Properties of three monoclonal antibodies that recognize an 80-kDa phytohemagglutinin-binding glycoprotein from porcine lymphocytes

• Published in: Biochemistry and cell biology Vol. 67; no. 4-5; p. 224
• Main Authors: Faucher, S, Cardin, J, Talbot, B, Dupuis, G
• Format: Journal Article
• Language: English
• Published: Canada 01.04.1989
• Subjects: Animals; Antibodies, Monoclonal - analysis; Antibodies, Monoclonal - isolation & purification; Blotting, Western; Carrier Proteins; Cloning, Molecular; Cross-Linking Reagents; Enzyme-Linked Immunosorbent Assay; Glycoproteins - analysis; Lymphocytes - analysis; Molecular Weight; Phytohemagglutinins; Precipitin Tests; Receptors, Mitogen - analysis; Swine
• ISSN: 0829-8211
• DOI: 10.1139/o89-034

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.