Clinical usefulness of a trypsin radioimmunoassay kit

From the clinical use of RIA-gnost trypsin kit, the following results were obtained. 1. Standard curve showed a steep and good curve was shown. 2. Incubation: The condition for the first incubation was set at the room temperature for 10-24 hours and that for the second incubation at the room temperature for 3-5 hours. With these settings, satisfactory results were obtained. 3. Reproducibility and recovery: The C.V. of the reproducibility and the recovery were considered superior, and the values were below 10% and +/- 3%, respectively. 4. Correlation between trypsin and serum elestase-1: An excellent positive correlation (coefficient of correlation r = 0.889) was shown. 5. Serum trypsin concentration of normal and pancreatic diseases: The normal range was from 100 to 500 ng/ml. Acute pancreatitis rose obviously. Diabetes mellitus and chronic pancreatitis was below 500 ng/ml and the pancreatic cancer showed a tendency to scatter in the range of 50-1,250 ng/ml. The above results indicated that serum trypsin can be easily measured with high precision by using this method. Thus the method is considered useful for the diagnosis of pancreatic diseases.     

Clinical evaluation of serum neuron-specific enolase levels in patients with lung cancer

Serum neuron-specific enolase (NSE) levels were studied in 105 patients with malignant neoplasms (lung cancer 38, others 67), 13 patients with various benign diseases and 7 healthy adults. The mean serum NSE level in adult control subjects was 7.4 +/- 0.8 ng/ml, and cut off level was decided 10 ng/ml. Serum NSE levels were elevated in 14/38 (37%) of patients with lung cancer and in 14/67 (21%) of patients with the other malignant neoplasms. In patients with benign diseases, serum NSE level was elevated only in one patient with pituitary adenoma. In 7 patients with small cell lung cancer, the positive rate was higher (86%) than in those with non-small cell lung cancer (26%), and serum NSE levels were higher than 25 ng/ml except one case. There was no correlation between serum NSE and CEA (carcinoembryonic antigen) levels in patients with small cell lung cancer, also in patients with lung cancer. The measurement of serum NSE level seemed to be useful for diagnosis in patients with small cell lung cancer.     

Development of radioimmunoassay of intact laminin and its clinical evaluation as a tumor marker.

The concentration of laminin including laminin variants in serum samples was measured by a double-antibody radioimmunoassay using intact laminin. The mean level in 92 normal subjects (47 men and 45 women) was defined as 1 unit (U)/ml, and the cut-off value (2 S.D. above the mean) was 1.37 U/ml. Mean laminin level in 391 patients with various malignancies was 1.50 +/- 0.86 U/ml. Laminin levels were elevated in various cancer patients, and in 45.0% (176/391) the values exceeded the cut-off level; in patients with cancer of the stomach or pancreas, positivity rate exceeded 60%. Mean laminin concentration for 130 pregnant women (2.06 +/- 0.65 U/ml) was also significantly higher than that of normal controls, but concentrations for patients with various benign diseases were within a low range (0.55 +/- 0.29-1.10 +/- 0.29 U/ml). In the stomach or pancreas cancer patients, a positive correlation between laminin level and tumor progression or course of the disease was observed. These findings indicate that serum laminin level is potentially useful in the diagnosis and monitoring of certain cancers.     

Fundamental studies on thyroid stimulating hormone immunoradiometric assay kit (TSH RIABEAD II)

We have reported fundamental studies on the TSH immunoradiometric assay, using TSH RIABEAD II kit (Dainabot). The sensitivity of the assay was 0.03 mu IU/ml and its C.V. was 27.2%. Intra- and inter-assay C.V. were less than 5%. Dilution test and recovery test were good. Serum TSH level was 0.3-4.0 mu IU/ml in normal subjects, less than 0.03 mu IU/ml in untreated Graves' disease and subacute thyroiditis. Therefore, it was found that the clear difference exist in serum TSH levels between normal subjects and patients with untreated Graves' disease. There was a well correlation on the serum TSH levels between this method and TSH radioimmunoassay kit (Amerlex TSH, r = 0.983). Especially, the measurement of serum TSH levels, using immunoradiometric assay kit, was useful for the diagnosis of patients with Graves' disease.     

Immunochemical measurement and immunohistochemical detection of membrane-associated placental tissue protein 1

Using the avidin-biotin binding system, an enzyme immunoassay procedure was developed to measure the membrane-associated placental tissue protein 1 (MP1) in serum. The standard curve covered the range from 10 to 1000 ng/ml of MP1. The intra- and inter-assay coefficient of variations (C.Vs) were less than 5 and 10%, respectively. Recoveries of MP1 added to serum ranged between about 96 and 101%. The MP1 serum level was over 10 and under 112 ng/ml in non-pathological men, and under 240 ng/ml in non-pathological women. The MP1 level in the ovulatory phase was higher than in other phases of the menstrual cycle. In pregnancies during 6-39 weeks, the MP1 level ranged from 10 to 540 ng/ml, and it increased during the third trimester of gestational age. In benign gynecologic diseases, the MP1 concentration in serum ranged from 10 to 215 ng/ml. The MP1 levels in benign diseases were compared with those in ovarian malignancies, in endometrial carcinoma, and in uterine cervical cancer. The immunohistochemical location of MP1 was detected in the cell membrane of ovarian cystadenocarcinoma.     

Detection of ferritin in human serum with a MAP-H(2)O(2)-HRP voltammetric enzyme-linked immunoassay system

A voltammetric enzyme-linked immunoassay based on new system of m-aminophenol (MAP)-H(2)O(2)-horseradish peroxidase (HRP) has firstly been developed and used for the detection of HRP, labelled HRP and ferritin in human serum. HRP or labelled HRP catalyzes the oxidation reaction of MAP with H(2)O(2), the product of which produces a sensitive voltammetric peak at potential of -0.46 V (vs. SCE) in Britton-Robinson (BR) buffer solution. By using this voltammetric peak, HRP can be measured with a detection limit of 9.5x10(-1) mU/l and a linear range of 2.5-2.5x10(2) mU/l. The detection limit to ferritin is 0.25 ng/ml and the linear range 0.25-320 ng/ml. The processes of the electro-reduction of the product of the enzyme-catalyzed reaction have been investigated in detail.     

Elaboration of a radioimmunoanalytical method for the determination of serum ferritin

A radioimmunoanalytical (RIA) method was elaborated for the determination of ferritin in human blood serum in clinical practice. Placental ferritin separated from the human after-delivery placental and antibodies against the human placental ferritin obtained by the immunization of rabbits with this antigen were used. The whole complex of basic conditions and parameters of the RIA method was tested including the estimation of the region of normal values and clinical tests. The method elaborated was compared with the commercial kit Ferritin RIA Amersham, code IM 1051, chosen as reference kit. The results of the determination of control parameters as well as ferritin levels obtained by the method elaborated exert good agreement with the reference kit and correspond to requirements for routine RIA practice.     

Serum ferritin in patients with malignant neoplasms

Serum ferritin levels were studied in 264 patients who were consisted of 153 patients with various malignant neoplasms and 111 patients with various benign diseases. Positive rate of serum ferritin in all patients with malignant neoplasms was 35%. Hepatomas and pulmonary cancer showed relatively high positive rate, respectively 65% and 42%. In patients with benign diseases, hepatic diseases showed the high positive rate (52%) and the other benign diseases was low positive rate (11%). The relationship between serum ferritin and alpha-feto-protein in patients with hepatomas and other liver diseases was low. And the relationship between serum ferritin and CEA (carcinoembryonic antigen) in patients with malignant neoplasms of gastrointestinal tract was also low. It seemed that the measurement of serum ferritin levels will be of low value in the differentiation of the patients with malignant neoplasms.     

Basic and clinical evaluation of a new assay procedure "SCC RIABEAD" for estimation of squamous cell carcinoma related antigen

We examined the efficacy of a new commercial assay procedure (SCC RIABEAD) for estimation of squamous cell carcinoma related antigen (SCC). Intraassay and interassay variance were 4.0-14.6% and 4.6-17.0% respectively. Recovery and dilution tests gave satisfactory results. The normal range was under the level of 1.63 ng/ml. The patients with uterine cervical cancer or squamous cell carcinoma of the lung showed high positive rates. The values of SCC measured by SCC RIABEAD were well-correlated to those by SCC RIAKIT. However, SCC RIABEAD showed lower values in low SCC level and higher values in high SCC level than SCC RIAKIT.     

A study on postsplenectomy changes of serum tuftsin level using a radioimmunoassay protocol

In this article, postsplenectomy changes of serum tuftsin level were studied on both human subjects and rabbits by using a self-established radioimmunoassay protocol. Antituftsin antibodies were raised in rabbits and roosters.¹²⁵I-tuftsin was prepared through an Iodogen method. The characteristics of the RIA were satisfactory with a detecting range of 0.5–100 ug/ml and the lowest detection limit of 400ng/ml. Serum tuftsin levels of splenectomized subjects were measured and compared with control groups. The serum tuftsin level from 30 postsplenectomy human beings was 406±179 ng/ml ( ± s) while that from a control group of 40 healthy blood donors was 557±256 ng/ml; the serum tuftsin level of 10 postsplenectomy rabbits was found to be 206±75 ng/ml while that of a control group of 10 normal rabbits was 318±96 ng/ml. The results showed that serum tuftsin level decreased markedly after splenectomy.     

Evaluation of radioimmunoassay of the blood tobramycin level for the purpose of setting the dosage based on the clinical pharmacokinetic theory and a comparison of radioimmunoassay with other assay methods

With the purpose to use for therapeutic drug monitoring (TDM), blood concentrations of tobramycin (TOB) in each patient were measured by radioimmunoassay (RIA). A RIA kit of TOB (Clinical assay-Japan Travenol) was evaluated for precision and recovery, in that partial improvement of the method was made, in order to measure low level of TOB. The RIA was compared with high-performance-liquid-chromatography (HPLC), bioassay (BA) and 2 kinds of enzyme immunoassay (EIA) (EMIT and SLFIA). The RIA of TOB revealed high precision (1.8-2.4% in C.V.) and high reproducibility (5.0-6.9% in C.V.). It was found that this RIA kit can be used for measuring low level of serum TOB concentrations by a modification of the method. The total range of measurable blood level is from 0.1 to 16.0 micrograms/ml. The nearly one to one correspondence was observed between RIA and other 4 methods, when 154 samples obtained from 18 cases were measured. A representative case of TDM for TOB was demonstrated, in which predicted concentrations agreed fairly well with actual measured values at steady state. It was concluded that the RIA kit is useful for clinical application of TDM for the adequate dosage regimen of TOB. Modification of the method for rapid assay of a small number of samples will increase the clinical usefulness.     

Basic evaluation of highly sensitive thyroid stimulating hormone immunoradiometric assay kits

Usefulness of three kinds of TSH kits by immunoradiometric assay (IRMA) was evaluated. They were able to measure low levels (less than 0.1 microIU/ml) in serum thyroid stimulating hormone (TSH) with incubation of short time (4 hours). In particular, RIABEAD II kit had a highly specific affinity for TSH and the normal range (+/- 2 S.D.) using it showed from 0.20 to 3.50 microIU/ml in 150 normal subjects. In patients with hyperthyroidism and in patients with hypothyroidism, the values of TSH were lower and higher than those of normal subjects, respectively. Another kits showed similar results. These results indicate that these TSH-IRMA kits are useful to evaluate serum TSH levels exactly.     

Purification and determination of human prostate specific antigen in the serum

A human prostate specific antigen (PA) has been purified from an extract of prostatic tissue obtained during operation for benign prostatic hypertrophy (BPH). The antigen, which can be demonstrated a single component by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), has an apparent molecular weight of about 34,000 and has lower mobility for the positive pole than prostatic acid phosphatase (PAP). Double antibody radioimmunoassay (RIA) for PA in serum was developed with the antiserum raised in rabbit against partially purified PA. In normal serum of 30 controls the concentration were studied by the RIA. The normal upper limit of the serum PA levels in assay was set at 2.5 ng/ml. Elevated levels were observed in serum from 19 out of 21 untreated patients with prostatic carcinoma and 9 out of 23 patients with BPH, but latter less than 10 ng/ml. The results indicate that the PA is a potentially useful marker as well as PAP for prostatic cancer.     

Determination of the antimitotic agents N-desacetylcolchicine, demecolcine and colchicine in serum and urine

In an effort to characterize the pharmacokinetic behavior of the antimitotic agent N-desacetylcolchicine a selective, sensitive high-performance liquid chromatographic method was developed for the determination of N-desacetylcolchicine, demecolcine and colchicine in serum or urine. To 0.5 ml of serum or 0.1 ml of urine diluted to 0.5 ml were added 50 microliters demecolcine (2 micrograms/ml) which serves as the internal standard. The sample was extracted using a C2 reversed-phase solid extraction column. N-Desacetyl-colchicine, colchicine and the internal standard were eluted from the column with methanol. The combined eluates were evaporated to dryness and the residue was reconstituted with water. The reconstituted sample was injected into a C18 reversed-phase column and eluted using a mobile phase consisting of 0.1 M potassium dihydrogenphosphate, 5 mM 1-pentanesulfonic acid in methanol and acetonitrile with a final pH of 6.0, at a flow rate of 1.5 ml/min. N-Desacetylcolchicine, colchicine and the internal standard were detected using a variable-wavelength ultraviolet detector at 254 nm. The limit of detection was 0.4 ng/ml for desacetylcolchicine and 4.0 ng/ml for colchicine. The method is linear over a concentration range of 1.0-200 ng/ml. The method has been shown to be a rapid, reliable method to monitor N-desacetylcolchicine levels in clinical trials in cancer patients.     

Modified nucleosides in human serum.

Methylated purines and pyrimidines derived from the degradation of transfer ribonucleic acid have been shown to be excreted in abnormal amounts in the urine of patients with cancer. Recent technology developed by Gehrke and Kuo has allowed the separation and quantification of modified nucleosides in serum using reversed-phase high-performance liquid chromatography with diode-array measurement. Serum levels of ten modified nucleosides were measured in 37 normal healthy adults to establish normal values and to correlate activity with age and sex. In addition, serum levels of patients with several malignancies were measured to determine activity in these diseases. Levels of modified nucleosides in normal individuals were consistently reproducible and showed no significant variation among males versus females or with age. Patients with malignant diseases showed consistent elevations and these were highest in patients with more advanced disease. The evidence of no significant differences in the mean levels of modified nucleosides in serum with age or sex in normal adults and elevations in patients with malignancies demonstrate the potential value of modified nucleosides as cancer biomarkers.     

增强化学发光酶免疫分析法测定血清中的铁蛋白

本文采用对碘苯酚增强的Ｌｕｍｉｎｏｌ－Ｈ２Ｏ２－ＨＲＰ化学发光反应体系作为免疫分析的最终检测手段，建立了一种新的铁蛋白的免疫分析方法，与酶联免疫分析法相比，该方法具有灵敏度高，线性范围宽等特点。方法的相对标准偏差３．７％，标准曲线线性范围为０．１２－２０００ｎｇ／ｍｌ，用该方法对血清中的铁蛋白进行了测定，其结果与ＥＬＩＳＡ法所得结果一致。     

Fundamental and clinical evaluation of an immunoradiometric assay procedure "Amerwell AFP" for estimation of serum alpha-fetoprotein

The efficacy of assay procedure for estimation of serum alpha-fetoprotein (AFP) was examined on "Amerwell AFP" immunoradiometric assay (IRMA) using monoclonal AFP antibody. The condition of incubation was most satisfactory for 2 hours at 37 degrees C. Coefficients of variance for intraassay and interassay were 5.1-10.0% and 8.4-9.9% respectively. Dilution test gave satisfactory results. The binding capacity of microplate-well tagged monoclonal AFP antibody with AFP antigen was satisfactory for assay reactions. This method showed a good correlation to the AFP RIA bead (Dinabot Co.) method. The normal range (reference value) was within the level of 5.0 IU/ml due to Hoffmann's method in the examination of 860 subjects. Estimation of AFP with "Amerwell AFP" IRMA kit was a feasible routine method of clinical application for tumor marker.     

Myocardial injury occurs earlier than myocardial inflammation in acute experimental viral myocarditis

Endomyocardial biopsy often fails to show myocardial inflammation for patients with clinically suspected myocarditis. The serum isoforms of troponin T (cTnT) level is a very sensitive marker of myocardial injury and it is elevated even in the absence of myocardial inflammation. We investigated the correlations for myocardial injury, virus titers and inflammation in acute viral infection. Using the murine coxsackievirus group B3 (CVB3) myocarditis model, the histopathologic findings and virus titers in mouse hearts were compared with the serum cTnT levels measured by ELISA at various time points. Viable virus titers in the hearts peaked at 3 days after infection (8.22 +/- 0.13 log10 PFU/100 mg of heart); they decreased at day 7 and no viable virus was detected from day 14. Myocardial inflammation was minimal at day 3, peaked at day 7 and markedly decreased at day 14. The individual serum TnT levels were significantly increased at day 3 (7.37 +/- 1.46 ng/ml), persisted to day 7 (0.73 +/- 0.08 ng/ml), and normalized at day 14. Serum cTnT levels were correlatable with virus titers in the heart (r = 0.744, P <0.01), but the serum cTnT levels were not correlated with the degrees of inflammation. Using the less myocarditic strain of CVB3, similar relationships were observed between the changes for the serum cTnT levels and the heart virus titers. During the course of viral infection, myocardial injury precedes the pathologic evidence of inflammation, and the elevated cTnT levels provide evidence of myocardial injury even in the absence of any histologic findings of myocarditis.     

Two-dimensional high-performance liquid chromatography at low ng/ml levels of the anti-proliferative agent B859-35 in serum with automated sample clean-up, solid-phase trapping and ultraviolet detection.

An automated non-chiral high-performance liquid chromatographic method is described for the determination of the new anti-proliferative agent B859-35 in serum. This method employs sample clean-up of 1 ml of biofluid by liquid-solid extraction with the AASP (Advanced Automatic Sample Preparation) system. First separation is achieved on a LiChrospher-60-RP-Select-B column. A fraction of this elute is then collected by solid-phase trapping. Thereafter, the final chromatogram is developed on a narrow-bore Hypers1-CPS column and quantified with ultraviolet detection at 230 nm. The limit of quantitation of the assay is 250 ng/ml. Linearity was proven in the range 0.25-100 ng/ml. Typical figures for precision at these concentrations are 7.4 and 3.3%, and for accuracy 8.0 and 1.3%, respectively. An application of this method to the study of pharmacokinetics of B859-35 in serum samples of cancer patients is given.     

Determination of sialic acids in human serum by reversed-phase liquid chromatography with fluorimetric detection.

A simple, rapid and highly sensitive reversed-phase liquid chromatographic method has been developed for the determination of sialic acids in human serum. The sialic acids, released by hydrolysis of serum, are converted in borate buffer with malononitrile to highly fluorescent compounds. The reaction mixture is separated isocratically within 5 min using an octadecyl-bonded silica column and a mobile phase of methanol and ammonium acetate buffer (15:85, v/v; pH 5.5). Measurement of the fluorescence intensity of the reaction mixture at 434 nm with irradiation at 357 nm allowed determination of 30-1000 ng/ml of sialic acids with high reproducibility. The limit of detection was 2 ng/ml. Intra-day and inter-day coefficients of variation for assaying 300 ng/ml N-acetylneuraminic acid (NANA) were 1.5% (n = 9) and 2.6% (n = 7), respectively. The recoveries of NANA were 98.5-101.1% for serum. The method has been used for clinical determinations.