A bispecific F(ab')2 fragment recognizing both human thyroid-stimulating hormone (TSH) and alkaline phosphatase (AP) was prepared by disulfide bond exchange between F(ab')2 fragments of IgG1 mAb against TSH and AP. AP was polymerized by glutaraldehyde, and a sandwich enzyme-linked immunosorbent assay for TSH was developed by using the AP polymers and the bispecific F(ab')2 fragment. In this assay, the preparation of covalently linked AP-mAb conjugates was not needed, and the interaction of mAb with non-specific proteins was greatly reduced. The sensitivity for TSH increased in proportion to the degree of AP polymerization, and the lower detection limit obtained with the AP trimer was 0.5 muU/ml. The use of the bispecific F(ab')2 fragment allows us to use monomers and polymers of AP and thereby regulates the sensitivity of the assay.

Alkaline Phosphatase↗

PubMedDate not supplied

Memory effect of caerulein and its analogs in active and passive avoidance responses in the rat.

The memory effects of caerulein (CER) and its analogs ([des-Gln2]-CER and [Leu5,Nle8]-CER) were compared with that of cholecystokinin octapeptide (CCK-8) using active and passive avoidance responses in rats. In the active avoidance test, single subcutaneous (SC) injection of CER and its analogs immediately after the learning trials at doses of 10 and 100 ng/kg prevented extinction of learned task for 10 days, and at a dose of 1000 ng/kg for at least 15 days, but the effect of CCK-8 was somewhat weaker. In the saline control group, the number of responses decreased after 5 days. In the passive avoidance response, electroconvulsive shock (ECS)-induced amnesia was partially prevented by CCK-8 at doses of 100 and 1000 ng/kg SC, while CER and its analogs at doses of more than 100 ng/kg totally prevented the ECS-induced amnesia. Intraperitoneal administration of scopolamine caused complete amnesia which was also partially prevented by CCK-8, while CER could totally prevent the amnesia following SC injection of 2 micrograms/kg. These results indicate that CER and its analogs are more effective than CCK-8 for preventing experimental amnesia.

Amino Acid Sequence↗

PubMedDate not supplied

Metabolism of a mutant insulin by a receptor-mediated process and an insulin degrading enzyme.

The metabolism of a mutant insulin, [LeuB25]-insulin, was studied in vitro and in vivo. Porcine or mutant insulin (4 micrograms/kg body weight) was injected i.v. into streptozotocin-induced diabetic rats and their plasma glucose and insulin levels were determined. The half-lives of porcine and mutant insulin were 3 min and 18 min, respectively. The ability of the mutant insulin to lower the blood glucose levels was 38% of that of normal when the glucose levels at the nadir were compared. Receptor-mediated degradation of the mutant insulin assessed by chromatography of the degraded materials in the media after incubation with cells was less compared with that of porcine insulin (4% vs. 24%). The media containing the insulin degrading enzyme (IDE) of IM-9 cells and rat livers degraded porcine and mutant insulin to the same extent. These results suggest that the decreased clearance of insulin is due to the decreased receptor binding and the decreased receptor-mediated degradation, but is not due to the decreased degradation by IDE.

Animals↗