Preparation and characterization of125I labeled progesterone derivatives for the development of a radioimmunoassay for progesterone

Preparation and purification of radioiodinated progesterone derivatives for the development of a radioimmunoassay of progesterone is described. We have standardized two procedures for preparing radioiodinated progesterone conjugate. In the first procedure¹²⁵I labeled histamine was conjugated to progesterone 11 hemisuccinate by the mixed anhydride method. In the secod procedure, tyrosyl methyl ester was conjugated to progesterone 11 hemisuccinate and iodination of the conjugate was carried out. Purification of the iodinated products was carried out by solvent extraction and thin layer chromatography techniques. The radiochemical purity of the tracers prepared by both methods were more than 95%. Labeled progesterone derivatives prepared were used for developing a radioimmunoassay procedure. The non-specific binding of the tracer was about 2–3%, while up to 80% binding could be obtained in the presence of excess antibody. The radioiodinated tracer could be used up to four months in the assay.     

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.     

Development of a direct radioimmunoassay for serum progesterone

A direct radioimmunoassay for the measurement of progesterone in human serum is described. Progesterone 11-hemisuccinate was conjugated to tyrosine methyl ester (TME) by the mix anhydride method and then iodinated using chloramine-T. The radiochemical purity of different batches of¹²⁵I-progesterone was greater than 95% and showed 70–75% binding with excess antibody. Progesterone 11-hemisuccinate was coupled with BSA and injected to rabits. Antisera collected after three booster injections and having aK value of 1·10⁹1/M was selected for the assay. Significant reduction in binding with antibody was seen when hormone free serum was used in the assay system. Various blocking agents were tried to reduce the serum effects and none of them were found satisfactory. From a series of optimization experiments, an assay was developed without the use of blocking agents. This assay used a much higher concentration of antibody along with lower amount of serum sample (50 l). The optimized assay has a sensitivity of 0.5 nj/ml and a working range of 0.5 to 100 ng/ml. Serum samples were analyzed analysis showed good correlation between the results obtained from the present system and the DPC kit. (Y=0.93X+0.5,r=0.93, forn=25).     

Preparation of radiolabeled clonidine for use in radioimmunoassay

The preparation of stable and immunoreactive radioiodinated clonidine for the development of a radioimmunoassay of clonidine is described. Tyrosylmethyl ester (TME) conjugate of carboxylic acid derivative of clonidine was radioiodinated using chloramine T as the oxidizing agent. The radioiodinated clonidine was purified by thin layer chromatography, gel filtration and solvent extraction. Radiochemical purity, specific activity and immunoreactivity were determined. The stability of the labeled compound during storage was tested in various solvents at different temperatures.     

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.     

Preparation of Monomeric Affinity-Purified Fab'-ß-D-Galactosidase Conjugate for Immunoenzymometric Assay

Abstract

A Simpler method for the preparation of monomeric affinity-purified Fab'-ß-D-galactosidase conjugate is described. Rabbit (anti-human IgG) serum was subjected to successive processes of pepsin digestion to convert IgG to F(ab')_2′ reduction with 2-mercaptoethy on a column of human IgG-Sepharose 4B. The affinity-purified Fab' thus obtained without using gel filtration was reacted with excess of maleimide groups introduced into ß-D-galactosidase from Escherichia coli. The monomeric Fab'-ß-D-galactosidase conjugate formed was separated from unconjugated Fab' by gel filtration and from unconjugated ß-D-galactosidase by affinity chromatography on a column of goat (anti-rabbit IgG) IgG-Sepharose 4B. By immunoenzymometric assay technique for human IgG, the monomeric conjugate was compared with a monomeric conjegate prepared by a previously reported complexmethod and non-monomeric conjugate which contained 3.7 Fab' molecules per ß-D-galactosidase molecule. The present monomeric conjugate provided as sensitive a dose-response curve as the previously reported monomeric conjugate and a more sensitive dose-response curve than the non-monomeric conjugate.     

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).     

TIRF-based biosensor for sensitive detection of progesterone in milk based on ultra-sensitive progesterone detection in water

We report on recent advances of our immunoassay for the hormone progesterone in cows milk. Detection is based on total internal reflectance fluorescence (TIRF), the binding-inhibition assay with an immobilized progesterone derivative, and a commercially available monoclonal antibody to progesterone as biological recognition element. The fully automated River Analyzer (RIANA) biosensor for unattended, cost-effective, and continuous monitoring of environmental pollution therefore was adapted for sensitive determination of progesterone in milk. First, the sensitivity and robustness of the existing progesterone assay for water analysis were improved, resulting in a detection limit (LOD) of only 0.2 pg mL^–1 and a quantification limit (LOQ) of only 2.0 pg mL^–1. These extraordinary results are the lowest detection and quantification limits for progesterone determination using biosensors yet reported in the literature. Second, the accurate indicator of ovulation was calibrated and detected in three different types of milk (UHT milk, fresh milk, and raw milk). For commercial milk and randomly procured raw milk nominal levels of progesterone are typically in the range 5–15 ng mL^–1. Limits of detection (LOD) achieved for added progesterone (i.e. spiked samples) were between 45.5 and 56.1 pg mL^–1 depending on milk type. Having in mind the 1:10 dilution factor, these results are still a success. For the first time a commercially available antibody was incorporated into an immunoassay for progesterone detection in bovine milk, giving a detection limit below 1 ng mL^–1 for a fully automated biosensor. Thus the outstanding progress made with this biosensor in environmental monitoring and water analysis has now been successfully adapted to milk analysis for use in the field of reproduction management.Dedicated to the memory of Wilhelm Fresenius.     

Preparation of Sn-β-alanine: A new freeze-dried kit to be labelled with99mTc for liver scintigraphy

Sn-alanine kits were prepared in lyophilized form containing 7.02·10^–2 M -alanine and 5.5·10^–4M stannous chloride dihydrate. The optimal pH value of the preparation was found to be equal to 4.3–5.1. The radiochemical purity and the stability of^99mTc-alanine were assessed by gel filtration column scanning techniques (GCS) and thin layer chromatography, and the labelling yield of the complex was higher than 95%. The organ distribution data in mice showed that more than 90% of the injected dose had been accumulated in the liver. However, a negligible amount of radioactivity was detected in the non-target organs. The stability of^99mTc-alanine was followed for 5 hours and the Sn-alanine kit was stable for at least 3 months.     

An Improved Method for the Conjugation of FAB' to B-D-Galactosidase from Escherichia coli

Abstract

An improved method for the conjugation of Fab' to B-D-galactosidase from Escherichia coli was developed. The enzyme with thiol groups was treated with excess of N, N'-o-phenylene-dimaleimide to introduce maleimide groups, and the maleimide-enzyme was reacted with thiol groups in the hinge of Fab' to form Fab'-B-D-galactosidase conjugate. Gel filtration of Fab' was required only once, and the recovery of Fab' in the conjugate was 71–81%, while in the previous method, gel filtration was required twice and the recovery was 31–38 %. Although the enzyme activity was decreased slightly (8–12%) by the dimaleimide treatment, the conjugate was as useful for sandwich enzyme immunoassay as that obtained by the previous method. 1gG also could be conjugated to B-D-galactosidase more efficiently in the present method than in the previous one.     

Time-resolved fluoroimmunoassay of progesterone by Eu-labelled protein-A

Summary A standard curve for progesterone using a solid phase time-resolved fluoroimmunoassay was obtained. The 11-hydroxyprogesterone hemisuccinate bonded to ovalbumin was adsorbed to the wells of polystyrene microtiter strips. After incubation of scalar quantities of free progesterone (from 12 to 5000 pg) with specific antibody and an excess of Eu-labelled protein-A, the wells were washed and the europium bound to the solid phase was measured as a 2-naphthoyltrifluoroacetone chelate using a time resolved fluorometer.

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Zeitaufgelöster Fluorescenzimmunoassay von Progesteron mit Hilfe von Eu-markiertem Protein-A

     

Analytical method development for a specific assay method for monomethoxypoly(ethylene glycol)succinimido carbonate (mPEG-SC): A comparison of spectrophotometric, titrimetric and chromatographic methods

Three different quantitative methods, which rely on base-mediated reactions for the purity assessment of the hydrolytically unstable activated ester, mPEG-SC (1) are discussed. In a spectrophotometric assay, controlled base-catalyzed hydrolysis of mPEG-SC (1) affords N-hydroxysuccinimide and its concentration can be determined by UV analysis. Reaction of mPEG-SC (1) with benzylamine and a non-aqueous back titration of the remaining benzylamine can also be used for purity determination of mPEG-SC (1). HPLC analysis of derivatized forms of mPEG-SC (1) affords a potential specific assay for these polymers. The relative attributes and shortcomings of these methods are elaborated. Although the titration assay is intermediate in specificity, the greater precision of the method makes it the preferred method at this stage of development of an HPLC-ELSD or HPLC-UV assay method. The percent relative standard deviation (%R.S.D.) of the titration method was 2.9% as opposed to 8.8% for the HPLC assay. The accuracy of the titration method was shown to be 101 ± 4.0% and was sufficient to justify a blending operation according to FDA cGMP guidelines.     

Statistical Evaluation of the Quality Control Results of 99mTc(Sn)-DPD

The results of the quality control of ^99mTc-DPD produced during five consequtive years are statistically evaluated. Radiochemical purity of the kit determined in 75 batches was 98.3±1.3%. TLC on silica gel with the mixture of acetone and methanole (1:1, v/v) was used to determine the content of free pertechnetate. The labeled complex and ^99mTc-hydrolyzate was separated by using ascending paper (Schleicher & Schull) chromatography and lN NaCl as the mobile phase. Reliable results were obtained showing that the content of the impurities ^99mTc-pertechnetate and ^99mTc-hydrolyzate is 1.7±1.3% and 3.4±2.1%, respectively. The biodistribution depends on the quantity of DPD. For the animal experiments its content should be 70–80 g/kg b.w. The experiments revealed that the mean value of bone distribution was 8.8±1.9%/g and in muscles 0.043± 0.42%/g. The uptake in liver and kidneys was below 3%, i.e., 0.65±0.29 and 1.71±0.68%/organ, respectively. The bone/muscle ratio should be at least 160. The analysis shows that the obtained values are arranged around their, statistically allowed, mean values.     

Radiolabelling optimisation of 7-bromo-5-[123I]-iodo-4-oxo-1,4-dihidroquinoline-2-carboxylic acid: a potential tracer for NMDA SPECT studies

The synthesis of 7-bromo-5-[¹²³I]-iodokynurenic acid is described. The tracer was prepared using a nucleophilic non-isotopic exchange reaction from the corresponding bromo derivative. Optimisation of the reaction parameters and HPLC purification were performed and the radiotracer was obtained in a chemical and radiochemical purity >95% and a specific activity of 235 Ci/mol.     

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.     

A new approach for quantitative analysis of l-phenylalanine using a novel semi-sandwich immunometric assay

Here, we describe a novel method for l-phenylalanine analysis using a sandwich-type immunometric assay approach for use as a new method for amino acid analysis. To overcome difficulties of the preparation of high-affinity and selectivity monoclonal antibodies against l-phenylalanine and the inability to use sandwich-type immunometric assays due to their small molecular weight, three procedures were examined. First, amino groups of l-phenylalanine were modified by “N-Fmoc-l-cysteine” (FC) residues and the derivative (FC-Phe) was used as a hapten. Immunization of mice with bovine serum albumin/FC-Phe conjugate successfully yielded specific monoclonal anti-FC-Phe antibodies. Second, a new derivatization reagent, “biotin linker conjugate of FC-Phe N-succinimidyl ester” (FC(Biotin)-NHS), was synthesized to convert l-phenylalanine to FC-(Biotin)-Phe as a hapten structure. The biotin moiety linked to the thiol group of cysteine formed a second binding site for streptavidin/horseradish peroxidase (HRP) conjugates for optical detection. Third, a new semi-sandwich-type immunometric assay was established using pre-derivatized l-phenylalanine, the monoclonal anti-FC-Phe antibody, and streptavidin/HRP conjugate (without second antibody). Using the new “semi-sandwich” immunometric assay system, a detection limit of 35 nM (60 amol per analysis) and a detection range of 0.1–20 μM were attained using a standard l-phenylalanine solution. Rat plasma samples were analyzed to test reliability. Intra-day assay precision was within 6 % of the coefficient of variation; inter-day variation was 0.1 %. The recovery rates were from 92.4 to 123.7 %. This is the first report of the quantitative determination of l-phenylalanine using a reliable semi-sandwich immunometric assay approach and will be applicable to the quantitative determination of other amino acids.     

Effect of experimental conditions on the in vitro stability of99mTc (Sn)-pyrophosphate

The in vitro stability of^99mTc (Sn)-PyP as a function of experimental conditions of the preparation of the kit and time elapsed after labeling has been tested. The preparation was protected by using nitrogen-purged reactant solutions and kit vials and by ascorbic acid. The samples under nitrogen are stable for 6 h when the content of^99mTc-pertechnetate raises to 5%. The best stability was achieved by addition of 5 g of ascorbic acid per ml of the kit (content of^99mTc-pertechnetate about 0.5%). To accelerate the decomposition, exogenous hydrogen peroxide was used. In this case it was found that the presence of 10 g of ascorbic acid inhibits the effect both of oxygen and peroxide (6 g H₂O₂/ml of the kit). Radiochemical purity of^99mTc (Sn)-PyP remains practically unchanged for 6 h (content of^99mTc-pertechnetate about 0.5%).     

Large-scale synthesis of α-amino acid-N-carboxyanhydrides

Hetero- and homopolymers prepared from α-amino acid-N-carboxyanhydrides (NCAs) monomers are widely useful products. The preparation of pure NCA monomers has been extensively studied in the past. Purification methods including repeated crystallizations, extraction, and flash column chromatography have been devised. However, these methods are not easily amendable to large-scale NCA preparations. This article describes the synthesis of numerous highly purified NCAs on a >100?g scale using a simple filtration step through diatomaceous earth (celite). The resulting NCAs provided polyethylene glycol (PEG)–amino acid triblock polymers devoid of low-molecular-weight by-products that were routinely observed when unfiltered batches of NCAs were used. Also disclosed is the preparation of NCAs at ambient temperature. Traditionally, NCA reactions using a phosgene source are heated. This study shows these reactions can be driven by the slight exotherm that forms upon reagent mixing. This eliminates the need for an external heating source, simplifying large-scale reactions.     

Partial purification, and some properties and reactivities of cetraxate benzyl ester hydrochloride-hydrolyzing enzyme

Debenzylating enzyme from Aspergillus niger enzyme (commercial crude cellulase) catalyzes the hydrolysis of cetraxate benzyl ester hydrochloride (2), a precursor of the antiulcer agent (1). The enzyme was highly purified by three kinds of chromatographies (hydrophobic, ion exchange, gel filtration) with a recovery of 36%. The content of the debenzylating enzyme was about 0.1% in the crude cellulase, but the enzyme showed no cellulase activity. The purified enzyme was inactivated by Hg2+, and diisopropyl phosphorofluoridate (DFP). It was a monomer with a molecular weight of about 35,000, and its isoelectric point was estimated to be 5.3. It showed a debenzylating activity for the phenylpropionic acid benzyl ester moiety of various benzyl ester derivatives, and the benzyl ester of phenylalanine or that of tyrosine was also well hydrolyzed.     

Quality control of radioimmunoassay for TSH, LH, FSH, C-peptide, prolactin based on NIH-computer program

As there has been no data available on the variation of RIA through a year, we assessed within kit and component of between assay variation of TSH, LH, FSH, C-peptide and PRL kit through a year (more than 12 sequential assays). The average of within kit variation was 6.9% (ranged from 4.7 to 9.8%) and that of component of between assay variation was 11.2% (ranged from 8.9 to 14.5%). These values were almost similar to those obtained for the within kit variation and component of between assay variation employing single lot of kit. These results warrant that quality of kits produced by manufacturers was homogeneous throughout the observation period.