A solid-phase enzyme immunoassay of thromboxane B2

A solid-phase enzyme immunoassay for thromboxane B2 was developed using a conjugate of thromboxane B2 and beta-galactosidase. Anti-thromboxane B2 IgG was bound to a polystyrene tube, and the enzyme-labeled and unlabeled thromboxane B2 were allowed to react in a competitive manner with the immobilized antibody. Then, the specifically bound beta-galactosidase was assayed fluorimetrically, and the enzyme activity was correlated with the amount of unlabeled thromboxane B2. By using a calibration curve, thromboxane B2 was determined in the range of 20 fmol-14 pmol. 2,3-Dinor- and 2,3,4,5-tetranor-thromboxane B2 cross-reacted with thromboxane B2 to the extents of 18.6% and 0.4%, respectively. Most prostaglandins and their metabolites tested showed cross-reactivities of less than 1%. In application of the method to human blood and urine, an octadecylsilyl silica column was utilized for extraction and concentration of thromboxane B2. The crude extract contained a substance(s) which disturbed the enzyme immunoassay and gave an apparently high value of thromboxane B2, and the interfering substance was separated from thromboxane B2 by reverse-phase HPLC. Various amounts of authentic thromboxane B2 added to the purified material from human plasma could be determined by the enzyme immunoassay with a recovery of about 80% and the results correlated well with the values obtained by radioimmunoassay (r = 0.979). When the extract from human urine was analyzed by reverse-phase HPLC, the 2,3-dinor metabolite rather than thromboxane B2 was the predominant compound detected by the enzyme immunoassay.     

Enzyme immunoassay of thromboxane B2

An immunoassay for thromboxane B2 was developed in which the hapten molecule was labeled with beta-galactosidase. The immunoprecipitate formed after competition between enzyme-labeled and unlabeled thromboxane B2 was subjected to a fluorometric assay of beta-galactosidase. Thromboxane B2 was detectable in the range of 0.1-30 pmol. Both enzyme immunoassay and radioimmunoassay showed essentially the same cross-reactivities with other prostaglandins and their metabolites when the same antibody was used. Known amounts of thromboxane B2 were added to human plasma, and the sample was applied to an octadecyl silica column. The extract was analyzed by enzyme immunoassay to examine the correlation between the added (x) and measured (y) thromboxane B2 (y = 1.09x + 11.07 pmol/ml, r = 0.99). A satisfactory correlation was observed between radioimmunoassay (x) and enzyme immunoassay (y) (y = 0.92x + 4.64 pmol/ml, r = 0.96). The validity of enzyme immunoassay was also confirmed by gas chromatography-mass spectrometry of a dimethylisopropylsilyl ether derivative of thromboxane B2 methyl ester. The method was applicable to the assay of thromboxane B2 produced from endogenous precursor during thrombin-induced aggregation of human platelets.     

Enzyme immunoassay measurement of the urinary metabolites of thromboxane A2 and prostacyclin

We have used a recently developed enzyme immunoassay (EIA) method for measuring urinary concentrations of TXB2, 6-keto PGF1 alpha, 2,3-dinor-TXB2, 2,3-dinor-6-keto PGF1 alpha and 11-dehydro-TXB2 using acetylcholinesterase from Electrophorus Electricus coupled to TXB2, 6-keto PGF1 alpha and 11-dehydro-TXB2. Urinary PGI2 and TXA2 breakdown products and their metabolites were extracted from 3-40 ml of urine corresponding to 100 mumoles creatinine. Measurements were performed after Sep-Pak extraction and thin layer chromatography separation in a system that allows separation between dinor- and parent derivatives. Because of the relatively high cross reactivity (10-15%) of the anti-TXB2 serum with 2,3-dinor TXB2 and the anti-6-keto PGF1 alpha serum with 2,3-dinor-6-keto PGF1 alpha, measurements were done using 3 antisera (anti-TXB2 and anti-6-keto PGF1 alpha diluted 1/50,000, anti 11-dehydro-TXB2 diluted 1/200,000). The reproducibility of the technique was assessed by measuring the same urine stored frozen in aliquots together with each series of samples (Coefficient of variation 6-12% (n = 20), depending on the compound). In addition, the use of a different solvent system for the thin layer chromatography did not affect the results although the migration of the compounds was modified significantly. Determination of the urinary excretion of TXB2 and prostacyclin metabolites in 17 healthy individuals by this method provided results in agreement with those obtained by other methodologies. In addition, comparisons made between EIA and gas chromatography/mass spectrometry analysis showed good correlation between the urinary metabolites as determined by each technique (r = 0.98).     

A light initiated chemiluminescent immunoassay for procalcitonin

Human procalcitonin （PCT） is the prohormone form of calcitonin which was first identified in 1981 . It is a glycopmtein composed of 116 amino acids without hormonal activity and is mainly produced by parafollicular cells （C cells） in the thyroid gland. PCT is enzymatically cleaved into lower molecular weight peptides, including the N-terminal with 57 amino acids, C-terminal with 21 amino acids, and calcitonin with 32 amino acids. The concentration of PCT is very low in normal human plasma, but is increased in the plasma of patients with sepsis and infection . Furthermore, PCT is mainly induced during severe systemic inflammation caused by bacterial infections, but not by non-bacterial infection. Therefore, PCT is considered as an important marker of serious systemic infection and septicemia.     

A rapid enhanced chemiluminescent immunoassay for allergen-specific IgE antibodies

An enhanced chemiluminescent immunoassay is described for the measurement of allergen-specific IgE antibodies. The assay is demonstrated for pollen from four grass species. A comparison was made between results obtained by this method and those obtained by the radioallergosorbent (RAST) procedure; a high degree of correlation (r = 0.95) was found for D. glomerata specific IgE. The assay is rapid and can be carried out in under 1 hour. The advantages of the luminescent assay as compared with the RAST procedure are discussed.     

Fractional conversion of thromboxane B2 to urinary 11-dehydrothromboxane B2 in man

Thromboxane (TX) B2, the chemically stable hydration product of pro-aggregatory TXA2, undergoes two major pathways of metabolism in man, resulting in the formation of 2.3-dinor-TXB2 and 11-dehydro-TXB2, respectively. We have measured the excretion of the latter during the infusion of exogenous TXB2 over a 50-fold dose range in order to examine the fractional conversion of TXB2 to urinary 11-dehydro-TXB2 and to re-assess the rate of entry of endogenous TXB2 into the circulation. Four healthy male volunteers received 6-h intravenous infusions of the vehicle alone and TXB2 at 0.1, 1.0 and 5.0 ng.kg-1.min-1 in random order. They were pretreated with aspirin 325 mg/d in order to suppress endogenous TXB2 production. Urinary 11-dehydro-TXB2 and 2,3-dinor-TXB2 were measured before, during and up to 24 h after the infusions and in aspirin-free periods, by means of NICI-GC/MS-validated radioimmunoassays. Aspirin treatment suppressed urinary 11-dehydro-TXB2 by 91%. The fractional elimination of 11-dehydro-TXB2 was independent of the rate of TXB2 infusion and averaged 6.8 +/- 0.7%, as compared to 6.4 +/- 0.9% for 2,3-dinor-TXB2. Interpolation of 11-dehydro-TXB2 values obtained in aspirin-free periods onto the linear relationship between the quantities of infused TXB2 and the amount of metabolite excreted in excess of control values (y = 0.0058x, r = 0.94, P less than 0.001) permitted calculation of the mean rate of entry of endogenous TXB2 into the circulation as 0.12 ng.kg-1.min-1. We conclude that: (a) urinary 11-dehydro-TXB2 is at least as abundant a conversion product of exogenously infused TXB2 as 2,3-dinor-TXB2; (b) its excretion increases linearly as a function of the rate of entry of TXB2 into the circulation up to approx. 40-fold the calculated rate of secretion of endogenous TXB2; (c) the latter is consistent with previous estimates based on monitoring of the beta-oxidation pathway of TXB2 metabolism.     

Circulating and urinary thromboxane B2 metabolites in the rabbit: 11-dehydro-thromboxane B2 as parameter of thromboxane production

The metabolism of thromboxane B2 was studied in the rabbit. The aim of the study was to identify metabolites in blood and urine that might serve as parameters for monitoring thromboxane production in vivo. [5,6,8,9,11,12,14,15-3H8]-Thromboxane B2 was administered by i.v. injection to rabbits, and blood samples and urine were collected with brief intervals. The metabolic profiles were visualized by two-dimensional thin layer chromatography and autoradiography, and the structures of five major metabolites were determined using chromatographic and mass spectrometric methods. In urine the major metabolites were identified as 11-dehydro-TXB2 and 2,3,4,5-tetranor-TXB1, and other prominent products were 11-dehydro-2,3,4,5-tetranor-TXB1, 2,3-dinor-TXB1 and 2,3-dinor-TXB2. In the circulation, TXB2 was found to disappear rapidly. The first major metabolite to appear was 11-dehydro-TXB2, which also remained a prominent product in blood for the remainder of the experiment (90 min). With time, the profile of circulating products became closely similar to that in urine. TXB2 was not converted into 11-dehydro-TXB2 by blood cells or plasma. The dehydrogenase catalyzing its formation was tissue bound and was found to have a widespread occurrence: the highest conversion was found in lung, kidney, stomach and liver. The results of the present study suggest that 11-dehydro-TXB2 may be a suitable parameter for monitoring thromboxane production in vivo in the rabbit in blood as well as urinary samples, and possibly also several tissues. This was also demonstrated in comparative studies using radioimmunoassays for TXB2 and 11-dehydro-TXB2.     

Identification of the major urinary metabolite of thromboxane B2 in the monkey.

Thromboxane B2 (TxB2) was biosynthesized from prostaglandin endoperoxides (PGG2, PGH2) using guinea pig lung microsomes and infused into an unanesthetized monkey. Urine was collected and TxB2 metabolites were isolated by reversed phase partition chromatography and high performance liquid chromatography. A major metabolite (TxB2-M) was found to be excreted in greater than two-fold abundance relative to other metabolites. Its structure was determined by gas chromatography-mass spectrometry to be dinor-thromboxane B2. In vitro incubation of TxB2 with rat liver mitochondria yielded a C18 derivative with a mass spectrum identical to that of TxB2-M, substantiating that the major urinary metabolite of TxB2 in the monkey is a product of a single step of beta-oxidation.     

Fractional conversion of thromboxane A2 and B2 to urinary 2,3-dinor-thromboxane B2 and 11-dehydrothromboxane B2 in the cynomolgus monkey

Following the intravenous administration of thromboxane (TX) B2, the stable hydration product of TXA2, to human and nonhuman primates the most abundant urinary metabolites are 2,3-dinor-TXB2 and 11-dehydro-TXB2. However, it is not known whether fractional conversion of TXB2 to its enzymatic metabolites is an accurate representation of TXA2 metabolism. Thus, we have compared the metabolic disposition of synthetic TXA2 and TXB2 via the beta-oxidation and 11-OH-dehydrogenase pathways in vivo in the monkey. TXA2 or TXB2 (20 ng/kg) was intravenously administered to four cynomolgus monkeys pretreated with aspirin in order to suppress endogenous TXA2 production. Urinary TXB2, 2,3-dinor-TXB2 and 11-dehydro-TXB2 were measured before, during and up to 24 h after thromboxane administration by means of reversed-phase high-performance liquid chromatography radioimmunoassay. Aspirin treatment suppressed urinary 2,3-dinor-TXB2 and 11-dehydro-TXB2 by approx. 75%. A similar fractional conversion of TXA2 and TXB2 into 2,3-dinor-TXB2 and 11-dehydro-TXB2 was found. These results suggest that TXA2 is hydrolyzed to TXB2 prior to enzymatic degradation and that metabolites of the latter represent reliable indices of TXA2 biosynthesis. Due to the variability in the conversion of thromboxanes into 2,3-dinor-TXB2 and 11-dehydro-TXB2, the measurement of both metabolites seems to represent a more reliable index of acute changes in TXA2 production.     

A channel-resolved approach coupled with magnet-captured technique for multianalyte chemiluminescent immunoassay

A concept of channel-resolved multianalyte immunoassay (MAIA) and a semi-automated flow-through chemiluminescent (CL) MAIA system coupled with magnet-captured technique were proposed for rapid quantitation of different analytes in a single run. Using alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA) and carcinoma antigen 125 (CA 125) as model analytes. They were firstly incubated in the mixtures of capture antibodies-immobilized paramagnetic microspheres (PMs) and corresponding alkaline phosphatase-labeled antibodies under stir and pumped into three parallel detection channels, the PMs were simultaneously captured by magnet, and the CL signals from the three channels were then sequentially collected with the aid of optical shutters to perform quantitative detection. AFP, CEA and CA 125 could be rapidly assayed in the ranges of 1.0-40microg/l, 0.20-30microg/l and 1.0-50kU/l with the detection limits of 0.60microg/l, 0.080microg/l and 0.70kU/l at 3sigma, respectively. After manual dispensing of specimen and reagents the whole assay process could be completed in 18min. The assay results of clinical serum samples with the proposed method were in acceptable agreement with the reference values. This system, based on the designed channel-resolved strategy and magnet-captured technique provides a semi-automated, reusable, simple, sensitive, rapid and low-cost approach for MAIA without using of expensive array detector.     

Development of radioimmunoassay for thromboxane B2

A simple method for the preparation of rat liver urate oxidase is described. The enzyme was purified from rat liver homogenate by cell fractionation, detergent treatment, alkali treatment, and affinity chromatography on 8-aminoxanthine-bound Sepharose 4B. This enzyme preparation had a specific activity of 9.1 U/mg of protein and was purified about 1000-fold from the liver homogenate. After sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by staining with Coomassie brilliant blue, this preparation yielded one protein band at a position corresponding to a molecular weight of 33,000.     

Enhanced chemiluminescent sandwich enzyme immunoassay for hen egg lysozyme

A sensitive chemiluminescent sandwich-type enzyme immunoassay for hen egg lysozyme was developed. The assay was performed on polystyrene microtitre plates using immobilized specific polyclonal rabbit antibody against lysozyme, a peroxidase conjugate and the H₂O₂/luminol-enhanced chemiluminescence detection reagent. The chemiluminescent signal was detected using either a microplate luminometer, or photographic film in a camera luminometer. The detection limit for lysozyme was 0.3 ng/mL, and this was three times lower than that obtained using a colorimetric method with H₂O₂ and o-phenylendiamine as substrates. Recovery of the assay was 97–112% and the relative standard deviation ranged from 3.6% to 10.3%. The immunoassay overcame interference from the food sample matrix when lysozyme, used as a bacteriostatic agent, was measured.     

Development and validation of chemiluminescent immunoassay for vitellogenin in five salmonid species.

A highly sensitive and specific chemiluminescent immunoassay (CLIA) was developed for quantification of vitellogenin (Vg) in five salmonids. The CLIA for salmon Vg was performed using the two-site method, with anti-masu salmon beta'-component as primary antibody and chemiluminescent acridinium-labeled anti-rainbow trout lipovitellin F(ab)'(2) as the second antibody. Using cutthroat trout Vg as the standard, the working range of the CLIA was from 60 pg to 500 ng Vg/ml. Intra- and inter-assay coefficients of variation ranged from 3.04 to 6.67% and 3.23 to 5.86%, respectively. For the various salmonid species, serially diluted samples of serum from vitellogenic fish ran parallel to their purified Vg standard curve in the CLIA. In male cutthroat trout maturing during the 4 months before spawning, serum Vg levels ranged from 1.56 to 8000 ng/ml. High levels of Vg in some individuals may have resulted from temporary elevation of estradiol-17beta levels in the same fish during December or January (1-2 months before spawning). This is the first report on changes in serum Vg levels in maturing male trout using CLIA, the most sensitive assay for Vg yet developed.     

Metabolism of thromboxane B2 in the monkey.

[3H8]Thromboxane B2 was biosynthesized and infused into an unanesthetized monkey. Several urinary metabolites were isolated and their structures elucidated using gas chromatography-mass spectrometry. In addition to the major urinary metabolite, dinor-thromboxane B2, a series of metabolites resulting from dehydrogenetion of the alcohol group at C-11 were identified: 11-dehydro-thromboxane B2, 11-dehydro-15-keto-13,14-dihydro-2,3-dinor-thromboxane B2, and 11-dehydro-15-keto-13,14-dihydro-19-carboxyl-2,3,4,5-tetranor-thromboxane B2. 6-(1,3-dihydroxypropyl)-7-hydroxy-10-oxo-3-pentadecaenoic acid was also identified. Three mono-O-ethylated metabolites were formed from thromboxane B2, which in this study was infused in an ethanolic solution. A small quantity of thromboxane B2 was excreted unchanged into the urine.     

Structure and quantitative determination of the major urinary metabolite of thromboxane B2 in the guinea pig.

2,3-Dinor-thromboxane B2 was the major urinary metabolite of thromboxane B2 in the guinea pig. The structure was assessed mainly by mass spectrometric analysis of a number of derivatives of the metabolite and by chemical degradation by oxidative ozonolysis. A method for quantitative determination of 2,3-dinor-thromboxane B2 in guinea pig urine based on multiple ion analysis and octadeuterated 2,3-dinor-thromboxane B2 as internal standard was developed. The basal excretion of the metabolite was 65 +/- 36 (S.D.) ng/kg x 24 h (n = 19; range 19--140 ng). This level corresponded to an endogenous synthesis of 543 +/- 300 ng of TXB2. No increase in the excretion was seen after anaphylaxis, in contrast to what has earlier been reported for PGF2 alpha.     

Metabolism of thromboxane B2 in the cynomolgus monkey.

[5,6,8,9,11,12,14,15-3H8]-Thromboxane B2 was injected into the saphenous vein of female cynomolgus monkeys, and blood samples were withdrawn from the contralateral saphenous vein. The compound was eliminated from the circulation with a half-life of about 10 min after an initial rapid disappearance. Some more polar products appeared with time, and also small amounts of material less polar than thromboxane B2; however, the dominating compound in all blood samples was unconverted thromboxane B2. About 45% of the given dose of tritium was excreted into urine in 48 hrs. Several metabolites of thromboxane B2 were found. The major urinary metabolite was identified as dinorthromboxane B2 (about 32% of urinary radioactivity). Unconverted thromboxane B2 was also found in considerable amounts (13% of urinary radioactivity). It is concluded that 1) dehydrogenation at C-12 is not a major pathway in the degradation of this compound, in contrast to metabolism at the corresponding C-15 alcohol group of prostaglandins; 2) after having gained access to the circulation, thromboxane B2 is the main circulating compound; however, assay of thromboxane B2 in plasma will be complicated or precluded by large artifactual production of the compound by platelets during sample collection.     

Plasma thromboxane B2 in haemodialysed patients

Plasma thromboxane B2 (TXB2) concentration was measured in 7 cases of terminal renal failure before and after haemodialysis. The TXB2 levels were higher in the investigated group than in the control group (p less than 0.05). Haemodialysis induced a further increase in the TXB2 concentration. Increased thromboxane production may play a part in the pathogenesis of accelerated atherosclerosis in uraemic patients treated with chronic haemodialysis.