A Method to Assess Radiochemical Purity of Compounds Measured by Radioimmunoassay

Abstract

A method is described for assessing radiochemical purity of compounds measured by radioimmunoassay. This method is applicable in cases when chromatography is used prior to the radioimmunoassay proper. In individual fractions of the chromatographic zone containing the compound assayed, both radioactivity (e. g. in DPM) and mass (e. g. in pg) are measured. The measurement of mass is performed using radioimmunoassay. Radiochemical purity is assumed, if the radioactivity/mass ratio (specific activity) in individual fractions of the zone is constant. The constancy of specific activity is tested statistically using a regression analysis. A radioimmunoassay of progesterone in a blood plasma pool collected during the follicular phase of the menstrual cycle was used to demonstrate the practical features of the assessment of radiochemical purity. Radiochemical pure progesterone could be obtained only after repeated chromatography. Purification to radiochemical purity resulted in a significantly decreased estimate of progesterone contents in the plasma pool investigated. A value of 344 pg/ml was obtained, in contrast to the value of 422 pg/ml found prior to the repeated chromatography. The former value can be considered an accurate and valid estimate.     

Properties of bacterial luciferase/NADH : FMN oxidoreductase and firefly luciferase immobilized onto sepharose

NADH : FMN oxidoreductase and bacterial luciferase have been efficiently coimmobilized onto Sepharose 4B. This luminescent immobilized enzyme system can be used to assay NADH. The assay is rapid and sensitive with a lower limit of detection of 0.2 pmol/assay tube. The intra-assay precision was 3.5% at 2 × 10^-5 M and 5.8% at 2 × 10^-6 M NADH. Light intensity was proportional to NADH concentration from 0.2 to 1000 pmol. Added serum and certain dehydrogenases were found to be inhibitory; however, inhibition could be eliminated by a combination of heat treatment and dilution. Firefly luciferase has also been immobilized onto both Sepharose 4B and CL 6B. The detection limit for ATP using this immobilized enzyme was 0.2 pmol and the assay was linear from 0.2 to 2000 pmol. The intra-assay precision was 4.8% at 2 × 10^-4 M and 3.2% at 1 × 10^-5 M ATP. The immobilized enzymes remained fully active when rapidly frozen in the presence of glycerol and DTT. Such preparations could be stored for at least two months with no loss of activity. A variety of different compounds were used to block any remaining reactive groups on the Sepharose following immobilization of the enzymes. Glycine, 2-aminoethanol, and ethylenediamine were examined. The preparations where ethylenediamine was used as a blocking agent exhibited better activity and stability than the others.     

Crystallization of markoffian copolymers

An equilibrium theory is proposed for crystallization of (A, B) binary copolymers whose comonomeric unit sequences are statistically described by conditional pair probabilities P_AA, P_AB, P_BA, and P_BB. These are linked to the product of the reactivity ratios by r = r_Ar_B = (P_AAP_BB)/(P_ABP_BA). Three cases are considered here, (i) B units are rejected from the crystals, (ii) cocrystallization of A and B comonomeric units is possible in the full range of compositions within a single crystal structure (copolymer isomorphism), (iii) cocrystallization takes place either in a poly(A)-type or in a poly(B)-type structure, depending on composition (copolymer isodimorphism). For case (i) crystallization the theory demonstrates, according to expectation, that alternating copolymers (r = 0) produce the largest melting point depression, whereas in case (ii) they give rise to the smallest composition difference between the crystals and the liquid. The theory developed here further illustrates that for binary copolymers which are isodimorphic (case iii), a phase diagram is obtained similar to that for a classical binary system of small molecules.     

Chromatographic behaviour of monoclonal antibodies against wild-type amidase from Pseudomonas aeruginosa on immobilized metal chelates

The aim of this work was to devise a one‐step purification procedure for monoclonal antibodies (MAbs) of IgG class by immobilized metal affinity chromatography (IMAC). Therefore, several stationary phases were prepared containing immobilized metal chelates in order to study the chromatographic behaviour of MAbs against wild‐type amidase from Pseudomonas aeruginosa. Such MAbs adsorbed to Cu(II), Ni(II), Zn(II) and Co(II)–IDA agarose columns. The increase in ligand concentration and the use of longer spacer arms and higher pH values resulted in higher adsorption of MAbs into immobilized metal chelates. The dynamic binding capacity and the maximum binding capacity were 1.33 ± 0.015 and 3.214 ± 0.021 mg IgG/mL of sedimented commercial matrix, respectively. A K_D of 4.53 × 10⁻⁷ m was obtained from batch isotherm measurements. The combination of tailor‐made stationary phases of IMAC and the correct selection of adsorption conditions permitted a one‐step purification procedure to be devised for MAbs of IgG class. Culture supernatants containing MAbs were purified by IMAC on commercial‐Zn(II) and EPI‐30–IDA–Zn(II) Sepharose 6B columns and by affinity chromatography on Protein A‐Sepharose CL‐4B. This MAb preparation revealed on SDS–PAGE two protein bands with M_r of 50 and 22 kDa corresponding to the heavy and light chains, respectively. Copyright © 2011 John Wiley & Sons, Ltd.     

Radioimmunoassay for Thromboxane B2

Abstract

A radioimmunoassay for a prostaglandin endoperoxide metabolite, thromboxane B₂, was developed. The antibodies were very specific for this compound, and the method had a sensitivity of 10 pg. Platelets from a human subject were aggregated by addition of collagen and the synthesis of prostaglandin endoperoxides with time was monitored by parallel assay of prostaglandin E₂, F_2α ^and thromboxane B₂.     

Chemluminescence immunoassay for estriol in saliva

Estriol is determined in whole saliva of pregnant women by a direct solid-phase chemiluminescence immunoassay. The assay uses a monclonal antibody raised against estriol-6-crboxymethyloxime/bovine serum albumin and the homologous chemiluminescent marker conjugate estriol- 6-carboxymethyloxime/aminopentylethylsiolominol (E3/APEI). The anti-estriol antibody is bound to the wells of a microtitration plate via a second antibody directed against the monoclonal antibody; 50 μl of saliva and 12.5 pg of E3/APEI per well are used. The incubation time is 10 min at room temperature. The calibration graph covers 5–750 pg of estriol and the detection limit is 4.5 pg (0.31 nmol l^-1. Mean recovery of added estriol is 98%. Within-assay coefficient of variation is 11.8–5% for 0.21–6.5 nmol l^-1 E3, and the between-assay value is 15.7–6.9% for 0.27-3.5 nmol l^-1 E3. The correlation of E3 concentration in time-matched samples of saliva and sera from pregnant women was good (r = 0.934). Total assay time including calculation of results is 3 h for 40 saliva samples.     

Comparison Between Four Radioimmunoassays for Plasma Estriol

Abstract

We have compared the results obtained for plasma estriol (E₃) when four radioimmunoassay methods were used to measure this steroid during various stages of human pregnancy. The first method used a nonspecific antiserum combined with a chromatographic step. The second method utilizes as binding reagent a specific antibody against estriol combined with the same chromatographic step. The last two methods involve solvent extraction only, combined with the specific antiserum. Dichloromethane and ether respectively are used as solvent. The lowest levels of E₃ were obtained with methods I and II. With dichloromethane extraction the E₃ levels were comparable to those obtained with methods I and II. When using ether extraction the E₃ levels were in most cases two to four times higher. Even with highly specific anti- E₃ sera, chromatography is still required to achieve specificity.     

Automated direct high-performance liquid chromatographic assay for estetrol, estriol, cortisone and cortisol in serum and amniotic fluid.

An automated direct assay for the simultaneous determination of unconjugated estetrol, estriol, cortisone and cortisol in serum and amniotic fluid, using high-performance liquid chromatography with electrochemical detection and ultraviolet detection, has been developed. The analysis time is ca. 1 h. This system offers good reproducibility with low coefficients of variation (estetrol, 2.3%; estriol, 2.3%; cortisone, 2.6%; cortisol, 1.9%). Detection limits are low enough for routine determinations (estetrol and estriol, 150 pg; cortisone and cortisol, 5 ng). Comparison of the values measured by the present method and by radioimmunoassay revealed significant correlations for estetrol (r = 0.787, p less than 0.01), estriol (r = 0.957, p less than 0.01), cortisone (r = 0.956, p less than 0.01) and cortisol (r = 0.865, p less than 0.01). This system proved to be valuable in monitoring feto-placental function.     

Denitrification and removal of heavy metals from waste water by immobilized microorganisms

Pseudomonas fluorescens, immobilized on soft polyvinyl chloride granules containing up to 35% softeners as carbon source, was used for simultaneous removal of nitrate and heavy metals. In typical continuous column operation, a 100 mg/L nitrate input solution was reduced to a 20 mg/L output at a feeding rate of 1500 mL/h, with a capacity of 14 kg/day/m³, and with an efficiency of 79%. In the same column, Pb(NO₃)₂ concentration was reduced from 1.0 to 0.05−0.1 mg/L and ZnSO₄ concentration was reduced from 10 to 5 mg/L.Pseudomonas aeruginosa immobilized on an O₂ plasma-treated melt blown polypropylene web was used for removing 95% of a 1.7 nCi PuCl₄ activity from a nuclear plant waste water in a batch operation.     

Determination of Estrogens by High Performance Liquid Chromatography with Pre-Label Derivatization with 2-(4-Carboxy)-5,6-Dimethylbenzimidazole (II): Shortening of Analysis Time and Application to Monitoring Estrogen in Serum From In-Vitro Fertilization Embryo Transfer (IVF-ET) Patients

Abstract

We report a sensitive high performance liquid chromatography (HPLC) method for determination of free and conjugated estrogens (estrone, estradiol and estriol) by a fluorescent pre-labeling regent, 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole, with modification of previous work. The modified method was also tried, in preliminary work, for diagnosis of the in-vitro fertilization embryo transfer (IVF-ET) process. The reagent volumes were changed to one-tenth, derivatization conditions were changed to mild conditions at 40 C, and a solid-phase extraction process by SEP-PAK could be omitted after restudy of reaction conditions. As a result analysis time could be shorted within 40 min. The proposed HPLC method was applied to monitoring of free and conjugated estrogens in the patients who attend in-vitro fertilization embryo transfer (IVF-ET). The subsequent increase of free and conjugated estrone, estradiol and estriol was observed with the progress of follicle growth following ovulation stage in the IVF-ET process. We tried to plot estrogens for assist of clinical diagnosis of IVF-ET. The free estrone: 200-600 pg/ml, estradiol: 200-600 pg/ml and estriol: 100-300 pg/ml, conjugate estrone: 1000-5000 pg/ml, estradiol: 3000-8000 pg/ml and estriol: 2000-7000 pg/ml) in the patients without hormone disease were observed before human chorionic gonadotropin stimulation (hCG) on IVF-ET process. It was expected that free estrogen values, especially E1 and E3 could be use as validation products for diagnosis of hormone disease in IVF-ET process.     

Monomer-isomerization polymerization. XVIII. Monomer-isomerization copolymerizations of 4-phenyl-2-butene with 4-methyl-2-pentene and 3-heptene with Ziegler-Natta catalyst

4-Phenyl-2-butene (4Ph2B) undergoes monomer-isomerization copolymerization with 4-methyl-2-pentene (4M2P) and 2-and 3-heptene (2H and 3H) with TiCl₃–(C₂H₅)₃Al catalyst at 80°C to produce copolymer consisting exclusively of 1-olefin units. For comparison the copolymerization of 4-phenyl-1-butene (4Ph1B) with 4-methyl-1-pentene (4M1P) and 1-heptene (1H) was carried out under similar conditions. The composition of the copolymers obtained from these copolymerizations was determined from the ratios of optical densities D₁₃₈₀ and D₁₆₀₀ of infrared (IR) spectra of their thin films. The apparent monomer reactivity ratios for the monomer-isomerization copolymerization of 4Ph2B with 4M2P, 2H, and 3H in which the concentration of olefin monomer in the feed was used as internal olefin and those for the copolymerization of 4Ph1B with 4M1P and 1H were determined as follows: 4Ph2B(M₁)-4M2P(M₂); r₁ = 0.90, r₂ = 0.20, 4Ph1B(M₁)-4M1P (M₂); r₁ = 0.40, r₂ = 0.70, 4Ph2B(M₁)-2H(M₂); r₁, = 0.45, r₂ = 1.85, 4Ph2B(M₁)-3H(M₂); r₁ = 0.50, r₂ = 1.20, 4Ph1B(M₁)-1H(M₂); r₁ = 0.55, r₂ = 0.75. The difference in monomer reactivity ratios seemed to originate from the rate of isomerization from 2- or 3-olefins to 1-oletins in these monomer-isomerization copolymerizations.     

Reactivity ratios and microstructure determination of vinyl acetate–alkyl acrylate copolymers by NMR spectroscopy

(Vinyl acetate)/(ethyl acrylate) (V/E) and (vinyl acetate)/(butyl acrylate) (V/B) copolymers were prepared by free radical solution polymerization. ¹H-NMR spectra of copolymers were used for calculation of copolymer composition. The copolymer composition data were used for determining reactivity ratios for the copolymerization of vinyl acetate with ethyl acrylate and butyl acrylate by Kelen-Tudos (KT) and nonlinear Error in Variables methods (EVM). The reactivity ratios obtained are r_v = 0.03 ± 0.03, r_E = 4.68 ± 1.70 (KT method); r_v = 0.03 ± 0.01, r_E = 4.60 ± 0.65 (EV method) for (V/E) copolymers and r_v ? 0.03 ± 0.01, r_B ? 6.67 ± 2.17 (KT method); r_v = 0.03 ± 0.01, r_B = 7.43 ± 0.71 (EV method) for (V/B) copolymers. Microstructure was obtained in terms of the distribution of V- and E-centered triads and V- and B-centered triads for (V/E) and (V/B) copolymers respectively. Homonuclear ¹H 2D-COSY NMR spectra were also recorded to ascertain the existence of coupling between protons in (V/E) as well as (V/B) copolymers. © 1995 John Wiley & Sons, Inc.     

Determination of Mercury at the Picogram per Milliliter Level Using Immobilized Horseradish Peroxidase

Abstract

New test method and test device for the mercury (II) determination at the pg/mL level were developed based on the mercury inhibitory action on horseradish peroxidase immobilized on solid supports – in the cells of the polystyrene plate and on the chromatographic paper. The reactions of o-dianisidine, 3,3′,5,5′-tetramethylbenzidine and o-phenylenediamine oxidation by hydrogen peroxide were used as the indicator reactions. The mercury inhibitory effect increased in the presence of thiourea. Under the elucidated optimal conditions the calibration curves for the mercury determination showed a linear relationship between the peroxidase inhibition degree and the mercury concentration in the range of 0,1–1000 pg/mL. The mercury detection limits were 0,1–10 pg/mL in dependence on the concrete indicator reaction. The analysis completed in 15 min. The proposed test device was applied to the mercury determination in underground waters of Moscow region. The mercury content obtained was coincident with that obtained by atomic-fluorescent method with cold vapour.     

Prediction of drug bioavailability in humans using immobilized artificial membrane phosphatidylcholine column chromatography and in vitro hepatic metabolic clearance

This study reports a rapid screening method for the prediction of oral drug bioavailability in humans based on combined immobilized artificial membrane (IAM) chromatographic capacity factor (k_IAM) and in vitro stability in hepatic microsomes. The fraction of drug absorbed (F_a) in humans was predicted for a set of 15 structurally diverse commercial drugs based on k_IAM values using a mobile phase consisting of acetonitrile: Dulbecco's phosphate‐buffered saline. The hepatic intrinsic clearance (CL ) was calculated from in vitro disappearance half‐life, and the oral bioavailability was predicted using in vitro hepatic clearance (CL_h) and F_a. Significant correlations were observed for the relationships between predicted hepatic extraction ratios (ER_h) and actual presystemic metabolism (r = 0.854) and between predicted and observed oral bioavailabilities (r = 0.805; p < 0.01). The IAM capacity factor together with the hepatic microsomal disappearance half‐life may be useful in identifying compounds with high oral absorption potential in early drug discovery processes. Copyright © 2009 John Wiley & Sons, Ltd.     

Monomer-isomerization polymerization. XVII. Monomer-isomerizations copolymerizations of 2-butene with 3-heptenes and 4-methyl-2-pentenes with Ziegler-Natta catalyst

2-Butene(2B) copolymerizes with 3-heptene(3H) and 4-methyl-2-pentene(4M2P) by a monomer-isomerization copolymerization mechanism in the presence of TiCl₃–(C₂H₅)₃Al catalyst at 80°C to yield the copolymers of 1-olefin units. By comparison, the copolymerization of 1-butene(1B) with 4-methyl-1-pentene(4M1P) was also carried out under similar conditions. The composition of the copolymers obtained from these copolymerizations was determined from the ratios of optical densities D₇₂₃/D₁₃₈₀ and D₁₁₇₀/D₁₃₈₀ in their infrared (IR) spectra. The apparent monomer reactivity ratios for the monomer-isomerization copolymerization of 2B with 3H and 4M2P, in which the concentration of olefin monomer in the feed was used as 2-olefin, were determined as follows: cis-2B(M₁)/3H(M₂); r₁ = 4.00, r₂ = 0.20: trans-2B(M₁)/3H; r₁ = 3.50, r₂ = 0.20; 4M2P(M₁)-trans-2B(M₂): r₁ = 0.05, r₂ = 9.0. These results indicate that the isomerization of 2-olefins to 1-olefins was important to monomer-isomerization copolymerization.     

Automated direct assay system for the measurement of sex steroid hormones in serum using high-performance liquid chromatography

An automated direct assay system using high-performance liquid chromatography was developed for the simultaneous measurement of estradiol, estrone, progesterone, 17 alpha-hydroxyprogesterone, 20 alpha-hydroxyprogesterone, testosterone and androstenedione in biological fluids. A comparison between the values measured by this method and by radioimmunoassay revealed good correlation for estradiol (r = 0.938, p less than 0.001) and progesterone (r = 0.903, p less than 0.001). Estradiol and estrone could be analysed above the level of 250 pg/ml, and progesterone, 17 alpha-hydroxyprogesterone, 20 alpha-hydroxyprogesterone, testosterone and androstenedione could be analysed above the level of 5.0-7.5 ng/ml. The method was applied to the clinical appraisal of placental function and maturation of ovarian follicles.     

Twentyfold increase in alkaline phosphatase activity by sequential reversible activation of the enzyme followed by coupling with a copolyme of ethylene and maleic anhydride

Alkaline phosphatase, APase, (EC 3.1.31) from calf intestine, after shifting the equilibrium by effector molecules towards the dimeric form of the enzyme, was coupled (ratio 1:2, protein: copolymer) to a copolymer of ethylene and maleic anhydride, EMA. The water-soluble APase-EMA was separated from APase and the unbound EMA by DEAE-cellulose ion exchange chromatography. The specific activity of the APase-EMA, compared to APase, increased 26-fold at pH 7.1 and 10-fold at pH 8.6. The pH optimum of APase-EMA was shifted down from pH 9.5 (native APase) to 8.6. This change could be interpreted in terms of polyelectrolyte theory. APase-EMA retained 50–70% of its optimum activity in the pH range 7–8, while APase retained only 5–15% of its optimum activity within the same pH range. Its isoelectric point, pI, was 4.2 (APase 6.0) and it migrated on polyacrylamide gel electrophoresis in a single band, anodic movement twice as fast as APase. Parallel with the kinetic measurements, the reactive-enzyme sedimentation method was used to measure S_20,w values. S_20,w values obtained for APase-EMA, activated APase, and APase dialyzed against wafer were 6.56S, 6.46S, and 5.17S, respectively. Molecular weights, M_r, were determined by equilibrium sedimentation: the values obtained were 180,000, 160,000, and 84,500. M_r values of APase-EMA and APase (native) estimated by Sepharose-4B gel filtrations were essentially the same. The above-mentioned values remained unchanged for APase-EMA after intensive dialysis against water, whereas for the activated APase, separation from the effector molecules caused the equilibrium to shift back to the monomeric, very slightly active enzyme with concomitant changes of S_20,w to 5.15 and M_r to 82,000.     

Comparative Study of the Determination of Parabens in Shampoos by Liquid Chromatography with Amperometric and Coulometric Detection

The simultaneous determination of four para‐hydroxybenzoic acid esters (parabens) in shampoos was studied by liquid chromatography (LC) with amperometric (LC‐AD) and coulometric (LC‐CD) detection. The parabens were separated on an ODS C18 reversed column by isocratic elution with a mobile phase based on methanol‐0.1 M acetic acid (60 : 40%, v/v) with 0.02 M NaClO₄ at a flow rate of 0.8 mL min^?1. The limit of detection (S/N>3) for the analytes was in the 15–25 pg (injected mass) range at an applied potential of 1.20 V vs. Ag/AgCl using the LC‐AD and in the 2–3 pg range at a potential of 0.790 V vs. Pd using the LC‐CD. The peak ratio of the internal standard peak (IS: 4‐hydroxybenzoic acid sec‐butyl ester) versus the analyte peak was found to be related to the amount injected from 0.1 ng to 100ng (r=0.996–0.999) with the LC‐AD and from 0.050 ng to 100 ng range (r=0.999–1.000) with the LC‐CD. The relative standard deviation (RSD, n=10) was comprised between 1.8 to 3.5% by LC‐AD ( 5 ng injected) and between 2.0 to 2.4% by LC‐CD (0.5 ng injected). The determination of four most used parabens in ten different shampoos was successfully realized.     

ImmobilizedE. coli Alkaline Phosphatase

We have immobilized E.coli alkaline phosphatase (EC 3.1.3.1) by linking it covalently to sepharose 4B. This preparation has several advantages over the soluble enzyme. The immobilized enzyme is easily separable from other constituents in incubation mixtures. The immobilized enzyme can be reused repeatedly and is more stable than the soluble enzyme to heat treatment in the presence of 10 mM Mg²⁺. The insoluble and soluble phosphatases removed 75 and77%, respectively, of the inorganic phosphorus from casein. The immobilized enzyme inactivated two enzymes believed to be active in the phosphorylated state, acyl-CoA : cholesterol acyltransferase (ACAT) by 39% and NADPH-cytochrome P-450 reductase by 89%. The utility of immobilized alkaline phosphatase for studying the phosphorylation and dephosphorylation of soluble or membrane-bound enzymes and proteins is discussed.     

Immunochemical Detection of Microquantities of Bacterial Formate Dehydrogenase

Abstract

Two immunochemical methods were developed for detection of NAD⁺?dependent formate dehydrogenase (EC 1.2.1.2, FDH) isolated from the methylotrophic bacteria Pseudomonas sp. 101:1) the dot-blot analysis using rabbit polyclonal antibodies; and 2) the indirect competitive ELISA using poly- or monoclonal mouse antibodies. The first method was used for screening the bacterial gene bank, the sensitivity is 5 and 1 pg enzyme per sample using the anti-rabbit antibodies - horse radish peroxidase conjugate or the biotinylated anti-rabbit antibodies and avidin - peroxidase conjugate, respectively. The second method was applied for precise determination of FDH concentration in cell-free extracts of selected recombinant clones. Mouse polyclonal antibodies to bacterial FDH have exibited a rather high affinity binding also to FDH from the methylotrophic yeast Candida methylica. In the indirect competitive ELISA the sensitivity of bacterial FDH determination is 1 ng per sample.