Production and use of monoclonal antibodies to phosphotyrosine-containing proteins

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A hybridoma secreting monoclonal antibodies that bind to PT-residues of PT-proteins was isolated from a fusion of Sp 2/0 cells with spleen cells of mice immunized with the ABP analog of the target antigenic determinant.
Male 8-weeks old Balb/c mice were immunized intradermally and in their footpads with 100 .
mu.
g of Keyhole Limpet Hemocyanin (KLH) carrier derivatized with about 30 ABP groups per 100,000 MW of KLH and emulsified in complete Freunds adjuvant.
The mice were injected again about 3 months later with the same antigen.
Four days later, spleens were removed from four mice killed by ether and cell suspensions prepared.
The spleen cells (1.
6.
times.
10.
sup.
8) from immunized mice and the Sp 2/0 fusing partner cells (8.
times.
10.
sup.
7) were each washed three times with serum free Dulbecco's Modified Eagles (DME) media, then mixed together and centrifuged at 400.
times.
g for 5 minutes at room temperature.
The cell pellet was resuspended in 0.
2 ml DME and re-centrifuged at 400.
times.
g for 10 minutes.
The spleen and Sp 2/0 cells were fused using polyethylene glycol (MW=1500, Behringwerke).
Fused cells were grown in HAT medium.
Hybrids that secrete anti-ABP antibody were detected by a solid phase radioimmunoassay.
Wells of plates (Dynatech) were coated overnight with ABP-BSA (50 .
mu.
l of 2 .
mu.
g ml.
sup.
-1 solution in PBS), then washed with PBS and a 1% BSA solution in PBS.
Twenty-five microliters of 1% of BSA and 20 .
mu.
l of culture fluid were pipetted into each well and incubated for 3 hours at 37.
degree.
C.
The wells were next washed 2-times with PBS, and 50 .
mu.
l of a solution containing .
sup.
125 I-labeled rabbit anti-mouse Ig (20 .
mu.
g ml.
sup.
-1, 2.
5.
times.
10.
sup.
4 cpm ng.
sup.
-1) (Gateway, affinity purified on a normal mouse Ig-Sepharose 4B immunosorbent and labeled by the chloramine T method) were added to each well and incubated 4 hours at 37.
degree.
C.
The wells were washed three times with PBS, and remaining .
sup.
125 I was determined in a scintillation spectrometer