Medial Iliac Lymph Node Research Articles

Generation of a rat monoclonal antibody specific for importin alpha3/Qip1.

Importin alpha, which mediates the nuclear import of nuclear localization signal (NLS)-containing proteins, is a member of nuclear transport factors. Importin alpha binds directly NLS and functions as an adapter for accessing the importin beta-dependent import pathway. To date, several isoforms of importin alpha have been identified and classified into three subfamilies in higher eukaryotes. In this study, we report on the production of a rat monoclonal antibody (MAb) against importin alpha3/Qip1, a member of the importin alpha family, using a rat medial iliac lymph node method. The MAb 3D10 produced, reacted with both recombinant and endogenous importin alpha 3/Qip1. Immunoblotting analysis revealed that MAb 3D10 exclusively recognizes importin alpha3/Qip1 among members of the importin alpha family, in various mammalian cells.

Preparation of monoclonal antibody against d-3-methoxy-4-hydroxyphenylglycol

We have been trying to prepare a specific monoclonal antibody against an endogenous 3-methoxy-4-hydroxyphenylglycol (MHPG) which will be a diagnostic marker for mental diseases such as depression. In our previous paper, we discriminated d- and l-MHPG separated from its racemate by chiral chromatography judging from their optical rotations and retention times in comparison with those of purified d-MHPG from human urine. In this paper, a d-MHPG-specific monoclonal antibody was prepared to develop an enzyme immunoassay (EIA). An adequate amount of d-MHPG as hapten was purified by chiral chromatography using a Ceramospher Chiral RU-1 column. The hapten was conjugated to human serum albumin as a carrier protein by a Mannich reaction with formaldehyde. This antigen solution was mixed with an equal volume of Freund's complete adjuvant and injected into 3 WKY/NCrj rats. After the titer of antisera increased, medial iliac lymph node cells from a rat in the highest titer were fused with SP2/0 myeloma cells. Hybridomas were screened by competitive EIA remarking recognition of dl-MHPG and cloned by a limited dilution method. After the fourth cloning, a monoclonal cell line was established. The monoclonal antibody in a culture medium was examined on EIA with 25 competitors including d-MHPG. Nineteen analogs showed little cross reactivity. Only 5 compounds showed weak cross reactivity. This concluded that the monoclonal antibody was reasonably specific for d-MHPG and should be useful for practical EIA.

Comparison of local and systemic responsiveness of lymphocytes in vitro to Bovicola ovis antigen and Concanavalin A in B. ovis-infested and naive lambs

The in vitro proliferation assay was used to determine lymphocyte responsiveness to soluble antigen of B. ovis and to Concanavalin A (Con A) in peripheral blood, spleen and various lymph nodes from B. ovis-infested and naive lambs. From March to July, an assay of monthly blood samples showed generally higher proliferative responses to antigen and Con A in B. ovis-infested than naive lambs. The proliferative response of cells from the skin-draining prescapular lymph nodes to B. ovis antigen was significantly higher in B. ovis-infested than naive lambs. Responses of cells from the medial iliac, mediastinal and mesenteric lymph nodes (which do not receive lymph from the skin) and spleen showed no significant differences between groups. Within the B. ovis-infested lambs, the response of cells from the prescapular lymph node was significantly higher than that from any other lymphoid organ examined. Responsiveness of the prescapular, medial iliac and mesenteric lymph node and spleen cells to Con A was not significantly different between groups, while mediastinal lymph node cells showed a significantly higher response in B. ovis-infested lambs. The data indicate that the antigen-specific cellular immune response is operating mainly locally, at the level of the skin and draining lymph nodes. Responses to the T cell mitogen Con A did not support non-specific immunodepression as reported in other ectoparasite/host systems.

A novel method of preparing rat-monoclonal antibody-producing hybridomas by using rat medial iliac lymph node cells.

A novel method of preparing hybridomas producing rat monoclonal antibodies was established. The enlarged medial iliac lymph nodes from rats injected via hind footpads with an emulsion of antigen and Freund's adjuvant were used for cell fusion. Ovalbumin was used as a representative antigen. The incidence of hybridomas producing antibody of interest with this method was about 10 times higher than that of hybridomas with the conventional method using mouse or rat spleen cells. The average percentages of hybridomas producing IgG1, IgG2a, IgG2b and IgG2c were 37.1%, 47.0%, 15.9% and 0.0%, respectively. A single injection with antigen was sufficient for immunization in this method.