Antibody Growth and Purification Protocol

Antibody Purification

Buffers/Reagents
Protein A or Protein G (Sepharose or Agarose)
Bio-Rad econo columns
Bind Buffer 1L
   0.53g NaH2PO4-H2O
   0.87g Na2HPO4
   8.77g NaCl
   pH to 7.0
Elution Buffer 1L
   7.51g glycine
   pH to 3.0
Neutralization buffer
1M Tris-HCl pH 9.4

Hybridoma Growth

1.   Most hybridomas can be grown in either DMEM or hybridoma media. I usually use DMEM. When in doubt, use DMEM.
a.   If a small percentage of contaminating bovine IgG is not a problem, then proceed to step 6.
b.   Some cells need to be started in DMEM prior to change to hybridoma media after thawing
2.   The following 3 steps MUST be completed as quickly as possible to prevent FBS contamination since these steps ARE NOT STERILE.
3.   Filter FBS through whatman paper or pre-filters to remove precipitated lipoproteins.
4.   Run FBS through protein A column to remove bovine IgG (this step can be skipped if small percentage of bovine IgG is ok). See below for details of setting up column.
5.   Add FBS to media and filter media, or filter FBS through 0.22M filter prior to use. Hybridoma media offers no benefit if bovine IgG stripped FBS is used.
6.   Start cells in 100mm dish till almost confluent, freeze cells in 45% FBS 10%DMSO 45% media.
7.   Expand cultures to largest flasks manageable (I usually use 8 T-175 flasks). The more flasks the less time it takes to generate supernatant. Volume in Flasks should be around 35mL.
8.   Alternatively roller bottles can be used to generate higher cell densities. The special hybridoma flasks contaminate easily and have membrane ruptures and offer no benefit.
9.   Grow cultures to confluency and high cell density. DMEM should be turning yellow, but DOES NOT always depending on the hybridoma. Some hybridomas die before they turn the media yellow, it is always good to split the 8 flasks into two groups and passage them on different days just to be safe, I always keep a 100mm dish going.
10.   Harvest supernatants from flasks and wash out as many cells as possible. Leave behind 2 mL per flask and fresh media. Re-use flasks until they contaminate. Gentamicin usually kills most fungal growths that commonly infect hybridoma cultures.
11.   Spin supernatants down and pour supernatants without cells into fresh bottle and store at -20 until 1 liter builds up.

Purification
1.   Add azide to thawed supernatant. I usually add 1mL per liter of a 30% azide solution. Purifications should be done at RT since this guarantees highest binding and the azide prevents contamination and will be dialyzed out at the end.
2.   Supernatant pH should be between 7.0 and 7.5, this works well for both A and G, contrary to what is being taught.
3.   All buffers and supernatants MUST BE FILTERED 0.45uM before starting to prevent column clogging. I usually pre-filter several times with whatman paper before 0.45 filtering.
4.   Decide on whether to use protein A or protein G based on species (most murine antibodies should use protein G, hamster should use protein A)
5.   Wash column out prior to addition of protein beads with dH2O to remove particulate matter
6.   Break off tip using razorblade 2/3 of the way down tip, the normal bio-rad hole is too small and will cause a very slow flow
7.   Fill column with appropriate amount of beads. Typically protein-A or G will hold up to 20mg IgG per mL column. More beads equals slower flow rate.
a.   1-2 mL of bead volume (2-4mL 50% slurry) is usually enough per liter of supernatant
8.   Keep column covered but not closed to prevent dust and debris from entering.
9.   After beads settle, add entire econo-column volume water,let drip.
10.   Add entire column volume binding buffer, let drip.
11.   Add supernatant being careful not to disturb bead bed.
12.   Collect 1st drippings and re-circulate again after 1st run is complete.
13.   Typical flow rate should be between 0.5 and 1mL per minute, faster dripping decreases binding ability of beads. Bigger columns drip slower.
14.   After supernatant is recirculated, wash resin with binding buffer until A280 is less than 0.05.
15.   Setup microfuge rack with 12 tubes each containing 100uL 1M Tris-HCl pH 9.4 to neutralize acid. Some IgGs can be de-activated by acid.
16.   Fill up column with elution buffer and collect 1mL fractions. Most of antibody will be in fractions 2-6. Spec each fraction, blank to 90% elution buffer, 10% neutrilazation buffer.
17.   Pool fractions containing antibody and spec again. Concentrate or dilute so that concentration is between 1-2mg/mL. Typical molar extinction coefficient of IgG is between 1.3 and 1.4. So an absorbance of 2 would be divided by 1.4 to get concentration of antibody.
18.   Yield of 1L Hybridoma supernatants can be as little as 1mg to 19mg from personal experience.
19.   Dialyze antibody to 1x PBS and filter sterilize.
a.   Typically you would want to dialyze to 1:10,000
b.   I usually do 1:100 or 1:1000 two times.
20.   Store at 4 C and add azide if necessary, no azide if being used in-vivo