Solid Phase Screening of Nanoantibody Libraries-Nano Antibody Special Topic-Tekbiotech-Yeast Display Service,Phage display technology

Solid Phase Screening of Nanoantibody Libraries

As the application scope of phage antibody library continues to expand, the antigen form that can be used to screen antibodies is also diverse, which can be pathological sections, intact cells, and even organs of living animals. The specificity and affinity of antibodies obtained by phage library screening can be optimized and adjusted. The use of this technology can not only effectively avoid the specific human antibodies obtained during the immunization process, but also overcome the shortcomings of low efficiency of monoclonal antibody preparation, poor stability of hybridoma in vitro culture, and time-consuming and labor-intensive methods.

Ⅰ．Principle of Solid Phase Screening

Solid phase screening is to coat the antigen on a solid organic medium (such as an ELISA plate, an organic membrane or an immunotube), and by incubating the antigen with the antibody library, the specific phage antibody can bind to the antigen. This method is simple, the technology is mature, and it is relatively easy to obtain high-affinity antibodies. The efficiency of screening is related to the purity of the target antigen. The liquid phase screening method mainly coats the biotinylated antigen on magnetic beads coupled to anti-biotin, and then uses the phage library for multiple rounds of screening.

II. Solid Phase Screening Steps:

(1) Dilute the antigen with 50mmol/L sodium bicarbonate buffer (pH 10.0), coat with enzyme-linked immunosorbent assay (ELISA) plate, 100μL per well, and place at 4℃ overnight

(2) Seal with PBS/5% milk for 1 hour the next day, put it into the phage library, incubate at 37℃ for 2 hours, wash 6 times with PBS/0.05% Tween (PBST), and add 200μL elution buffer to each well to elute the bound phage, and then neutralize with 2mol/L Tris-HCl.

(3) Measure the phage titer and amplify it for the next round of screening. Following the "adsorption-elution-amplification" method, a total of 3 rounds of elution were carried out. The subsequent 2 rounds of screening gradually reduced the amount of antigen coating, resulting in a high enrichment of specific phages.

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Figure 1 Solid-phase screening process

III. Types and Selection of Solid-phase Carriers

Common Carrier Types	Plastic Products	Microparticles	Membrane Carriers
Binding method	The most commonly used plastic product is polystyrene, and antigens or antibodies are bound to this carrier by non-covalent or physical adsorption.	It is easy to form chemical coupling with antigens or antibodies, and has a large binding capacity. It can be evenly dispersed in the entire reaction solution, so the reaction speed is faster.	Common membrane carriers include nitrocellulose membrane (Nc), nylon membrane and glass cellulose membrane, which adsorb antibodies/antigens by non-covalent bonds.
Introduction	There are disadvantages such as small binding capacity, high elution adsorption efficiency and unevenness. Currently, non-covalent and chemical coupling covalent adsorption methods are commonly used to improve it.	Can be made into magnetized microparticles	Widely used in qualitative or semi-quantitative spot ELISA

IV. Advantages of Solid Phase Screening

(1) Antibodies can be obtained without in vivo immunization. The screening target can be any antigen, which is easy to mature and modify.

(2) The phage antibody library is a unity of genotype and phenotype, and can combine the antibody selection ability and the phage amplification ability. It is an extremely effective screening system.

V. Common Strategies for Solid-phase Screening of Phage Libraries

Epitope screening: Epitope is a determining site on the surface of an antigen. The main function of this site is to determine the binding characteristics and folding state of the antigen and antibody. It can be a continuous amino acid residue in the primary structure of the antigen, i.e., a linear epitope, or it can be discontinuous, but contains certain amino acids necessary for binding to the receptor, i.e., a conformational epitope. Since spatial epitopes are difficult to simulate, for some receptors that are not easy to purify, a known linear epitope in the extracellular region of the receptor can be purified, which is much easier than purifying the full-length membrane receptor, or directly chemically synthesized linear neutralizing peptides.

VI. Optimization Strategy for Solid Phase Screening of Phage Libraries

Antigen Concentration

Washing Conditions

There is an effective concentration for the antigen used for screening. When the antigen concentration is too high, nonspecific adsorption increases greatly, which increases the number of screening rounds and the workload; at the same time, it may cause high-affinity antibodies to bind and not be dissociated, resulting in clone loss. When the antigen concentration is too low, functional antibodies with low affinity may not bind and cannot be enriched and screened.

If the washing stringency is too high, those phage antibodies with functional but low affinity will be washed away; if the stringency is too low, it will lead to an increase in nonspecific binding.

VII. Experimental Process of Cell Screening

Tek Biotech has complete upstream and downstream docking services, including antigen and antibody expression and purification, antibody humanization, recombinant antibody expression and other services. It has mature and stable technology in library construction. The phage display technology provides a library capacity of 10^8-10^9 for the construction of the first-level immune library, and the insertion rate meets >95%. The affinity of the antibodies obtained by screening is generally at the nM-pM level. It has a variety of screening systems, including direct screening, competition, negative screening, cell screening, etc., and can provide VHH screening, scFv sequence screening, Fab antibody screening, etc. to meet the different needs of different customers. High product delivery standards: for immune libraries, deliver pre- and post-immunization serum, antibody display library, screening elution products, nano antibody sequences with complementary degree of CDR region, QC quality control standards (including RNA extraction, cDNA preparation, etc.).

If you are interested in carrying out phage display antibody projects, or other related services, such as antibody sequencing, scFv, Fab and Fc fusion proteins, please contact us!

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