Epitope Mapping Service with Random Mutagenesis and Yeast 2-Hybrid

Top panel: diagram illustrating the reverse Y2H screen. A (bait): scFv of antibody; B (prey): antigen. When there is antibody-antigen interaction, the DNA repressor protein (REP) will suppress cell growth. When mutations in antigen disrupt antibody-antigen interaction, REP suppression is relieved, which leads to cell growth and the turn-on of other reporter genes. Bottom panel: an example of using reverse Y2H to map the epitope of Pembrolizumab in human PD1 protein. Note that Mutant #4 carries disruptive mutation indicative of epitope location. Sequencing 10 disruptive mutants allows pinpointing the locus of a potential epitope in human PD1 ectodomain.

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Epitope Mapping with Random Mutagenesis and Yeast 2-Hybrid

Name: Epitope Mapping with Random Mutagenesis and Yeast 2-Hybrid
SKU: BCABEM
Price: 4950.00 USD
Availability: InStock

$4,950.00

Item Cat No.: BCABEM

Antibody: Monoclonal Antibody

Application: Epitope mapping

Reactivity: Human, rabbit, rodent

BiCell Scientific’s epitope mapping service provides high-resolution epitope mapping results with yeast 2-hybrid approach combined with random mutagenesis and alanine inserting scanning technique.

Epitope mapping is the process of experimentally identifying the binding site, or epitope, of an antibody on its target antigen protein. Epitope mapping is the most tedious and error-prone experiment because not all epitopes are linear. Conformational epitopes are formed by the 3D structure that amino acids form but not their primary sequence.

BiCell Scientific Inc takes advantage of a natural selection approach in yeast 2-hybrid system by selecting the mutations that abolish the antibody-to-antigen interaction. BiCell Scientific Inc utilizes 2 mutagenesis techniques to map the epitope: (1) random mutagenesis to pinpoint the epitope locus; (2) alanine insertion mutagenesis to delineate the boundary of the epitope.

Molecular cloning of CDRs (CDR1, CDR2, and CDR3) from IgG heavy chain and light chain into yeast 2-hybrid vector pY2H, expressing the scF_v form of fused F_v domains from heavy chain and light chain.
Transform pY2H-scF_v (bait) into Y187 yeast strain.
Molecular cloning of antigen protein into pY2H (prey) vector.
Perform random mutagenesis on antigen (>9 mutations per kb) with Mutazyme II DNA polymerase.
Perform yeast 2-hybrid screen with different growth stringency levels.
Recover stringency grown yeast cells and plate them to form single colonies.
Clone and sequence the antigen DNA from recovered yeast cells to locate the epitope site.
Perform site-directed mutagenesis on antigen to introduce alanine insertion to every amino acid site surrounding the potential epitope site.
Perform yeast 2-hybrid screen with different growth stringency levels.
Recover stringency grown yeast cells and plate them to form single colonies.
Clone and sequence the antigen DNA from recovered yeast cells to define the boundary of the epitope.

Customers will receive the following deliverables at the end of each project:

Fully mapped epitope sequence
Mutagenesis report with a full spectrum of mutations that abolish antibody-antigen interaction

For Research Use Only. Not for use in clinical diagnostics or therapeutics.

Molecular cloning of CDRs (CDR1, CDR2, and CDR3) from IgG heavy chain and light chain into yeast 2-hybrid vector pY2H, expressing the scF_v form of fused F_v domains from heavy chain and light chain – 1-2 weeks
Molecular cloning of antigen protein into pY2H (prey) vector – 1-2 weeks
Perform random mutagenesis on antigen (>9 mutations per kb) with Mutazyme II DNA polymerase – 2-3 weeks
Perform yeast 2-hybrid screen with different growth stringency levels – 2-3 weeks
Recover stringency grown yeast cells and plate them to form single colonies – 1-2 weeks
Clone and sequence the antigen DNA from recovered yeast cells to locate the epitope site – 2-3 weeks
Perform site-directed mutagenesis on antigen to introduce alanine insertion to every amino acid site surrounding the potential epitope site – 1-2 weeks
Perform yeast 2-hybrid screen with different growth stringency levels – 2-3 weeks
Recover stringency grown yeast cells and plate them to form single colonies – 1-2 weeks
Clone and sequence the antigen DNA from recovered yeast cells to define the boundary of the epitope – 2-3 weeks

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Epitope Mapping with Random Mutagenesis and Yeast 2-Hybrid

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