Masao Kono

Regulatory role of atrial natriuretic polypeptide in water drinking in rats

Intracerebroventricular (i.c.v.) administration of synthetic alpha-human atrial natriuretic polypeptide (alpha-hANP) decreased the water intake of dehydrated rats. Anti-ANP antiserum, which can neutralize endogenous ANP, potentiated the water intake induced by water deprivation or angiotensin II (AII) injection in rats. These findings suggest that endogenous ANP in rat brain antagonizes the action of AII and plays an important role in the maintenance of drinking behavior.

Relationships among islet cell antibodies, residual β-cell function, and metabolic control in patients with insulin-dependent diabetes mellitus of long duration: Use of a sensitive C-peptide radioimmunoassay

Relationships among islet cell antibodies (ICA), residual beta-cell function, and metabolic control were studied in 60 insulin-dependent diabetics (IDDs) of long duration (6 to 31 years). Sensitive C-peptide immunoreactivity (CPR) and ICA assays with limits of 0.017 nmol/L and 5 Juvenile Diabetes Foundation (JDF) U, respectively, demonstrated that baseline (0.16 +/- 0.02 nmol/L, mean +/- SE, n = 26), as well as maximum CPR values (0.34 +/- 0.05 nmol/L), during 100-g oral glucose tolerance tests (OGTT) in ICA-positive IDDs were significantly higher than corresponding values in ICA-negative ones (baseline values, 0.10 +/- 0.01 nmol/L, P less than .05; maximum values, 0.20 +/- 0.04 nmol/L, P less than .01, n = 34). Negative correlation was observed between increment of serum CPR and metabolic control indices, including fasting blood glucose (FBG) and HbA1c levels (P less than .05). In addition, ICA-positive insulin-dependent diabetes mellitus (IDDM) patients had lower values of FBG (8.2 +/- 0.4 mmol/L, P less than .01 v ICA-negative IDDs) and HbA1c (9.2% +/- 0.2%, P less than .05 v ICA-negative IDDs) than ICA-negative ones (FBG, 9.9 +/- 0.4 mmol/L; HbA1c, 9.8% +/- 0.2%). These results indicate that minute CPR responses to OGTT detected by sensitive methods may represent residual pancreatic beta cells, which may contribute to ICA generation and good metabolic control in IDDs of long duration.

Clinical usefulness of serum phospholipase A2 determination in patients with pancreatic diseases.

A new kit for radioimmunoassay of serum phospholipase A2 (PLA2) with monoclonal antibody (S-0932, Shionogi, Osaka, Japan) was used to examine PLA2 levels in patients with various diseases. Patients with acute pancreatitis showed significantly increased serum PLA2 levels. In patients with chronic pancreatitis, significant correlations were observed between the levels of factors evaluated by the secretin test and serum PLA2 levels. In patients with pancreatic cancer, serum PLA2 levels varied with disease severity. Serum PLA2 concentrations were within the normal range in patients with other malignant tumors, diabetes mellitus, and chronic liver diseases but were increased in patients with chronic renal failure. S-Sepharose column analysis of sera showed a small peak of pro-PLA2 and a large peak of PLA2 in sera from patients with severe acute pancreatitis, but a large peak of pro-PLA2 in healthy controls and patients with other diseases. On G-100 gel filtration, high-molecular-weight PLA2 immunoreactivity was detected in sera of patients with chronic renal failure, whereas a single peak of PLA2 immunoreactivity coinciding with that of standard PLA2 was detected in sera of patients with acute pancreatitis. These results suggest that (a) measurement of serum PLA2 is clinically useful for diagnosis and monitoring of pancreatitis, (b) active PLA2 in the circulation is dominant in severe acute pancreatitis, and (c) the kidney may be the main site of PLA2 degradation or excretion.

A Highly Specific and Sensitive Radioimmunoassay of Caerulein, An Analogue of Cholecystokinin-8

A highly specific and sensitive competitive radioimmunoassay was developed for caerulein (CLN), an analogue of cholecystokinin-8 (CCK-8), in plasma and brain. Antiserum was produced in rabbit by immunization with N delta-[CLN-(1-6)]-ornithine amide conjugated with bovine serum albumin by the glutaraldehyde method. N alpha-[CLN-(1-6)]-lysine amide was labelled with 125I-Bolton & Hunter reagent and used as a labelled antigen after purification by high-performance liquid chromatography. This assay was highly specific for CLN, and cross reactivities for other related peptides, CCK-4, CCK-8, gastrin-I, and gastrin-(14-17), were not observed (less than 0.01%). The limits of determination in biological specimens after CLN administration were 11 pg/ml in human plasma and rat plasma and 80 pg/g in rat brain. This study showed that the slight structure difference between hapten and 125I-labelled antigen is important to the assay performance.

A sensitive two-site enzyme immunoassay for human pancreatic secretory trypsin inhibitor (PSTI) using monoclonal antibodies

By using monoclonal antibody against human pancreatic secretory trypsin inhibitor (PSTI), we developed a highly sensitive, simple, and reliable two-site enzyme immunoassay system. The minimum amount of PSTI detected by this EIA is approximately 10 pg/ml when a 100 microliter aliquot of the sample is used. Good reproducibilities of within- and between-assay series and excellent recovery of exogenous PSTI from serum were observed. The correlation between the values obtained by the EIA and RIA methods was given by the linear regression equation, y = 1.09x + 4.6, for which the correlation coefficient (r) was 0.980 (n = 20). Antigenicity of the trypsin-PSTI complexes was found to be approximately 10% of that of PSTI. From these results, it seems that our recently developed EIA system for PSTI is useful in clinical testing for quantitation of PSTI in body fluids, for biochemical studies on synthesis and secretion of PSTI, and also for study of pathophysiological mechanisms involved in the development of acute pancreatitis and certain malignant neoplasms.

Preparation and characterization of labelled interferon-γ and the development of radioreceptor assay for interferon-γ

Abstract A 125 I-labelled recombinant interferon-γ (IFN-γ) was prepared by the lactoperoxidase—glucose oxidase method. The specific activity of the labelled IFN-γ was 31 Bq U −1 and its molecular weight, immunoreactivity and receptor binding ability remained the same after the labelling. Using the labelled IFN-γ and FL5-1 cells from human amniotic membrane, a radioreceptor assay was developed. Natural IFN-γ, recombinant IFN-γ and the labelled IFN-γ were observed to bind to the same binding sites on the cells with similar affinity ( K d = 1.3–2.2 × 10 −10 M). The radioreceptor assay was more specific than a bioassay because the labelled IFN-γ did not compete with IFN-α or IFN-β. It was much more sensitive (90 pM) than conventional competitive radioimmunoassay (300 pM) using the same labelled IFN-γ, and as sensitive as immunoenzymometric assay (60 pM). The radioreceptor assay should be useful not only for research on IFN-γ but also for the determination of biological activity in process control and/or quality control of IFN-γ manufacturing.

Simultaneous Immunoenzymometric Assay for Antibodies Against Human Interleukin-2 and Human Serum Albumin in Rat Serum

A simultaneous immunoenzymometric assay for anti-human interleukin-2 antibody and anti-human serum albumin antibody in rat serum was developed. Two antigen-immobilized polystyrene balls were immersed in a diluted serum sample in an assay tube and then the antibodies on the balls were made to react with a horseradish peroxidase labelled anti-rat IgG antibody in the same tube after washing. The enzyme activity of each ball was measured by fluorometry. Not only were the sensitivity (70 ng/ml each), assay recovery (100-101%), and precision (C.V. = 5-13%) comparable to those of conventional immunoenzymometric assays using one antigen-immobilized ball but the assay was also much more feasible for mass routine assays. Thus, conventional immunoassays can be replaced by this convenient simultaneous method.