.

For more details, please visit AlpVHHs website www.alpvhhs.com