Iodination of placental ferritin for RIA

For purposes of radioimmunoanalytical determination of serum ferritin, conditions for antigen iodination and separation were searched for, which could provide a satisfactory radiochemical purity and specific activity, high immunoreactivity and stability of the resulting labeled product, necessary for an acceptable expiration of the RIA kit. Two iodination methods (chloramine and conjugation methods) were tested, and a three-step procedure was elaborated for iodination and separation by gel column chromatography. The iodinated antigen obtained —¹²⁵I-placental ferritin with IR_max of about 80%,¹²⁵I^–<8%, specific activity of about 0.6MBq/g and stability for the expiration period of 3 to 4 months — is quite satisfactory for the RIA applications.     

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.     

A secondary antibody format chemiluminescence immunoassay for the determination of estradiol in human serum

A competitive immunoassay for estradiol (E2) based on secondary antibody format was established. The donkey anti-rabbit IgG was used as the secondary antibody to coat micro-plates, and the horseradish peroxidase (HRP)-luminol-H₂O₂ chemiluminescent system with high sensitivity was chosen as the detection system. The addition of sodium trichloroacetate (CCl₃COONa) in the enzyme buffer as a replaceable packing material can realize directly analysis of E2 in human serum without extraction, which improved reproducibility and resolution of the assay. Additionally, the method showed specific recognition of estrogen, without cross-reaction for the major steroids (estrone (E1), estriol (E3), dihydrotestosterone (DHT), androstenedione, testosterone (T)) commonly found in human serum. The chemiluminescence immunoassay with secondary antibody can be applied to detect E2 with good precision at concentrations as low as 1.48 pg mL⁻¹. The proposed method has been successfully applied to the determination of E2 in 97 human sera and showed a good correlation compared with the commercially radioimmunoassay (RIA) kit with a correlative coefficient of 0.9881. This method has exhibited great potential in the fabrication of diagnostic kit and can be used in the clinical analysis of E2 in human serum.     

Radioimmunoassay kit formulation and its validation for serum progesterone using progesterone radiotracer purified by gel filtration

Purification of the radioiodinated progesterone tyrosine methyl ester conjugate by gel filtration and the development, optimization and clinical validation of a direct radioimmunoassay (RIA) of progesterone using this radiotracer are described. High purity radiotracer is essential for the error free performance of any RIA. Progesterone 11 hemisuccinate was conjugated to tyrosine methyl ester (TME) by the mixed anhydride method and this conjugate was then radioiodinated by the chloramine-T method. Purification of the radioiodinated product was carried out by gel filtration. About 12 batches of the radiotracer were prepared and purified. The purification by gel filtration gave reproducible elution pattern and purity. The radiotracer thus purified was found to have consistent quality as compared to that of any other purification methods. Non-specific binding of the radiotracer was found to be <5% in second antibody-polyethylene glycol based separation. 'Zero standard' binding was found to be consistently 50-60% in all the batches of radiotracer. The radiochemical purity of all the batches of radiotracer was >95% as checked by paper electrophoresis. The stability (retention of the immunoreactivity) of the radiotracer was two to three months. No appreciable changes in the assay characteristics were observed during this period. The assay involved 3 hours incubation of progesterone antibody with individual standards or sample and radiotracer at room temperature. The optimized assay was then validated for internal and external quality control parameters. A RIA kit was then formulated with this radiotracer for estimation of progesterone in serum. The assay kit consisted of lyophilized individual standards ranging from 0.25 to 50 ng/ml. The clinical performance of the developed kit was compared with that of a commercial ELISA kit and a correlation of 0.94 was observed.     

Preparation of Tyr-C-peptide from genetically altered human insulin precursor

C-peptide radioimmunoassay (C-peptide RIA) is widely used in determination of pancreatic B-cell secretion activity.¹²⁵I labeled TyrC-peptide is indispensable in C-peptide RIA kit. Herein we discuss a way of obtaining recombinant Tyr-C-peptide. Arg32Tyr human proinsulin mutant (R32Y-proinsulin) gene was constructed by site-directed mutagenesis and overexpressed inEscherichia coli. Purified R32Y-proinsulin was converted to insulin and Tyr-C-peptide by trypsin and carboxypeptidase B codigestion. Tyr-C-peptide was isolated through reverse-phase HPLC (RP-HPLC) and identified by C-peptide RIA and amino acid analysis.     

Studies on aldosterone RIA kit using the two antibody method

Two antibody method of aldosterone RIA kit was investigated. This kit was measured directly without extracting the aldosterone. On fundamental studies, stability of standard curve, reproductivity, recovery test and dilution test were obtained good results. On clinical studies, the diurnal variation of serum aldosterone concentration in normal child and adult was correlated well to that of concentration of cortisol and ACTH. The concentration of three hormones was the highest in the early morning and it was the lowest in midnight.     

Ultrasensitive and Simultaneous Determination of Arsenic and Antimony in Clinical Samples by Atomic Fluorescence Spectrometry

The determination of trace elements in human fluids facilitates the identification of metabolic disorders and physiological abnormities in clinical diagnosis. Elemental concentrations in serum, urine, and saliva are often low and highly prone to contamination. Consequently, a simple and ultrasensitive detection technique is required. In this study, a method was developed for the simultaneous determination of trace arsenic and antimony in clinical samples by hydride generation atomic fluorescence spectrometry. The experimental parameters that affect the generation, delivery, and atomization of volatile arsenic and antimony hydrides were investigated. The limits of quantification were calculated to be 0.0037 nanogram per milliliter and 0.0070 nanogram per milliliter for arsenic and antimony, respectively. The method was successfully applied for the analysis of fortified human serum, urine, saliva, and certified reference materials. The results obtained by the reported method were not statistically different from the certified values at the 95 percent confidence level.     

Fundamental studies, reference values and relationship to menstrual cycle of prolactin RIA BEAD II

We have tried fundamental studies, reference values and relationship to menstrual cycle on Prolactin RIA BEAD II kit which has a method of IRMA using monoclonal antibody. On clinical studies, we investigated change of serum prolactin level during the menstrual cycle and relationship to other hormones (LH, FSH, estradiol, progesterone). It was the result that prolactin level of follicular phase was lower than that of preovulatory phase and luteal phase. We conclude that change of prolactin level during the menstrual cycle is related with change of estradiol level.     

A fundamental and clinical study of ferritin 'Eiken' radioimmunoassay kit

We have measured serum ferritin level using double antibody radioimmunoassay kit (Eiken ICL) and evaluated the characteristics of the kit and clinical usefulness. Satisfactory results were observed in standard curve, reproducibility, dilution and recovery test. In clinical evaluation, we have measured in normal subjects and patients with various diseases. The range in normal males and females were 13.0-158.7 ng/ml and 7.3-73.0 ng/ml, respectively. Serum ferritin level was elevated in patients with hepatoma, biliary cancer, lung cancer and other malignant diseases. Measurement of serum ferritin value would be useful in the monitoring of cancer patients.     

A comprehensive evaluation of the kinetic method applied in the determination of the proton affinity of the nucleic acid molecules

The determination of proton affinity (PA) of 2'-deoxyadenosine (dA) is used as a case study for the evaluation of possible drawbacks in the determination of the PA of the nucleic acid molecules by the kinetic method. The observed Delta PA among the different values obtain for dA by applying this procedure in its different extensions was 0.64 Kcal/mol, which is within the uncertainties of any theoretical or experimental approach. It was demonstrated that nucleosides can be generally used as reference compounds to measure the PA of an unknown nucleoside. The evaluation of Delta Delta S value for two competing reaction channels taken by proton-bound heterodimers formed by two nucleosides provides clear information on the reference base which has to be discarded from the set of reference compounds used for the estimation of an unknown PA. The PA of dA obtained with the most elaborated kinetic method (237.00 +/- 0.07 kcal/mol) is consistent with the value of 237.0 kcal/mol obtained by a simple treatment of the relative intensities of the product ions generated by two couples of the proton bound dimers formed by the nucleoside and two reference amines. The kinetic method can be, therefore, confidently used to assess the proton affinity of the multifunctional molecules such as nucleosides and nucleobases.     

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.     

Determination of estradiol in human serum using magnetic particles-based chemiluminescence immunoassay

A magnetic particles (MPs)-based chemiluminescence immunoassay (CLIA) with high sensitivity, specificity, and reproducibility was proposed for the evaluation of estradiol (E₂) in human sera. The MPs coated with secondary antibody were used as dispersed solid phase for the immunoassay, and the horseradish peroxidase (HRP)-luminol-H₂O₂ chemiluminescent system with high sensitivity was chosen as the detection system. The method showed specific recognition to E₂, without cross-reaction for the major steroids, including estrone (E₁), estriol (E₃), dihydrotestosterone (DHT), androstenedione, and testosterone (T), which was commonly found in human serum. The addition of sodium trichloracetate (Na-TCA) in the enzyme buffer as a blocking agent contributed to the realization of direct analysis of E₂ in human serum without extraction. Besides, the effects of several physicochemical parameters, including the dilution ratios of E₂-6-HRP conjugate and anti-E₂ polyclonal antibody, immunoreaction time, chemiluminescent (CL) substrate volume, volume of MPs, and CL reaction time, were studied and optimized. The proposed method had a detection limit of 2.51 pg mL⁻¹ with a larger working range of 15-1000 pg mL⁻¹. The inter-assay and intra-assay coefficient of variation (CV) were both less than 15%. The average recoveries of three different spiked concentration samples were 93.3, 106 and 101%, respectively. The method has been successfully applied to the determination of E₂ in 105 human sera and showed a good correlation compared with the commercial radioimmunoassay (RIA) kit with a correlative coefficient of 0.9892. This method has exhibited great potential in the fabrication of diagnostic kit and could be used in the clinical analysis of E₂ in human serum.     

Comparison Of Highly Sensitive Sandwich Enzyme Immunoassay And Radioimmunoassay For Human Ferritin

Abstract

A highly sensitive sandwich enzyme immunoassay (EIA) for human ferritin was developed using rabbit anti-ferritin IgG-coated polystyrene balls and affinity-purified rabbit anti-ferritin Fab' labelled with β-D-galactosidase from Escherichia coli and compared with the corresponding sandwich radioimmuno assay (RIA). The specific and nonspecific binding of labelled anti-ferritin to the polystyrene balls were examined in relation to the amount of labelled anti-ferritin used per tube, and the highest sensitivity of each immunoassay (0.2 amol/tube in EIA and 2.5 amol/tube in RIA) was obtained by using the minimal amount of the corresponding labelled anti-ferritin (0.71 fmol in EIA and 4.5 fmol=4436 cpm in RIA) which gave a reliable calibration curve. The sandwich RIA was less sensitive, largely because the specific radioactivity of ¹²⁵I-labelled anti-ferritin used was not sufficiently high.     

Electrochemical detection of human ferritin based on gold nanorod reporter probe and cotton thread immunoassay device

In this study, a natural cotton thread immunoassay device combined with gold nanorod (GNR) reporter probe is developed for the rapid, sensitive and quantitative electrochemical determination of human ferritin, a lung cancer related biomarker. Human ferritin as an analyte and a pair of monoclonal antibodies are used to demonstrate the proof-of-concept on the cotton thread immunoassay device. An enhancement of the sensitivity is achieved by using gold nanorod as an electroactive report probe compared with a traditional gold nanoparticle (GNP) report probe. The device was capable of measuring 1.58 ng/mL ferritin in 30 min by anodic stripping voltammetry (ASV) testing, which meet the requirement for clinical diagnosis.     

Evaluation and comparison of high-sensitivity immunoradiometric assay kits for thyroid stimulating hormone

Fundamental and clinical characteristics of 3 kinds of high-sensitivity immunoradiometric assay (IRMA) kits for thyroid stimulating hormone (TSH). i.e., RIA BEADS II (kit A), TSH kit Daiichi II (kit B) and Ab tube TSH 'Eiken' (kit C) and one conventional radioimmunoassay (RIA) kit, i.e., TSH kit Daiichi (kit D), were studied. In the recovery test and the reproducibility test, there was no significant difference between the 4 kits. The sensitivities of kits A, B and C were much higher than that of kit D, and those IRMA kits were sensitive enough to distinguish hyperthyroidism from normal samples. For low concentrations of TSH (less than 5 microU/ml), the data from kits D, B, C and A tended to show higher values in that order. The correlation between the data measured by kits B and D, and the tendency of kit A toward lower values agreed well with other reports.     

Optimization of the gravimetric determination method of nickel as dimethylglyoximate for nickel raw materials

The method parameters of an almost one century old method for analyzing nickel as dimethylglyoximate were critically examined for the analysis of nickel raw materials and many of the method parameters were observed to have a significant effect on the Ni recovery. Thus, because the Ni precipitation method parameters vary a lot in analytical literature and also in practice, the obtained Ni results by different methods are not comparable. During this study it was found that the double precipitation worked out perfectly in eliminating the effects of impurity elements. The residual Ni content in the filtrates should also be measured to obtain accurate and precise Ni results. In complexing the impurity elements, tartaric acid, stabilized by acetic acid, turned out to be effective, and when added to the sample solution before ammonium addition, the best pH conditions for homogenous Ni precipitation with dimethylglyoxime were obtained. The optimized Ni determination method described was found to be accurate and highly reproducible when tested with Ni concentrates and standard reference materials containing high Ni concentrations.     

Gluten determination by gliadin enzyme-linked immunosorbent assay kit: interlaboratory study

An interlaboratory study with 10 participants was performed to obtain validation and performance data for an enzyme-linked immunosorbent assay (ELISA) kit developed for quantitative gluten determination in foods. The ELISA kit used for this study is based on 2 monoclonal and 1 polyclonal antibody developed by Immunotech, a Beckman Coulter Co. This kit did not show any false positive results or cross-reactivity with oat, rice, maize, and buckwheat. The gliadin standard from the Working Group on Prolamin Analysis and Toxicity was included in the kit as reference material for calibration. All participants obtained a gliadin ELISA kit with Standard Operational Procedure and a form for recording test results. The study included 13 samples labeled as "gluten-free" and 2 samples spiked by wheat flour. Seven samples had gliadin content below the limit of quantitation (LOQ) of the method, and 1 sample exceeded the highest calibration level. Gliadin content in the range from 10 to 157 mg/kg (1st day) and from 11 to 183 mg/kg (2nd day) was found in 7 samples (including 2 spiked samples). Results of these samples were used for further statistical analysis and evaluation. The Cochran, Dixon, and Mandel statistical tests were applied for detection of outliers. The LOQ of the kit was estimated.     

Human proinsulin-C-peptide radioimmunoassay method; stability of the assay kit

We have been studies time sequent stability each on standard human-C-peptide, human-C-peptide antiserum, 125I-tyrosyl human-C-peptide and the assay kit (all reagent) which is necessary in human proinsulin-C-peptide radioimmunoassay(RIA). Also we measured using this assay system, human proinsulin C-peptide in blood after oral administration of glucose to normal subject. Standard human-C-peptide and human-C-peptide antiserum were very stable on storage at 4 degrees C, 125I-tyrosyl human-C-peptide was unstable as compared with the former two, The stability of the assay kit was influenced by the stability of 125I-tyrosyl human-C-peptide, and was stable at 4 degrees C for ten weeks after preparation. Three lots of the assay kit prepared at different period showed almost same stability. We think this assay system using the assay kit is satisfactory in respect of stability. The measured values of human proinsulin-C-peptide in blood, using this assay system, showed insulin secretory reaction after oral administration of glucose.     

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。     

Determination of trace elements in human serum by inductively coupled plasma atomic emission spectrometry with flow injection

An analytical method for the simultaneous determination of some trace elements (Au, Fe, Mg, Li, Sr, Zn) in human serum by inductively coupled plasma atomic emission spectrometry (ICP-AES) with flow injection is described. Physical interference caused by the change of sample viscosity is discussed. When 100 μl of serum was injected, the relevant recoveries of > 99% for Li, > 98% for Cu and Mg, > 95% for Fe were obtained for an NIST SRM with R.S.D. > 1.3% using optimized flow injection parameters. The prepared lyophilized control serum for routine analysis in clinical laboratories was analyzed and verified for the validity of the technique employed in this experiment using NIST SRM 909 as a primary reference material.