Properties of three monoclonal antibodies that recognize an 80-kDa phytohemagglutinin-binding glycoprotein from porcine lymphocytes.
Monoclonal antibodies (mAbs) directed against porcine splenocyte phytohemagglutinin receptor glycoproteins were produced in BALB/c mice. Three antibody-producing, stable hybridomas were cloned and expanded in the peritoneal cavity of BALB/c mice. The mAbs (A7, B1, and H3) were purified and belong to the IgG2 subclass of immunoglobulins (kappa light chain). Each 125I-labeled mAb bound to purified porcine splenocytes with an (apparent) affinity KA congruent to 10(8) M-1 (Scatchard analysis). The number of (apparent) binding sites was 5 x 10(4) sites/cell in the case of B1 and H3, and approximately 15 x 10(4) sites/cell for A7. Immunoprecipitation experiments showed that the three mAbs recognized a single antigenic protein of Mr 80 kilodaltons (gp80). In addition, each mAb recognized a different epitope of gp80, as observed by Western blot analyses. Assessment of the relative ability of anti-gp80 mAbs to stimulate porcine splenocytes as determined by [3H]thymidine incorporation showed weak (A7 and B1) or no (H3) mitogenic activity. Cross-linked anti-gp80 mAbs were not mitogenic, except in the case of B1. In contrast, each anti-gp80 mAb (cross-linked or untreated) showed synergistic mitogenic properties when used in combination with a suboptimal concentration of phytohemagglutinin. The mechanism involved in this synergistic effect is discussed.
Department of Biochemistry (Faculty of Medicine), University of Sherbrooke, Que., Canada.
S
S
Faucher
J
J
Cardin
B
B
Talbot
G
G
Dupuis
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000302:isolation & purification; D015153:Blotting, Western; D002352:Carrier Proteins; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000032:analysis*; D008214:Lymphocytes / Q000032:analysis*; D008970:Molecular Weight; D010835:Phytohemagglutinins; D011233:Precipitin Tests; D011975:Receptors, Mitogen / Q000032:analysis*; D013552:Swine
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D006023:Glycoproteins; D010835:Phytohemagglutinins; D011975:Receptors, Mitogen