Standard Operating Procedure

Monoclonal Antibody Production and Purification

Hazards:

DIMETHYLSULFOXIDE (DMSO): combustible, causes irritation, harmful if swallowed; keep away from eyes, skin, clothing; keep in a tightly sealed container. Inhalation may cause nasuea.

LIQUID NITROGEN: liquid form can cause severe burns. Avoid contact

Protection:

lab coat

gloves (when measuring G418 and DMSO)

insulating gloves when going into the LN2 tank

Waste:

Plastic disposables used for tissue culture should be autoclaved.

Drain all solutions from flasks before putting into waste.

When flasks in hoods are filled with liquid waste, dispose of contents down the drain with water running, rinse flask, add enough bleach to cover the bottom, and put back in place.

Pasteur pipets DO NOT go in trash. Put them in sharps container next to each hood.

General Cleanliness:

Wipe down hood before and after use with 70% Ethanol (spray bottle).

Don’t leave paper towels, spills, extra equipment, etc. in the hood.

Wash hemacytometer immediately after use with ethanol and dry.

Monoclonal Antibody Supernatant Production and Purification

Supernatant Production

1. Cell lines are frozen in liquid nitrogen freezer.

Look up which cell line is needed in LN₂ log.

2. Remove one vial of cells and quick-thaw in water bath (37⁰). Resuspend cells in 10 ml of RP10, spin for 5’, remove sup and resuspend in 10 ml RP10. Transfer cells to petri dish. Count cells to determine density and viability.

16 squares in microscope field= x 10^4 cells/ml

Dilute cells 1:2 in Trypan blue (10ul) and count the number of viable cells in 16 squares

# cells x 2 = ## x 10^4 cells/ml

Example: 28 cells X 2 = 56 x 10^4 cells / ml or 0.56 x 10^6 cells / ml

Viability = # alive cells x 100 (dead cells stain blue from dye)

total # cells

3. Monitor cells daily - check density and viability. Split cells (usually 1: 5 or 1 : 10 when they reach a density of around 0.5 to 1.0 x10⁶ /ml. Plan your week accordingly.

Split cells like this:

1 dish -- 2 dishes -- 10 dishes -- roller bottle with 300-500 ml RP10

Once roller bottle is 0.5 - 1.0 x 10^6 cells / ml, split half (150-250 mls) into second roller bottle and fill both to 1 L RP10. (If over a weekend and you are concerned about cells possible overgrowing, put different amounts of cell sup into each roller bottle. )

4. Allow cells to grow in roller bottles until they reach a density of about 1 x 10^6 cells / ml in 2 L total media. At this density, spin cells for 10 min at 1200 rpm. The cells can be spun down in Falcoln 2075 bottles with the blue top. Discard supernatant and resuspend pellets in 1 L total of RP-0 (RPMI with 25 ml SC but no FCS). I usually pour the sup from 1st spin into a flask to discard, then resuspend the 1st spin in RP-0 and put it into the now empty 1st roller bottle. Using the same Falcoln 2075 bottles, I spin down the 2nd roller bottle, discard that sup, then put these resuspended pellets into the same roller bottle as the first spin. At the end, you will have all cells resuspended 1 L RP-0 in one roller bottle.

Everything to this step needs to have been carried out aseptically.

5. Cells will soon start to die in this media. When viability reaches 20-30%, spin cells for 20 min at 5000 rpm in JA-10 rotor (Yogi lab centrifuge). Cells can be spun down in plastic autoclaved bottles. Filter supernatant through 45uM bottle top filter. Discard pellet. Add Na-azide (1:100) and refrigerate.

Affinity Chromatography using Protein G or Protein A Sepharose

1. Antibodies bind differently to different beads (Protein A or Protein G). Check chart to see which beads bind best to the type of antibody to be purified.

2. Some columns are already made. If a column is not made for a particular antibody, make a column as follows: Wash KONTES column with PBS. Add approx 1.4 mL of Protein G or Protein A. Let the beads settle, preferably overnight. Wash column with 10 mL PBS [Column supplies are on the top shelf across from blue hood.] If using pre-made column stored in Na-azide, wash w/ 15 mL PBS before use.

3. Add 4 mL supernatant to column. Assemble gravity flow system. Adjust flow rate to around 1 mL / min. Let supernatant flow overnight. Allow tubing to hang below the column to ensure that the column will not dry out. Collect 1 mL sample of Pre-Flow supernatant. This may be tested to determine how much antibody is present in the supernatant.

4. The antibody is now stuck onto the beads in the column. Wash column with around 15 mL PBS. To obtain antibody, elute ten 1 mL fractions of 0.1 M Citric Acid pH 2.5 over the column. First add 100 UL of 2 M Tris buffer pH 8.0 into ten microcentrifuge tubes. Add 1 mL of Citric Acid to the column and collect this in tube #1. Continue to add and collect 1 mL fractions. Wash column with 10-15 mL PBS after all fractions are collected. If first time over the column does not yeild much antibody, try messing with supernatant pH to improve binding. Do this by adding 1M Acetate Buffer pH 4.5 until you see a slight color change.

BCA Assay (Protein detection) Pierce kit

1. This assay will determine which fractions contain the antibody. It is performed in a 96 well flat bottom plate.

2. Add 100 ul of ddH20 into wells A1-A12 and B1-B12. Add 100 ul of BSA (Bovine Serum Alubumin) from kit into wells A1 and B1. Do a 1:2 serial dilution from A1 to A 11 and B1 to B11 changing tips between wells. Toss the remaining 100ul from A11 and B11. A12 and B12 are left as blanks.

3. Add 90ul of ddH20 into wells C1-C10. Add 10 ul from each microcentrifuge tube into wells C1-C10.

4. Mix 2.5 mL of Reagent A, 2.4 mL of Reagent B, and 100 ul of Reagent C in a 15 mL conical tube. Mix thoroughtly. Add 100 ul solution into each well. Incubate plates at 37 degrees for 22-25 min.

5. Row A and B should be the same colors. Row C will show color changes if antibodies are present (antibody is usually present in fractions 2-5). Use ELISA plate reader to detect the absorbancies of each well.

6. Use cricket graph program to get graph and equation to determine actual concentration.

7. If fractions are between 0.5-1 mg/mL, they can be combined and concentrated to get around 1 mg/mL. To do this, use Centricon tubes and spin at 3000 rpm for 3-6 minutes, then retest in BCA assay.

Dialysis

1. If antibody is present, it needs to be put in dialysis against PBS to get rid of any impurities.

2. Dialysis tubing is stored in the cold room. Make sure to always wear gloves when handling tubing so as not to damage the tubing.

3. Cut off various lengths of tubing (4-7 inches) and place in 500 mL ddH20 with stir bar. Bring water to a boil on hot plate/stirrer. Once the water starts to boil, pour it out and add 500mL cold ddH20. Spin for 5 min, pour off water and add another 500mL cold ddH20, spin for 5 min, repeat one more time.

4. Attach orange clip to one end of tubing and squeeze out PBS from tube. Add fraction with antibody into tubing. Attach orange clip at opposite end to seal tube.

5. Put tubing into 1000 mL beaker and fill with PBS. Let spin at slow speed in cold room for at least an hour. Repeat 2x. (one time should be overnight)

6. Transfer antibody to small plastic tubes for storage at -80. Remember to record placement of tubes in log. Unconjugated Ab’s should be stored in 1.25 mL freezer vials with colored tops (Maureen’s bench). Store in 500ul aliquots, do not add Na Azide. Bio/FITC Ab’s, store in 0.6 mL microcentrifuge colored tubes in 100ul aliquots--add Na azide @ 1:100. Label tubes with Ab name, how purified (Ex.-protein G purified), concentration, date, your initials.

BCA Graph

1. Take BCA assay plate to ELISA plate reader (rm 6-241)

2. Turn on plate reader - tip right hand corner in the back.

3. Click on plate reader software icon (Delta soft 3 program)

4. Go to plate

Read plate

5. Change wavelength to 562 (single)

6. Hit OK

7. Plate is read, Plate & Template appears on the screen

8. Zero template by going to

Template

Edit Template

Type in ‘#’ in box A12 or B12, whichever is less

Close screen

9. New Plate & Template should now be zero adjusted (Box A12 or B12 should be green)

10. Save file onto floppy disk

11. Close plate reader and turn off

Cricket graph:

1. Open up Delta Soft program on MHC (it’s saved in your folder)

2. Go to Analysis. Open up file you want to analyze. Print

3. Open up ‘BCA’, a cricket graph file.

4. Acording to OD of antibody, insert appropriate range of OD numbers using the standard from the albumin values. You should set 5 data pounts.

5. Go to Graph

New graph

Scatter (OD values on the horizontal, ug/ml on the vertical

6. For best curve fit

Options

Curve fit

Methods-Linear, Cofficients displat ‘r’

Each cell line so cells will be split at different times.