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PubMed1994

The local lymph node assay: developments and applications.

The murine local lymph node assay is a predictive test method for the identification of contact allergens in which sensitizing activity is measured as a function of induced proliferative responses in lymph nodes draining the site of application. In this article the development and validation of the assay are described and comparisons with guinea pig predictive test methods discussed. In addition we examine the advantages and limitations of the method and consider new opportunities and applications of the assay in the context of the toxicological evaluation of sensitizing potential.

Allergens↗

PubMed2005

Assay development and data analysis of receptor-ligand binding based on scintillation proximity assay.

In this paper, we described the optimization of a generic binding assay to measure ligand-receptor interactions for peroxisome proliferator-activated receptors (PPARs). The assay is based on scintillation proximity assay, in which a protein is coated on scintillant-incorporated beads, and a radiolabeled ligand stimulates the beads to emit a signal by binding to the immobilized protein. An intrinsic binding affinity of unlabeled ligands is determined by competitive displacement of the radioligand. The protein coating and ligand binding are achieved in one step by simply mixing ligands, protein and beads in sequence. No additional steps of pre-coating and washing of beads are required. Protein is captured on beads effectively by electrostatic interactions, thus no affinity labeling of protein is required. In data analysis, ligands are grouped into two classes based on their binding affinities. For tight binding ligands, an equation is derived to accurately determine the binding affinity. Otherwise a general equation applies. This quantitative and high throughput assay provides a tool to screen a large library of molecules in search of potent ligands.

PPAR gamma↗

PubMed1995

Host cell contaminant protein assay development for recombinant biopharmaceuticals.

The efficiency and consistency of a biopharmaceutical purification process determines drug quality, including which specific types and concentrations of residual host cell or process contaminants may remain. Commercial reagents and generic analytical methods are available for quantitating most of these contaminants. However, no generic assay is available for quantitation of the specific contaminant host cell proteins (HCPs) which are unique to a novel purification process. Because of this, proprietary reagents and assays must be developed for the quantitation of process-specific HCPs in each biopharmaceutical drug. The need to develop proprietary reagents which are both sensitive to, and specific for, potentially complex mixtures of unique contaminant proteins has defined what is acceptable methodology for development of quantitative HCP assays. Within the biopharmaceutical industry this need is most often satisfied by the development of multi-analyte HCP immunoassays based upon the null cell mock purification model. Confidence in the quantitative nature of a given HCP assay, and the validity of analytical measurement obtained by the assay, is dependent upon empirical demonstration of the unique stoichiometry of the HCP assay reagents. In conjunction with other analytical and validation methods, an HCP immunoassay may be thought of as a necessary quantitative tool for the optimization and validation of biopharmaceutical purification process efficiency and consistency, rather than as an end in itself.

Drug Contamination↗

PubMed2004

Assay development for high throughput screening of p21(Waf1/Cip1) protein expression in intact cells using fluorometric microvolume assay technology.

The p21(Waf1/Cip1) protein represents a broad-acting cyclin-dependent kinase inhibitor that plays a key role in cell cycle regulation. Furthermore, p21(Waf1/Cip1) protein has been described as a direct participant in regulating genes involved in growth arrest, senescence and aging. In response to genotoxic insults (e.g., following chemotherapeutic treatment), p21(Waf1/Cip1) protein accumulates mainly through p53-mediated transcriptional activation and is also regulated at the post-transcriptional level. In tumor cells, p53 is frequently mutated leading to reduced p21(Waf1/Cip1) protein induction that may contribute to resistance to treatment by DNA-damaging agents. In order to identify compounds capable of restoring p21(Waf1/Cip1) protein level, we have developed a 96-multi-well plate-based high throughput screening assay in intact cells using the Applied Biosystems Fluorometric Microvolume Assay Technology (FMAT) macro-confocal system. Briefly, following incubation with test compounds, human MCF7 breast carcinoma cells were fixed and p21(Waf1/Cip1) protein was detected using anti-p21(Waf1/Cip1) monoclonal antibody and anti-mouse IgG conjugated to the red fluorescent dye Alexafluor 633. FMAT provides a set of raw images at a high magnification, in which fluorescence concentrated in a cell is detected as a specific signal. The mean fluorescence of a population of cells is calculated independently of the number of cells as with a classical FACS analysis. This is of particular interest for screening anticancer drugs that may affect cell number and therefore may impact on the readout. This assay was validated using reference compounds such as camptothecin and actinomycin D, known inducers of p21(Waf1/Cip1) protein.

Belgium↗

PubMed1997

The EMIT Cyclosporine Assay: development of application protocols for the Boehringer Mannheim Hitachi 911 and 917 analyzers.

OBJECTIVE: The purpose of this work was to develop applications for the EMIT Cyclosporine (CsA) Assay on the Hitachi 911 and 917 analyzers. METHODS AND RESULTS: Instrument settings were optimized to arrive at the following assay characteristics on the Hitachi 917. Limit of sensitivity was 50 micrograms/L. Intra-assay coefficients of variation (CV) were 8.1% (n = 20; mean = 62 micrograms/L) and 4.2% (n = 20; mean = 315 micrograms/L), while interassay CVs were 13.0% (n = mean = 73 micrograms/L) and 5.7% (n = 43; mean = 391 micrograms/L). Recoveries of 95-104% were obtained by spiking aliquots of 3 whole blood patient pools of known CsA concentrations with CsA. Serial dilutions of 3 patient specimens demonstrated linear relationships between expected and actual CsA concentrations (r = 0.99, 0.99, 0.98; regression lines: y = 1.19x -17.1; y = 0.75x + 18.0; y = 1.01x + 3.7). Specimen carryover was not evident. Calibration stability is at least 10 days. Comparable assay characteristics were found for the Hitachi 911. Sequentially-collected trough whole blood specimens from renal (n = 3), liver (n = 3) and heart (n = 4) transplant patients prescribed CsA were collected up to 78 days post-transplant and analyzed by EMIT on the Hitachi 917 and also by fluorescence polarization immunoassay (FPIA) and high performance liquid chromatography (HPLC). The following linear regression equations were produced for the renal [EMIT = 0.801 (TDx) + 4.98, r = 0.91, Sy/x = 32, n = 37; EMIT = 0.877 (HPLC) + 56, r = 0.87, Sy/x = 38, n = 37]; liver [EMIT = 0.808 (TDx) - 27, r = 0.94, Sy/x = 42, n = 37; EMIT = 0.953 (HPLC) + 44, r = 0.89, Sy/x = 57, n = 37] and heart [EMIT = 0.820 (TDx) - 24, r = 0.94, Sy/x = 31, n = 45, EMIT = 0.956 (HPLC) + 54, r = 0.91, Sy/x = 38, n = 45] patient samples. FPIA values average 32% more than EMIT-derived CsA concentrations on the Hitachi 917, which in turn averaged 15% more than HPLC values. In addition, these levels were compared intra-individually. CsA concentrations within all patients were significantly higher (p < 0.05, paired t-test) by FPIA compared to EMIT and by FPIA compared to HPLC. Although CsA concentrations within most patients were significantly higher (p < 0.05) by EMIT compared to HPLC, levels determined in 4 transplant patients (1 renal, 1 liver, 2 heart) were not different. CONCLUSION: Development of applications for the EMIT CsA Assay on two highly automated, random access instruments, the Hitachi 911 and Hitachi 917, enhances the versatility of the immunoassay for routine therapeutic drug monitoring of this immunosuppressant in the clinical setting.

Chromatography, High Pressure Liquid↗

PubMed1992

Virulence of Mycobacterium tuberculosis for guinea pigs: a quantitative modification of the assay developed by Mitchison.

One of the more accepted methods of assay of virulence of tubercle bacilli is one developed by Mitchison in which guinea pigs were infected by the intramuscular route with 1.0 mg of tubercle bacilli freshly harvested from Lowenstein Jensen medium and in which virulence was based on a subjective score of the extent of gross disease in the animal 6 weeks after infection. Due to the practical difficulties involved in such an assay when routinely performed, the following modifications were made: frozen stocks of the culture were prepared prior to the date of infection, the inoculum was quantitated by means of colony forming units, and virulence was based on the number of tubercle bacilli recovered from the spleen of infected animals 6 weeks after infection. A significant correlation was obtained between the findings from the Mitchison assay of virulence and the modification. The latter assay is recommended for its quantitative and objective features.

Animals↗

PubMed1999

A newly developed assay for melatonin using cells expressing human mel-1a receptor.

We have developed a radioreceptor binding assay (RRA) method for melatonin using membranes from Chinese hamster ovary cells that can stably express human mel-1a receptors. We measured melatonin levels in plasma samples collected every 4h for 24h using the RRA and radioimmunoassay (RIA) methods, simultaneously. There was a statistically significant correlation between the melatonin levels measured by the two methods, this newly developed method providing a sensitive bioassay. As it is possible to circumvent the cross-reactivity usually occurring in the RIA method, this method may be an important tool for detecting bioactive substances relative to the mel-1a receptor.

Animals↗

PubMed1990

Immunoradiometric assay for cytomegalovirus-specific IgG antibodies: assay development and evaluation in blood transfusion practice.

An immunoradiometric assay (radio-immunosorbent test; RIST) for the detection of IgG antibodies to human herpesvirus 4 [human cytomegalovirus (CMV)] has been developed. The technique utilizes CMV antigen passively adsorbed to a polyvinyl microtitration plate and a radiolabelled murine monoclonal anti-human IgG antibody to detect binding of human antibody to the 'solid phase' reagent. The assay was optimized, and its specificity confirmed by testing paired acute and convalescent sera from patients with acute CMV or other human herpesvirus infections. To determine the assay's sensitivity 1433 blood donor sera were examined. The RIST was more sensitive than a standard complement fixation (CFT), in that 53% of these sera were positive by RIST and 48% positive by CFT. There were 1303 concordant results, 88 sera positive only by RIST and 19 sera were only positive by CFT. These discrepant results remained after an attempt to exclude false positive reactivity; their significance is discussed. Use of a monoclonal anti-human IgG antibody in the RIST reduced non-specific binding to the control uninfected cell antigen such that blood donor sera could be tested in the assay using only a CMV antigen without generating an unacceptable false positive rate.

Animals↗

PubMedDate not supplied

Assay development for a portable fiberoptic biosensor.

The fiberoptic biosensor with tapered optical probes has been developed to perform rapid and sensitive fluoroimmunoassays. A number of assays for biologic analytes were developed using a laboratory breadboard device that employed a large, 514 nm argon ion laser. These assays, with limits of detection of 5-50 ng/ml for protein antigens, showed promise for clinical use because of their demonstrated lack of matrix effects from plasma, seru, or blood. However, such a large device was impractical for on-site diagnostics, so a new, portable, multichannel biosensor was developed. To test this new biosensor, which uses 635 nm laser diodes, the assays were converted to use the cyanine dye, Cy5. The detection antibodies were labeled with Cy5 and assays performed to detect the F1 antigen of Yersinia pestis and the protective antigen of Bacillus anthracis. The limit of detection was found to improve by a factor of 10 for each assay. The portable biosensor was then evaluated in a blind test containing F1 antigen spiked into 30 of 173 serum samples. One hundred percent detection was achieved for samples with 100 ng/ml or more F1 antigen, with a specificity of 88%.

Animals↗

PubMed2005

A buccal cell model comet assay: development and evaluation for human biomonitoring and nutritional studies.

The comet assay is a widely used biomonitoring tool for DNA damage. The most commonly used cells in human studies are lymphocytes. There is an urgent need to find an alternative target human cell that can be collected from normal subjects with minimal invasion. There are some reports of buccal cells, collected easily from the inside of the mouth, being used in studies of DNA damage and repair, and these were of interest. However, our preliminary studies following the published protocol showed that buccal cells sustained massive damage and disintegrated at the high pH [O. Ostling, K.J. Johanson. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem. Biophys. Res. Commun. 123 (1984) 291-298] used, but that at lower pH were extremely resistant to lysis, an essential step in the comet assay. Therefore, the aims of this study were to develop a protocol than enabled buccal cell lysis and DNA damage testing in the comet assay, and to use the model to evaluate the potential use of the buccal cell model in human biomonitoring and nutritional study. Specifically, we aimed to investigate intra- and inter-individual differences in buccal cell DNA damage (as strand breaks), the effect of in vitro exposure to both a standard oxidant challenge and antioxidant treatment, as well as in situ exposure to an antioxidant-rich beverage and supplementation-related effects using a carotenoid-rich food. Successful lysis was achieved using 0.25% trypsin for 30 min followed by proteinase K (1mg/ml) treatment for 60 min. When this procedure was performed on cells pre-embedded in agarose on a microscope slide, followed by electrophoresis (in 0.01 M NaOH, 1mM EDTA, pH 9.1, 18 min at 12 V), a satisfactory comet image was obtained, though inter-individual variation was quite wide. Pre-lysis exposure of cells to a standard oxidant challenge (induced by H2O2) increased DNA strand breaks in a dose related manner, and incubation of cells in Trolox (a water soluble Vitamin E analogue) conferred significant protection (P<0.05) against subsequent oxidant challenge. Exposure of buccal cell in situ (i.e. in the mouth) to antioxidant-rich green tea led to an acute decrease in basal DNA strand breaks. In a controlled human intervention trial, buccal cells from 14 subjects after 28 days' supplementation with a carotenoid-rich berry (Fructus barbarum L.) showed a small but statistically significant (P<0.05) decrease in DNA strand breaks. These data indicate that this buccal cell comet assay is a feasible and potentially useful alternative tool to the usual lymphocyte model in human biomonitoring and nutritional work.

Antioxidants↗

PubMed1997

Measurement of tacrolimus (FK506) and its metabolites: a review of assay development and application in therapeutic drug monitoring and pharmacokinetic studies.

Tacrolimus (FK506, Prograf) is a macrolide immunosuppressant used for the prevention of organ rejection after transplantation. Tacrolimus demonstrates considerable interindividual variation in its pharmacokinetic profile. This has caused difficulty in defining the optimum regimen and has highlighted the need for therapeutic drug monitoring. Several assay methods for the measurements of tacrolimus in biological specimens have been developed. These assay methods were used for therapeutic drug monitoring and/or pharmacokinetic studies. Two commercially available immunoassays, based on the same monoclonal antibody to tacrolimus, have been used for therapeutic drug monitoring of tacrolimus in whole blood. For pharmacokinetic studies, the assay methods were used to measure tacrolimus and its metabolites in very low concentrations in selected biological matrixes to determine the metabolic and pharmacokinetic profiles of this drug.

Biological Assay↗

PubMedDate not supplied

Parathyroid-hormone-related peptide immunochemiluminometric assay. Developed with polyclonal antisera produced from a single animal.

An immunochemiluminometric assay of parathyroid-hormone-related peptide (PTHrP) was developed with purified antisera produced from a single goat immunized with (1-86) PTHrP. One batch of purified antibodies was labeled with acridinium ester used as tracer antibodies; a second batch of the purified antibodies was immobilized onto plastic bead. Sensitivity of the assay was 0.1 pmol/L. The assay had no cross-reactivity with PTH. Seventy-five percent of healthy individuals had undetectable PTHrP (mean +/- S.D. were 0.73 pmol/L +/- 0.6; n = 110). The 95 percent upper reference limit was 2.0 pmol/L. Five of the seven patients with humoral hypercalcemia associated with malignancy (HHM) of solid-tumor had PTHrP greater than 2.0 pmol/L. The test efficacy in detecting HHM was 71 percent, which is similar to previous extraction radioimmunoassay (RIA) or immunoradiometric assay (IRMA). The new assay lowered the detection limit to 0.1 pmol/L, which is a great improvement from that of RIA at 2.0 pmol/L and IRMA at 1.0 pmol/L; however; it did-not improve the test efficacy in detecting patients with HHM. It may indicate the patients with HHM only have elevated plasma PTHrP in 70 percent of them. Technically, the method can be used as a model to develop chemiluminescent esoteric test for research or reference laboratories.

Animals↗

PubMed2004

Performance evaluation of a specific IgE assay developed for the ADVIA centaur immunoassay system.

OBJECTIVE: To develop and evaluate a liquid phase immunoassay for accurate determination of allergen-specific IgE (sIgE) as a useful tool in the diagnosis of allergy patients. DESIGN AND METHODS: A fully automated, quantitative sIgE assay was developed for the ADVIA Centaur technology platform using a unique calibration method based on a recombinant reference allergen. Compared to most other IgE-assays, the assay employs a reverse sandwich architecture using monoclonal mouse anti-human IgE antibody covalently bound to paramagnetic particles in the solid phase and capturing the sample IgE. Bound sIgE reacts with liquid biotin-labeled allergen, which is detected as chemiluminescence using acridiniumester-labeled streptavidin. RESULTS: The ADVIA Centaur sIgE assay (Centaur assay) has exclusive reactivity to human IgE and performs with excellent linearity in the assay range 0.35-100 kU/L and high precision (imprecision within-run <2.6%, between-run <4.9%, and total imprecision <7.1%). The analytical sensitivity is <0.10 kU/L. Using Pharmacia CAP system FEIA (CAP) as a comparative method, positive/negative concordance was 94% at 0.35 kU/L cut-off, and the Centaur assay has a sensitivity of 90% and a specificity of 98%. Validation of the assay in a general population sample (The Copenhagen allergy study) revealed that sIgE was highly associated with a clinical diagnosis of inhalation allergy. CONCLUSIONS: The Centaur assay is an allergen-specific assay for measurement of IgE without interference from other types of immunoglobulins or nonspecific IgE. The assay performs with a linear reaction, high assay range, and good reproducibility. The assay correlates well with the CAP system and is in agreement with clinical diagnosis.

Allergens↗

PubMed2000

Application of imprinted synthetic polymers in binding assay development.

The first part of the review describes a method for the synthesis of molecularly imprinted polymers for use in binding assays. The method considers the many factors involved that affect the recognition properties of the materials and describes an approach to screening and optimization of these factors. The second part describes the development of binding assays using such polymers. This includes the use of different labels, the effect of solvent and buffer, the scale of the assay (amount of solid polymer), and how these influence the quality of the assay in terms of sensitivity, selectivity, and speed of analysis.

Adsorption↗

PubMed2002

Pharmacokinetics of CAMPATH-1H: assay development and validation.

CAMPATH-1H is a humanised monoclonal antibody against the CD52 antigen which is being developed for treatment of chronic lymphocytic leukaemia (CLL), autoimmune disease and prevention of transplant rejection. Measurement of antibody serum levels is important for optimising dose regimens but difficult owing to the low concentration compared with normal human IgG. After consideration of various methods, a suitable assay was developed based on indirect immunofluorescence. Test samples were incubated with target cells (HUT-78, a human T cell line) and the CAMPATH-1H was detected by binding of a fluorescent-labelled anti-human Ig using a flow cytometer. Robustness of the assay was demonstrated under a range of experimental conditions. Because of the low affinity of CAMPATH-1H, only a weak signal was seen at low concentrations. The limit of detection was 0.15 microg/ml and the limit of quantitation was 0.25 microg/ml. Since serum samples were diluted at least 1:2, the lowest concentration which can be measured in patient serum was 0.5 microg/ml. The overall precision (coefficient of variation) was +/-13% and the overall accuracy (bias) was +9%. There was a low incidence of false-positive results (<2%) in normal or pre-treatment patient serum. Quantitative recovery was obtained from serum samples spiked with CAMPATH-1H and stored under a variety of conditions, including being treated at 56 degrees C for 30 min and frozen and thawed up to four times. This validated assay is suitable for the measurement of CAMPATH-1H levels in clinical trials and the same principles may be applied to any other cell-binding monoclonal antibody.

Alemtuzumab↗

PubMed1999

Propofol hydroxylation by dog liver microsomes: assay development and dog breed differences.

Pharmacokinetic studies indicate that clearance of propofol, an anesthetic agent, is slower in greyhounds compared with other dog breeds. Biotransformation of propofol to 2,6-diisopropyl-1,4-quinol (4-hydroxypropofol) by cytochrome P-450 in the liver is proposed as a critical initial step in the elimination of this drug in dogs. Breed differences in the activity of this enzyme could therefore explain pharmacokinetic differences. An in vitro propofol hydroxylase assay was developed and then used to compare enzyme activities in liver microsomes from male greyhound, beagle, and mixed-breed dogs (five each). HPLC of incubate identified only one NADPH-dependent metabolite, which had a chromatographic retention time and UV absorbance, fluorescence, and mass spectra that were identical with authentic 4-hydroxypropofol standard. HPLC with fluorescence detection provided a highly sensitive quantitation method for 4-hydroxypropofol with a quantitation limit of 8 ng/ml using optimized excitation/emission wavelengths (288 nm/330 nm, respectively). Estimates of apparent K(m) and V(max) for propofol hydroxylation by microsomes from a male beagle dog were 7.3 microM and 3.8 nmol/mg/min, respectively. At a substrate concentration of 20 microM, propofol hydroxylase activity was significantly lower (p =.032) in greyhound microsomes (1.7 +/- 0.4 nmol/mg/min) compared with beagle microsomes (5.1 +/- 1.3 nmol/mg/min) but was not statistically different (p =.42) compared with mixed-breed microsomes (3.1 +/- 1.2 nmol/mg/min). These results indicate that there are breed differences in propofol hydroxylase activity and that deficient hydroxylation of propofol by one or more hepatic cytochrome P-450 isoforms may contribute to slow pharmacokinetic clearance of propofol by greyhounds.

Anesthetics, Intravenous↗

PubMed2003

Toward an international standard for PCR-based detection of food-borne thermotolerant Campylobacters: assay development and analytical validation.

As part of a European research project (FOOD-PCR), we developed a standardized and robust PCR detection assay specific for the three most frequently reported food-borne pathogenic Campylobacter species, C. jejuni, C. coli, and C. lari. Fifteen published and unpublished PCR primers targeting the 16S rRNA gene were tested in all possible pairwise combinations, as well as two published primers targeting the 23S rRNA gene. A panel of 150 strains including target and nontarget strains was used in an in-house validation. Only one primer pair, OT1559 plus 18-1, was found to be selective. The inclusivity and exclusivity were 100 and 97%, respectively. In an attempt to find a thermostable DNA polymerase more resistant than Taq to PCR inhibitors present in chicken samples, three DNA polymerases were evaluated. The DNA polymerase Tth was not inhibited at a concentration of 2% (vol/vol) chicken carcass rinse, unlike both Taq DNA polymerase and DyNAzyme. Based on these results, Tth was selected as the most suitable enzyme for the assay. The standardized PCR test described shows potential for use in large-scale screening programs for food-borne Campylobacter species under the assay conditions specified.

Base Sequence↗

PubMed1989

A murine local lymph node assay for the identification of contact allergens. Assay development and results of an initial validation study.

The development of an alternative predictive test for the identification of contact sensitizing chemicals is described. The method is based upon the fact that, following epicutaneous application, sensitizing chemicals initiate a primary immunological response in the draining lymph node(s) which is characterized by lymphocyte proliferation. Experimental conditions for the measurement in vitro of the induced lymph node cell proliferative response have been optimized. On the basis of the data presented a local lymph node assay was developed in which CBA/Ca strain mice were exposed daily, for 3 consecutive days, to various concentrations of the test chemical, or to vehicle alone, on the dorsum of the ear. Lymph node activation was measured subsequently as a function of increased node weight, the frequency of large pyroninophilic cells and lymphocyte proliferation in the presence or absence of an exogenous source of interleukin 2 (IL-2). The results of a validation study are reported in which 22 well-characterized sensitizing chemicals of varying potency were examined. With the exception of three chemicals where water was used as the application vehicle, positive responses, defined as a substantial increase in lymphocyte proliferative activity, were recorded with all these test materials. Under the conditions employed non-sensitizing chemicals, including non-sensitizing irritant chemicals, failed to influence the immunological status of the draining lymph node. Taken together, the data suggest that the local lymph node assay provides the basis for a rapid and cost-effective alternative to the currently available guinea pig predictive test methods. The local lymph node assay may be of particular value for the evaluation of coloured or irritant chemicals.

Allergens↗