Immunogen, Freund's adjuvant, Freund's incomplete adjuvant, saline, syringe

Female Balb/C mice aged 6-8 weeks, microplate, anti-mouse enzyme-labeled secondary antibody

Total volume: 200 μL per mouse; Immunogen dose: 20 μg per mouse per injection; Adjuvant dose: 100 μL per mouse per injection. Inject every two weeks, and after four immunizations, collect blood to test antibody titer. Fusion can be performed when the titer reaches 10^5. Administer a booster immunization before fusion. (Use Freund's complete adjuvant for the first immunization and Freund's incomplete adjuvant for subsequent immunizations.)

Indirect ELISA: The known antigen is adsorbed onto a solid-phase carrier to form a solid-phase antigen. An enzyme-labeled anti-antibody is then used to detect antibodies in the sample.

(1) Coating: Coat the plate with the antigen/antibody corresponding to the antibody/antigen to be tested, at a concentration of 100 ng/well, 100 μL per well. For example: For 1 plate (100 wells), you will need 100 ng * 100 = 10 μg. Take 10 μL of a 1.0 mg/mL solution (10 μg / 1.0 mg/mL = 10 μL) and dilute it in 10 mL of CBS buffer. Incubate at 4°C overnight.

(2) Washing: Wash the plate using a plate washer with PBST, 300 μL/well, 3 times.

Blocking: Prepare 1% BSA in PBST, add 200 μL/well, and block at room temperature for 1 h.

Washing: Wash the plate with PBST using a plate washer, 300 μL/well, 3 times to remove excess BSA.

(3) Primary antibody labeling: Add the antibody or antigen to be detected, 100 μL/well, and incubate at room temperature for 2 h.

(4) Washing: Wash the plate with PBST using a plate washer, 300 μL/well, 3 times.

(5) Enzyme-labeled secondary antibody: Use HRP-IgG (horseradish peroxidase-conjugated secondary antibody) or another secondary antibody, diluted in 1% BSA before use, 100 μL per well, and incubate at room temperature for 1 h.

(6) Washing: Wash the plate with PBST using a plate washer, 300 μL/well, 3 times.

(7) Color development: Add TMB, 50–100 μL per well, and incubate at room temperature for 12 min.

(8) Stop reaction: Add 1 M H₂SO₄, 50–100 μL per well.

(9) Reading: Measure OD450 using a microplate reader.

Mice of the same strain as the immunized mice are used, typically 6-10 week-old Balb/c mice.

(1) Cervical dislocation is performed, followed by immersion in 75% alcohol for 3-5 minutes for disinfection.

(2) The skin is cut open with sterile scissors to expose the peritoneum.

(3) 6-8 mL of culture medium is injected using a sterile syringe.

(4) The peritoneal cavity is repeatedly flushed, and the lavage fluid is aspirated.

(5) The fluid is transferred to a 10 mL centrifuge tube and centrifuged at 1200 rpm for 5-6 minutes.

(6) The cells are resuspended in culture medium containing 20% newborn calf serum (NCS) or fetal calf serum (FCS), and adjusted to a concentration of 1×10⁵/mL.

(7) The cell suspension is added to a 96-well plate at 100 μL/well.

(8) The plate is placed in a 37°C CO₂ incubator for culture.

Feeder cells are typically prepared one day before cell fusion. One mouse can yield 5-8×10⁶ peritoneal macrophages. When using mouse thymocytes as feeder cells, the cell concentration is adjusted to 5×10⁶/mL; for mouse splenocytes, 1×10⁶/mL; and for mouse fibroblasts (3T3), 1×10⁵/mL—all plated at 100 μL/well.

The myeloma cell line should be of the same strain as the immunized animal to ensure a high hybrid fusion rate and facilitate the inoculation of hybridoma cells into the peritoneal cavity of mice of the same strain for large-scale McAb production.

(1) Harvest SP2/0 myeloma cells in the logarithmic growth phase by centrifugation at 1000 rpm for 5 minutes. Discard the supernatant, resuspend the cells in incomplete medium, and count them. Take the required number of cells and wash them twice with incomplete medium.

(2) Simultaneously, prepare an immune spleen cell suspension and wash it twice with incomplete medium.

(3) Mix the myeloma cells and spleen cells at a ratio of 1:10 or 1:5 in a 50 mL plastic centrifuge tube, and wash once with incomplete medium at 1200 rpm for 8 minutes.

(4) Discard the supernatant and use a pipette to remove any residual liquid to avoid affecting the PEG concentration.

(5) Gently tap the bottom of the centrifuge tube to slightly loosen the cell pellet.

(6) Perform fusion at room temperature:

① Within 30 seconds, add 1 mL of pre-warmed 45% PEG (Merck, MW 4000) containing 5% DMSO while stirring continuously.

② Allow the reaction to proceed for 90 seconds; if the room temperature is low (e.g., in winter), extend the time to 120 seconds.

③ Add pre-warmed incomplete medium to terminate PEG activity, adding sequentially 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, and 10 mL at 2-minute intervals.

④ Centrifuge at 800 rpm for 6 minutes.

⑤ Discard the supernatant and gently resuspend the cells in approximately 6 mL of 20% FBS-RPMI 1640 medium. Avoid vigorous pipetting to prevent dissociation of fused cells.

⑥ Based on the number of 96-well plates used, supplement with complete medium (10 mL per 96-well plate).

⑦ Dispense the fused cell suspension into 96-well plates pre-seeded with feeder cells (100 µL/well) and culture in a 37°C, 5% CO₂ incubator. Typically, one 96-well plate contains 1 × 10⁷ spleen cells.

The standard protocol involves maintaining the culture in HAT selection medium for two weeks, then switching to HT medium for another two weeks, before finally transitioning to regular culture medium.

During the screening of hybridoma cells through selective culture, only a small fraction of the resulting hybrid cell lines can secrete specific antibodies against the immunogen. Generally, when hybridoma cells cover approximately 1/10 of the well bottom area, specific antibody detection can be initiated to screen for the desired hybridoma cell lines.

(1) Prepare feeder cell suspension (same as pre-fusion preparation).

(2) Count cells from positive wells and adjust cell density to 1-5×10³/mL.

(3) Aliquot 130 cells into 6.5 mL of complete medium with feeder cells (20 cells/mL). Distribute 100 μL/well to rows A, B, and C (2 cells/well). Dilute the remaining 2.9 mL cell suspension with 2.9 mL of complete medium containing feeder cells (final 10 cells/mL), then distribute 100 μL/well to rows D, E, and F (1 cell/well). Further dilute the remaining 2.2 mL with 2.2 mL of complete medium with feeder cells (final 5 cells/mL), and plate 100 μL/well to rows G and H (0.5 cells/well).

(4) After 4-5 days of culture, small cell clones become visible under an inverted microscope. Add 200 μL of complete medium to each well.

(5) By days 8-9, cell clones become macroscopically visible and should be promptly subjected to antibody testing.

The cryopreservation method for hybridoma cells is the same as that for other cell lines. In principle, each ampoule should contain at least 1×10^6 cells. However, for the original well of hybridoma cells, the number may vary depending on the culture conditions. For cells cultured in a 24-well plate, one well can be frozen in one ampoule when the cells have fully covered the bottom of the well.

1. In vitro, hybridoma cells can be cultured in large quantities using rotating culture tubes, and monoclonal antibodies can be harvested from the supernatant. However, this method yields a low antibody concentration, typically 10–60 μg/mL in the culture medium. For large-scale production, the cost becomes relatively high.

2.In vivo inoculation of hybridoma cells to produce ascites or serum.

① Solid Tumor Method: Hybridoma cells in the logarithmic growth phase are inoculated subcutaneously on the back of mice at a density of 1–3×10^7/mL, with 0.2 mL injected per site (2–4 sites in total). When the tumor reaches a certain size (usually after 10–20 days), blood can be collected, and the serum may contain monoclonal antibodies at a concentration of 1–10 mg/mL. However, the amount of blood that can be collected is limited.

② Ascites Preparation: The standard procedure involves intraperitoneally injecting 0.5 mL of Pristane or liquid paraffin into BALB/c mice. After 1–2 weeks, 1×10^6 hybridoma cells are injected intraperitoneally. Ascites typically develops 7–10 days after cell inoculation. The health of the mice and signs of ascites formation should be closely monitored. Once the ascites volume is maximized but before the mice succumb, they are euthanized, and the ascitic fluid is aspirated into a test tube using a pipette. Generally, 1–10 mL of ascitic fluid can be obtained per mouse. Alternatively, a syringe can be used to withdraw the ascitic fluid, allowing for multiple collections. The monoclonal antibody concentration in the ascites can reach 5–20 mg/mL. This is currently the most commonly used method. Additionally, the ascites-derived cells can be cryopreserved and later revived for intraperitoneal inoculation into mice, resulting in faster ascites production and higher yields.

(1) Pack the Protein A resin into a column. Approximately 10mg of antibody can be purified per 1mL of resin.

(2) Replacement: Use 15 column volumes (CV) of purified water to replace the 20% ethanol storage solution in the chromatography column.

(3) Equilibration: Equilibrate the column with 15 CV of phosphate equilibration buffer.

(4) Loading: Filter the sample through a membrane before loading. Circulate the sample through the column 3 times to allow antibody binding to the resin.

(5) Re-equilibration: Wash away unbound proteins using 15 CV of phosphate buffer.

(6) Elution: Elute the antibody with 5 CV of glycine elution buffer.

(7) After elution, the eluate contains purified antibody. Adjust the pH using neutralization buffer.

(8) Post-use column treatment: First replace the glycine with phosphate buffer, then replace the phosphate buffer with purified water, and finally store the resin in 20% ethanol.

(9) Determine the concentration of purified antibody using a protein quantification instrument.

(1) Prepare separation gels and stacking gels of different concentrations according to the formulation scheme of the separation gel. Inject the separation gel into the glass plate sandwich, cover the top with purified water to keep the gel surface flat, and prepare the stacking gel after the separation gel has polymerized.

(2) After preparing the stacking gel, pour off the small amount of water on the surface of the separation gel, then add the stacking gel and insert the sample comb.

(3) Non-reducing polyacrylamide gel electrophoresis

Dilute the electrophoresis sample with purified water to a total loading amount of 5 μg per well. If mixing with 4× non-reducing SDS-PAGE loading buffer, use a volume ratio of 7.5 μL sample + 2.5 μL buffer.

(4) Reducing polyacrylamide gel electrophoresis

Dilute the electrophoresis sample with purified water to a total loading amount of 5 μg per well. If mixing with 5× reducing SDS-PAGE loading buffer, use a volume ratio of 8 μL sample + 2 μL buffer, then heat in a 100°C water bath for 10 minutes.

(5) Remove the comb and load the samples. Load 5 μL of protein marker standard.

(6) Connect the power supply and run at a constant voltage: 80-100 V for the stacking gel and 120-150 V for the separation gel.

(7) Remove the gel, cut off the stacking gel, place it in a Petri dish, add purified water, heat until boiling, then stop and discard the water.

(8) Staining: Add 20–25 mL of rapid staining solution (enough to cover the gel), heat to boiling, and maintain for 8 minutes. Then recover the staining solution.

(9) Destaining: Add an appropriate amount of purified water, heat to boiling for 5 minutes, then change the water and repeat 2–3 times.

(10) Observe and record the results using a gel imaging system, then save the data.