Production of Monoclonal Antibodies

[Cellbiogal]

Production of Monoclonal Antibodies

Injection protocol (4 week protocol):


Day 0: 1st immunization: mix in buffer dissolved proteins with Freund's complete adjuvant at a ratio of 1:1; use about 100 g per mouse (see protocol for polyclonal antibodies).

Day 14: 2nd injection: for 2nd to 4th injection use Freund's incomplete adjuvant.

Day 21: 3rd injection

Day 28: 4th injection


Test of Immunization by Immunoblot and/or Immunofluorescence:

Day 30: collect some blood from the tail vein; separate serum and use for immuno-test.

* The desired titer of antibodies should be:

(a) immunoblot: better than 1:300 (prefered 1:1000)

(b) immunofluorescence: better than 1:20 (refered 1:50)


Preparation of Media and Solutions:

* DMEM medium
* DMEM medium + 10% Fetal Calf Serum + 10 M mercapto ethanol
* HT medium
add HT supplement, 50x stock (Gibco #41065-012) to DMEM/FCS
* HAT hybrid selection medium
add HAT supplement, 50x stock to culture medium (Gibco # 21060-017); 2 ml + 98 ml culture medium)
* Lysis medium
* A: 0.83 g NH4Cl/100 ml, pH 7.2.
* B: 2.06 g Tris/100 ml, pH 7.2.
* Mix 9 ml A with 1 ml B

Culture of X63-Ag 8.653 Non-secreting Myeloma Cells:
o Start the cell culture of the myeloma cells about 7 to 10 days before cell fusion.
o Test for HAT deficiency by cultering some cells in HAT medium.

Sterile Tools for Fusion: 3 sterile Petri dishes; sterile 15 ml conical tubes; sterile 50 ml conical tubes; at least 2 sterile forceps; at least 2 sterile scissors; sterile wire mesh screen (0.5 mm mesh size; can be obtained at a local metall store as stainless steel, wire screen.); sterile Pasteur pipettes; calculator; several sterile boats; sterile blue & yellow tips; haematocytometer; DMEM medium, HT, & HAT medium; DMSO; Tryptophan Blue; sterile 3 cc plastic syringe; PEG/DM SO solution (Sigma cat.# P-7306)

Fusion Protocol:


spleen of one mouse .653 myeloma cells
1. Remove spleen from mouse under sterile condition (left hand side).
2. Remove fat tissue and wash 2-times with DMEM medium in Petri dish.
3. Mince spleen with scissors and press through a 0.5mm wire screen using 3 cc syringe plunger.
4. Wash screen with 10 ml EGELS medium.
5. Transfer cells to 15 ml conical tube and allow chunks to settle for about 5 min.
6. Transfer supernatant to new 15 ml conical tube.
7. Spin at 1300 rpm for 5 min.
8. Discard supernatant.
9. Resuspend in 10 ml lysis buffer (to lyse red blood cells); A: 0.83 g NH4Cl/100 ml, pH 7.2; B: 2.06 g Tris/100 ml, pH 7.2. Mix 9 ml A with 1 ml B.
10. Allow to lyse for 10 min and spin at 1,300 rpm for 5-10 min. 1. Judge the quality of the myeloma cells: a smooth, round shape is essential for a good fusion.
11. Resuspend in 10 ml DMEM medium; take an aliquot and dilute it 1:50 for cell count; spin at 1,300 rpm for 5 min. 2. Spin at 1,200 rpm for 5 min.

count cells:
12. Resuspend in 20 ml DMEM medium.
13. Cell count: _____ x 108 in 10 ml culture medium.

F1. 2 ml Hybri-Max fusion medium (Sigma P-7306) dilute with DMEM medium to 47% PEG.
F2. Combine 1 x 108 spleen cells (10 ml) with 2 x 107 myeloma cells at a ration of 5:1 and centrifuge at 1,200 rpm for 5 min.
F3. Discard supernatant, loosen pellet by rubbing the tube along the metal grid of the hood.
F4. Add fusion medium, resuspend pellet gently and let stand for exactly 1 min.
F5. Add 10 ml DMEM medium dropwise while shaking. Add additional 10 ml DMEM medium slowly while mixing.
F6. Spin at 1,200 rpm for 5 min, discard supernatant.
F7. Resuspend in 10 ml HAT medium. Determine total number of cells: _____ x 108.
F8. Dilute to 0.5 x 106 cell/ml. 280 ml HAT medium.
F9. Plate into ____ 96-well plates; 200 l per well.

Protocol by Walter Steffen.

[sergbio]

Hi Cellbiogal!

What do you mean under 100g per mouse for 1st immunization. Can you explain what quantity of recomb protein is optimal for immunization scheme ( or it is individual for each protein!

Thanks!

[admin]

I think she means make the following:

Dissolve your protein with Freund's complete adjuvant at a ratio of 1:1
(50% / 50%)

Then just weigh up to 100 g.

Welcome to the forums
Sergbio!

[sergbio]

Hi admin!

Thank you for explanation, but I think there is a mistake ( not 100 g but 100mkl, because it is very difficult to imagine 100g\per mouse (where mouse weight is near 20g)
)

[sergbio]

HI!
Is anybody know about a source of antigen for PREPARATION of HUMAN CD8 Mab HYBRIDOMA? ( I mean recombinant protein or fused peptides or anything else?)

According to ATCC collection of cell lines and hybridomas, for immunization researchers use lymphocytes / But what kind of antigen they use in Elisa for clone selection? ( CD8 as I know a complex of 8 peptides with Mw near 25kDa)

Thank you!

[remo]

Hi
Iwant to know about Genomic Antibody technique