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PubMed1992

New ultrasensitive assay development by using monoclonal antibodies for detecting prostate-specific antigen.

The Serono Maia Clone prostatic-specific antigen (PSA) kit incorporates an immunoradiometric assay for the measurement of PSA in the serum. The method can be used over a range of 0-100 ng/ml without dilution. Standard concentrations are 0, 0.4, 1, 5, 20, and 100 ng/ml. Up to date, 373 normal men, 89 normal women, 117 prostatic carcinoma, 98 other carcinoma, and 85 benign prostatic hypertrophy have been tested. The aim of this study is to evaluate the sensitivity and specificity of a new immunoassay method for the determination of PSA, that could be able to evaluate low levels of PSA, resulted undetectable with other methods. This ability could be useful in patients treated with hormone-suppressive therapy or after radical prostatectomy. We have collected all low values present in samples examined. With the Serono Maia Clone PSA kit only 26.7% of these have been evaluated as 'out' values as opposed to 46.5% with the Hybritech kit.

Antibodies, Monoclonal↗
PubMed2000

Screening for risk factors for thrombosis using a new generation of assays developed to evaluate the functionality of the protein C anticoagulant pathway.

These results, obtained in a small series of patients, suggest that both the ProC Global assay and the PCP Test would be suitable, using well-defined cut-off levels, to identify all the carriers of the Factor V Leiden mutation and all the patients with a protein C deficiency or with combined defects of the protein C pathway. For both assays, however, the sensitivity for protein S deficiency was below 60%. These results in selected patients are congruent with those previously reported in the literature about the ProC Global assay, the PCP Test, and other assays evaluating the functionality of the protein C anticoagulant pathway. All demonstrated a weak sensitivity to protein S deficiency, suggesting that protein S plays only a minor role as an APC cofactor in such global assays. A major discrepancy between the two evaluated assays was obtained in the group of patients without abnormality of the protein C pathway. Actually, using the ProC Global assay, more than 40% of the patients had a decreased PCAT-NR while presenting with none of the three tested abnormalities, whereas none of the studied patients had a ratio below 1.80, and only 5 of 143 (3.5%) had a ratio below 2.00 when using the PCP Test. The observation that around 40% of the control patients had a decreased PCAT-NR could suggest the influence of currently unknown defects of the protein C/protein S pathway on the ProC Global assay. It could also be hypothesized that the higher factor VIII levels already reported in patients with a history of thrombosis than in controls had a significant role in the low responsiveness, but this parameter was not tested in the authors' series. In that connection, it is also well established that elevated factor VIII levels both shorten the APTT and reduce the anticoagulant effect of heparin when evaluated using APTT. Actually, some of the samples investigated in this study were obtained during the acute phase of thrombosis. It is not possible to draw out the hypothesis of an association of biologically undetectable minor changes in various factors involved in the protein C anticoagulant pathway; all the individual factors would remain within their normal ranges. Finally, because 40% of the patients without abnormalities of the protein C pathway had a decreased PCAT-NR, the question arises whether the ProC Global assay might in itself be a biologic marker of thrombophilia, independent of its sensitivity for abnormalities of the protein C anticoagulant pathway. In that connection, the correlation between the result of the ProC Global assay and the risk for thromboembolism was recently evaluated by two different groups. In both cases, the preliminary results suggested that a decreased response to the ProC Global assay might be an independent risk factor for venous thrombosis. The two global assays could therefore have distinctly different applications. If the global assays are used in the hemostasis laboratory to screen for abnormalities of the protein C pathway, and thus to rationalize the use of specific assays, the PCP Test should be chosen, because of its high specificity. Because only 3.5% of the control patients had a ratio below 2.00 (and none had a ratio below 1.80), the PCP Test could be accurately used as a first-step assay in the laboratory screening for these abnormalities of the protein C anticoagulant pathway. Using such a flow chart, the specific assays for APC resistance or the identification of the factor V Leiden mutation and protein C would be performed only in case of a ratio below a cut-off defined using receiver operating characteristic (ROC)-analysis in unselected patients. Because of the weak sensitivity of this assay to both constitutional and acquired protein S deficiencies (below 15% using 1.80 as the cut-off level, or 60% using 2.00), the measurement of this parameter had to be performed in all cases. If, on the other hand, the assay is used to screen for risk factors for thrombosis, the ProC Global assay could b

Adolescent↗
PubMed2003

1,536-well assay development and screening using whole cell displacement binding and laser scanning imaging.

A screen of a GPCR against Pharmacopeia's combinatorial libraries was performed using 1,536-well plates in a 1.5-microl assay volume with an LSI that was specially modified to enable detection at these volumes. The screen encompassed approximately 4 x 10(6) compounds. The assay uses a CHO cell line that expresses human CXCR1. The plate format chosen was the Corning 1536 low-profile wafer plate. The performance of the screen is evaluated, and the necessity to obtain cytotoxicity data from the same well is described.

Animals↗
PubMed2001

Embryonic development assay with Daphnia magna: application to toxicity of aniline derivatives.

An assay system using Daphnia magna embryos was applied to investigate the adverse effects of aniline derivatives. The data were compared with our previous data for chlorophenols. This new assay provides useful information to evaluate the toxicity of chemicals and the differences in sensitivity between the life stages. The effects of 15 aniline derivatives on embryonic development of D. magna embryos were determined. At the start of exposure, 2-6-h old eggs (between stages 1 and 2, round in shape, diameter approx. 400 microm), were used. In control and solvent control groups, embryonic development from an egg to a free-swimming animal proceeded completely within 3 days with more than 90% hatchability. Median effective concentrations (EC50s) to reduce the numbers hatched were determined and gross morphological abnormalities of hatched animals recorded. Anilines induced no obvious morphological abnormalities and no developmental delay although premature deaths occurred. However, they affected the number of embryos hatching in a dose-dependent manner. In addition, this embryo assay was more sensitive to aniline derivatives (except for aniline) than acute juveniles immobilization assay. Ratios of 48-h EC50 (juvenile)/3-day EC50 (embryo) for eight anilines were greater than 5.0. Particularly, the ratios of 4-methyl-, 4-ethyl- and 3-methylaniline were 77, 23 and 11, respectively. EC50s for embryos and juveniles were poorly correlated (r = 0.41). This indicated that the sensitivities of the two life stages were different to the effects of anilines. EC50s were poorly correlated (r = -0.097) with the log Kow (1-octanol/water partition coefficient). These results were compared with previous results for phenols.

Aniline Compounds↗
PubMed2004

Determination of lipoic acid in rat plasma by LC-MS/MS with electrospray ionization: assay development, validation and application to a pharamcokinetic study.

A simple, sensitive and specific LC-MS/MS method for the determination of lipoic acid was developed and validated over the linearity range 5-1000 ng/mL (r2 > 0.99) with 200 microL rat plasma using rosigliatzone as an internal standard (IS). The assay procedure involved a simple one-step liquid-liquid extraction of lipoic acid and IS from plasma into ethyl acetate. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40 degrees C. The residue was reconstituted in the mobile phase and injected onto a Hichrom RPB column (4.6 x 250 mm, 5 microm). Separation of lipoic acid and IS was achieved with a mobile phase consisting of 0.05 M formic acid:acetonitrile (40:60, v/v) at a flow rate of 1.0 mL/min. The API-3000 LC-MS/MS was operated under the multiple reaction monitoring mode (MRM) using the electrospray ionization technique. Positive and negative ion acquisition within the same chromatographic run was used in the present method. For lipoic acid a pseudo-molecular ion transition pair was acquired in negative polarity, whereas for IS the transition pair was acquired in positive polarity. Quantitation was determined for both analyte and IS in MRM scan mode. Absolute recovery of lipoic acid and IS was >70 and 97%, respectively. The lower limit of quantification (LLOQ) of lipoic acid was 5.0 ng/mL. The inter- and intra-day precision in the measurement of quality control (QC) samples 5, 15, 400 and 800 ng/mL were in the range 2.18-5.99% relative standard deviation (RSD) and 0.93-13.77% RSD, respectively. Accuracy in the measurement of QC samples was in the range 87.40-114.40% of the nominal values. Analyte and IS were stable in the battery of stability studies, viz. bench-top, auto-sampler and freeze-thaw cycles. Stability of lipoic acid was established for 1 month at -80 degrees C. The application of the assay to a pharmacokinetic study in rats confirmed the utility of the assay.

Animals↗
PubMed2005

Different real time PCR approaches for the fine quantification of SNP's alleles in DNA pools: assays development, characterization and pre-validation.

Single nucleotide polymorphisms (SNPs) are becoming the most common type of markers used in genetic analysis. In the present report a SNP has been chosen to test the applicability of Real Time PCR to discriminate and quantify SNPs alleles on DNA pools. Amplification Refractory Mutation System (ARMS) and Mismatch Amplification Mutation Assay (MAMA) has been applied. Each assay has been pre-validated testing specificity and performances (linearity, PCR efficiency, interference limit, limit of detection, limit of quantification, precision and accuracy). Both the approaches achieve a precise and accurate estimation of the allele frequencies on pooled DNA samples in the range from 5 % to 95 % and don't require standard curves or calibrators. The lowest measurement that could be significantly distinguished from the background noise has been determined around the 1 % for both the approaches, allowing to extend the range of quantifications from 1 % to 99 %. Furthermore applicability of Real Time PCR assays for general diagnostic purposes is discussed.

Alleles↗
PubMedDate not supplied

C-peptide immunochemiluminometric assay developed from two seemingly identical polyclonal antisera.

Two goats were immunized with synthetic C-peptide. Their antisera were immunopurified separately on a C-peptide affinity column. The purified antibodies from one goat were labeled with acridinium ester; the other goat's antibodies were immobilized on plastic beads. Standards were synthetic C-peptide. The new immunochemiluminometric assay (ICMA) for C-peptide was compared with our routine radioimmunoassay (RIA) by using Novo's 125I-labeled C-peptide and Cambridge Medical's antiserum. Good correlations were found between the new ICMA and the RIA (r = 0.951; ICMA = 1.07 RIA + 147; n = 112). The new ICMA showed good recoveries (91 percent to 108 percent) of added C-peptide and parallelism of diluted specimens. The incubation time was shortened from 48 hours for RIA to five hours for ICMA. In addition, it was shown that polyclonal antisera from two animals immunized by the same antigen and purified by the same affinity column can be used to prepare immunometric assays.

Animals↗
PubMedDate not supplied

The role of the independent clinical laboratory in new assay development and commercialization.

Most would agree that these are exciting times in the field of laboratory medicine. As the body of scientific knowledge expands and research activities, such as those catalyzed by the sequencing of the human genome, bring us closer to the promise of personalized medicine, the clinical laboratory industry will have increasing opportunities to partner with owners of intellectual property to develop and commercialize new diagnostic tests. The large, independent clinical laboratories are particularly well positioned to commercialize important new tests, with their broad market penetration, infrastructure, and the scale to run esoteric tests cost-effectively.

Biological Assay↗
PubMed1998

Up-regulation of luciferase gene expression with antisense oligonucleotides: implications and applications in functional assay development.

HeLa Tet-Off cells were transfected transiently as well as stably with a recombinant plasmid (pLuc/705) carrying the luciferase gene interrupted by a mutated human beta-globin intron 2 (IVS2-705). The mutation in the intron causes aberrant splicing of luciferase pre-mRNA, preventing translation of luciferase. However, treatment of the cells with a 2'-O-methyl-oligoribonucleotide targeted to the aberrant splice sites induces correct splicing, restoring luciferase activity. The effects are sequence-specific, depend on the concentration of the oligonucleotide, and can be modulated by the pretreatment of the cell line, Luc/705, with tetracycline. Thus, the cell line provides, among others, a novel functional assay system superior to other procedures that are based on protein down-regulation. In particular, the system would be ideal in assessing the cellular delivery efficiency of antisense oligonucleotides.

Alternative Splicing↗
PubMed1986

Determination of endotoxins on hypodermic needles by means of a chromogenic Limulus amoebocyte lysate assay. Development of a test model.

A test model was developed in order to describe the determination of endotoxins on hypodermic needles in a reliable and reproducible manner. As in all in-vitro experiments one has to be very careful about extrapolating data to in-vivo situations. However by choosing a hydrophilic (Salmonella typhimurium ATCC 14028) and a hydrophobic bacterium (Acinetobacter calcoaceticus var. anitratus RR 8212/113), one could hope to obtain a quite representative idea about the extraction of contaminating gram-negative micro-organisms. Using the proposed extraction procedure and a chromogenic LAL assay as detection system, it was possible to achieve a quantitative idea about the extraction of endotoxins on hypodermic needles. Extracting needles at 19 degrees C during 83 to 98 min produced the highest yield as to the detection of endotoxins from hydrophilic strains. For the detection of endotoxins from hydrophobic strains, the highest yield values were obtained when the extraction was performed at temperatures between 64 and 80 degrees C during 96 to 120 min. However it seems necessary to perform the extractions at least three times in order to obtain reproducible results. In conclusion, using the extraction procedure as described, it is possible to measure endotoxin-like activity, on the inner and outer surface of hypodermic needles in a simple but accurate way, using water as extraction fluid and a chromogenic Limulus Amoebocyte Lysate assay as detection system.

Acinetobacter↗
PubMed1991

Use of newly developed assays for protein C and plasminogen in horses with signs of colic.

Protein C content and plasminogen activity were measured in plasma from 100 horses with signs of colic. Data were analyzed by grouping horses 4 ways. Each horse was allotted to 1 of 2 outcome groups (survivors and nonsurvivors), 1 of 3 broad-category diagnosis groups (inflammatory disorders, strangulating obstructions, and all other gastrointestinal disorders), and 1 of 2 clinical management groups (medical and surgical). In a fourth grouping, all horses (although numbers of horses included in each subgroup were small) were assigned either to specific diagnostic groups that had high expectation for activated hemostasis (intestinal ischemia, endotoxemia, jugular thrombosis, peritoneal adhesions, and laminitis) or to a control group, in which active hemostasis was unlikely. Within 2 to 24 hours after admission, nonsurvivors developed lower protein C content than did survivors. Protein C content and plasminogen activity became low during hospitalization in horses with strangulating obstructions and in horses having surgery. The results from the grouping by specific diagnosis must be considered pilot data because the numbers of horses in each subgroup were small. Although not statistically significant, trends were noticed in protein C and plasminogen: (1) horses with intestinal ischemia and endotoxemia developed low protein C content and plasminogen activity, (2) protein C content became low in horses that developed peritoneal adhesions or laminitis, and (3) plasminogen activity became low in horses that developed jugular thrombosis. Low protein C content or low plasminogen activity, or both, may be useful as predictors for outcome and for these specific complications of equine colic.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Improved troponin T ELISA specific for cardiac troponin T isoform: assay development and analytical and clinical validation.

The first generation of troponin T ELISA (TnT 1) can yield false-positive results in patients with severe skeletal muscle injury. Therefore, a cardiac-specific second-generation troponin T ELISA (TnT 2) was developed, in which the cross-reactive antibody 1B10 has been replaced by a high-affinity cardiac-specific antibody M11.7. No cross-reactivity of TnT 2 was observed with purified skeletal muscle troponin T (1000 micrograms/L) or in test samples from 43 marathon runners and 24 patients with rhabdomyolysis and highly increased creatine kinase. TnT 2 was increased > 0.2 microgram/L in 5 of 40 patients with renal failure and in 4 of 20 muscular dystrophy patients. The detection limit is 0.012 microgram/L. Day-to-day imprecision (CV) within the range 0.19-14.89 micrograms/L was < 5.8%. In 4955 patients without myocardial damage, 99.6% had TnT < 0.10 microgram/L. Assay comparison (TnT 1 vs TnT 2) over the whole concentration range (i.e., in 323 samples from AMI-suspected patients) showed a slope, intercept, and standard error of estimate (Sey) of 1.18, 0.01 micrograms/L, and 0.81 microgram/L, respectively.

Animals↗
PubMed1989

A rapid, sensitive method for the simultaneous quantitation of estradiol and estradiol conjugates in a variety of tissues: assay development and evaluation of the distribution of a brain-enhanced estradiol-chemical delivery system.

A rapid and sensitive method that permits the simultaneous quantitation of estradiol (E2) and the E2 conjugate [estradiol 17-(1,4-dihydrotrigonellinate)] in biological tissues from rats is described. This method development was necessitated by the design of this E2-chemical delivery system (E2-CDS) which permits the predictable metabolism of the E2-CDS to a charged quaternary ion (E2-Q+) and its subsequent hydrolysis to slowly liberate E2. Based upon in vitro determinations of the rates of oxidation of E2-CDS to E2-Q+ and hydrolysis of E2-Q+ to E2, we anticipated that concentrations of E2-Q+ would be several orders of magnitude higher in tissues than E2. Three steps were used to extract and prepare samples for the radioimmunoassay (RIA) of estradiol. The first step is homogenization and extraction of the biological samples with an organic solvent; the second step is the base catalyzed hydrolysis of the E2 conjugate in 1 N NaOH; and the third step utilizes solid-phase extraction (SPE) with C18 reversed-phase columns which provide a means of achieving a rapid and precise extraction and separation of E2. The analysis of plasma samples does not require the initial solvent extraction. All purified E2 samples were then reconstituted in the assay buffer and assayed by RIA for E2. The application of this procedure to the determination of E2-Q+ and E2 in biological materials was assessed for specificity, reliability and recovery in vitro by using tissues and plasma from rats and in vivo evaluations of the distribution of E2 and E2-Q+ were done using rats treated with 1 mg E2-CDS/kg. Our in vitro analyses revealed that E2 and E2-Q+ could be differentially extracted with a high recovery and that both compounds could be specifically and reliably assayed in brain, plasma, anterior pituitary, liver, kidney, lung, heart and adipose tissue. In vivo analyses revealed that following a single i.v. injection of E2-CDS, brain levels of E2 exceeded serum levels by 30-, 41-, and 82-fold while brain levels of E2-Q+ exceeded serum levels by 33-, 70-, and 294-fold at 1, 7 and 14 days, respectively. Collectively, these data indicate that E2-Q+ and E2 can be reliably quantitated in a variety of tissues following the administration of an E2-chemical delivery system.

Animals↗
PubMedDate not supplied

The assay development of a molecular marker for transmissible spongiform encephalopathies.

The transmissible spongiform encephalopathies are a group of neurodegenerative diseases which include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, and bovine spongiform encephalopathy (BSE) in cattle. Two-dimensional electrophoresis of proteins was previously used to identify two marker proteins, 130/131, which are selectively present in the cerebrospinal fluid (CSF) of patients with CJD and not in patients with other dementias. The recent characterization of these proteins by amino acid sequencing has identified them as members of the 14-3-3 family of proteins. Polyclonal antibodies against 14-3-3 (all isoforms), 14-3-3 gamma, 14-3-3 beta, and 14-3-3 theta are immunoreactive with a 30 kDa marker band from CJD CSF. Silver staining of two-dimensional electrophoresis separated BSE CSF proteins does not identify a similar marker. However, 14-3-3 immunoreactivity is found in cattle CSF when these proteins are blotted to polyvinylidene difluoride but not when blotted to nitrocellulose.

14-3-3 Proteins↗
PubMedDate not supplied

Affinity ligand selection from a library of small molecules: assay development, screening, and application.

A facile and cost-effective process for screening synthetic libraries for an affinity ligand is described. A high throughput 96-well plate filtration method was designed to screen both discrete compounds and mixtures of compounds attached to a solid support. Human serum albumin (HSA) was used as a target protein to demonstrate the proof of concept. Detection and quantitation by fluorescence was accomplished with the use of fluorescamine to conjugate the protein in the filtrate. It is found that mixtures demonstrating low average binding reflect an overall lower hit rate of the components, whereas deconvolution of mixtures with high protein binding consistently provides a high hit rate. This differs from many of the previous experiences screening solid-phase mixtures in which high false positive rates are noted to occur. A total of 100K compounds were tested: 25K as discrete samples and 75K as mixtures. An overall hit rate of 8% was observed. Secondary screening of compounds measured specificity, recovery, and dynamic binding capacity. The effectiveness of the method is illustrated using an affinity column made with a representative lead compound. A similar purity was achieved in a single-step purification of HSA from serum as compared to that obtained by two steps of ion-exchange chromatography. The process for primary screening of a large number of compounds is simple, inexpensive, and applicable to any soluble target protein of known or unknown function from crude mixtures and may have additional utility as a generic chemical affinity tool for the functional characterization of novel proteins emerging from proteomics work.

Chromatography, Affinity↗
PubMed2003

tRNA modification by S-adenosylmethionine:tRNA ribosyltransferase-isomerase. Assay development and characterization of the recombinant enzyme.

The enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase catalyzes the penultimate step in the biosynthesis of the hypermodified tRNA nucleoside queuosine (Q), an unprecedented ribosyl transfer from the cofactor S-adenosylmethionine (AdoMet) to a modified-tRNA precursor to generate epoxyqueuosine (oQ). The complexity of the reaction makes it an especially interesting mechanistic problem, and as a foundation for detailed kinetic and mechanistic studies we have carried out the basic characterization of the enzyme. Importantly, to allow for the direct measurement of oQ formation, we have developed protocols for the preparation of homogeneous substrates; specifically, an overexpression system was constructed for tRNA(Tyr) in an E. coli queA deletion mutant to allow for the isolation of large quantities of substrate tRNA, and [U-ribosyl-(14)C]AdoMet was synthesized. The enzyme shows optimal activity at pH 8.7 in buffers containing various oxyanions, including acetate, carbonate, EDTA, and phosphate. Unexpectedly, the enzyme was inhibited by Mg(2+) and Mn(2+) in millimolar concentrations. The steady-state kinetic parameters were determined to be K(m)(AdoMet) = 101.4 microm, K(m)(tRNA) = 1.5 microm, and k(cat) = 2.5 min(-1). A short minihelix RNA was synthesized and modified with the precursor 7-aminomethyl-7-deazaguanine, and this served as an efficient substrate for the enzyme (K(m)(RNA) = 37.7 microm and k(cat) = 14.7 min(-1)), demonstrating that the anticodon stem-loop is sufficient for recognition and catalysis by QueA.

Catalysis↗