I. Fundamental Principles of Monoclonal Antibody Production Using Hybridoma Technology

Monoclonal antibodies (mAbs) are highly homogeneous antibodies produced by a single B cell clone, targeting a specific antigenic epitope.  They are typically generated using hybridoma technology, which is based on cell fusion techniques.  This method involves fusing antigen-sensitized B cells (capable of secreting specific antibodies) with myeloma cells (possessing unlimited proliferative potential) to create B cell hybridomas.

In this process, mouse myeloma cells (which can be cultured and expanded indefinitely in vitro) are fused with B cells from inbred mice immunized with the target antigen.  The resulting hybrid cell line combines the myeloma cells' ability to proliferate endlessly with the B cells' capacity to produce and secrete specific antibodies.By isolating and culturing individual hybridoma cells, a single-cell clone (monoclonal line) is established.  These hybridomas can then be grown in culture or injected into mouse peritoneal cavities to produce large quantities of highly concentrated, uniform antibodies.  These mAbs exhibit consistent structure, amino acid sequence, and specificity.  As long as no mutations occur, the antibodies secreted over time will maintain identical structure and function—a level of reproducibility unattainable with other methods.

II. Production Process of Monoclonal Antibodies

2.1 Immunization of Experimental Animals

(1) Primary Emulsification: Mix the antigen (150–200 μg per mouse) with an equal volume of Freund’s Complete Adjuvant (FCA). Emulsify the mixture by repeatedly pushing and pulling through a medical three-way stopcock and syringes until the emulsified mixture forms a droplet that remains intact on the water surface without rapid dispersion.

(2) Primary Immunization: Use a multi-point injection method, prioritizing subcutaneous injection sites in the limbs and armpits, followed by the back.

(3) Subsequent Immunizations: Mix the antigen (80–100 μg per mouse) with an equal volume of Freund’s Incomplete Adjuvant (FIA). The emulsification process and immunization procedure are identical to the primary immunization.

(4) Blood Collection and Serum Titer Testing: After the fourth immunization, collect blood from the tail vein. Use the serum as the primary antibody in an indirect ELISA to determine the serum titer of the mice. If the OD value is ≥1 at a serum dilution of 12,800, the sample meets the fusion criteria.

(5) Final Boost Immunization: Administer the antigen in its original concentration (200–250 μg per mouse) via intraperitoneal injection. Cell fusion can be performed 3–5 days later.

2.2 Cell Fusion

(1) Resuspend SP2/0 cells and immune mouse splenocytes separately in incomplete 1640 medium, then count using a hemocytometer. Mix the cells at a ratio of splenocytes : SP2/0 cells = 1:3 in a 50 mL sterile centrifuge tube. After thorough mixing, add incomplete 1640 medium up to 40 mL.

(2) Centrifuge at 1500 rpm for 5 min. (During this step, prepare a 37°C water bath for warming the cell fusion tube.)

(3) After centrifugation, carefully discard the supernatant and residual liquid along the tube wall (sterile absorbent paper strips can be used to remove excess liquid). Gently tap the mixed pellet of SP2/0 cells and immune splenocytes to loosen it, then immerse the bottom of the centrifuge tube in the 37°C water bath.

(4) Retrieve 1 mL of pre-warmed fusion agent PEG1450 from the incubator and add it dropwise to the cell pellet over 60 seconds, while gently rotating the tube to ensure even distribution.

(5) Let the mixture incubate undisturbed for 45 seconds. Then, take pre-warmed incomplete 1640 medium (37°C) and add 1 mL dropwise over 60 seconds to dilute the PEG fusion agent (while rotating the tube). Next, add another 1 mL dropwise within 30 seconds, followed by slowly adding the remaining 45 mL of medium in a controlled manner.

2.3 Determination of Serum Antibody Titer in Immunized Mice

(1) After four immunizations, collect blood from the tail vein of the mice.

(2) Incubate the whole blood at 37°C for 1 hour, followed by 4°C for 1 hour (or overnight at 4°C). Centrifuge at 3000–4000 rpm for 5–10 min, then transfer the supernatant to a new, clean EP tube to obtain the immune mouse serum.

(3) Randomly select serum from one mouse to determine the optimal antigen coating concentration using a checkerboard titration. The antigen coating concentration gradient is typically set between 0.25–10 μg/mL, and the initial serum dilution factor is usually 400× or 500×.

(4) Measure the OD450nm by indirect ELISA and plot the results as a line graph. The optimal coating concentration is determined when:The starting point (highest OD450nm value) no longer increases significantly with higher antigen coating concentrations.The entire curve shows a smooth downward trend.

(5) Using the optimal coating concentration, measure the serum antibody titers of the other mice. A serum dilution factor of 12,800× with an OD450nm ≥ 1.0 is considered sufficient to meet the fusion criteria.

III. Advantages and Limitations of Monoclonal Antibodies

Advantages of Monoclonal Antibodies:

(1) Hybridomas can survive and proliferate "indefinitely" in vitro, continuously producing highly specific and homogeneous antibodies, provided no genetic mutations occur in the cell line.

(2) Highly specific and uniform antibodies can be obtained in large quantities using relatively impure antigens.

(3) Since a theoretically "unlimited" supply of homogeneous antibodies can be produced, they are suitable for immunological assay techniques that rely on labeled antibodies, such as IRMA and ELISA.

(4) Due to their high specificity and single biological function, monoclonal antibodies can be used for in vivo radioimmunoimaging and immunotargeted therapy.

Limitations of Monoclonal Antibodies:

(1) The inherent affinity and restricted biological activity of monoclonal antibodies limit their scope of application. Since monoclonal antibodies cannot perform precipitation or agglutination reactions, many detection methods cannot be carried out with them.

(2) The reaction intensity of monoclonal antibodies is weaker compared to polyclonal antibodies.

(3) The preparation process is technically complex, time-consuming, and labor-intensive, making monoclonal antibodies more expensive.

IV. Applications of Monoclonal Antibodies

1．Diagnostic Reagents in Laboratory Medicine

As diagnostic reagents in clinical laboratories, monoclonal antibodies are widely used in techniques such as enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), immunohistochemistry (IHC), and flow cytometry due to their high specificity, purity, and homogeneity. Moreover, the use of monoclonal antibodies has significantly contributed to the development of commercial diagnostic kits.

2．Protein Purification

Monoclonal antibodies serve as crucial ligands in affinity chromatography. The monoclonal antibody is adsorbed onto an inert solid-phase matrix (such as Sepharose 2B, 4B, 6B, etc.) and packed into a chromatography column. When a sample passes through the column, the target antigen specifically binds to the immobilized monoclonal antibody, while other components remain unbound. After thorough washing of the column, the bound antigen is eluted by altering the ionic strength or pH of the elution buffer. The eluate is then collected, yielding the purified antigen.

3．Targeted Cancer Therapy and Radioimmunoimaging Technology

By conjugating monoclonal antibodies specific to tumor antigens with chemotherapeutic drugs or radioactive agents, the antibodies can deliver these therapeutic substances directly to target tissues to kill cancer cells—an approach known as targeted cancer therapy. Additionally, when labeled with radioisotopes, monoclonal antibodies can be administered to patients for radioimmunoimaging to aid in tumor diagnosis.Currently, most monoclonal antibodies are murine-derived, and heterologous animal serum may cause allergic reactions in humans. Therefore, developing human-human monoclonal antibodies or humanized antibodies is crucial. However, significant progress in this area has yet to be achieved.

KMD Bioscience has been dedicated to antibody preparation research for many years. Monoclonal antibodies are produced by monoclonal hybridoma cells formed through the fusion of activated B lymphocytes with myeloma cells. With our experienced technical team in antibody preparation, we have accumulated substantial expertise and specialized techniques in antibody development. Coupled with our well-established antibody production facilities, we are capable of providing customized monoclonal antibody services across various species for our clients.

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