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PubMed1999

The value of analytical assays that are stability-indicating.

It is essential to validate an assay with regards to its precision, accuracy, reproducibility, selectivity and robustness. In pharmaceutical research, in addition to these requirements, an assay is often required to be proven beyond doubt to be stability-indicating. A stability-indicating assay is one that can accurately and selectively differentiate an intact drug from its potential decomposition products. An assay that fails to meet this requirement would produce stability data that are inaccurate and misleading. This concept could be applied to other fields of research, such as clinical laboratory chemistry where accurate and reliable clinical data are imperative in the diagnosis and following-up of diseases. In this study, we employed an amperometric HPLC assay developed for the measurements of urinary catecholamines and metanephrines to illustrate the validation process of this assay with regards to its stability-indicating capacity. The implications of this study are also discussed.

Catecholamines↗

PubMed1998

Optimisation of the polymerase chain reaction and dot-blot hybridisation for detecting cytomegalovirus DNA in urine: comparison with detection of early antigen fluorescent foci and culture.

Rapid, sensitive and specific assays are required for the diagnosis of CMV infection following transplantation. We describe our experience in developing assays for detecting CMV in urine. Conventional preparation of probes cloned after amplification in E. coli led to contamination with E. coli nucleic acids; these hybridised to E. coli DNA present in urine and produced false positive results. Two CMV probes (Hind III and gL) hybridised to human DNA despite high stringency; these probes were thus unsuitable for detecting viral nucleic acids in clinical samples. A PCR derived probe from the immediate early gene of CMV detected dot-blotted CMV DNA specifically. Optimal preparation of urine for detection of CMV DNA was as follows; four freeze/thaw cycles and ultracentrifugation before in vitro proteinase K/SDS treatment, phenol:chloroform extraction, heat denaturation and direct application onto a nylon membrane. However, dot-blot hybridisation was a poor test for CMV in urine; it had low sensitivity and specificity compared with virus isolation and DEAFF. Single round PCR of a 293 bp region of CMV DNA was sensitive and specific to CMV targets. However, undiluted urine contained PCR inhibitors that could only be partly removed by using PEG precipitation. PCR of CMV DNA from urine was specific but was insensitive compared to conventional culture and DEAFF. A significant proportion of urine samples were toxic in conventional culture and DEAFF tests but, PCR of CMV DNA from urine is insensitive and despite its specificity is unlikely to be advantageous in clinical practice even when DEAFF or culture prove unreliable.

Antigens, Viral↗

PubMed1996

Characterizing replication intermediates in the amplified CHO dihydrofolate reductase domain by two novel gel electrophoretic techniques.

Using neutral/neutral and neutral/alkaline two-dimensional (2-D) gel techniques, we previously obtained evidence that initiation can occur at any of a large number of sites distributed throughout a broad initiation zone in the dihydrofolate reductase (DHFR) domain of Chinese hamster ovary (CHO) cells. However, other techniques have suggested a much more circumscribed mode of initiation in this locus. This dichotomy has raised the issue whether the patterns of replicating DNA on 2-D gels have been misinterpreted and, in some cases, may represent such noncanonical replication intermediates as broken bubbles or microbubbles. In an accompanying study (R. F. Kalejta and J. L. Hamlin, Mol. Cell. Biol. 16:4915-4922, 1996), we have shown that broken bubbles migrate to unique positions in three different gel systems and therefore are not likely to be confused with classic replication intermediates. Here, we have applied a broken bubble assay developed from that study to an analysis of the amplified DHFR locus in CHO cells. This assay gives information about the number and positions of initiation sites within a fragment. In addition, we have analyzed the DHFR locus by a novel stop-and-go-alkaline gel technique that measures the size of nascent strands at all positions along each arc in a neutral/neutral 2-D gel. Results of these analyses support the view that the 2-D gel patterns previously assigned to classic, intact replication bubbles and single-forked structures indeed correspond to these entities. Furthermore, potential nascent-strand start sites appear to be distributed at very frequent intervals along the template in the intergenic region in the DHFR domain.

Animals↗

PubMed1988

The application of biochemical criteria to the assessment of macrofilarial viability.

Previous attempts to assess nematode viability have been critically reviewed and the need to apply more objective biochemical criteria emphasized. The practicalities of assay development have been discussed with regard to sensitivity, selectivity and methodological considerations. The biochemical basis and assay methology for six assays (adenine leakage, adenine uptake, leucine uptake, 14CO2 evolution, lactate output and MTT reduction) that we have recently evaluated are detailed. The viability of Acanthocheilonema viteae females exposed for 120 h in vitro to 17 standard compounds (at 10 microM) has been assessed using these six assays and compared relative to motility indices from the micromotility meter. It was concluded that, despite the slightly superior sensitivity of the 14CO2 evolution assay, the MTT reduction method was most suitable for field use due to its technical and practical simplicity, and its applicability to fragments of onchocercal tissue. It was suggested that, in the absence of a better in vitro assay, the feasability of using MTT reduction together with histology should be assessed in a validation exercise with Onchocerca gibsoni.

Animals↗

PubMed1993

Relation of plasma and red blood cells reduced haloperidol concentrations to haloperidol reductase activity assayed in red blood cells in psychiatric population.

1. Haloperidol (HAL) reductase activity in red blood cells (RBC) was determined by a newly developed assay method in 120 blood samples from 75 Japanese psychiatric patients receiving HAL. 2. Plasma concentrations of HAL and reduced haloperidol (RHAL), a reductive metabolite of haloperidol, were also measured in these samples. 3. RBC concentrations of HAL and RHAL were measured in 62 of these samples. 4. No significant correlations were found between HAL reductase activity in RBC vs plasma or RBC RHAL/HAL ratios, which may represent activity of the enzyme metabolizing HAL into RHAL. 5. RHAL concentrations were three times higher in RBC than in plasma, though HAL concentrations were at the same level in both tissues. This may reflect accumulation of RHAL in RBC.

Adolescent↗

PubMed1986

Screening complex hazardous wastes for mutagenic activity using a modified version of the TLC/Salmonella assay.

10 complex hazardous wastes were tested for mutagenic activity using a modified version of the TLC/Salmonella assay developed by Bjørseth et al. (1982). This fractionation/bioassay scheme couples thin-layer chromatography (TLC) with the Salmonella/mammalian-microsome (Ames) assay for the detection of mutagenic constituents in complex mixtures. Crude (unadulterated) hazardous wastes and selected hazardous waste extracts were fractionated on commercially available cellulose TLC plates. Mutagenicity testing was performed in situ by applying a single overlay of minimal growth agar, tester strain TA98 or TA100, and the optional metabolic activation system directly onto the developed chromatogram. A mutagenic effect was indicated either by the appearance of localized clusters of revertant colonies or by an increase in total revertant growth vis-à-vis control plates. 7 of 10 hazardous wastes (including tars, emulsions, sludges, and spent acids and caustics) demonstrated mutagenic activity when tested by this method. To assess the sensitivity of the modified TLC/Salmonella assay, 14 Salmonella mutagens from a wide range of chemical classes and polarities were tested. Selected compounds included heterocyclics, aromatic amines, alkylating agents, antitumor agents, a nitrosamine and a nitroaromatic. 11 of the 14 mutagens were positive in this test system. The 3 compounds refractory to analysis included a polycyclic aromatic hydrocarbon and two volatiles.

Chromatography, Thin Layer↗

PubMed1998

The value of synthetic linear epitope analogues of La/SSB for the detection of autoantibodies to La/SSB; specificity, sensitivity and comparison of methods.

In a previous study it was shown that La/SSB contains four linear epitopes, p147-154, p291-302, p301-318 and p349-364. The aim of the present study was to investigate the value of the synthetic epitope analogues of the La/SSB autoantigen for the detection of antibodies to La/SSB, in comparison with recombinant La and fragments of this protein. A total of 122 sera with anti-La/SSB activity, from patients with primary Sjögren's syndrome (pSS) or systemic lupus erythematosus (SLE), were tested in various peptide-based assays. In addition, 62 sera from pSS or SLE patients with other autoantibody specificities and 95 sera from healthy individuals were used as controls. The autoantibody specificity was identified by counter immunoelectrophoresis and immunoblot. The peptide-based ELISA assays presented sensitivities ranging from 78% to 88-8% and specificities from 69% to 94-3%. Dot blot assays exhibited sensitivities ranging from 93-6% to 97%, but remarkably lower specificities from 56% to 88%. The most sensitive and specific peptide 349GSGKGKVQFQGKKTKF364 was synthesized and attached on a tetramer sequential oligopeptide carrier SOC4 and used for immunoassay development. Assays based on the recombinant native La protein, the La-C terminal (215 aa), and the N-terminal of La with a mutation at base pair 640 (nine adenines instead of eight) were also developed and compared with the SOC4 peptide-based assay. Of anti-La-positive sera, 88.1% were reactive with both the synthetic peptide SOC4-(349-364aa) and the recombinant La protein. Eighty-three percent of sera were reactive with the La N-terminus and 67.8% of sera were reactive with the La C-terminus. Using sera that were anti-Ro-positive but anti-La-negative, 37% were reactive with the recombinant protein, 26% with the La N-terminus, 33% with the La C-terminus and only 11 % with the synthetic peptide. Our results suggest that the synthetic peptide epitopes exhibit high sensitivity and specificity for the detection of anti-La/ SSB antibodies in ELISA and dot blot techniques. The peptide SOC4-(349-364aa) has the same sensitivity for the detection of anti-La/SSB antibodies as the recombinant protein.

Amino Acid Sequence↗

PubMed2006

The calcium-binding domain of the stress protein SEP53 is required for survival in response to deoxycholic acid-mediated injury.

Stress protein responses have evolved in part as a mechanism to protect cells from the toxic effects of environmental damaging agents. Oesophageal squamous epithelial cells have evolved an atypical stress response that results in the synthesis of a 53 kDa protein of undefined function named squamous epithelial-induced stress protein of 53 kDa (SEP53). Given the role of deoxycholic acid (DCA) as a potential damaging agent in squamous epithelium, we developed assays measuring the effects of DCA on SEP53-mediated responses to damage. To achieve this, we cloned the human SEP53 gene, developed a panel of monoclonal antibodies to the protein, and showed that SEP53 expression is predominantly confined to squamous epithelium. Clonogenic assays were used to show that SEP53 can function as a survival factor in mammalian cell lines, can attenuate DCA-induced apoptotic cell death, and can attenuate DCA-mediated increases in intracellular free calcium. Deletion of the highly conserved EF-hand calcium-binding domain in SEP53 neutralizes the colony survival activity of the protein, neutralizes the protective effects of SEP53 after DCA exposure, and permits calcium elevation in response to DCA challenge. These data indicate that the squamous cell-stress protein SEP53 can function as a modifier of the DCA-mediated calcium influx and identify a novel survival pathway whose study may shed light on mechanisms relating to squamous cell injury and associated cancer development.

Amino Acid Sequence↗

PubMed1998

Development of a sensitive enzymeimmunoassay for oxytocin determination in bovine plasma.

A highly sensitive and specific second antibody enzymeimmunoassay (EIA) on microtiterplates for oxytocin determination in bovine plasma using the biotin-streptavidin amplification system was developed. Biotin was coupled to oxytocin and used to bridge between streptavidin-peroxidase and the immobilized oxytocin antiserum in the competitive assay. The assay was carried out directly in 200 microliters of bovine plasma. Oxytocin standards prepared in hormone-free plasma were used. The sensitivity of the assay was 0.25 pg/well which corresponded to 1.25 pg/ml plasma; the 50% relative binding was seen at 2.8 pg/well. Plasma volumes for the assay ranging from 50 to 200 microliters did not influence the shape of the oxytocin standard curve; however a distinct drop in the OD450 was observed with higher plasma volumes. The oxytocin antiserum used in the assay showed no significant cross-reaction with other octapeptides tested. The assay was compared with a radioimmunoassay (RIA) procedure employing prior solvent extraction of plasma samples. The oxytocin concentrations assayed by EIA and RIA in plasma samples obtained from four cows before, during and after milking were highly correlated and very similar (r = 0.97). Hence the assay developed offers an attractive alternative to the RIA since no prior laborious plasma extraction is needed. Further, the assay has the distinct advantage of being non-radioactive in nature.

Animals↗

PubMed1997

Development of an assay for urinary free cortisol determination on the Technicon Immuno 1 system.

OBJECTIVES: To develop and evaluate a method using an ethyl acetate extraction procedure for the determination of urinary free cortisol on the Technicon Immuno 1 system from Bayer Corporation. DESIGN AND METHODS: We tested the assay precision, linearity, and correlation with the Urinary Kallestad Quanticoat Cortisol radioimmunoassay. We also studied the efficiency of the extraction procedure, performed a cross-reactivity study with different cortisol metabolites, and determined the reference values. RESULTS: The assay shows within-run CVs varying from 1.6 to 5.3% and between-day CVs from 2.7 to 6.1% for urinary free cortisol concentrations from 58 to 1097 nmol/L. The assay demonstrates an excellent linearity and a very good correlation with the Kallestad Quanticoat Cortisol assay (slope = 0.94, y-intercept = 29 nmol/L, Sy/x = 54 nmol/L, r = 0.996). The reference values were estimated at 42-281 nmol/d. The extraction procedure shows an average recovery of 99.0% and minimal interference with the cortisol metabolites tested with the exception of cortisone. CONCLUSIONS: The evaluation shows that the developed assay has the analytical characteristics required for its utilization in a clinical laboratory.

Cross Reactions↗

PubMed1993

Comparison of 5 microplate colorimetric assays for in vitro cytotoxicity testing and cell proliferation assays.

This paper describes a critical comparative evaluation of 5 miniaturised colorimetric assays applicable to cytotoxicity testing of anti-tumour drugs (and other toxins) in vitro. Each assay shows a different linear range for optical density versus cell number, a different sensitivity to change in cell number and a different minimum detectable cell number; the values of these parameters vary with experimental conditions and with cell line used. All the methods gave good correlation with viable cell number (determined by colony forming efficiency) in toxicity assays after 3 or 4 days of treatment, but they underestimated cell death after 2 days. Toxicity levels for individual chemicals (in a standard 6-day assay) are similar for the different assays, irrespective of the mechanism of action of the chemical being tested. Two of the more recently developed assays (APNaOH and SRB) were found to be very sensitive under the conditions examined.

Antineoplastic Agents↗

PubMed2005

Can platelet function tests predict the clinical efficacy of aspirin?

"Aspirin resistance" and "aspirin nonresponsiveness" are terms used both to describe both the failure of aspirin to protect subgroups of individuals from severe vascular events and to evoke an appropriate inhibition of platelet function. Several studies utilizing a broad range of platelet function tests have shown that some subgroups of individuals exhibit a reduced or completely missing antiplatelet response to aspirin. The clinical significance of aspirin nonresponsiveness for the prediction of clinical endpoints remains, however, to be determined. Thus far, only three prospective clinical trials have demonstrated a possible relationship between aspirin nonresponsiveness and subsequent vascular events. Most platelet function tests used in respective clinical studies cannot be reliably performed in clinical routine and are not interchangeable for monitoring antiplatelet treatment. There is a need for a simple and reliable assay for predicting the clinical efficacy of antiplatelet therapy. Recent data demonstrate that none of the currently developed assays, including the PFA-100 system, are presently able to accomplish these objectives.

Adenosine Diphosphate↗

PubMed1999

Monoclonal antibodies to progesterone: characterization and selection for enzyme immunoassay in bovine milk.

Thirty-one stabile murine monoclonal antibody (MAb) producing cell lines to progesterone were generated by using a short and a long immunization protocol. Long-term immunization with high doses of 11alpha-hydroxyprogesterone-hemisuccinate-bovine serum albumin (11alpha-OH-P-HS-BSA) antigen led to very good antibody response in Balb/c mice. The donor mouse produced antiserum with a high titre of 1/250,000. Eleven MAbs were selected for further characterization since they showed high sensitivities (<35 pg/well to inhibit 50% of the tracer) in bridge homologous enzyme immunoassay (EIA). The results were compared to the donor mouse polyclonal antiserum. The MAbs and the donor mouse antiserum were generally found to be highly specific, when tested with 30 different steroids. Employing MAb 9C11, with affinity constant, K(alpha), to 11alpha-OH-P-HS of 1.1 x 10(10) M(-1), a bridge heterologous microtitre plate EIA for milk progesterone was developed, using the second-antibody coating technique and horseradish peroxidase (HRP) as an enzyme label. The assay is simple and convenient to use, as it permits direct addition of undiluted milk samples, at the same time maintaining high sensitivity, high precision, and a wide range of optical density (OD) values. The major advantage of the assay developed, compared to previously published direct addition milk progesterone immunoassays, is that progesterone concentrations, measured by the EIA, were not influenced by changing milk fat concentrations, even when milk samples containing up to 10% of milk fat were used for analysis.

Animals↗

PubMed2006

Development of an ex vivo organ culture model using human gastro-intestinal tissue and Campylobacter jejuni.

Campylobacter jejuni is an important food-borne pathogen. However, relatively little is understood regarding its pathogenesis, and research is hampered by the lack of a suitable model. Recently, a number of groups have developed assays to study the pathogenic mechanisms of C. jejuni using cell culture models. Here, we report the development of an ex vivo organ culture model, allowing for the maintenance of intestinal mucosal tissue, to permit more complex host-bacterium interactions to be studied. Ex vivo organ culture highlights the propensity for C. jejuni to adhere to mucosal tissue via the flagellum, either as discrete colonies or as multicellular units.

Campylobacter jejuni↗

PubMed1981

Enzyme immunoassay for human chorionic gonadotropin and its clinical application.

The assay for hCG is important in gynecological and obstetrical fields and has been performed by RIA and radioreceptor assay (RRA). But these procedures use radioisotope which limits popular use. Recently enzyme immunoassay (EIA) has been developed in which procedure the enzyme has been used for labelling in place of radioisotope. The labelling enzyme is stable and has no adverse effect on human beings and its activity is easily measured. In this study, EIA for hCG has been developed. beta-D-Galactosidase (beta-Gal) is preferred because an infinitesimal amount of beta-Gal is easily measured, and MCAE of conjugating agent is used to prevent self-coupling. As solid phase sheep red blood cells were used at first, but now silicone rods are being used because of easy handling. To remove serum interference, sequential competitive method is carried out and 0.5 mIU/ml to 200 mIU/ml of hCG is measurable in 12 h. Coefficients of variation are satisfied. This newly developed assay has been used clinically in diagnosis of brain metastasis of trophoblastic disease, early diagnosis of pregnancy and prognosis of threatened abortion and is now being tried for follow-up of trophoblastic disease.

Abortion, Threatened↗

PubMedDate not supplied

[The use of a polymerase chain reaction method for the diagnosis of Mycoplasma pneumonia].

The assay system for the diagnosis of Mycoplasma pneumoniae infections, based on polymerase chain reaction (PCR), has been developed. The sensitivity of the detection of M. pneumoniae DNA in analysis of different clinical samples has been established. The study has shown the possibility of using the newly developed assay system for the detection of M. pneumoniae in the material obtained from patients with atypical forms of pneumonia and from Mycoplasma carriers.

Adult↗

PubMed2002

Development of internal controls for the Luminex instrument as part of a multiplex seven-analyte viral respiratory antibody profile.

The ability of the Luminex system to simultaneously quantitate multiple analytes from a single sample source has proven to be a feasible and cost-effective technology for assay development. In previous studies, my colleagues and I introduced two multiplex profiles consisting of 20 individual assays into the clinical laboratory. With the Luminex instrument's ability to classify up to 100 distinct microspheres, however, we have only begun to realize the enormous potential of this technology. By utilizing additional microspheres, it is now possible to add true internal controls to each individual sample. During the development of a seven-analyte serologic viral respiratory antibody profile, internal controls for detecting sample addition and interfering rheumatoid factor (RF) were investigated. To determine if the correct sample was added, distinct microspheres were developed for measuring the presence of sufficient quantities of immunoglobulin G (IgG) or IgM in the diluted patient sample. In a multiplex assay of 82 samples, the IgM verification control correctly identified 23 out of 23 samples with low levels (<20 mg/dl) of this antibody isotype. An internal control microsphere for RF detected 30 out of 30 samples with significant levels (>10 IU/ml) of IgM RF. Additionally, RF-positive samples causing false-positive adenovirus and influenza A virus IgM results were correctly identified. By exploiting the Luminex instrument's multiplexing capabilities, I have developed true internal controls to ensure correct sample addition and identify interfering RF as part of a respiratory viral serologic profile that includes influenza A and B viruses, adenovirus, parainfluenza viruses 1, 2, and 3, and respiratory syncytial virus. Since these controls are not assay specific, they can be incorporated into any serologic multiplex assay.

Adenoviridae↗

PubMed2005

Development of capture assays for different modifications of human low-density lipoprotein.

Antibodies to malondialdehyde (MDA)-modified low-density lipoprotein (LDL), copper-oxidized LDL (oxLDL), Nepsilon(carboxymethyl) lysine (CML)-modified LDL, and advanced glycosylation end product (AGE)-modified LDL were obtained by immunization of rabbits with in vitro-modified human LDL preparations. After absorption of apolipoprotein B (ApoB) antibodies, we obtained antibodies specific for each modified lipoprotein with unique patterns of reactivity. MDA-LDL antibodies reacted strongly with MDA-LDL and also with oxLDL. CML-LDL antibodies reacted strongly with CML-LDL and also AGE-LDL. oxLDL antibodies reacted with oxLDL but not with MDA-LDL, and AGE-LDL antibodies reacted with AGE-LDL but not with CML-LDL. Capture assays were set with each antiserum, and we tested their ability to capture ApoB-containing lipoproteins isolated from precipitated immune complexes (IC) and from the supernatants remaining after IC precipitation (free lipoproteins). All antibodies captured lipoproteins contained in IC more effectively than free lipoproteins. Analysis of lipoproteins in IC by gas chromatography-mass spectrometry showed that they contained MDA-LDL and CML-LDL in significantly higher concentrations than free lipoproteins. A significant correlation (r=0.706, P<0.019) was obtained between the MDA concentrations determined by chemical analysis and by the capture assay of lipoproteins present in IC. In conclusion, we have developed capture assays for different LDL modifications in human ApoB/E lipoprotein-rich fractions isolated from precipitated IC. This approach obviates the interference of IC in previously reported modified LDL assays and allows determination of the degree of modification of LDL with greater accuracy.

Animals↗