W hen an appropriate level o f response was detected the animals w ere sacrificed and the spleen aseptically dissected from the carcass, placed in a sterile Petri dish w ith 2ml o f R P M I and teased apart using sterile forceps. This material w as then layered onto the top o f a further 20ml o f RPM I and clumps o f cells allowed to settle out o f suspension. The remaining cell suspension was then transferred to a sterile Universal tube and the cells gently pelleted at lOOOrpm in a bench top centrifuge. The supernatant w as aspirated from the pellet, which w as then gently tapped loose and treated w ith 1ml o f red blood cell lysis buffer and gently mixed in the pipette. The cells w ere then added to 20ml o f RPM I and w ashed. The resultant
<
2
Serum A Serum B Serum C
N orm al m ouse serum Rabbit anti-PI-TP
1
0
10
100
1000
D ilution factor
F ig 5.3 S creen in g fo r th e p re se n c e o f P I-T P specific a n tib o d ie s in th e s e ra o f im m u n ised a n im a ls p r io r to u se in fusion to p ro d u c e a n tib o d y se c re tin g h y b rid o m a s.
This graph show s the results from an ELISA to determ ine the relative
responses o f the m ice to the injected antigen. M aterial for assay w as prepared from blood obtained by tail bleeds o f the im m unised mice. T he resultant sera w ere then diluted as illustrated in the figure above, and assayed in an ELISA utilising im m obilised PI-TP as the capture phase for the antibodies. The binding o f antibodies w as detected using a goat anti-m ouse- H RPO conjugate, and O PD substrate. On the basis o f these data tw o anim als w ere selected at random , and sacrificed, one for fusion, and the other for possible later use. Polyclonal rabbit anti-PI-TP antibody w as included as a positive control.
cell pellet w as again gently tapped loose and resuspended in 10ml o f R P M I - any clumps o f cells w ere removed. The cells w ere counted and left to stand until required.
The JK fusion partner cells w ere counted and the necessary volum e to give a 1:4 ratio o f JK:splenocytes calculated. The cells w ere then taken and pelleted by gentle centrifugation and washed in 20ml o f RPM I, after which they w ere resuspended in 10 ml o f R PM I and transferred to the splenocyte tube and all th e cells present pelleted at 1000 rpm for 10 minutes. The pellet w as loosened and 1ml o f a 50% w /v solution o f P E G (mw 3000-3700) w as added dropw ise with gentle shaking over one minute, prior to the addition o f 1ml o f R PM I in similar fashion. A fiirther 20 ml o f R P M I w as added, again w ith gentle agitation over a period o f four minutes. The tube containing the cells w as then inverted three time and the cells pelleted at 800 rpm for eight minutes. The supernatant w as aspirated, the cell pellet resuspended in R PM I supplem ented w ith 10% foetal calf serum, L-Glutamine and Penicillin/Streptomycin, and the w hole incubated at 37 °C for three to four hours.
At the end o f the fusion procedure the cells w ere then plated o u t onto 96 well sterile tissue culture plates, which had previously been seeded w ith a feeder layer o f M RC-5 cells. After being allowed to grow for 4 days in H A T supplem ented medium lOOpl o f medium w as removed from the wells and replaced. A fter 7 days the supernatants from the plated w ere screened for appropriate antibodies as described for the m ouse sera. These procedures w ere conducted by the staff o f the M onoclonal A ntibody U nit at University College London, M essrs. Terry Jow ett and Alan O 'Shea
Positive cultures w ere then subject to tw o round o f cloning by limiting dilution to provide pure hybridomas and expanded sufficiently to form the basis o f frozen stocks. Three clones o f each hybridoma w ere selected on the basis o f th e titre achieved and frozen dow n in PCS supplemented with 8% tissue culture grade D M SG (from Sigma).