1) 1st injection: 15 µg Antigen in 200 µl adjuvant, injected subcutaneously.
2) 2nd injection: 2 weeks later; boost with 15 µg of Antigen in adjuvant. (If time
permits, boost again a month later).
3) 7 - 10 days later bleed the mouse and test serum using the assay which will be used
for screening. If titre is not high enough, boost again two weeks after previous
boost.
2) Separate the clot from the sides of the tube (ringing) using a pasteur pipette. Place clot at 4šC O.N.
3) Spin at 10000 x g for 10 min. at 4šC to separate the serum.
4) Serum can be stored at -20šC after adding Glycerol to 50%.
If monoclonal antibodies are desired, once you have a "good" polyclonal and a reliable screening method proceed to the next step.
2) Myeloma fox-NY
Origin: Robertsonian, 8-azaguanine resistant, HPRT -, APRT -.
(mice have resistance to drug and expression of heavy chain on the same
chromosome).
3) Macrophage-derived .i.J774A.1
Maintenance of the cells:
Stock solutions:
€IMDM: JRH Scientific 51-47178.
€.i.FCS;: Fetal Bovine Serum- defined, Hyclone A-1111-L (no heat-inactivation needed) or any other brand that has been sampled and checked in advance.
€Insulin: 100X stock= 20 I.U./ml
Dissolve 80 mg of Insulin (25 I.U./mg) in 90 ml of ddH2O by adjusting to pH
3.0 with 1-2 drops of 6N HCl. After Insulin has dissolved readjust pH to 7.2
with 0.5 M Na2HPO4, bring volume to 100 ml and filter sterilize with 0.2 µ
membrane, keep frozen.
€Oxaloacetic Acid: 100X stock = 0.1 M
Filter sterilize, keep frozen.
€D(+) Glucose: 100X stock = 45%
Filter sterilize, keep frozen.
€Transferrin: 1000X stock = 1 mg/ml
Dissolve 1 mg human transferrin for each ml of IMDM. Make a 2 mg/ml
FeCl3.6H2O or 1.2 mg/ml FeCl3 in 0.001 M HCl. Add 1µl Fe solution to each ml
of transferrin solution. Mix, let stand on ice for 30 min. Filter sterilize, store at
4šC.
€HT supps: 100X stock = 1.36 mg/ml (H) , 0.73 mg/ml (T).
Weight out 68 mg hypoxanthine ( Sigma H-9377) and 36 mg of thymidine (Sigma
T-9250 ), combine. Add 1N NaOH (a few drops to 1 ml) and stir until the
powder is in solution. Bring to a final volume of 50 ml with ddH2O. Filter
sterilize, store at -20šC.
€Supplements: 25X stock (store at -20šC).
Mix equal volumes of the following:
L- Glutamine (200mM = 100X) , Gibco 320-5030.
Penn-Strep (100X), Gibco 600-5070PG.
Non-essential amino acids (10 mM = 100X), GIBCO 320-11404G.
Sodium pyruvate (100 mM = 100X), Sigma S 8636.
€2-Mercaptoethanol: 1000X stock (5 x 10 -2). (Store at 4šC.)
€AT supplement : 50X stock , Sigma A-7422. (Store at -20šC.)
€.i.PCM;: Parents Conditioned Medium.
Medium is collected from the the apropriate parent myeloma after the cells have
reached high density, then spun and filtered. Aliquot and keep at -20šC.
€MCM: Macrophage Conditioned Medium. (used instead of feeder cells)
Seed macrophages at a density of 1.5x105 cells/ml in the medium described on
next page. Add 2.5 µg/ml LPS which induces differentiation.
Collect sup after 2-3 days, or when medium is getting too yellow. Induce 2 more
times , each time with 1 µg/ml LPS and collect sup after 2 days each.
Pool the sups, filter and use as recomended. (Could be aliquoted and stored at
-20C.)
Preparation of Media:
for P3X63-Ag8.653: IMDM complete
to 400 ml of IMDM add:
5 ml 100X Insulin
5 ml 100X Oxaloacetic acid
5 ml 100X D(+) glucose
0.5 ml 1000X transferrin
0.5 ml 1000X 2- Mercaptoethanol
5 ml 100X HT
20 ml 25X supplements
75 ml FCS ( final 15% )
100 ml PCM ( Parent conditioned medium ).
for fox-NY: IMDM or RPMI (JRH Scientific, 51-50178).
10 % FCS
1X AT supplement
transferrin
supplements
PCM, when available from this cell line .
for J774A.1: out of convenience we have been using the same medium as the one for the P3X63-Ag8.653 ( = Ag8 ), without the PCM.
Growth conditions:
All cell lines mentioned above grow at 37šC, 7% CO2 .
The Myelomas optimal density is 3.5x105/ml.
The Macrophages optimal density is 1.5x105/ml. When expanding them , use a
"policeman" to scrape them; this is easier to do if they are growing in petri dishes at
this stage.
Freezing Hybridoma / Myeloma / macrophages.
.i.freezing cells;
Freezing solution: 90% FCS + 10% DMSO, ice cold.
1) Spin down 107 cells (106 minimum) at 1200 rpm for 5 min.
2) Aspirate medium.
3) Resuspend in 1 ml of ice cold freezing solution.
4) Transfer vial to an insulated freezing box and place at -70šC for at least 1 hr. (could
be for a couple of days).
5) Transfer the vial from the -70šC to the liquid nitrogen tank and log the entry in the
freezer log.
Thawing cells:
.i.thawing cells;
1) Take vial out of liquid nitrogen tank and thaw it immediately in a 37šC bath (about
1 min).
2) When there is still a small piece of ice left, dilute the cells by transfering them into a
conical tube containing 10 ml of the growth medium at 37šC.
3) Spin at 1200 rpm for 5 min.
4) Aspirate medium and resuspend cells in 5 ml of medium, in a 25cm2 flask.
Cell Fusion and Selection
.i.cell fusion;
Solutions:
€IMDM
€IMDM complete with MCM instead of PCM.
€PBS
€.i.PEG 50%; ( w/v ): ( PEG 4000 "gas chromatography grade" Art.9727 - Merck).
Sold by Bryant Laboratories, (415)526-3141, or MC/B Manufacturing Chemists,
Inc.
€.i.Aminopterin;: Sigma, A 5159 (50X ), use it as 100X. ( final = 2x10-7 M ).
1) Prepare a T-150 flask with 170ml of IMDM complete with MCM instead of PCM (= IMDM - m ), and keep it in the incubator for fused cells.
2) Isolation of spleen cells :
a ) Sacrifice mouse by cervical dislocation. Immerse mouse in 70% Ethanol.
b ) Remove spleen ( on the left side ) and transfer into a small petri dish which
contains IMDM at room temp. Clean fat from the spleen and transfer the
spleen into an empty dish.
c ) Using sharp tipped forceps, one end is punctured. A curved forceps is used
to hold down the intact end, and the spleen is gently rubbed towards the
opened end with another set of forceps. The cells from inside the spleen will
ooze out with very little damage. Stop the process when you are left with a
nearly empty, transparent skin. Collect the cells by rinsing with IMDM.
d) Transfer cell suspension to a 15ml conical tube and let the cell debris settle out
(approx. 5 min.).
e) Remove the cell suspension (without disturbing the settled cell debris) and
transfer to a 50ml conical tube. Add an additional 30ml of IMDM and pellet
the cells at 1200 rpm for 5 - 10 min.
f) Aspirate the medium, resuspend pellet and wash again with 30ml of IMDM.
One immunized spleen has aprox. 108 cells. After this wash the cells are
ready for the fusion.
3) Myeloma cell preparation:
It is essential that the .i.myeloma;s be free of debris, rounded and refractive
under phase contrast, and that they are harvested in log or late log phase growth
(between 3.5 and 9 x105 cells/ml).
a) Thaw cells 7 days before scheduled fusion. Myeloma do not grow well after
being in culture for more than a few weeks.
b) Make sure you refreeze cells for future use.
c) We have been using a ratio of 2 spleen cells : 1 myeloma cell. However,
workers have been using a ratio from 1 to 10 spleen cells per myeloma
successfuly. For one spleen we harvest 5x107 cells. It is advisable to do this
spin at the same time that the second wash of the spleen cells is done.
4) The fusion: .i.fusion;
a) The washed myeloma and spleen cells are pooled in 30ml of PBS (room temp.)
and spun gently at 1000 rpm for 10 min.
b) Aspirate the PBS and resuspend pellet gently by tapping the tube. Volume
should be approx. 0.8 ml.
c) Set a timer.
d) Add an equal volume of PEG solution, slowly, dropwise, with gentle tapping,
over 1.5 min. at room temp. Then gently wiggle the tube for 1.5 min.at 37šC.
Some cell clumping will be evident.
e) The suspension is spun at 1000 rpm for 3-4 min. (at this point you should see
the different layers of cells with PEG on top).
f) Slowly add 37šC IMDM to 10 ml, without disturbing pellet. After adding ,
swirl the tube gently to mix and dilute the PEG. Do not disturb the cells.
g) Spin at 1000 rpm for 5 min.
h) Aspirate medium, resuspend cells by tapping. Slowly add 5 ml of 37šC
IMDM-m.
i) Bring to 20 ml and add to the flask in the incubator. (If using feeder cells, add
them at this point; 106 cells/ml).
j) Add Aminopterin (2ml to 200 ml of medium). (Some workers will leave the
cells at this point for 24 hours before adding Aminopterin. We add the drug
immediately.)
k) Seed the cells in 96 well microtiter dishes, 250 µl per well, 8 plates per fusion.
First clones may be seen in 7-10 days. First screen will usually start after 2 weeks,
with a second and third, if necessary, a few days later.
Screening
ELISA
.i.ELISA;
Materials:
€ Plates - 96 well Dynatech Immulon, type 2. (Fisher 17-0221-199).
€ Adsorbtion buffer 10X = 1M NaHCO3 pH 9.6 ( 80ml 2M Na2CO3 + 170ml 2M
NaHCO3 + 250ml ddH2O ) or PBS.
€ PBS, TBS
€ PBS + 0.1% Tween 20 or TBS + 0.1% Tween 20.
€ Blocking solution = 2% BSA (type V ) in PBS. (Add 0.02% azide for longer storage.)
€ Elisa buffer = 2% BSA + 0.1% Tween 20 in PBS ( azide optional ).
€Diethanolamine Ph 9.8 ( 48.5 ml diethanolamine + 0.1 gm Na Azide + 0.05 gm
MgCl2.6H2O in 400 ml ddH2O. Adjust pH to 9..8 with concentrated HCl. Bring to
500 ml., store at 4šC in dark bottle).
€ Enzyme linked antibody = Alkaline phosphatase Goat anti mouse. ( Zymed
62-6422 ).
€ Substrate = Sigma 104 phosphotase substrate tablets ( Sigma 104 -105 ).
Protocol:
1) ADSORBTION OF ANTIGEN
a) Dilute Ag to 10 µg/ml in 1X adsorbtion buffer.
b) Add 100 µl of Ag solution to each well. (A good control would be to leave an
empty well with no Ag for every well that contains it.)
c) Leave O.N covered with saran wrap , at 4šC.
2) BLOCKING
a) Wash unbound Ag by inverting the plates and flicking the wells dry.
b) Rinse by adding PBS to each well and inverting it again (use squirt bottle).
c) Repeat the rinse twice.
d) Add 100 µl of blocking solution to every well, leave 1 hr at room Temp or O.N
at 4šC.
3) PRIMARY ANTIBODY
a) Add the antibody to be tested :
Sup of cells = 25 µl , mix well by pipeting up and down (10 times).
serum, ascites = 1:500 to 1:1000 dilution. Do dilutions in blocking solution.
b) Leave 1 hr at room temp or O.N at 4šC.
4) SECONDARY ANTIBODY
a) Wash unbound antibody 4 times with PBS + 0.1% Tween 20.
b) Add 100 µl of enzyme linked antibody to all wells. Do the appropriate
dilutions in the Elisa buffer. (ex: AP is 1000X).
c) Leave 1 hr at room temp or O.N at 4šC.
5) SUBSTRATE
a) Dissolve substrate in Diethanolamine (for AP substrate, warm 37šC, 5 min.)
to concentration of 1 mg/ml.
b) Wash plate 4 times with PBS + 0.1% Tween 20 (use TBS instead of PBS for
AP).
c) Add 100 µl of substrate to every well.
d) Watch color development. This could take from a few seconds to 20 min.
e) If needed, stop the reaction by adding 50 µl of 4M NaOH.
f) Read absorption in Elisa reader at correct wavelength (for AP system 405
nm).
Expanding Hybridoma Clones
1) Transfer the srongest positive clones to a 24 well plate by scraping the adhering
cells with a truncated yellow tip.
2) Bring the volume to 1 ml using the IMDM-m medium.
3) When cells reach confluency, freeze them ( about 106 cells per full well ). Scrape
them using a blue tip.
4) Add 1 ml of IMDM-m to cells left in the well, let them grow and repeat the freezing.
5) While the cells are growing, save their sup at each freezing. Use the sup to do the
other tests required such as western, immunofluorescence and others. (If sup is not
used under sterile conditions, add 0.02% azide and keep at 4šC for months.)
6) Transfer the cells to a 25 cm2 flask in 4 ml of IMDM-m and continue to expand as
desired. The cells at this point are "addicted" to the macrophage supplement. It is
possible to wean them by gradually reducing the amount of MCM. We do it only if
very large volumes are needed.
7) After deciding which one are the best clones, it is advisable to reclone them to
ascertain their homogeneity (next chapter).
8) The subclones should be checked by all the assays which were used to test the
originals . The subclones should be frozen at least twice.
9) If convinced that the subclone is monoclonal, checking the isotype is possible . (We
have been using a kit from Zymed - MONOAB-ID).
10) If not convinced that the hybrydoma is monoclonal, reclone a second time.
Cloning of Hybridoma
.i.cloning of hybridoma;
1) Make an accurate cell count of cells in log phase (3 X 105 - 1 X 106 cells / ml).
2) Determine the volume needed which will give you 100 cells.
3) Add the 100 cells to 25 ml of IMDM-m and plate 250 ml of the suspension in each
well. Therefore, each well should get 1 cell delivered into it.
4) Clones should be visible and ready to screen in 10 to 14 days.
Ascites Production
.i.ascites;
1) 7 days before injection of cells, inject 0.5ml of Pristane (Tetramethyl - pentadecane,
Sigma T-7640), intraperitoneally.
2) Prepare cells in log phase.
3) On day of injection, spin 1-2 x106 cells per mouse.
4) Resuspend in IMDM or PBS at 1-2 x106 cells/0.5ml (do not leave the cells without
serum for more than 1 - 2hr). Transfer cells to syringe using a 16G needle.
5) Inject 0.5ml per mouse, using a 20G needle, intraperitonealy.
6) Collect ascites when animals are ready (swollen abdomen), 10 days to 15 days
after injection.
7) Spin fluid at 3000g for 10 min. to remove the cells. If there is an oil layer, remove it
and discard. Carefully remove the supernatant from the cells.
8) For storage, add sodium azide to 0.02%. Store at -20šC.
Literature
1. Antibodies - A Laboratory Manual; D. Lane, Ed Harlow.
2. Monoclonal Antibodies: Principles and Practice; James W. Goding.
3. Monoclonal antibody technology; Ailsa M. Campbell.