We adapted one of the in-vitro immunization methods to induce antibody responses and confirmed the success of the immunization by enzyme-linked immunosorbent assay (ELISA) without hybridization. We have previously reported several methods of in-vitro immunization using different conditioned media. Here we introduce another method of in-vitro immunization using a mixture of three types of supernatant (thymocytes, and adherent and non-adherent splenocytes of mouse). Splenocytes were immunized in-vitro by a recombinant human growth hormone (rhGH) with the above conditioned media, and the results by ELISA showed a much higher optical density than the other in-vitro immunization methods that we had previously reported. Humoral responses as a result of in-vitro immunization to soluble antigens were easily confirmed by ELISA using the above-conditioned media. This finding indicates that any other conditions thought to be critical by other researches may not be essential for in-vitro immunization.
Animals ; *Antibody Formation ; Antigens/immunology ; Cell Adhesion ; Female ; Growth Substances/*immunology ; Immunization ; Lymphocyte Activation/immunology ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins/immunology ; Spleen/cytology/*immunology ; T-Lymphocytes/*immunology

This study was performed to prove the hypothesis that oncogene expressions would have the same patterns with those of cellular growth to growth factors in FRTL-5 cells. Ribonucleic acids of FRTL-5 were extracted at 15', 30', 60' and 120' after administration of growth factors to quiescent FRTL-5, and blotted to the nitrocellulose membrane. They were hybridized with radiolabelled c-fos, c-myc and beta-actin probes. Hybridized dot blots were autoradiographed and the amount of radioactivity was measured by densitometry. Densitometric readings were used as the indices of oncogene expressions. Expressions of c-fos and c-myc were more prominent in combined administrations of TSH (10 mU/ml) and IGF-I (100 ng/ml) or IgG of Graves' disease (Graves' IgG; 1 mg/ml) and IGF-I than in combined administration of TSH and Graves' IgG. IgG of primary myxedema suppressed oncogene expressions by TSH or Graves' IgG, but not by IGF-I. From the above results, it was suggested that expressions of c-fos and c-myc to growth factors would have similar patterns with those of cell growth to growth factors in FRTL-5, and the actions of TSH and Graves' IgG would be manifested through same signal transduction system, but IGF-I would be manifested by its own.
Animal ; Cell Division/drug effects/genetics ; Cell Line/cytology/physiology ; Gene Expression/drug effects/immunology ; Graves' Disease/immunology ; Growth Substances/genetics/*pharmacology ; Immunoglobulin G/pharmacology ; Insulin-Like Growth Factor I/pharmacology ; Myxedema/immunology ; Proto-Oncogene Proteins c-fos/*genetics ; Proto-Oncogene Proteins c-myc/*genetics ; RNA/analysis ; Rats ; Rats, Inbred F344 ; Support, Non-U.S. Gov't ; Thyroid Gland/cytology ; Thyrotropin/pharmacology ; Time Factors