Antibody Selection for Phage Display ELISA Identification-Special Topic on Phage Display Technology-Tekbiotech-Yeast Display Service,Phage display technology

Antibody Selection for Phage Display ELISA Identification

No matter what method is used to prepare antibodies, or what kind of antibodies are prepared. Functional verification of the prepared antibodies is very important, and the premise of functional verification is to obtain high-purity proteins. Usually, the method we use to verify the function is indirect ELISA detection. The principle of indirect ELISA will not be repeated here, but we need to determine the enzyme-labeled antibody used, which is usually the antibody of the label protein carried by the expressed protein. Here we take phage surface display as an example to illustrate the selection of ELISA identification antibodies for antibody preparation.

First, let's introduce the surface display related carriers:

pADL-10 Low-Display Phagemid Vector Series:

The phagemid pADL-20 Vector Series:

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pADL-100 Maximum Display Phagemid Vector Series:

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fADL Multivalent Phage Vector Series:

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pHAL30:

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phen:

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pCANTAB:

Only some vector maps are shown here, but through the composition of these vectors, we can understand that when using phage display technology to prepare antibodies, the selected phage or phagemid surface display vectors usually carry tag proteins——

His tag: This fusion tag is composed of six histidine residues and can be inserted at the C-terminus or N-terminus of the target protein. It is the most commonly used tag for protein purification. The target protein fused with the His tag can be purified using a nickel column to increase the purity of the protein.

Hemagglutinin (HA) tag: This tag contains 9 amino acids and the sequence is YPYDVPDYA. It is derived from the antigenic determinant on the surface of the influenza virus hemagglutinin. It has little effect on the spatial structure of the target protein and is easy to fuse and express to the N-terminus or C-terminus of the target protein. It is easy to use Anti-HA antibodies for ELISA and other detection.

Myc tag: This tag contains 10 amino acids and the tag sequence is EQKLlSEEDL. It can be fused and expressed to the N-terminus or C-terminus of the target protein. These 10 amino acids can still be used as antigen epitopes to identify their corresponding antibodies when expressed in different protein frameworks. Therefore, Myc tag has been successfully applied in Western-blot hybridization technology, immunoprecipitation and flow cytometry to detect the expression of target recombinant proteins.

Sometimes two tags are even added, His+HA and His+Myc, to perform two affinity chromatography purifications to obtain high-purity, high-specificity and even high-solubility target proteins.

In addition to tag antibodies, M13 antibodies are sometimes used for phage surface display. Next, we will introduce some commercial products of M13 recombinant antibodies: M13 Major Coat Protein (RL-ph1, IgG2b kappa light chain monoclonal antibody) against M13, M13 enzyme-labeled antibody (Anti-M13 (HRP) Antibody), M13 g8p antibody and HRP enzyme-labeled M13 antibody (arigo). Enzyme-labeled antibodies can be directly used for ELISA detection, reducing the use of secondary antibodies, saving time and cost. If the indirect ELISA method is used to detect enzyme-labeled secondary antibodies, there is no need to add labels during the construction of the vector. Phage ELISA detection can be performed directly without worrying about the impact of the introduction of labels on antibody expression.

Tek Biotech has a deep reserve of antibody research and development knowledge. Our scientists can provide customers with a more systematic antibody downstream engineering service, including antibody purification, antibody pairing (ELISA custom development), antibody-antigen binding kinetics research, antibody sequencing and antibody modification, etc. One-stop technical services save customers valuable time and money.

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