Monoclonal antibody preparation technology

Monoclonal antibody preparation technology

Standardized protocol for the preparation of monoclonal antibodies (McAb-sop)

Chapter 1: Immunization of mice

Chapter 2: Cell Fusion

Chapter III: Cell Construction

Chapter IV: Identification of cell strains

Chapter 5: Ascites Preparation

Chapter 6: Antibody purification and identification (omitted)

Chapter 1. Immunization of mice (BALB/c)

Mouse immunization is a key link in the preparation of monoclonal antibodies, and quality-controlled mouse immunization is the only guarantee for the preparation of high-quality monoclonal antibodies.

A mouse immunization two-way program: According to the immunological principle, the mouse immunization program is designed as follows:

1. Short-term immunization program:

First time: 1d 100ug (soluble Ag) + equal volume of complete adjuvant. Intraperitoneal injection. 0.4ml / only

Second: 7d 100ug + equal volume of incomplete adjuvant intraperitoneal injection. 0.4ml / only

Third time: 12d 50ug IV 0.3ml / only

(Enhanced) Fourth: 15d 20ug IV 0.3ml / only

Fusion within the 17th to 24th

2 long-term immunization program:

First time: 1d 100ug+ equal volume complete adjuvant intraperitoneal injection (IP) 0.4ml / only

Second: 14d 100ug+ equal volume incomplete adjuvant IP 0.4ml / only

Third time: 21d 100ug IP or IV 0.4ml / only

Fourth time: 25d 50ug IV 0.3ml / only

(Enhanced) Fifth: 28d 20ug IV 0.3ml / only

Fusion within the 30th~37d.

Note: a. Before the fusion (ie, before booster immunization), the serum titer of the immunized rat must be determined. The short-range immunization titer must be ≥1:8000, and the long-range immunization ≥1:32000, before the fusion can be performed. b. After the completion of the immune cycle Mice need to complete the fusion within a week. Otherwise the quality of the fusion cannot be guaranteed. two. Mouse immune quality control standard

1. Immune mouse serum titer short range ≥ 1:8000, long range ≥ 1:32000;

2. The mice need to complete the fusion within one week after immunization;

3. The spleen of the immunized mice was significantly larger than the spleen of the blank mice and was more adhesive.

Immune mouse selection: The immune type is: baleb/c mouse; sex. Female is good: Size: 6-7 weeks old: Weight: 20g;

Robust and lively.

Chapter two. Cell fusion

one. Preparation before fusion

1. Spleen cells: The spleens were taken out under aseptic conditions in a rat that met the immunological control standard, and a single spleen cell suspension was prepared, and the spleen cells of each mouse were about 100-300 million. Sterility is strictly guaranteed during the preparation process. The spleen cells can be prepared by selecting a grinder + mesh or by blowing with a sterile syringe.

2. SP2/0 cell preparation: SP2/0 cells were ventilated 4 days before fusion and screened twice with 8-Ag, changed every other day, and replaced with ordinary medium 1640, 10% calf serum, SP2/ one day before fusion. The number of 0 cells is preferably between 20 million and 35 million. The cells are in a stage of division, and aging cells are not suitable for use.

3. PEG1450 preparation: take PEG1450 dry powder high pressure, add an equal volume of PBS dissolved at 60 ° C can be used, a fusion of 0.8ml 50% PEG1450.

4. HAT medium preparation: HAT1640 medium. 20 ml/plate of 20% calf serum prepared according to the number of plating plates.

5. Stop solution 15-20ml: 1640 medium or PBS without Hepes (pH 7.4)

2. Integration:

The spleen cell suspension was prepared and washed with PBS (pH 9.4) and mixed into SP2/0 cells. The spleen cells were mixed with SP2/0=10:1 to 5:2 and centrifuged. After centrifugation at 1200 rpm×5 min, the cells were mixed and dried. Knock the pine cell mass. Add PEG1450 0.8ml in a 37 °C water bath and add it in 50 seconds. After the addition, react in a 37 °C water bath for 2 min. Add the stop solution and add 15-20 ml in 5 min. Add the stop solution slowly along the tube wall. Join. The action should be gentle. It should not be blown to avoid separation of the newly fused cells.

3. After the fusion, add the sample:

After termination of the fused cells, centrifugation was performed at 900 rpm x 8 min and blotted dry to prevent residual PEG. After centrifugation, the fused cells were transferred to HAT medium, and 200 ul/well was dropped, and the whole process should be prevented from blowing off the fused cells.

4. Change the liquid:

The cells to be fused were replaced with HT medium after culturing for about 7 days in HAT (i.e., after unfused SP2/0 and spleen cells died). 200 ul / well on the 8th to 10th day of testing.

third chapter. Cell formation

1. Fusion board detection:

The cells to be fused to the medium change to a medium size of about 10,000 cells or more to start the test (see the attached ELISA method for the detection method). After the ELISA quality control (ie, negative control <1.0, positive control>1.0), select positive holes (generally OD450 ≥ 0.5) for subcloning.

2. Subcloning methods and tests:

Pick all the cells in the positive well of the fusion plate and add 8-10ml HT medium, and mix the 4-6 longitudinal rows. After the subclones grow for 5-7 days, the clones grow up (about 10,000 cells/well). Detection (by ELISA method).

3. Monoclonal plating and testing:

A well count with a high positive value for subcloning was selected for limiting dilution of 4:2:1: 0.5 cells with four gradients and 1 positive cell line/block. When the monoclonal growth is 7-10 days, the monoclonal cells are detected to a medium size (10,000/well), and the monoclonal cells are ≥8/strain (generally 12 wells are detected). The same monoclonal is performed again.

four. The cell line is established:

After two times of monoclonal detection, the three wells/strains were picked out and expanded into 24 well plates; after 24 cells were overgrown, cross-Ag identification and stability identification were performed (ie, the culture supernatant was tested again to see if it was still After the identification of the monoclonal antibody, one of the strains was selected. The OD450 box height was expanded and cultured in a cell bottle, and frozen, 5 plants/plant, ≥ 2 million/cell.

Chapter Four. Cell line identification

One of the same batch must be revived for identification after the cell strain has been frozen.

Identification criteria: 1. The number of viable cells recovered is ≥ 1 million cells per branch;

2. There are viable cells in living cells ≥500,000/plant;

3. In the resuscitation cells, there must be no microorganisms other than the cells of the cell line (eg, bacteria, fungi, mycoplasma, etc.).

4. After the resuscitation cells grow to a certain number, select the well-grown cells for monoclonal counting and test whether the monoclonal antibody-secreting ability is all positive or has antibody secretion.

5. The cell culture supernatant also needs to be tested by ELISA to determine whether the antibody is secreted and cross-antigen is reviewed at the same time, and whether it is a monoclonal antibody by western-boltting.

chapter Five. Ascites preparation and cell line expansion

Preparation of ascites: After the cell strain is qualified, the cells in the culture flask are collected. The method of ascites in mice is as follows:

1. Mouse selection: 10 weeks old baleb/c female mice, the hair of the mice is shiny, and the activity is about 30 grams.

2. Prepare the mice before the ascites: select the rats in the sensitized mice for 7 to 30 days to choose paraffin oil (or other mineral oil) 0.5ml / IP (abdominal) injection.

3. Ascites: Collect qualified hybridoma cells in PBS (pH 7.4) or saline (sterile), 2 million to 5 million mice. IP

4. Ascites collection: Ascites can be produced 5-7 days after hybridoma cells are injected into the abdominal cavity of mice. (Mouse status: Abdominal awning, mice with wilting, not thinking about drinking) At this time, about 2-5 ml/piece can be taken with a sterile syringe. Characteristics of ascites: bloody or milky white or yellowish, fatty, slightly thick. Usually taken once every other day, each mouse can be sacrificed three times. )

Ascites preservation: Ascites was taken and placed at 4 ° C overnight. Centrifuge at 500 rpm for 10 min. Store the supernatant for 4 hrs (3-7 days) for short-term storage, -20 °C for the first half of January, and -80 °C for long-term June.

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