Simple potentiometric method for determination of specific activity of carrier-free125I with the aid of ion-selective membrane electrode

A simple potentiometric method for determination of specific activity of carrier-free¹²⁵I (NaI) has been elaborated. The method is based on the measurement of tracer amounts of iodide present in the¹²⁵I preparate, by using an ion-selective membrane electrode. The method presented is suitable in practice for rapid estimation of specific activity of carrier-free¹²⁵I preparation in cases where the knowledge of its numerical value is necessary.     

Comparison study between direct and indirect labeling of estradiol for radioimmunoassay purpose

In the present study, estradiol (E₂) which contains an aromatic phenol ring in its chemical structure was chosen for direct and indirect labeling techniques using ¹²⁵I and chloramine-T oxidation method. In direct technique the hydrogen atom in phenol ring was replaced by a radioiodine atom directly, which in indirect one, histidine methyl ester (HME) was labeled firstly with ¹²⁵I using chloramine-T method then conjugated with E₂. The ¹²⁵I-labeled materials were purified using HPLC technique. The comparison study between the two obtained tracers was carried out in terms of radiochemical purity, binding percent, specific activity, non specific binding, binding displacement, shelf life using a radioimmunoassay (RIA) with specific antibody. The results indicated that indirect iodination of estradiol will cause significant increasing in studied parameters when combined with specific antibody than direct one.     

Radioimmunoassay of Human Follicle Stimulating Hormone /HFSH/

A method for determining Human FSH in blood/serum by RIA is described. The radioiodination of FSH with¹²⁵I is carried out under carefully controlled conditions such as, amount of initial activity of¹²⁵I take for iodination, the reaction volume, and the reaction time, etc. The tracer FSH obtained thus is with minimum damage and optimum specific activity which is ideally suited for RIA. The shelf-life of the tracer is enhanced by the addition of benzyl alchol. The tracer can be conveniently stored at+4–6°C upto 10 weeks, avoiding the repeated freezing and thawing process. Antiserum to FSH is raised in rabbits by repeated injections via intramuscular route. The method utilizes polyethylene glycol /PEG/ as the separation system. Using this method, a number of control samples of men and women of reproductive age group are screened. This sensitive assay has a good validity and has an inter-assay variation less than 15%.     

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.     

Determination of the specific activity of carrier-free125I preparations by neutron activation analysis

Chemical methods are unsuitable for the determination of the specific activity of commerical¹²⁵I preparations because of the unknown chemical state of the iodine in solutions more than a few weeks old.¹²⁵I and¹²⁷I were determined in samples from seven different manufacturers by instrumental neutron activation analysis; the specific activities found ranged from about 45% to more than 90% of that of the truly carrierfree product. Correlation of the specific activities with the¹²⁶I:¹²⁵I ratios indicates contamination of one of the products with stable iodine during the manufacturing process. Part of this paper was presented at the 1968 Internat. Conf. on Modern Trends in Activation Analysis.     

A novel method for the simultaneous determination of125I and14C activities using liquid scintillation counting

The viability of a dual label liquid scintillation technique has been investigated. To avoid the need for two procedures, gamma counting for¹²⁵Iodine (¹²⁵I) and liquid scintillation counting for¹⁴C. Since the¹²⁵I spectrum covers almost as wide a range of pulse heights as¹⁴C, conventional dual label methods would result in very low¹⁴C counting efficiencies. The conventional dual label technique has ben modified to increase the¹⁴C counting efficiency and to accomodate the consequent additional spillover of¹²⁵I counts into the upper window. This dual label technique has been applied to the determination of¹²⁵I and¹⁴C activities in blood samples. The accuracy of the method has been tested, and its advantages and limitations are discussed.     

Untersuchungen über die Verwendung von125J-markiertem Testosteron bei Proteinbindungsmethoden

The newly synthesized¹²⁵I-testosterone-3-carboxy-methyloxime-tyramide was tested for its applicability in protein binding methods. The binding of the radioactive steroid derivative to sex hormone binding globulin (SHBG), albumins and testosterone antibodies was examined. Furthermore the adsorption to florisil, charcoal and amberlite was studied. The results show:

1. ¹²⁵I-testosterone is bound to antibodies obtained by immunization with testosterone-3-oxime-albumin conjugate similarly to³H-testosterone and unlabelled steroid.
2. ¹²⁵I-testosterone and³H-testosterone show identical binding to florisil, charcoal and amberlite.

Therefore the¹²⁵I-labelled hormone is suitable in competitive protein binding methods using testosterone antibodies and it can displace the tritium labelled hormone. Since¹²⁵I-steroids have higher specific activities the sensitivity of the method is increased. Furthermore the practicability is enhanced. Special problems arise through the relatively short half-life of the isotope and the changing specific activity. Another difficulty is its decreased chemical stability when compared with the tritiated steroid.     

Radioimmunoassay of human placental lactogen

A sensitive and specific radioimmunossay procedure (RIA) has been developed for the measurement of Human Placental Lactogen (HPL). Pure HPL has been labelled with¹²⁵I and a specific activity of 100 μCi/μgm of HPL has been attained. Dextran-coated charcoal has been employed to separate the bound from the free hormone in radioimmuno-assay. The sensitivity of this technique has been found to be 0.2 ng of HPL. Intraassay and inter assay variations have been found to be less than 10%. This procedure has been adopted to establish the normal range of HPL in pregnant women at different periods of gestation, and to evaluate risk pregnancies.     

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.     

Determination of in-core power in low energy research reactors by measurement of16N and18F in the primary coolant

The measurement of¹⁶N and¹⁸F activity in the primary coolant of the JASON Argonaut reactor has been used to monitor in-core reactor power. The¹⁶N is produced by the¹⁶O(n, p)¹⁶N reaction and the 6.1 MeV photopeak was measured on-line using a BGO detector adjacent to the primary coolant circuit. These data provided a relative measure of power stability during steady state operation and a measure of linearity at different power levels. The¹⁸F is produced in the primary coolant by the¹⁸O(p, n)¹⁸F reaction and aliquots of primary coolant were sampled from the reactor dump tank for off-line radiochemical analysis. The¹⁸F was separated as trimethylfluorosilane and the activity was determined by measurement of the 0.511 MeV annihilation photopeak using a NaI(TI) detector. The measured¹⁸F activity was used to determine actual in-core reactor power using both ab-initio calculations and by comparison of results with a calibrated power reactor. The¹⁸F data also provided a method of nomalising the¹⁶N data for direct monitoring of in-core reactor power in JASON.     

Quality assurance of iodine-124 produced via the nuclear reaction124Te (d, 2n)124I

For more than a year,¹²⁴I (T=4.15 d) has been produced routinely with a compact cyclotron by irradiation of¹²⁴TeO₂ with 14 MeV deuterons, followed by dry distillation of the iodine radioisotopes formed from irradiated target materials. The following by-products have been measured and compiled in each charge: 13.2-d¹²³I, 60-d¹²⁵I, 13.0-d¹²⁶I, 12.4-h¹³⁰I and 8.02-d¹³¹I. The data show that after 45 h decay time, the sum of the activities of these nuclides is less than 5% of the¹²⁴I activity. Observation of this limit has been required by the Swiss Regulatory Agencies for a PET study of cell proliferation in human brain tumors using [¹²⁴I] IUdR.     

Preparation and Evaluation of 125I-Aflatoxin B1

Aflatoxin B₁ (AfB₁), present in fungus infested crops is highly carcinogenic and is measured by immunoassays. ¹²⁵I labeled aflatoxin B₁ is a key reagent for development of radioimmunoassay (RIA) which exhibits less interference and better sensitivity than other immunoassays. Since AfB₁ lacks suitable functional groups for radiolabeling, an oxime derivative of AfB₁ was synthesised and evaluated by UV-spectrophotometry and ¹H NMR spectroscopy. ¹²⁵I-histamine was conjugated to AfB₁ oxime by mixed anhydride method and purified by solvent extraction followed by TLC. The tracer obtained was immunoreactive, stable as ethanolic solution and could be used in RIA.     

A Rapid Method of Quantitating Steroids Resulting from the Incubation of Gonadal Tissues with Radioactive Precursors

Abstract

A rapid method has been developed for the quantitation of steroid metabolites resulting from the incubation of specific gonadal cell types or gonadal tissue with radioactive precursors. The method involves the use of high performance liquid chromatography (HPLC) for separating the steroids and a flow-through radioactive detector (Flo-One HP) for quantitating the radioactive ³H precursor and metabolites in the presence or absence of ¹⁴C-steroid recovery tracers. A comparison is made between the results obtained directly by the Flo-One HP radioactivity detector and the fraction collection method, (counting aliquots from individual fractions in the liquid scintillation counter). In addition, the results using an electronic stream splitter in the analysis of a percentage of the effluent directly by Flo-One HP are evaluated. The remaining percentage is collected in a fraction collector and is used for further analysis (e.g. recrystallization, RIA, further purification and characterization).     

A rapid radioimmunoassay procedure for thyroxin

A sensitive thyroxin /T₄/ radioimmunoassay procedure with a short incubation time /10 min/ similar to a conventional stat test in clinical chemistry is described. The assay parameters such as concentration of antisera and¹²⁵I-T₄, and incubation temperature that affect the kinetics of antigen-antibody reaction have been studied. Within- and between-assay variations were less than 7% CV. Analysis of 45 serum samples by the proposed method and by a conventional assay gave similar results /Y=1.05X–0.187; r=0.990/.     

Preparation of carbon-11 labelled acetate and palmitic acid for the study of myocardial metabolism by emission-computerised axial tomography

Methods have been developed for the labelling of acetate and palmitic acid with the positron-emitting radionuclide,¹¹C (T=20.4 min). Labelling was achieved via carbonation of the appropriate alkyl magnesium bromide (methyl magnesium bromide or n-pentadecyl magnesium bromide) with¹¹C-labelled carbon dioxide produced by the¹⁴N(p, α)¹¹C nuclear reaction. The radiochemical yield and speed of each method of labelling are such that a radiochemically pure product is obtained in injectable form and in activity (>10 mCi) suitable for the study of myocardial metabolism by emission-computerised axial tomography. High pressure liquid chromatography and thin layer chromatography were used to assess the radiochemical purity of each radiopharmaceutical. The specific activity of¹¹C-labelled acetate was estimated by an enzymic procedure to be greater than 0.5 Ci/μmole.     

Development of radioimmunoassay

Therapeutic monitoring of theophylline can be accurately performed by radioimmunoassay (RIA). It is radioactive tracer as an essential reagent for the development of very sensitive RIA. Direct radiolabeling of theophylline with¹²⁵I is very difficult due to the absence of appropriate functional groups. Hence carboxylic acid of theophylline was tagged to tyrosine methyl ester and then radiolabeled. The derivatives of theophylline, bearing a propionic acid and butyric acid side chains at seventh and eight position of theophylline, were synthesised and coupled to tyrosine methyl ester. Theophylline-tyrosine methyl ester conjugates were labeled with¹²⁵I using chlora mine—T. Radiolabeled theophylline was purified by solvent extraction followed by thin layer chromatography. The purified radiolabeled compound were assessed for their radiochemical purity, specific activity and immunoreactivity. Stability studies of radiolabeled compounds were performed with different solvents at different temperatures. Theophylline serum samples analysed using developed and commercial kits showed the correlation coefficient of 0.961 (n=9).     

Neutron activation analysis of zeolite catalysts

A new method for the determination of aluminum and silicon has been developed for zeolite catalysts. In contrast to previous methods, thermal neutrons are used for the analysis of both elements, and cadmium absorbers are not needed. The silicon determination utilizes a one-hour irradiation to observe the³¹Si produced by the (n, ) reaction of³⁰Si. A 15-second irradiation is used for the²⁷Al(n, )²⁸Al reaction. The²⁸Al activity is corrected for the contribution from the²⁸Si(n,p)²⁸Al reaction by using the analyzed weight of silicon in the sample and the data for a silicon standard irradiated simultaneously with the zeolite and the aluminum standard. The quantitation limits are 0.012 g for silicon and 3.3×10^–5 g for aluminum. Sodium presents a significant interference, but this element can be removed by taking advantage of the ion exchange properties of these materials.     

Limitations in detection of 15N incorporation by mass spectrometry in protein-based stable isotope probing (protein-SIP)

The method of protein-based stable isotope probing (protein-SIP) has previously been shown to allow the modeling of carbon fluxes in microbial communities, thus tackling one of the key questions in microbial ecology. The method allows the analysis of stable isotope distribution in peptides, revealing metabolic activities of the species present in an ecosystem. Besides carbon, an application of protein-SIP with nitrogen is of interest for resolving the nitrogen fluxes in microbial communities. Thus, the sensitivity and reliability of a protein-SIP approach employing ¹⁵N was analyzed. For this, cultivations of Pseudomonas fluorescens ATCC 17483 with different ratios of ¹⁴N/¹⁵N were performed, from 10 % down to 0.1 % ¹⁵N. After incubation leading to complete labeling of biomass, proteins were extracted and separated by one-dimensional gel electrophoresis, followed by tryptic digest and UPLC Orbitrap MS/MS analysis. ¹⁵N relative isotope abundance (RIA) was calculated based on isotopic patterns from identified peptides in mass spectra. Proteomics data have been deposited to ProteomeXchange with identifier PXD000127. The distribution of ¹⁵N RIA values among peptides was analyzed in samples with different ¹⁵N amount, and potential causes for variations within individual samples of either technical or biological origin were investigated. Using a number of 50 peptides, significant differences (p?≤?0.05) in ¹⁵N incorporation were found between samples of different ¹⁵N RIA down to 0.1 %. The study demonstrates that protein-SIP using ¹⁵N is sufficiently sensitive for quantitative investigation of microbial activity in nitrogen cycling processes.     

Human proinsulin. C-peptide radioimmunoassay method. 125I labeling of human proinsulin. C-petide

125I-labelled human-C-peptide was prepared by chloramin T method, enzymic method and active ester method, respectively. Using respective 125I-labelled human-C-peptides in human proinsulin-C-peptide RIA, we compared the binding (Bo/T%) to antibody, displacement by standard human-C-peptide, the recovery test and stability. The usable 125I-labelled antigen for human proinsulin-C-peptide RIA could be prepared by chloramin T method and enzymic method wich labelled 125I to tyrosyl human proinsulin connecting peptide, and active ester method which conjugates 125I-labelled active ester to human proinsulin connecting peptide. The differences among those 125I-labelled antigens was not observed in displacement (B/Bo%) by standard human-C-peptide and the recovery test. In the case of constant preparation of 125I-labelled antigen for RIA, the enzymic method was the best from the viewpoint the reaction ratio is stable and stability of Bo/T% is good.     

Effect of the specific activity of the tracer on rat luteinizing hormone radioimmunoassay

Rat luteinizing hormone /LH/ was labelled with¹²⁵I by the Chloramine T method.¹²⁵I-LH, used as tracer in radioimmunoassay, was separated from the labelling reaction mixture by gel filtration. By using the proper protein/radioiodine ratio in the labelling reaction mixture the specific activity of¹²⁵I-LH was adjusted to 2.5–20.5 MBq g^–1. The influence of the specific activity on the assay parameters as well as on the tracer stability was investigated.