Enzyme immunoassay of a substance P-like immunoreactive substance in human plasma and saliva.

A sensitive and specific double-antibody enzyme immunoassay (EIA) for a substance P (SP)-like immunoreactive substance (SP-IS) was developed. For competitive reactions, the SP-antibody was incubated with SP standard (or sample) and beta-D-galactosidase labeled Tyr8-SP (delayed addition). Free and antibody-bound enzyme hapten were separated by using an anti-rabbit immunoglobulin G coated immunoplate. Activity of the enzyme on the plate was fluorometrically determined. The present immunoassay allows detection of 0.4 to 10 fmol/well of SP. Using the present EIA, SP-ISs in human saliva and plasma were determined. The level of SP-IS in human saliva was about 7 pmol/l, which was almost three times higher than that in human plasma.     

Enzyme immunoassay of angiotensin II in human plasma.

We established a highly sensitive double-antibody enzyme immunoassay (EIA) for angiotensin (ANG) II. For competitive reactions, the ANG II-antibody was incubated with ANG II standard (or sample) and beta-D-galactosidase-labeled ANG III (delayed addition). Free and antibody-bound labeled antigen were separated using an anti-rabbit immunoglobulin G (IgG) coated immunoplate. The enzyme activity on the plate was fluorometrically determined. The present immunoassay allows detection of 0.4 to 72 fmol/well of ANG II. Using the present EIA, ANG II-like immunoreactivity (-LI) in human plasma was determined. The level of ANG II-LI in human plasma from 10 healthy volunteers was 33.3 +/- 10.4 pmol/l.     

A fluorimetric determination of lecithin-cholesterol acyltransferase in blood plasma

A fluorimetric assay for lecithin-cholesterol acyltransferase in human plasma (normal and pathological) is described. The lowered cholesterol concentration in the enzyme reaction is determined fluorimetrically by using the cholesterol oxidase—peroxidase —tyramine system. The assay requires only 10 μl of plasma.     

Determination of an irreversible inhibitor of monoamine oxidase B (MDL 72974A) in human plasma and urine by gas chromatography-positive-ion chemical ionization mass spectrometry.

A sensitive and specific assay has been developed for the quantitative measurement in human plasma and urine of the irreversible inhibitor of monoamine oxidase B [(E)-4-fluoro-beta-fluoromethylenebenzene-butanamine HCl salt] (MDL 72974A) (I). This assay is based on gas chromatography-mass spectrometry with ammonia as the chemical ionization reagent gas. After addition of 1-fluoro-2-(4-chlorobenzene)-ethanamine HCl salt (MDL 71946A) as the internal standard, plasma (1 ml) and urine (100 microliter) samples were extracted using an automated solid-liquid extraction procedure on CN columns. The eluent was dried with a stream of nitrogen, and the residue was derivatized with pentafluoropropionic anhydride. Selected-ion monitoring of the [MNH4]+ ions m/z 361 (I) and 351 (internal standard) was used for quantification. The method yielded a linear response over the concentration range 0.25-100 pmol/ml in plasma with a limit of quantitation of 0.25 pmol/ml. The within-day reproducibility at a concentration of 5 pmol/ml was 4.6% and at a concentration of 50 pmol/ml was 1.3%. The day-to-day reproducibility was 5.2 and 7.0% at concentrations of 10 and 30 pmol/ml, respectively. The method was applied to the quantification of I in plasma and urine after the administration of 12-mg doses of I to a healthy male volunteer.     

Phosphorimetric microassay for succinyltrialanyl-p-nitroanilide-hydrolyzing enzyme activity in human serum

A phosphorimetric assay of succinyltrialanyl-p-nitroanilide-hydrolyzing enzyme activity in human serum is described. p-Nitroaniline formed enzymatically is extracted with ether and determined phosphorimetrically in an ether/ethanol glass. The method is precise and very sensitive, requiring as little as 5 μl of human serum. The limit of detection is 10 pmol.     

Specific binding of beta-endorphin to the isolated renal basolateral membranes in vitro

Binding of human beta-endorphin (beta-EP) to rat renal basolateral membranes was characterized using [125I]Tyr27-beta-EP ([125I]beta-EP) as a primary ligand. Ten millimolar of ethylenediaminetetra acetic acid (EDTA) completely inhibited the degradation of [125I]beta-EP in the incubation mixture at 4 degrees C, thus making it possible to quantitatively examine the [125I]beta-EP binding. The specific binding of [125I]beta-EP to the basolateral membranes was reversible and saturable, and a nonlinear least-squares regression analysis of a saturation isotherm revealed two different classes of specific binding sites. One class had an apparent dissociation constant (Kd) of 0.68 nM and a lower number of binding sites (33 fmol/mg protein), whereas the other class had a lower affinity (apparent Kd of 210 nM) and a higher number of binding sites (7.3 pmol/mg protein). Inhibition of the [125I]beta-EP binding by naloxone (10 microM) was approximately only 20%, and that by D-Ala2-D-Leu5-enkephalin (10 microM) was null, suggesting the major role of a non-opioid binding component in specific [125I]beta-EP binding to basolateral membranes. Moreover, a 50% inhibition by 10 microM of dynorphin(1-13) suggests that a certain region of the primary structure of beta-EP, excluding at least the NH2-terminal enkephalin sequence, is of particular importance for the [125I]beta-EP binding. These lines of evidence suggest the existence of two different classes of specific binding sites for beta-EP on the renal basolateral membranes, and the high-and low-affinity bindings may be attributed to opioid and non-opioid receptors, respectively, as judged by known characteristics of opioid and non-opioid receptors in other peripheral tissues.     

Micro-enzyme immunoassay of vasoactive intestinal polypeptide (VIP)-like immunoreactive substance in bovine milk

A sensitive and specific enzyme immunoassay for vasoactive intestinal polypeptide (VIP)-like immunoreactivity was developed with the use of synthetic carboxy-terminal (C-terminal) fragment (residue 11-28) of porcine VIP conjugated with beta-D-galactosidase and a second antibody-coated immunoplate. Using 4-methylumbelliferyl beta-D-galactopyranoside as a fluorogenic substrate, the minimum amount of VIP-like immunoreactive substance (VIP-IS) detectable by this method was 0.1 fmol/well (2.5 pmol/l). The level of VIP-IS in bovine foremilk was above 100 pmol/l, which was more than eightfold higher than that in normal milk.     

Immunoprecipitation and liquid chromatographic-mass spectrometric determination of the peptide glucose-dependent insulinotropic polypeptides GIP1-42 and GIP3-42 from human plasma samples. New sensitive method to analyze physiological concentrations of peptide hormones

The gastrointestinal peptide glucose-dependent insulinotropic polypeptide (GIP1-42) is one of the incretin hormones regulating glucose-induced insulin secretion from the endocrine pancreas. GIP1-42 is a substrate of the circulating enzyme dipeptidyl peptidase IV, which removes the N-terminal peptide Tyr-Ala resulting in the inactive polypeptide GIP3-42. Hither to existing immunoassays do not enable a separate quantification of active and inactive forms, respectively. Therefore, we developed a highly specific and sensitive LC-MS assay for the identification and quantification of GIP1-42 and GIP3-42. Total GIP was immunoprecipitated from crude plasma samples using a C-terminally directed antibody. Thus, peptides were purified and concentrated prior to LC-MS analysis. The present immunoprecipitation-LC-MS assay enables the quantification of active and inactive GIP over a concentration range from 5 to 350 pmol/l in human plasma samples. Since this range covers the basal and postprandial levels of GIP the method is applicable to the determination of concentration changes and changes in the ratio of active and inactive forms of GIP in human plasma.     

Highly sensitive assay for gamma-glutamyltranspeptidase activity by high-performance liquid chromatography with electrochemical detection

A highly sensitive assay for gamma-glutamyltranspeptidase activity involving high-performance liquid chromatography (HPLC) with electrochemical detection was devised. gamma-Glutamyl-DOPA, a new synthetic dipeptide, which consists of naturally occurring amino acids, was found to be a good substrate for gamma-glutamyltranspeptidase purified from Proteus mirabilis. Enzymatically formed DOPA was adsorbed on an aluminium oxide column, eluted with 0.5 M hydrochloric acid and determined by HPLC with electrochemical detection. The sensitivity limit of this method was 0.5 pmol of DOPA formed. Some properties of gamma-glutamyltranspeptidase purified from P. mirabilis were investigated using gamma-glutamyl-DOPA as a substrate. In the presence of 0.15 M glycylglycine, the KM value of the enzyme for gamma-glutamyl-DOPA was 0.013 mM, and the maximum velocity was 247 nmol/min per mg protein. This method was applied to the assay of the enzymatic activity in human serum.     

An ultramicro assay for human plasma prekallikrein activity by phosphorimetry

A highly sensitive method for the phosphorimetric assay of prekallikrein in human blood plasma is described. Prekallikrein is converted to kallikrein (active form) by reaction at 0°C with actin. p-Nitroaniline, formed enzymatically from H-d-prolyl-l-phenylalanyl-l-arginyl-p-nitroanilide, is extracted with ether and determined phosphorimetrically in a mixture of ether and ethanol. The method is precise and highly sensitive, requiring as little as 0.25 μl of human blood plasma. The limit of detection for p-nitroaniline formed enzymatically is 5 pmol.     

Fluorimetric assay for argininosuccinate lyase in human plasma and erythrocytes with a benzoin reagent

A fluorimetric method for the assay of argininosuccinate lyase in human plasma (15–150 units) and erythrocytes (40–1200 units) is established. The arginine formed enzymatically is quantified by means of its fluorescent reaction with benzoin. The method is rapid, simple and sensitive enough to allow the enzyme activity to be determined in as little as 100 μl of plasma or 12.5 μl of packed erythrocytes.     

Improved Sensitive Sandwich Enzyme Immunoassay for Secretory Immunoglobulin a in Human Serum

Abstract

To overcome the IgA interference in enzyme immunoassay for serum secretory IgA (SIgA), a new specific, simple and more sensitive sandwich enzyme immunoassay, fully free of the serum IgA interference, was developed. SIgA standards or samples were added into the wells of polystyrene plate coated with rabbit IgG antibody to human secretory component. After incubation, the wells were washed and then, horseradish peroxidase-labeled Fab′ fragment of goat IgG antibody to human α-chain was added and incubated. The wells were washed again to remove the unbound labeled antibody, and the enzyme activity specifically bound to the well was assayed using 3,3′, 5,5′ - tetramethylbenzidine and H₂O₂ as substrate. The enzyme reaction was stopped by addition of 2M H₂SO₄. The SIgA concentration was determined from the absorbance at 450 nm. The minimun detectable sensitivity was 6ng/ml. The assay system had very good selectivity overcoming the interference of IgA. As the result of high sensitivity, only small amount of sample (2 μ1 for serum) was needed for analysis. In this assay, no cross reactivity was found with purified human IgG, mIgA, IgM or free secretory component (FSC). The recovery of SIgA mixed with human sera or biles was 99.6–108.1%. The coefficients of within-assay and between-assay variation were 5.8–9.3% and 6.2–9.2% respectively. It correlated well with a liquid phase competitive radioimmunoassay for human serum SIgA (r=0.96, n=30, P<0.0l). The level of SIgA in normal human serum was 8.04±3.60 (SD) μg/ml (n=117) and increased significantly in patients with choledocho- lithiasis (57.35±49.70 μg/ml, n=15, P<0.0l). SIgA concentrations in bile samples were also determined by the 2 4′ assay under the condition that FSC did not, interfere with the assay.     

Novel and Sensitive Noncompetitive Enzyme Immunoassay (Hetero-Two-Site Enzyme Immunoassay) for Arginine Vasopressin

Abstract

A novel and sensitive noncompetitive enzyme immunoassay (hetero-two-site enzyme immunoassay) for arginine vasopressin in plasma is described. Plasma (0.3 ml) was diluted 1.3-fold with an appropriate buffer and filtered by centrifugation in a micro-concentrator with polysaccharide membrane to eliminate plasma proteins. Arginine vasopressin in plasma filtrates was biotinylated and trapped onto anti-arginine vasopressin IgG-coated polystyrene balls. After washing the polystyrene balls to eliminate other biotinylated substances, the biotinylated arginine vasopressin was eluted from the polystyrene balls with HCl and was reacted with anti-arginine vasopressin Fab′-peroxidase conjugate. The complex formed was trapped onto streptavidin-coated polystyrene balls. Peroxidase activity bound to the polystyrene balls was assayed by fluorometry. The detection limit of arginine vasopressin was 11 fg (10 amol)/tube. This was 45-fold lower than that by competitive enzyme immunoassay using the same antiserum as used in this study and 9 to 400-fold lower than those previously reported by competitive radioimmunoassays. The assay range of arginine vasopressin in plasma was 0.14–140 ng /l using 100 μl of plasma filtrates corresponding to 75 u1 o f plasma. Plasma levels of arginine vasopressin i n 8 healthy subjects aged 25–41 yr with, ad libitum water in take and normal activity approximately 4 h after breakfast were 0.72 ± 0.22 (SD) ng /l (range, 0.42–1.04 ng /l).     

Determination of cibenzoline in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of cibenzoline (CipralanTM) in human plasma and urine. The assay involves the extraction of the compound into benzene from plasma or urine buffered to pH 11 and HPLC analysis of the residue dissolved in acetonitrile-phosphate buffer (0.015 mol/l, pH 6.0) (80:20). A 10-microns ion-exchange (sulfonate) column was used with acetonitrile-phosphate buffer (0.015 mol/l, pH 6.0) (80:20) as the mobile phase. UV detection at 214 nm was used for quantitation with the di-p-methyl analogue of cibenzoline as the internal standard. The recovery of cibenzoline in the assay ranged from 60 to 70% and was validated in human plasma and urine in the concentration range of 10-1000 ng/ml and 50-5000 ng/ml, respectively. A normal-phase HPLC assay was developed for the determination of the imidazole metabolite of cibenzoline. The assays were applied to the determination of plasma and urine concentrations of cibenzoline and trace amounts of its imidazole metabolite following oral administration of cibenzoline succinate to two human subjects.     

Determination of urinary vanillactic acid and plasma dihydroxyphenylalanine as markers of non-secreting neuroblastoma by high-performance liquid chromatography with electrochemical detection

An accurate and precise isocratic high-performance liquid chromatographic technique for the analysis of urinary vanillactic acid (VLA) and plasma dihydroxyphenylalanine (DOPA), especially at low concentrations (pmol/l) for VLA and nmol/l for DOPA), is described. The compounds were purified in a single step, (on an anion exchanger for VLA and on aluminium oxide for DOPA), separated by ion-pair reversed-phase liquid chromatography, and detected electrochemically. A single analysis was complete within 18 min. Mean recoveries of 103 and 81% were obtained for VLA and DOPA, respectively, and the limits of detection were 42 and 76 pmol/l, respectively. The mean values of the intra-assay coefficient of variation were 14 and 7.1% for VLA and DOPA, respectively, and the mean values of the inter-assay coefficient of variation were 15.7 and 11.6%, respectively. Modifications of the retention times (between 2 and 42 min) induced by changes in the eluent were determined. Reference values for normal children and children with neuroblastoma or various tumours are given.     

A Fluorometric Assay of L-Aspartate: 2-oxoglutarate Aminotransferase in Human Serum with N-(9-Acridinyl) maleimide

Abstract

A new assay of L-aspartate:2-oxoglutarate aminotransferase (EC 2, 6, 1, 1) (glutamic-oxaloacetic transaminase: GOT) is reported. Substrate L-aspartate was replaced with L-cysteine sulfinic acid (CSA) which yields sulfite and pyruvate. The enzyme activity was determined by measuring the sulfite with the aid of a fluorescent reagent, N-(9-acridinyl)maleimide (NAM). The method presents a extremely sensitive GOT assay in the range of 4 to 140 U/l using less than 10 μl of human serum.     

Enzyme immunoassay of somatostatin (SS)-like immunoreactive substance in bovine milk

A sensitive and specific enzyme immunoassay (EIA) for somatostatin (SS)-like immunoreactivity (SS-LI) was developed with the use of beta-D-galactosidase labeled antigen. The minimum amount of SS-like immunoreactive substance (SS-IS) detectable by this method was 1.0 fmol/well (25 pmol/l). The level of SS-IS in bovine foremilk was about 20 pmol/l, and the level was unchanged after delivery. On the other hand, the levels of gastrin releasing peptide (GRP)-IS and vasoactive intestinal polypeptide (VIP)-IS in bovine foremilk were very high, but fell during 1 week after delivery to about 10% of those in foremilk.     

An automated analyzer for methylated arginines in rat plasma by high-performance liquid chromatography with post-column fluorescence reaction

A fully automated analyzer for methylated L-arginine metabolites [N,N-dimethyl-L-arginine (ADMA), N-methylarginine (NMMA) and N,N'-dimethyl-L-arginine (SDMA)] by high-performance liquid chromatography with post-column fluorescence derivatization was developed. This system consists of an on-line extraction, a separation on a reversed phase ion-pair chromatograph, a post-column derivatization by o-phthaladehyde (OPA) and thiol reaction, and fluorescence detection. NMMA, ADMA and SDMA were separated in 40 min with isocratic elution by a combination of octanoate and cyclohexane carboxylate as ion-pair reagents. The eluate was monitored at 450 nm with excitation at 337 nm. The calibration curves for NMMA, ADMA and SDMA showed linearity over the range from 0.05 micromol l(-1) (0.5 pmol on column) to 5.0 micromol l(-1) (50 pmol on column). This method does not require any time-consuming pre-treatment and requires only 10 microl of plasma sample for assay.     

Automated determination of polyamines by high-performance liquid chromatography with simple sample preparation

Recently, a new fully endcapped reversed-phase packing material, Inertsil, was introduced, especially suitable for the determination of basic compounds. We used this packing material to separate o-phthaldialdehyde (OPA) derivatives of amino acid derivatives completely from the OPA derivatives of spermine (SPM), spermidine (SPD), putrescine (PUT) and cadaverine (CAD). The obtained separation made the commonly used off-line extraction procedure redundant and thus an on-line sample clean-up was introduced. This enabled automation of the procedure resulting in a better reproducibility and a more efficient use of equipment. Furthermore, no studies are required to determine the extraction recovery.

The present method has a cycle time of 30 min. A linear response for each polyamine was found up to 250 pmol, with an R² ranging from 0.9981 (SPM) to 0.9998 (CAD). The limit of detection, calculated at a signal-to-noise ratio of 3, was 0.1 pmol, corresponding to a plasma concentration of 0.1 μmol/l. The coefficient of variation (CV) for the peak area was below 3% and for retention times below 0.5% (n=15).

In order to evaluate the applicability of the method, three different types of sample were chromatographed, e.g. urine (obtained from healthy human volunteers), pig plasma and sulfosalicylic acid homogenates of pig intestine biopsies. Tissue homogenates and urine-specimen could easily be quantitated, while plasma concentrations were just above the limit of detection, resulting in a plasma CV ranging from 4.8% (SPM) to 13.6% (SPD) and a tissue CV ranging from 2.1% (SPM) to 8.5% (CAD), The urinary CVs were not determined.

In conclusion, the present method provides an easy way to measure polyamine concentrations for most applications.     

Sensitive Determination of Piritramide in Human Plasma by High-Performance Liquid Chromatography

Abstract

A selective and sensitive method for the determination of piritramide in human plasma is described. After addition of 50 μl of 2 M ammonia and 20 μl of aqueous promethazine solution (100 ng/10 μ1) as an internal standard, 1 ml of plasma was extracted with 5 ml of toluene (extraction efficiency: 93.9 × 2.6%; mean × S. D.; n = 5). HPLC was performed with a phenyl hypersil NC-04 column, particle size 5 μm, 250 × 4 mm I. D.; mobile phase: 8 parts of acetonitrile and 2 parts of 10 mM potassium phosphate buffer (pH 3. 3). The flow rate was set to 2 ml/min and the column temperature was 22°C. The assay was linear in a concentration range of 3.75 ? 3000 ng/ml (r = 0.999), with a lower limit of detection of 3 ng/ml. The precision was determined using spiked plasma samples (15 ng/ml; 300 ng/ml), with coefficients of variation of 6.1 and 5.9% (intraday; n = 5) and 6.5 and 0.2% (interday; n = 3). In the range of 5.6 ? 1500 ng/ml, the accuracy of the assay was 2.82%. The method was used for the determination of piritramide plasma concentrations in patients receiving intra- or postoperative analgesia.