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.     

A simple method for the determination of the specific activity of125I-tracer used in radioimmunoassay

Knowledge of the specific activity of the¹²⁵I-tracer is essential for optimization and for calculation of RIA parameters. The specific activity of the¹²⁵I-thyroxin used in thyroxin radioimmunoassay /RIA/ has been determined by a simple method involving combination of RIA and displacement analysis. It has been compared with the value obtained by the conventional method based on radioiodination data. Our studies indicate that even for a non-protein hormone like thyroxin the specific activity of¹²⁵I-thyroxin derived from iodination data is not reliable. The specific activities obtained by displacement analysis were consistent with the experimental findings.     

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.     

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.     

Further study on NAA in characterizations of reference materials

A program was initiated at Chalk River Laboratories (CRL) to determine the physical, chemical and radiological properties of wastes intended for disposal in IRUS (Intrusion Resistant Underground Structure), a below ground vault to be constructed at CRL. One of the most restrictive radionuclides for IRUS is¹²⁹I, which has been assigned a maximum activity concentration in waste of 10⁶ Bq/m³. The limit of detection for radionuclides in waste has been set at 1% of the approximate maximum activity concentration, or 10⁴ Bq/m³ for¹²⁹I. A radiochemical instrumental neutron activation analysis method has been developed to determine¹²⁹I in two waste streams, incinerator ash and liquid feed to a bituminizer. Solid samples are spiked with¹²⁵I tracer, fused at 960°C with Li₂B₄O₇ in a platinum boat in a flowing oxygen stream inside a three zone tube furnace, and the volatilized I₂ is trapped on in-line charcoal filters. The charcoal filters are irradiated together with a filter containing a spiked¹²⁵I/¹²⁹I standard, in the NRU reactor, and then subjected to post-irradiation chemistry to remove⁸²Br interference. The¹²⁹I concentration in the sample is determined by comparing the activity of the activated¹³⁰I in the sample with that of the standard, and the chemical recovery for¹²⁹I is determined from the activity of¹²⁵I tracer. Limits of detection for¹²⁹I in solids are typically 0.005 Bq/g, based on a 4 hour counting period on a 10% efficient HPGe gamma-spectrometer at a source to detector distance of approximately 12 cm. This paper presents a summary of the method and the results from analysis of two waste streams.     

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).     

Radioiodination of recombinant human growth hormone and its use in radioimmunoassay

Radioimmunoassay (RIA) of human growth hormone (hGH) using¹²⁵I-labeled tracer prepared from DNA recombinant hGH (r-hGH) and characterization of the tracer in the assay system are described. The radioiodination of r-hGH resulted in high yield of immunoreactive tracer. The immunoreactive fraction could be purified by gel-filtration on sephadex G-75. The quality of radioiodinated tracer of r-hGH has been found to be same as that of the tracer obtained from pituitary hGH (p-hGH) with respect to immunoreactivity, assay sensitivity and RIA standard curve parameters.     

New types of125I-radioligands for RIA of steroid compounds

We prepared new¹²⁵I-radioligands of the arylhydrazone type for the RIA of steroid compounds and studied the effect of the chain length between¹²⁵I and the antigen on the antibodyantigen reaction.     

Adsorption of 125I on palladium coated silver wire

The adsorption of ¹²⁵I on palladium coated silver wires was studied in this paper. The experimental conditions, e.g., reaction volume, carrier concentration, reaction time, reaction temperature, pH of the reaction mixture, were systematically optimized to obtain quantitative adsorption of ¹²⁵I on palladium coated silver wires. The experiments were performed using potassium iodide 8–9 μg as carrier in a reaction volume of 100 μL incubated in ~50 °C water bath for ~1 h, and the pH of the reaction system was controlled at 2–2.5. The distribution of activity on palladium coated silver wire was uniform, and the source cores can be easily sealed by laser welding into titanium capsules.     

Preparation and isolation of a homogeneous125I-cortisol derivative

Tracer specificity plays an important role in the radioimmunoassay (RIA) of steroid homones. In this paper, we describe the preparation and purification of¹²⁵I-labeled cortisol derivative with a carboxymethyloxime-histamine bridge. The investigation on the method of purification showed that HPLC could be adopted for the routine preparation of a pure, homogeneous tracer. The retention time observed in HPLC for¹²⁵I-histamine-CMO-cortisol conjugate could be used as an index for qualitative and quantitative assessments.     

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.     

Thrittene radioimmunoassay: description and application of a novel method

In the present paper the development and application of a novel thrittene radioimmunoassay (RIA) are described. ¹²⁵I-labeling of Tyr⁽⁰⁾-thrittene was performed by the iodogen-method and the mono-iodinated peptide, as RIA tracer, was separated by reversed-phase high performance liquid chromatography (HPLC). The RIA results show that the antiserum used in the radioimmunoassay turned to be C-terminal specific, without significant affinity to other members of the somatostatin peptide hormone family. Detection limit of the assay was 0.2 fmol/ml. This highly specific and sensitive thrittene RIA was used to investigate the distribution of thrittene in the rat gastrointestinal tract and other tissue samples. Different areas of the gastrointestinal tract and other tissues were removed from rats and after extraction the samples were processed for thrittene radioimmunoassay. Highest concentrations were found in the duodenum samples followed by jejunum and ileum, however, all the examined tissues contained highly enough thrittene for the measurement.     

Radioimmunoassay of phenytoin

We describe a radioimmunoassay procedure for the measurement of phenytoin (5,5-diphenylhydantoin) in serum samples. Antiserum to phenytoin has been produced against phenytoin-valeric acid-bovine serum albumin conjugate.¹²⁵I labelled phenytoin-acetic acid-tyrosine methyl ester has been used as a tracer. The assay covers a range of 10–500 ng/cm³ and has a sensitivity of 0.25 ng. The assay is validated by specificity tests, precision profile and recovery tests.     

Preparation of 125I-cytarabine and its radiochemical evaluation: Model of radio-therapeutic agent

This paper adresses the development of a new radiopharmaceutical for cancer imaging and therapy. The optimization of the labeling of thymidine analogous, cytarabine, with ¹²⁵I is described. High radiochemical yield and purity 98% was obtained by reacting 50 mg cytarabine with ¹²⁵I in the presence of iodogen as oxidizing agent and 0.5M phosphate buffer of pH 7 at 65 °C for 30 minutes. Preliminary in-vivo study was done in non-tumor bearing mice. The results revealed that this new tracer, ¹²⁵I-cytarabine, has a high affinity to be localized in tissues of high proliferation rate, e.g., bone marrow 10%, 60-minute post administration. Also, the labeled compound was cleared quickly from most of the body organs and concentrated in bladder 55%, 60-minute post administration. These findings suggest that ¹²⁵I-cytarabine, allows imaging and treatment of cancer. ¹²⁵I-cytarabine meets most of the requirements to be used as a successful diagnostic and therapeutic agent: it is a low molecular weight molecule that diffuses readily in tissues, it will not induce an antibody response, thereby leading itself to repeated injection or continuous infusion.     

Direct radioimmunoassay of serum progesterone using heterologous bridge tracer and antibody

The standardisation of a direct radioimmunoassay for progesterone using an¹²⁵I labeled progesterone prepared by iodinating the tyrosine methyl ester (TME) conjugated to a progesterone hemiphthalate derivative and an antibody prepared using progesterone linked to bovine serum albumin through 11α hemisuccinate derivative is described. The hemiphthalate derivative of progesterone was prepared by reacting 11α-hydroxy progesterone with phthalic anhydride which was then conjugated to TME by using isobutyl chloroformate. The conjugate was iodinated with¹²⁵I using chloramine-T as oxidising agent and purified by thin layer chromatography. Radiochemical purity of the tracer was >95% in all batches. The tracer gave 70–75% binding with excess antibody. Assays were optimised with 8-anilino-1-naphthalene sulphonic acid (ANS) and sodium salicylate as blocking agents to release the progesterone from binding proteins. The assay optimised with sodium salicylate as blocking agent has a sensitivity of 0.25 ng/ml and a working range of 0.25–50 ng/ml, whereas the assay with ANS has a sensitivity of 0.75 ng/ml and a working range of 0.75–100 ng/ml. Serum samples were analysed and compared with the values obtained with a homologous bridge assay.     

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.     

Radioiodinated paroxetine, a novel potential radiopharmaceutical for lung perfusion scan

Paroxetine (a selective serotonin reuptake inhibitor) was successfully labeled with ¹²⁵I via direct electrophilic substitution reaction at ambient temperature. The reaction parameters studied were paroxetine amount, CAT amount, pH of the reaction mixture, reaction temperature, reaction time and in vitro stability of ¹²⁵I-paroxetine. ¹²⁵I-paroxetine was obtained with a maximum labeling yield of 94 ± 0.23% and in vitro stability up to 24 h. Biodistribution studies showed that maximum in vivo uptake of ¹²⁵I-paroxetine in lungs was 27.89 ± 1.03% injected activity/g tissue at 15 min post-injection and retention in lungs remained high up to 1 h, whereas the clearance from mice appeared to proceed mainly via the hepatobiliary pathway. ¹²⁵I-paroxetine is not a blood product and so it is more safe than the currently available ^99mTc-macroaggregated albumin (^99mTc-MAA), and its lung uptake is higher than that of the recently discovered ^99mTc(CO)₅I and ^99mTc-DHPM. As a conclusion, radioiodinated paroxetine could be used as a novel radiopharmaceutical for lung perfusion scan safer than the currently available ^99mTc-MAA and more potential than the recently discovered ^99mTc(CO)₅I and ^99mTc-DHPM.     

Development of a process for routine production of123I using the127I(p, 5n)123Xe→123I reaction

The object of this paper is to give details of a production method for¹²³I, now in routine use at Harwell. We employ the (p, 5n) reaction, irradiating a liquid target of di-iodomethane (CH₂I₂) spiked with additional iodine, with 58 MeV protons. A yield of ∼9 mCi/μAh is obtained; the only detectable radionuclidic impurity is¹²⁵I, present to the extent of ∼0.15% by activity at the time of separation of Xe from I.     

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.     

Chemical and nuclear interferences in neutron activation of129I and127I in environmental samples

A combination of neutron activation and gamma-ray coincidence counting technique is used to determine the concentration of both long-lived fission produced¹²⁹I and natural¹²⁷I in environmental samples. The neutron reactions used for the activation of the iodine isotopes are¹²⁹I(n, )¹³⁰I and¹²⁷I(n, 2n)¹²⁶I. Nuclear interferences in the activation analysis of¹²⁹I and¹²⁷I can be caused by production of¹³⁰I or¹²⁶I from other constituents of the materials to be irradiated, i.e. Te, Cs and U impurities and from the¹²⁵I tracer used for chemical yield determination. Chemical interferences can be caused by¹²⁹I and¹²⁷I impurities in the reagents used in the pre-irradiation separation of iodine. The activated charcoals used as iodine absorbers were carefully cleaned. Different chemical forms of added¹²⁵I tracer and¹²⁹I and¹²⁷I constituents of the samples can cause different behaviour of¹²⁵I tracer and sample iodine isotopes during pre-irradiation separation of iodine. The magnitude of the nuclear and chemical interferences has been determined. Procedures have been developed to prevent or control possible interferences in low-level¹²⁹I and¹²⁷I activation analysis. For quality control a number of biological and environmental standard samples were analyzed for¹²⁷I and¹²⁹I concentrations.