Specific Solid-phase Research Articles

Current clinical and laboratory practice for the investigation of the antiphospholipid syndrome: findings from the 2008 Australasian antiphospholipid antibody survey

The antiphospholipid syndrome (APS) is an autoimmune condition characterised by vascular thromboses and/or pregnancy morbidity, and its diagnosis currently requires laboratory evidence for the presence of antiphospholipid antibodies (aPL). aPL are identified using a large number of laboratory procedures based on one of two distinct test processes, namely 'solid' or 'liquid' phase assays. The former include anticardiolipin antibodies (aCL) and anti-beta-2-glycoprotein-I antibodies (aB2GPI), while the latter are centred on clot-based tests used to identify the lupus anticoagulant (LA). Depending on their clinical presentation, affected individuals might be seen by a variety of clinical specialties including general physicians and general practionners, with a potentially wide variation in the aPL assays requested. The current report summarises findings from the '2008 Australasian antiphospholipid antibody survey', a simple 5-step survey that assessed current clinical and laboratory practice in the investigation of APS. The survey was despatched via various clinical and scientific professional bodies. Responses were received from 130 scientific and clinical personnel, primarily haematology based (94/130; 72%) or immunology based (34/130; 26%). Most respondents (97/130; 75%) ordered or recommended tests for solid phase aPL testing, and most also attempted to grade these tests and their isotypes. Most were familiar with aCL and aB2GPI testing, and tended to request primarily IgG and IgM isotypes of these antibodies. Only a small number of respondents requested/recommended IgA isotype testing of these antibodies or the other solid phase aPL assays (e.g., anti-prothrombin). A similar number of respondents (104/130; 80%) also ordered or recommended tests for LA, and most also attempted to grade these tests and their isotypes. Some discipline-related biases were also evident, in that 32/34 (94%) of immunology-based respondents identified that they ordered or recommended specific solid phase tests for aPL, whereas only 62/94 (66%) of haematology-based respondents did so. In contrast, 83/94 (88%) of haematology-based respondents identified that they ordered or recommended specific LA test procedures, whereas only 18/34 (53%) of immunology-based respondents did so. To our knowledge, this report represents the first ever attempt to survey a wide range of clinical and scientific personnel regarding ordering and recommending practices for aPL testing, and provides a snapshot of current clinical and laboratory practice for the investigation of APS in Australia and New Zealand. Most respondents to our survey still consider the immunoglobulin G (IgG) aCL test to be a useful first-line solid phase aPL test, and the dilute Russell's viper venom time (dRVVT) assay to be the most useful LA test.

Prospects and limitations of post-transplantation alloantibody detection in renal transplantation

Antibody-mediated immunity is generally accepted to be a major cause of kidney allograft injury and loss. Post-transplantation monitoring for circulating alloantibodies was proposed to represent a useful noninvasive diagnostic approach to assess individual immunologic risks and to guide the implementation of specific therapeutic measures. This was supported by the recent establishment of highly sensitive and specific solid phase immunoassays for detailed characterisation of reactivity patterns. Nevertheless, imperfect associations of serologic results with biopsy-based criteria of antibody-mediated rejection, as well as the disputed long-term significance of circulating alloantibody detected in recipients with normal graft function, necessitate a careful interpretation of monitoring results.

Neutrophil and recombinant myeloperoxidase as antigens in ANCA positive systemic vasculitis.

Myeloperoxidase (MPO) is one of the major autoantigens recognized by anti-neutrophil cytoplasm antibodies. The association of this antigen with specific disease entities requires that there is a source of pure antigen present in large quantities. Further delineation of the molecular mechanisms involved in the antigen-antibody interaction requires the ability to manipulate the molecule. The expression of recombinant MPO in Chinese hamster ovary cells has produced a source of pure protein, suitable for molecular studies. We have shown that this protein is an antigen recognized by 95% of anti-MPO antibodies from patients with systemic vasculitis. This recombinant molecule will be of use in providing an additional specific solid-phase assay for these antibodies and further forms of this protein which mirror the antigenicity of native MPO more exactly may replace chemically purified antigen. It will also be of great value in studies examining the epitopes recognized by anti-MPO antibodies and in studies of immunoregulation and T cell activation.

Analysis of Degradation Products of Non-Persistent Pesticides in Soil Using Liquid Chromatography-Tandem Mass Spectrometry

P-561 Abstract: Non-persistent pesticides were developed to counteract the persistence and bioaccumulation concerns associated with persistent pesticides. By their nature, they degrade in the environment quickly releasing multiple degradation products. The currently applied model for assessing organophosphorus (OP) pesticide exposure is through measuring non-specific dialkylphosphates and OP-specific metabolites in urine as biomarkers of exposure. This model assumes that measuring the metabolites in the body are good markers of exposure to parent pesticides in the environment. In this assumption, there exists a direct correlation between concentrations of parent pesticide in the environment to concentration of metabolite formed in urine. However, the model is obscured by the fact that OP pesticide breakdown products in the environment are usually the same chemical as the metabolite measured in the body. The potential contribution of degradation products found in the environment to the metabolite concentration found in people needs to be assessed. In order to determine this correlation, analytical methods need to be developed to extract, quantitate and evaluate breakdown products in environmental matrices. A single method for analyzing two classes of non-persistent pesticide (OP and pyrethroids) breakdown products in soil was developed. Pesticide degradation products were extracted from soil using pressurized fluid extraction (Dionex ASE®) followed by analysis using liquid-chromatography with atmospheric pressure chemical (APCI) and electrospray ionization (ESI) and tandem mass spectrometry. Additional clean-up steps post ASE® extraction were evaluated to determine any enhanced sensitivity and selectivity of target analytes. Nonspecific, specific OP and pyrethroid degradation products were isolated using ion exchange (for non-specific) and reversed phase (specific and pyrethroids) solid phase extraction post ASE extraction and analyzed with ESI and APCI with detection limits in the sub parts per billion to low parts per billion range with recoveries ranging from 70–110%. The results of these investigations lead to a single robust method capable of analyzing degradation products from two classes of pesticides with excellent recovery, sensitivity, and precision. As a consequence of the development of this method, initiation of studies determining how much, if any, degradation product formed in the environment contributes to the concentration of biomarkers in urine.

Serum inhibin B and follicle-stimulating hormone levels as markers in the evaluation of azoospermic men: a comparison

Inhibin B is a glycoprotein hormone produced mainly by Sertoli cells of the testes in the adult male. It selectively suppresses the secretion of pituitary follicle-stimulating hormone (FSH) and has local paracrine actions in the testes. Its measurement is useful for investigating the role of inhibin B in male gonadal dysfunction. The objective of this study was to investigate the efficacy of serum inhibin B in men with nonobstructive azoospermia in comparison with FSH. Serum concentration of FSH was measured using microparticle enzyme immunoassay, inhibin B by specific solid phase sandwich enzyme-linked immunosorbent assay in men with nonobstructive azoospermia (n = 46) and control fertile men (n = 5). Mean inhibin B and FSH level was 104.6 pg ml(-1) and 4.0 mIU ml(-1) in control men whereas the value for nonobstructive azoospermic men was 17.06 pg ml(-1) and 31.1 mIU ml(-1) respectively. Inhibin B and FSH levels were significantly different in azoospermia than controls (P < 0.0001). There were six cases of nonobstructive azoospermia with normal inhibin B. Testicular histology did not find any evidence of spermatogenesis in three cases with normal inhibin B. This demonstrated that inhibin B was not a superior predictor for testicular function in our study.

Development of a Sensitive and Specific Solid Phase Extraction−Gas Chromatography−Tandem Mass Spectrometry Method for the Determination of Elenolic Acid, Hydroxytyrosol, and Tyrosol in Rat Urine

A novel gas chromatography-tandem mass spectrometry (GC-MS/MS) method was developed, using an ion trap mass spectrometer, for the simultaneous determination of olive oil bioactive components, elenolic acid, hydroxytyrosol, and tyrosol, in rat urine. Samples were analyzed by GC-MS/MS prior to and after enzymatic treatment. A solid phase extraction sample pretreatment step with greater than 80% analytical recoveries for all compounds was performed followed by a derivatization reaction prior to GC-MS/MS analysis. The calibration curves were linear for all compounds studied for a dynamic range between 1 and 500 ng. The limit of detection was in the mid picogram level for tyrosol and elenolic acid (300 pg) and in the low picogram level for hydroxytyrosol (2.5 pg). The method was applied to the analysis of rat urine samples after sustained oral intake of oleuropein or extra virgin olive oil as a diet supplement.

Element fractionation in soil from urban-industrialized areas using sequential extraction

The application of solid-liquid extraction is proposed to assess extractable fractions of components in soil. The application of a several step scheme could give a lot of information about mobility of metals associated with specific solid phases, especially after separation of top and bottom layers of studied soil. In this study, it was aimed to take into solution metal fractions of major (Ca, Mg, Mn, Fe) and trace elements (Cr, Co, Cu, Zn, As, Cd, Pb) from soil collected in urban areas. The fractions were defined by using chemical extraction operationally. The extraction behavior of studied elements in a six-step sequential extraction procedure is discussed with respect to the properties of the reagents used. Here, metal fractionation of four soil samples (Parabraun Erde), top (0-10 cm) and bottom (10-30 cm) layers, collected from German anthropogenically influenced areas are presented. Methods. The following reagents were used in the six-step extraction scheme: 0.01 mol L-1 ammonium acetate, 0.1 mol L-1 acetic acid, 0.05 mol L-1 hydroxylamine hydrochloride (pH 2, 0.1 mol L-1 ascorbic acid in oxalate buffer, hydrogen peroxide, and hot cone, nitric acid. In extracts, the studied elements were determined by ICP-MS and ICP-OES. The extractability of most of the major and trace elements is similar for all steps for both layers of soils. The results obtained for Ca, Mg, Mn and Fe indicate that the soils contain rather moderately soluble carbonate and Mn oxides which are present in the soil in an ‘aged’ form. The trace elements studied (Cr, Co, Ni, Cu, Zn, As, Cd and Pb) from manganese and iron oxides and organic matter are mainly leached. The studies indicate that the mobile fraction of elements is mainly bound to easily reducible Mn oxides, moderately reducible Mn and Fe oxides and organic matter. It has been found that the concentration and proportion of different metal fractions are similar for surface and mineral layers of these soils collected in areas with anthropogenic influence. The results of this study show that soils collected in anthropogenically influenced areas contain more trace elements and the elements are more mobile. The statistical estimation of results obtained after digestion without complete dissolution of soil matrix demonstrates that the digestion procedure is suitable for quantification of total content of studied soils using ICP methods.

Pb scavenging from a freshwater lake by Mn oxides in heterogeneous surface coating materials

Selective extraction techniques were used to assay the importance of specific solid phases in Pb binding by heterogeneous surface coating materials (biofilms) in Cayuga Lake, NY. Hydroxylamine hydrochloride (NH 2OH·HC1) was used to extract easily reducible Mn oxides, and sodium dithionite (Na 2S 2O 4) was used to extract Mn and Fe oxides in two sets of biofilm samples retrieved from the lake. Pb remaining after extraction was removed by extraction with 10% HNO 3, determined by analysis of Pb 208 using a sector field mass spectrometer with an inductively coupled plasma ion source (ICP-MS), and compared to the total extractable Pb. The results indicate that the greatest contribution to total Pb binding to the heterogeneous surface coating materials was from Mn oxides. Pb adsorption capacity of Mn oxides exceeded that of Fe oxides on a molar basis by approximately an order of magnitude. The high reactivity observed for natural Mn oxides indicates that they are biogenic in origin, consistent with expectations based on the relative biotic and abiotic rates of Mn(II) oxidation under circumneutral conditions. Collectively, these results confirm expectations based on prior observations of adsorption of added Pb by Cayuga Lake biofilms before and after selective extraction, and also confirm predictions for Pb phase association in the lake based on the behavior of laboratory surrogates for adsorptive surfaces.

SIMULTANEOUS DETERMINATION OF FENTANYL AND MIDAZOLAM IN PLASMA USING DIRECT SOLID-PHASE MICROEXTRACTION BEFORE GAS CHROMATOGRAPHY–MASS SPECTROMETRY ANALYSIS

ABSTRACT We developed a rapid, specific, and sensitive solid-phase extraction method followed by gas chromatography–mass spectrometry analysis for the determination of fentanyl, midazolam or both drugs simultaneously in plasma samples. Various solid-phase microextraction fibres, extraction and desorption conditions were tested in the search of the best linearity, sensitivity and clean-up. The optimal solid-phase microextraction conditions were obtained with a new 65-µm polydimethylsiloxane/divinylbenzene (PDMS/DVB) fibre. Because of the high extraction selectivity and efficiency, the quantification limit for fentanyl and midazolam could be down to 0.8 ng mL−1 and 1 ng mL−1, respectively, for a plasma volume of 1 mL. The method described is sensitive enough for analysed fentanyl and midazolam in plasma samples for toxicological screening and therapeutic drug monitoring.

Characterization of bisazo compounds employing ultrafast spectroscopy

Fluorenone-based bisazo compounds with different substituents have been investigated in various organic solvents and in a solid state by means of absorption, fluorescence and picosecond time-resolved absorption pump–probe spectroscopy. The bisazo compounds were found to coexist in azo–enol and hydrazone–quinone (HQ) tautomeric forms. Absorption spectra of the two forms were determined and excited-state properties were characterized. The azo–enol tautomeric form has a short, of tens of ps, excited-state lifetime and shows strong excited-state absorption, whereas stimulated emission is weak or absent. The HQ tautomeric form has a longer excited-state lifetime and shows sizeable stimulated emission. The main spectroscopic features of bisazo molecules in a solid state are similar to those in solutions, however, the excited-state relaxation is influenced by the specific solid phase effects. The lifetimes of the HQ form molecules in solutions differ by more than 10 times for bisazo compounds with different substituents and correlate with the photosensitivity of xerographic layers based on these compounds.

Speciation of Submicrogram per Liter Levels of Arsenic in Water: On-Site Species Separation Integrated with Sample Collection

Speciation of arsenic is crucial for assessing health implications from arsenic ingestion and for effective removal of arsenic from water. We report a method for the speciation of submicrogram per liter levels of arsenic in water. The method incorporates water sample collection with on-site arsenic species separation. The method is based on selective retention of arsenic species on specific solid-phase cartridges followed by selective elution and hydride generation atomic fluorescence analysis of the arsenic species. The use of a membrane filter, a resin-based strong cation-exchange cartridge, and a silica-based strong anion-exchange cartridge allows for the speciation of particulate arsenic and soluble arsenite, arsenate, monomethylarsonate, and dimethylarsinate species. Detection limit is on the order of 0.05 μg/L. The method is suitable for direct water sample collection and on-site separation of arsenic species by flowing a measured volume of water sample through the filter and cartridges connected in serial. A particular advantage of this approach is to maintain the integrity of original arsenic species in the sample. It overcomes the common problem of instability of arsenic species after water sampling and during sample storage and handling. Applications of the method are demonstrated to the speciation of arsenic in well water, raw (untreated) river water, bottled water, and a standard reference material (SRM 1643d). Results agree well with the certified values of the SRM.

Determination of Zinc in Environmental Samples by Solid Phase Spectrophotometry: Optimization and Validation Study

A simple and specific solid-phase spectrophotometric (SPS) determination of zinc in μg dm-3 level has been developed based on the reaction of Zn(II) with 4-(2-pyridylazo)resorcinol (PAR) in the presence of potassium iodide; the product was then fixed on an anionic exchanger. The absorbance of the gel, packed in a 1 mm cell, is measured directly. PAR and KI concentrations were optimized simultaneously using response surface methodology (RSM) from sequential experimental Doehlert designs. The advantages of this methodology are discussed, as opposed to univariate optimization, which is also used. The method was validated by obtaining the performance characteristics: linearity, detection and quantification limits, precision, robustness and selectivity, using suitable chemometric techniques; the trueness was confirmed on environmental samples (water and milk).

Comparison of Elimination of Bacteriophages MS2 and φX-174 During Sewage Treatment by Natural Lagooning or Activated Sludges. A Study on Laboratory-Scale Pilot Plants

Using appropriate laboratory pilot plants we studied phage elimination during sewage treatment by lagooning and activated sludges. Sewage fed into two types of pilot plant was seeded with a somatic coliphage (φX-174) or an F-specific RNA phage (MS2). Samples were taken from inside the biological treatment tanks to follow phages disappearance. The kinetics of their elimination were compared with that of an inert tracer, rhodamine B. Other samples were taken at the outflow to evaluate the efficiencies of phage removal for the two treatment processes. The elimination of MS2 in the absence of any specific solid phase was also studied. Results show that rapid adsorption on solid particles was less marked for rhodamine than for the phages. The regression coefficients calculated for MS2 and φX-174 were, respectively, 0.46 day−1 and 0.37 day−1 for lagooning, and 0.13 h−1 and 0.128 h−1 for activated sludges, whereas those of rhodamine were 0.069 day−1 and 0.024 h−1. Thus elimination of the phages was faster than that of the tracer. This reflects the influence of factors acting exclusively on the viral particles. The phages do not therefore behave as inert molecules. The efficiencies of removal of the two types of phage were comparable for the two treatment processes. The extent of elimination was of the order of 2 log. The study of the elimination of MS2 in the absence of any specific solid phase showed that the floc of the activated sludges system favoured elimination more than the sediments in the lagooning system.

Analysis of the DNA-binding activities of Myc/Max/Mad network complexes during induced differentiation of U-937 monoblasts and F9 teratocarcinoma cells.

The bHLHZip protein Max interacts with both the Myc and Mad family proteins forming heterodimers which specifically bind certain E-box DNA recognition sequences, thereby regulating transcription. Whereas Myc proteins actively promote cell proliferation, Mad complexes have the opposite function. Although the main regulation of this network seems to be the control of myc- and mad family gene expression, regulation at the level of DNA-binding and transactivation may also be in operation. Few studies on the DNA-binding activity of native Myc:Max or Max:Mad complexes have been reported mainly due to technical difficulties. To overcome these problems we have developed a specific and sensitive solid phase DNA-binding assay based on partial purification of native Myc, Max and Mad1 complexes by immunological methods. Using this technique we report that the DNA-binding activity of c-Myc-containing complexes is reduced during induced differentiation of U-937 monoblasts and F9 embryonic teratocarcinoma cells. In contrast, the DNA-binding of Mad1-containing complexes increases during monocytic differentiation. In general, the DNA-binding activity of c-Myc and Mad1 correlate with their expression. However, our studies of early kinetics of TPA-induced differentiation of U-937 cells as well as of late events during F9 differentiation suggest that post-translational regulation of Myc and Max DNA-binding may also occur. The solid phase DNA-binding assay may thus provide a tool to study the regulation of DNA-binding in more detail.

Solid-phase Extraction of a Triazine Herbicide Using a Molecularly Imprinted Synthetic Receptor

A molecularly imprinted synthetic polymer receptor selective for triazine herbicides was prepared by suspension polymerization, and was applied to the specific solid-phase extraction of simazine, one of the triazine herbicides, from water. The procedure involves the reversed-phase mode operation of the synthetic receptor to retain lipophilic agrochemicals, including simazine, in water, followed by affinity separation based on hydrogen bonding which exhibits selective retention of simazine, eluting the other undesired compounds. The solid-phase extraction yielded a 56 times concentrated simazine solution with a recovery rate of 91%.

Common polymorphism of the vitamin D receptor gene is associated with variation of peak bone mass in young finns.

Previous studies suggested a relation between polymorphism of the vitamin D receptor (VDR) gene and bone mineral density (BMD) at perimenopausal age. To enlighten the possible association of the VDR gene polymorphism and BMD, we studied young (20-29 years) adults whose BMD provides a measure of their maximal bone mass. After sequencing the DNA regions flanking the polymorphic BsmI site, we set up a specific solid-phase minisequencing technique to assay this allelic variation. BMD values were adjusted for age, sex, weight, physical activity, smoking, and calcium intake. Young subjects homozygous for the b allele (BsmI site present) had a significantly higher BMD in lumbar spine and femoral neck than those homozygous for the B allele (BsmI site absent). This data shows that the BsmI polymorphism of the VDR gene is associated with peak bone mass. The implication of this result regarding the prevention of osteoporosis deserves further attention.

Seroprevalence of hantavirus antibodies in Germany as determined by a new recombinant enzyme immunoassay

In order to elucidate the epidemiological importance of hemorrhagic fever with renal syndrome in Germany, the prevalence of antibodies against hantaviruses was determined in 13,358 sera from residents of various geographic regions, 1,284 sera from occupational risk groups and 287 sera from chronic hemodialysis patients. Serological investigations were performed using a highly specific transferable solid phase enzyme immunoassay based on the recombinant nucleocapsid proteins of a Hantaan and a Puumala serotype strain. The overall antibody prevalence was found to be 1.68%. In the serum panels from western and southern Germany, it was determined to be 1.83% on average in contrast to only 0.8% in the panel from eastern Germany. An endemic focus revealing an antibody prevalence of 3.12% was detected in a low-mountain area called Suebian Alb, which is located in the federal state of Baden-Württemberg. Occupational risk groups and a group of chronic hemodialysis patients showed a significantly elevated antibody prevalence ranging from 3.3% to 10%. The Puumala serotype was found to be the prevailing virus, but the percentage of sera predominantly recognizing the Hantaan nucleocapsid protein increased towards the south and the east and was significantly elevated in dialysis patients.

Mutation specific PCR and direct solid phase sequencing assay for the detection of hepatitis B virus pre-C/C mutants in anti-HBe-positive, chronic hepatitis B.

Sequence analysis of the HBV DNA from patients with anti-HBe+, chronic hepatitis B revealed that the lack of HBeAg is mostly due to a single G-->A transition at nucleotide position 1896, resulting in a translational stop codon. A point mutation-specific polymerase chain reaction (msPCR) for the detection of this genetic variant was established. Two serologically defined groups of patients with symptomatic chronic hepatitis B (HBeAg+ n = 14, anti-HBe+ n = 11) were included in this study. Viral DNA from 43 sera (26 eAg+/17 anti-HBe+) was amplified twice, using two different sets of PCR primers. Each set contained the same -strand primer, but the +strand primers differed at their 3'-end, thus being complementary only to the wild-type or to the mutant DNA. Unspecific amplification was ruled out by choosing high annealing temperatures (66 degrees C) and cloned HBV-DNA as specificity controls. Furthermore, the results were compared to our findings obtained with direct solid-phase sequencing of the amplified viral DNA. Using msPCR, we found the mutation in four of 26 of the eAg+ sera and in 17 of 17 of our anti-HBe+ samples. Mixed virus populations were identified in 13 of 21 cases. Compared to the sequencing results, msPCR is more sensitive and less time consuming for the detection of the stop codon and thus is suitable as a rapid and specific screening method.

Pathogenesis of posttransfusion viral hepatitis in children with beta-thalassemia.

The pathogenesis of posttransfusion hepatitis was determined in 14 children with beta-thalassemia. All had blood samples obtained in 1980 or 1981, were vaccinated against hepatitis B virus in 1983 and had another serum sample collected in 1989. Seven children had detectable antibodies against hepatitis C virus before vaccination, and all were positive in 1989. With specific solid-phase enzyme immunoassays, all children had antibodies against hepatitis B virus, X and polymerase antigens in 1981, and six had one or both antibodies in 1989. Hepatitis B virus infection was confirmed by means of polymerase chain reaction, which demonstrated virus DNA in 13 of the 14 children. The amplification products spanning the X/precore region were smaller than expected, suggesting mutations in this region. Cloning and sequencing of these products revealed deletions spanning part or all of the X gene. The results show that these children were infected with hepatitis B virus even without other markers in serum, that hepatitis B persists years after vaccination and that such infections are associated with the presence of X deletion mutants. Coinfection with hepatitis B and C viruses, the former containing a new class of variants, is common in children with beta-thalassemia.

Primer specific solid-phase detection of PCR products.

Primer specific solid-phase detection of PCR products.