I. Fernandes et al., A RAPID AND EFFICIENT PURIFICATION METHOD FOR HORSE IGG(T) USING A RAT MONOCLONAL-ANTIBODY, Brazilian journal of medical and biological research, 27(11), 1994, pp. 2599-2606
1. Louvain rats (IgK-1a) were immunized with horse IgG(T). To generate
mAb to IgG(T), popliteal lymph node cells taken from the immunized an
imals were fused to a non-secreting LOU/C immunocytoma (IR983F). The h
ybridomas were cultured in HAT-containing medium and cloned under limi
ting dilution conditions. Supernatants from the growing hybrids were s
creened by ELISA using plates coated with horse IgG(T) or IgGa+b+c. 2.
The anti-IgG(T) mAb obtained was named LO-HoGT-1 (LOU anti-horse IgG(
T)). It is an IgG2a rat antibody whose light chain allotype is IgK-1a,
and with an affinity constant of 2.9 x 10(10) M(-1). 3. Ascites was i
nduced in LOU (IgK-1b) rats by injecting the hybridoma cells and incom
plete Freund's adjuvant ip. To obtain purified mAb, ascitic fluid was
applied to a Sepharose anti-rat LOU IgK-1a chain column. 4. The purifi
ed mAb was then coupled to Sepharose. Immunoelectrophoretically pure I
gG(T) was obtained by passage of horse serum through this column. The
entire procedure took less than 30 min and resulted in a highly purifi
ed IgG(T).