Kyoichi Matsumoto

A highly sensitive enzyme-linked immunoassay for serum free prostate specific antigen (f-PSA).

Monoclonal antibodies (MAbs) were generated against human prostate specific antigen (PSA) to allow development of a sensitive free-PSA (f-PSA) assay. Of a total of 211, 12 could detect only f-PSA, the other 199 MAbs binding to both f-PSA and complex-PSA. In the present study, one MAb (no. 5C6) specific for f-PSA and another (no. 79) reacting with both forms were used to develop an enzyme-linked immunoassay (ELISA) for serum f-PSA. The detection limit was established as 0.008 microg/l (n = 20, mean of zero standard + 3 S.D.) and the average recovery of f-PSA was 93-102%. The within-run and between-day coefficient of variation (CV) varied from 5.4-7.4% and 4.8-6.5%, respectively. The cross-reactivity of the assay to PSA-alpha1-antichymotrypsin complex was determined to be < 0.4%.

Faecal chitinase 3-like-1: a novel biomarker of disease activity in paediatric inflammatory bowel disease

Aliment Pharmacol Ther 2011; 34: 941–948

Expression of pepsinogen II with androgen and estrogen receptors in human prostate carcinoma

The expression of pepsinogen II (PG II), an aspartyl proteinase usually involved in the digestion of proteins in the stomach, was immunohistochemically investigated in conjunction with androgen (AR) and estrogen receptor (ER) status in prostate adenocarcinomas. Of a total of 38 samples obtained from radical prostatectomies, 23 tumors (60.5%) were positive for PG II and there was a significant positive correlation to the expression of AR but not to ER. Cells positive for PG II were localized mainly to the peripheral zones of tumorous glands which, in normal prostate, are negative, and in areas also expressing AR. In addition, a significant correlation between AR and ER was detected in the prostate carcinomas examined, which suggests a hormone‐dependent status. On the basis of these results, PG II expression might be closely related to hormonal alterations associated with the development of prostate tumors.

ELISA for a complexed antigen with a monoclonal antibody blocking reaction with the free antigen—assay-specific for complexed prostate-specific antigen

We have developed an enzyme-linked immunoassay (ELISA) for serum complexed-antigen (prostate-specific antigen; PSA)(c-PSA) with simultaneous blocking of free-antigen (PSA)(f-PSA). The assay utilizes three different monoclonal antibodies (MAbs) recognising three distinct PSA epitopes. The detection limit was established as 0.19 microg/l (n=20, mean of zero standard+2S.D.) and the average recovery of f-PSA was 98-100%. The within-run and between-day coefficients of variation (CV) ranged from 2.1% to 3.2% and 2.8% to 6.3%, respectively. There was a good correlation between serum c-PSA measured by the present ELISA and PSA-alpha(1)-antichymotrypsin complex (PSA-ACT) concentrations (r=0.991). This method should provide a better tool for discriminating between benign and malignant prostatic disease.

Detection of endogenous LRP6 expressed on human cells by monoclonal antibodies specific for the native conformation

LRP6 is a cell surface molecule that plays a critical role in the Wnt signaling pathway, and is implicated in numerous human diseases. Studies of cellular signaling mediated by LRP6 have relied on overexpression experiments, due to the lack of good monoclonal antibodies (mAbs) reactive with native LRP6 ectodomain. By using native recombinant LRP6 ectodomain fragment produced in mammalian expression system, we succeeded in developing a panel of anti-human LRP6 mAbs. Selected mAbs were capable of staining endogenous LRP6 on cell surface by using flow cytometry and immunofluorescence microscopy, and enriching detergent-solubilized LRP6 from cell lysate by immunoprecipitation.

One-step enzyme-linked immunosorbent assay (ELISA) for measurement of serum free leptin

In man, circulating leptin levels are increased with obesity and are regulated by a complex of hormonal, feeding and body-weight changes. Accurate and precise methods to quantitate circulating serum free leptin (f-leptin) concentrations are needed for physiological and clinical studies. We developed a one-step enzyme immunoassay to measure human f-leptin in serum. The detection limit was 0.40 ng/ml. The recovery of leptin added to serum was 90.8-102.8%. The within-run and between-day coefficients of variation (C.V.) ranged from 2.8 to 7.7 and 5.7 to 9.7%, respectively, and the immunoassay had an overall recovery rate for serial dilution in the range of 94. 0-109.9%. Measured serum f-leptin concentrations in 201 adults correlated (r=0.449, P<0.001) directly with body mass index (BMI kg/m(2)), particularly when results were separated by gender (r=0. 709 for male, P<0.001; r=0.643 for female, P<0.001). We conclude that this one-step enzyme immunoassay is accurate for measuring f-leptin in human serum.

Inhibitory Effect of Potassium Citrate on Rat Renal Tumors Induced by N-Ethyl-N-hydroxyethylnitrosamine Followed by Potassium Dibasic Phosphate

Potassium dibasic phosphate (PDP) was administered at a concentration of 10% by weight in basal diet to unilaterally nephrectomized Wistar rats previously given 1000 ppm N‐ethyl‐N‐hydroxyethyl‐nitrosamine (EHEN) in the diet for 2 weeks. To study the effect of alkalinization on renal mineralization, some animals concomitantly received 5% potassium citrate (PC). Feeding PDP alone promoted adenomatous hyperplasias, which were regarded as preneoplastic lesions, as well as renal cell tumors in EHEN‐initiated rats, whereas the addition of PC to PDP diets reduced the promoting effect. Histopathology, serum biochemistry and urinalysis indicated retardation of renal calcium crystallization by PC. Two other phosphate salts, sodium phosphate (SP) and calcium phosphate (CP), were also administered. SP showed a slight promoting effect on adenomatous hyperplasias and a 2‐fold increase in the yield of renal cell tumors, while CP induced a clear reduction of both lesions, over EHEN alone. The promoting effects of both PDP and SP and the inhibitory effect of PC were somewhat correlated to 5‐bromo‐2′‐deoxyuridine labeling indices, the degree of nephropathy, and mineralization in the kidney. Immunohistochemically, the nephropathy induced by phosphate salts was not linked to αzu‐globulin. A pathogenesis for renal carcinogenesis is suggested in which nephropathy associated with mineralization enhances the development of renal cell tumors.

Construction of an Immunochromatographic Determination System for N1,N12-diacetylspermine

N1,N12‐diacetylspermine (DiAcSpm) is a recently identified tumor marker. Its concentration increases in the urine of cancer patients at early clinical stages. To utilize this characteristic feature and thus contribute to the early detection of cancer, we developed an immunochromatographic determination system for DiAcSpm.