I. Common Methods for Antigen Purification

The requirement for antigens is that the higher the purity, the better, especially for the antigens used in primary immunization. For cellular antigens, approximately 1×10^7 cells can be used for intraperitoneal immunization. Soluble antigens need to be mixed with complete Freund's adjuvant and thoroughly emulsified. If the antigen is purified by polyacrylamide gel electrophoresis, the gel band containing the antigen can be excised, ground, and directly used for animal immunization.

II. Notes on Mouse Immunization

Select healthy BALB/c mice that are syngeneic to the myeloma cells used, aged 8–12 weeks, with no restriction on gender. To avoid poor immune responses or mortality during immunization, 3–4 mice can be immunized simultaneously.

The immunization procedure and methods are largely similar to those used for polyclonal antiserum production but may vary depending on the animal, antigen form, and immunization route, with the ultimate goal of obtaining high-titer antibodies. The immunization interval is typically 2–3 weeks. Generally, the higher the serum antibody titer in the immunized animal, the greater the likelihood of obtaining high-titer specific antibodies after fusion. Moreover, the quality of the monoclonal antibodies (e.g., antibody concentration and affinity) is closely related to the serum antibody titer and affinity during the immunization process. Splenocytes are harvested for fusion 3–4 days after the final immunization.

III. Notes on Myeloma Cell Culture

The most critical factor in selecting a myeloma cell line is its syngeneicity with the B cells to be fused. If splenocytes are to be fused, various myeloma cell lines can be used, but the most commonly employed is the Sp2/0 cell line. This cell line exhibits excellent growth and fusion efficiency and, importantly, does not secrete any immunoglobulin heavy or light chains. The maximum cell density for optimal growth is 9×10⁵/mL, with a typical doubling time of 10–15 hours.For fusion, cells should be in the logarithmic growth phase, with good morphology and high viability (≥95%). Prior to fusion, myeloma cells should be adapted to culture in 8-azaguanine-containing medium. One day before fusion, the cells should be adjusted to a density of 2×10⁵/mL using fresh medium; by the next day, they will typically be in the logarithmic growth phase.

IV. Precautions for Cell Fusion Process

The basic steps involve mixing two types of cells, adding PEG to induce cell fusion, then diluting the culture medium to neutralize PEG's effect. After fusion, the cells are appropriately diluted and distributed into culture plate wells for incubation. Several key points require special attention during the fusion process:

① Cell Ratio: The ratio of myeloma cells to spleen cells can range from 1:2 to 1:10, with 1:4 being the most commonly used. Ensure both cell types exhibit high viability before fusion.

② Reaction Time: In the mixed cell suspension, add 4.5 mL of culture medium dropwise in the first minute. After a 2-minute interval, add another 5 mL of culture medium, followed by 50 mL of additional medium.

③ Culture Medium Composition: A well-prepared culture medium is crucial for fused cells, requiring strict formulation of fetal bovine serum, various ions, and nutrients. If fusion efficiency declines, promptly inspect the medium conditions.

V. Precautions for Monoclonal Antibody Cryopreservation

Positive selected cell lines should be cryopreserved as early as possible. The lower the storage temperature, the better—cell lines stored in liquid nitrogen exhibit only a slight decline in viability, whereas those stored at -70°C deteriorate more rapidly. Unlike microbial strains, cells require extra caution during cryopreservation. Dimethyl sulfoxide (DMSO) is the most commonly used cryoprotectant. Post-thaw cell viability typically ranges between 50% and 95%. If viability falls below 50%, it indicates potential issues in the freezing or thawing process.

VI. Methods for Purifying Monoclonal Antibodies

The purification methods for monoclonal antibodies (mAbs) are similar to those for polyclonal antibodies. Ascites fluid typically contains a higher concentration of specific antibodies compared to polyclonal antibodies in antiserum, resulting in better purification efficiency. The choice of purification method depends on the desired level of purity. Common techniques include salt precipitation, gel filtration, and ion-exchange chromatography, while simpler methods like acid precipitation are also occasionally used.Currently, the most efficient method for monoclonal antibody purification is affinity chromatography. This often involves coupling Staphylococcal Protein A or anti-mouse immunoglobulin antibodies to a solid support (most commonly Sepharose) to prepare an affinity column. The antibodies are bound to the column and then eluted, achieving a recovery rate of over 90%.

Protein A binds to IgG1, IgG2a, IgG2b, and IgG3 subclasses, as well as small amounts of IgM. The antibody concentration in the eluate can be roughly estimated using UV absorbance—for mouse IgG monoclonal antibodies, an A280 reading of 1.44 corresponds to 1 mg/mL. To maintain antibody activity, it is crucial to either pre-fill the collection tube with a neutralizing buffer or immediately add a neutralizing solution after low-pH elution.

Based on a well-established antibody platform system, KMD Bioscience offers comprehensive upstream and downstream services, including antigen design, synthesis and modification, animal immunization, phage library construction and screening, as well as downstream antibody modification and application validation.Additionally, we provide antigen preparation services, encompassing DNA synthesis, plasmid construction, protein expression, protein purification, peptide synthesis and modification, small molecule structural analysis, and conjugation with carrier proteins.Furthermore, our antibody team possesses years of experience in developing in vitro diagnostic-grade antibodies and antigens, with extensive expertise in antibody selection and validation. To meet diverse client needs, we can provide customized solutions and tailored screening strategies.

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