Antibody screening and identification are critical processes in the development of monoclonal and polyclonal antibodies for research, diagnostic, and therapeutic purposes. This guide outlines the steps involved in screening and identifying antibodies with high specificity and affinity for their target antigens.

Materials and Reagents

1. Antigen: Purified target protein or peptide.
2. Antibody library: Hybridoma cells, phage display library, or Bcell repertoire.
3. Cell culture media: Suitable media for growing hybridoma or bacterial cells.
4. ELISA reagents: Coating buffer, blocking buffer, washing buffer, substrate solution, and stop solution.
5. Flow cytometry reagents: Fluorochromeconjugated secondary antibodies and buffers.
6. Western blot reagents: SDSPAGE gels, transfer buffer, membranes, blocking buffer, primary and secondary antibodies, and detection reagents.
7. Screening plates: 96well or 384well plates for ELISA and cell culture.
8. Equipment: Incubator, centrifuge, plate reader, flow cytometer, and western blot apparatus.

Protocol

1. Library Preparation

1.1. If using a phage display library, prepare the library by infecting E. coli with the phage library and amplifying it.

1.2. For hybridoma technology, immunize mice with the target antigen and fuse their spleen cells with myeloma cells to generate hybridomas.

2. Screening for Binding Specificity

ELISA Screening:

2.1. Coat the wells of an ELISA plate with the target antigen diluted in coating buffer and incubate overnight at 4°C.

2.2. Block the wells with blocking buffer to prevent nonspecific binding.

2.3. Add antibodycontaining samples (e.g., supernatants from hybridoma culture or phage display eluates) to the wells and incubate.

2.4. Wash the wells to remove unbound antibodies.

2.5. Add enzymeconjugated secondary antibodies specific to the primary antibodies and incubate.

2.6. Add substrate solution and measure the color change using a plate reader.

Flow Cytometry Screening:

2.1. Incubate cells expressing the target antigen with antibodycontaining samples.

2.2. Wash the cells to remove unbound antibodies.

2.3. Incubate with fluorochromeconjugated secondary antibodies.

2.4. Wash the cells and analyze by flow cytometry to detect bound antibodies.

Western Blot Screening:

2.1. Separate the target antigen by SDSPAGE and transfer it to a membrane.

2.2. Block the membrane with blocking buffer.

2.3. Incubate the membrane with antibodycontaining samples.

2.4. Wash the membrane and incubate with enzymeconjugated secondary antibodies.

2.5. Detect bound antibodies using chemiluminescent or colorimetric substrates.

3. Identification of Positive Clones

3.1. Identify wells or samples showing strong and specific binding to the target antigen.

3.2. If using hybridomas, clone positive wells by limiting dilution to isolate monoclonal antibodyproducing cells.

3.3. For phage display, amplify positive phage clones and perform additional rounds of panning if necessary to enrich for highaffinity binders.

4. Characterization of Antibodies

4.1. Affinity Measurement: Determine the binding affinity of selected antibodies using surface plasmon resonance (SPR) or biolayer interferometry (BLI).

4.2. Specificity Testing: Test the antibodies against related antigens to confirm specificity using ELISA, western blot, or flow cytometry.

4.3. Epitope Mapping: Identify the specific region of the antigen recognized by the antibody using techniques like peptide scanning or alanine scanning mutagenesis.

5. Validation and Production

5.1. Validate the antibodies in relevant biological assays (e.g., immunoprecipitation, immunofluorescence, neutralization assays).

5.2. Scale up the production of validated antibodies using appropriate expression systems (e.g., hybridoma culture, transient transfection in mammalian cells, or recombinant expression in bacterial/yeast systems).

6. Purification

6.1. Purify the antibodies using Protein A/G affinity chromatography, followed by further purification steps such as ion exchange or sizeexclusion chromatography if necessary.

Conclusion

This protocol outlines the essential steps for screening and identifying highaffinity, specific antibodies using various techniques. The choice of method depends on the nature of the antigen and the available antibody library. Successful identification and validation of antibodies are critical for their application in research, diagnostics, and therapeutics.
Reference:
https://www.alpha-lifetech.com/phage-display-library-screening-service/