Monoclonal Antibodies

 

If titre is positive do one of the following 2 weeks later:

1. Boost tail vein with 20 ug- 50 ug of Antigen in PBS and proceed with fusion on day 4. OR
2. Boost subcutaneously with 50 ug - 100 ug of Antigen in PBS and proceed with fusion 4 days later. OR
3. Boost 3 days in a row with 15 ug Ag in PBS and proceed with fusion on 4th day.

Cell Lines:

1. Myeloma; P3X63-Ag8.653

   -	Origin: BALB/c, non secreting, 8-azaguanine resistant, HPRT -.

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 J774A.1

Maintenance of the cells:

Stock solutions:

-	IMDM:
-	Fetal Calf Serum-
-	Transferring: Iron saturated. 1000X stock = 1 mg/ml
-	HT supps: 50X from Sigma H0137 ( Store at -20° C ).
-	2-Mercaptoethanol: 1000X stock (5 x 10 -2). (Store at 4°C.)
-	AT supplement : 50X stock , Sigma A-7422. (Store at -20°C.)
-	Kanamycin Sulfate: 100X from Gibco-BRL 600-5160AG . (Store aliquots at -20° C)
-	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 ug/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 ug/ml LPS and collect sup after 2 days each.

Pool the sups, filter and use as recommended. (Could be aliquoted and stored at -20C.)

Preparation of Media:

for P3X63-Ag8.653: IMDM complete to 425 ml of IMDM add:

-	0.5 ml 1000X transferring
-	0.5 ml 1000X 2- Mercaptoethanol
-	10 ml 50X HT
-	5 ml 100X Kanamycin Sulfate
-	75 ml FCS ( final 15 )

for fox-NY: IMDM or RPMI

-	10 FCS
-	1X AT supplement
-	transferring
-	Kanamycin

for J774A.1: same medium as the one for the P3X63-Ag8.653 ( = Ag8 ).

Growth conditions:

-	All cell lines mentioned above grow at 37°C, 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.

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 -70°C for at least 1 hr. (could be for a couple of days).
5. Transfer the vial from the -70°C to the liquid nitrogen tank and log the entry in the freezer log.

Thawing cells:

1. Take vial out of liquid nitrogen tank and thaw it immediately in a 37°C bath (about 1 min).
2. When there is still a small piece of ice left, dilute the cells by transferring them into a conical tube containing 10 ml of the growth medium at 37°C.
3. Spin at 1200 rpm for 5 min.
4. Aspirate medium and resuspend cells in 5 ml of medium, in a 25cm2 flask.