A rapid and efficient purification method for horse IgG(T) using a rat monoclonal antibody.
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 animals were fused to a non-secreting LOU/C immunocytoma (IR983F). The hybridomas were cultured in HAT-containing medium and cloned under limiting dilution conditions. Supernatants from the growing hybrids were screened 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 induced in LOU (IgK-1b) rats by injecting the hybridoma cells and incomplete Freund's adjuvant ip. To obtain purified mAb, ascitic fluid was applied to a Sepharose anti-rat LOU IgK-1a chain column. 4. The purified mAb was then coupled to Sepharose. Immunoelectrophoretically pure IgG(T) was obtained by passage of horse serum through this column. The entire procedure took less than 30 min and resulted in a highly purified IgG(T).
Fernandes
I
I
Laboratório de Imunopatologia, Instituto Butantan, São Paulo, Brasil.
Cormont
F
F
Latinne
D
D
Bazin
H
H
Takehara
H A
HA
Mota
I
I
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002460:Cell Line; D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006736:Horses / Q000276:immunology*; D006825:Hybridomas / Q000276:immunology; D007114:Immunization; D007122:Immunoelectrophoresis; D007074:Immunoglobulin G / Q000276:immunology / Q000302:isolation & purification*; D008297:Male; D051381:Rats; D013997:Time Factors
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G