Materials Needed

Expression Vector: Contains genes for the antibody's heavy and light chains
Host Cells: CHO or HEK293 cells
Transfection Reagents
Culture Media
Purification Columns: Protein A/G


Recombinant Antibody Production Protocol

1. Antigen Selection: The first step is to identify the target antigen that the desired antibody should bind to. This is a critical step as it determines the specificity of the final antibody.

2. Antibody Library Construction: A library of antibody genes is created, typically by cloning the variable region genes from B cells of an immunized animal or a human donor. This creates a diverse pool of antibody sequences that can be screened.

3. Antibody Screening: The antibody library is screened to identify the clones that bind the target antigen with high affinity. This is often done using display technologies like phage display or yeast display.

4. Antibody Engineering and Optimization: The selected antibody sequences can be further engineered and optimized to improve properties like binding affinity, specificity, stability, and effector functions. Techniques like affinity maturation, humanization, and antibody fragment engineering can be employed.

5. Expression System Selection: The optimized antibody genes are then cloned into an appropriate expression system, such as bacterial (E. coli), yeast (Saccharomyces cerevisiae), or mammalian (CHO, HEK293) cells. The choice depends on factors like post-translational modifications, expression levels, and scalability.

6. Transient or Stable Expression: The antibody can be expressed either transiently (short-term) or stably (long-term) in the host cells. Transient expression is faster but yields lower amounts, while stable expression takes longer but produces higher quantities of the antibody.

7. Antibody Purification: The recombinant antibody is purified from the cell culture using techniques like affinity chromatography, ion exchange chromatography, or size exclusion chromatography. This step ensures high purity and homogeneity of the final product.

8. Antibody Characterization: The purified antibody is thoroughly characterized using various analytical methods, such as ELISA, Western blotting, and flow cytometry, to verify its specificity, affinity, and other key properties.

9. Scale-up and Manufacturing: Once the optimal antibody candidate is identified, the production process can be scaled up to generate larger quantities for further development and applications, such as therapeutic or diagnostic use.


Polyclonal Antibody Production

1. Immunization

A. Prepare Antigen:
Antigen Purification: Purify the antigen or prepare the antigenic peptide/protein.
Formulation: Dissolve the antigen in a suitable adjuvant (e.g., Freund’s Complete Adjuvant for initial immunization, Freund’s Incomplete Adjuvant for booster immunizations).

B. Immunization Protocol:
Animal Selection: Choose an appropriate animal (e.g., rabbits, goats, sheep).
Initial Immunization: Inject the antigen-adjuvant mixture into the animal (e.g., intramuscularly or subcutaneously).
Booster Shots: Administer booster injections at regular intervals (e.g., every 2-4 weeks) to enhance the immune response.

2. Blood Collection

A. Harvest Blood:
Collection: Collect blood from the immunized animal after the immune response is sufficiently developed (usually 2-4 weeks post-final boost).
Processing: Allow blood to clot, then centrifuge to separate serum from the clot.

B. Serum Preparation:
Serum Separation: Collect the serum from the centrifuged blood and store it at -20°C or -80°C for long-term storage.

3. Antibody Purification

A. Affinity Purification:
Prepare Affinity Column: Use a column with an antigen-specific ligand or antigen-bound to a solid support.
Apply Serum: Load the serum onto the column to capture specific antibodies.
Wash and Elute: Wash the column to remove non-specifically bound proteins, then elute the antibodies using an elution buffer.

B. Concentration and Buffer Exchange:
Concentration: Concentrate the purified antibodies using centrifugal concentration devices if needed.
Buffer Exchange: Dialyze or use a desalting column to exchange the buffer to a suitable storage buffer (e.g., PBS with sodium azide).

4. Quality Control

A. Test Specificity and Titer:
ELISA: Perform an enzyme-linked immunosorbent assay (ELISA) to test the antibody’s specificity and titer.

B. Verify Purity:
SDS-PAGE or Western Blot: Check the purity of the antibody preparation.


Monoclonal Antibody Production
1. Immunization and Cell Fusion
A. Immunization:
Antigen Preparation: Prepare and purify the antigen as described above.
Immunization: Immunize a suitable mouse strain (e.g., BALB/c) with the antigen using the same protocol as for polyclonal antibodies.
B. Cell Fusion:
Splenocyte Isolation: Harvest splenocytes from the immunized mouse.
Fusion: Fuse splenocytes with myeloma cells (e.g., NS0, SP2/0) using a fusion agent (e.g., polyethylene glycol).

2. Selection and Screening
A. Selection:
HAT Medium: Plate the fused cells in HAT (hypoxanthine-aminopterin-thymidine) selection medium to select hybridoma cells.
B. Screening:
ELISA or Other Assays: Screen hybridoma supernatants for specific antibody production against the target antigen.

3. Cloning and Expansion
A. Cloning:
Limited Dilution: Perform limiting dilution to isolate single hybridoma clones.
Clone Expansion: Expand positive clones in culture flasks or bioreactors.

4. Antibody Production
A. Culture:
Scale-up: Grow the selected hybridoma clones in larger volumes for high-yield antibody production.
Harvest: Collect and process the culture supernatant to obtain antibodies.

5. Antibody Purification
A. Affinity Purification:
Prepare Affinity Column: Use Protein A/G affinity chromatography to purify the monoclonal antibodies.
B. Concentration and Buffer Exchange:
Concentrate and Buffer Exchange: As with polyclonal antibodies, use concentration and buffer exchange methods as needed.

6. Quality Control
A. Test Specificity and Titer:
ELISA and Western Blot: Validate specificity and titer.
B. Assess Purity:
SDS-PAGE or HPLC: Confirm purity.


Notes

Ethics: Follow ethical guidelines for animal use and care.
Documentation: Keep detailed records of all procedures, reagents, and results.

The recombinant antibody production protocol leverages the power of genetic engineering and cell culture technologies to create highly specific, customizable, and scalable antibodies. This approach offers several advantages over traditional hybridoma-based methods, including the ability to generate fully human or humanized antibodies, improved production efficiency, and the potential for engineering enhanced antibody properties. Producing antibodies, whether monoclonal or polyclonal, involves several critical steps, from immunization to antibody harvesting.
Reference:
https://www.kmdbioscience.com/pages/antibodyplatform.html