Genji Kawano

Detection of monocyte chemotactic and activating factor (MCAF) in normal blood and urine using a sensitive ELISA

We developed a highly sensitive enzyme-linked immunosorbent assay (ELISA) for human monocyte chemotactic and activating factor (MCAF), an inflammatory cytokine that plays an important role in the recruitment of blood monocytes to areas of inflammation. The ELISA, which is based on a sandwich method using two newly-developed monoclonal antibodies, could quantitatively detect MCAF in the range between 2.5 pg/ml (50 fg/sample) to 300 pg/ml after incubation for a total of 2 h, and showed no cross-reactivity with various structurally-related IL-8 superfamily proteins. It was not affected by blood or urine components non-specifically, and thus was directly applicable to clinical specimens. When serum and urine samples from healthy subjects were measured, they all turned out to contain detectable levels of MCAF (more than 30 pg/ml). By gel-filtration column chromatography analysis, MCAF in the body fluids was eluted as a single peak at the position corresponding to the molecular weight of 10 kD, suggesting that it exists as a monomer form, free from carrier proteins. The established ELISA here is expected to be effectively used for the further investigations on the relationship of MCAF with various inflammatory diseases.

Enteric‐formulated lactoferrin was more effectively transported into blood circulation from gastrointestinal tract in adult rats

We have previously demonstrated that intestinally infused bovine lactoferrin (bLF) is transported into the blood circulation via the lymphatic pathway, not via the portal circulation. Therefore, in the present study, we further investigated whether intragastrically infused enteric‐formulated bLF (EF‐bLF) was more efficiently absorbed than bLF from the intestine in adult rats. The rats were randomly divided into three groups: 30 and 300 mg kg−1 non‐enteric‐formulated bLF (non‐EF‐bLF) groups and a 30 mg kg−1 EF‐bLF group. Thoracic lymph was collected from a thoracic lymph duct under general anaesthesia. Bovine lactoferrin was infused into the stomach or duodenal lumen via a needle for a period of over 1 min in a volume of 1 ml kg−1. The bLF transported into the lymph was assayed quantitatively by double‐antibody enzyme‐linked immunosorbent assay (ELISA). Following the intragastric administration of bLF, the three groups showed almost the same lymph flow, but the bLF concentration in the lymph fluid in the EF‐bLF group increased significantly and peaked 3 h after administration. With intraduodenal administration, the bLF concentration in the lymph fluid of the higher non‐EF‐bLF group was significantly higher than those of the other groups. The amount of absorbed bLF in the EF‐bLF group was, however, about 10 times higher than that in the lower non‐EF‐bLF group, when it was administered intragastrically. These data show that enteric‐formulated bLF is less susceptible to gastric pepsin and is more efficiently absorbed from the intestine than is non‐enteric‐formulated bLF.