Changes In Assay Conditions Research Articles

Principles of Analytic Validation of Immunohistochemical Assays: Guideline Update.

In 2014, the College of American Pathologists developed an evidence-based guideline to address analytic validation of immunohistochemical assays. Fourteen recommendations were offered. Per the National Academy of Medicine standards for developing trustworthy guidelines, guidelines should be updated when new evidence suggests modifications. To assess evidence published since the release of the original guideline and develop updated evidence-based recommendations. The College of American Pathologists convened an expert panel to perform a systematic review of the literature and update the original guideline recommendations using the Grading of Recommendations Assessment, Development and Evaluation approach. Two strong recommendations, 1 conditional recommendation, and 12 good practice statements are offered in this updated guideline. They address analytic validation or verification of predictive and nonpredictive assays, and recommended revalidation procedures following changes in assay conditions. While many of the original guideline statements remain similar, new recommendations address analytic validation of assays with distinct scoring systems, such as programmed death receptor-1 and analytic verification of US Food and Drug Administration approved/cleared assays; more specific guidance is offered for validating immunohistochemistry performed on cytology specimens.

Method-induced variation in the bacterial cell surface hydrophobicity MATH test

Bacterial cell surface hydrophobicity is a relevant property in determining the ability of bacteria to adhere to inert surfaces. This property has been measured using the microbial adhesion to hydrocarbon (MATH) test. Several reports in the literature establish the percentage of adhesion to hydrocarbons (PoAtH) value produced by the MATH test for a broad variety of bacteria. Discrepancies in PoAtH values reported for the same strain of a specific microorganism suggest that some method-induced variation may exist, as different research teams employ different versions of the assay. The objective of the present study was to compare the performance of different versions of the MATH test as reported in the literature, to quantify the magnitude of the method-induced variation on PoAtH values. The study demonstrated that PoAtH values are influenced twice as much by variations in the employed assay than by actual differences in cell surface composition or architecture. The two L. reuteri strains studied responded differently to changes in assay conditions showing 40 and 70% method-dependent variation for strain ATCC 53609 and 55730, respectively. These results highlight the need to properly standardize the MATH test to enable comparison of PoAtH values produced by independent research teams.

Characterisation of an in vivo Pig-a gene mutation assay for use in regulatory toxicology studies

Integration of in vivo genotoxicity testing into standard toxicology studies presents multiple advantages as it reduces animal use and costs, accelerates data generation and provides concurrent data that are useful for interpreting results. The in vivo Pig-a assay is a mammalian gene mutation assay that utilises peripheral blood and thus has a high integration potential. Although inter-laboratory reproducibility has been well demonstrated, further characterisation is required for this assay. In this study, we evaluated intra-laboratory reproducibility of the in vivo Pig-a gene mutation assay (MutaFlow® kit) in rats through the conduct of an assay characterisation prior to subsequent use in Good Laboratory Practices (GLP) toxicology studies. To evaluate intra-laboratory reproducibility, intra-assay and inter-assay variability, ruggedness, robustness and blood storage stability were assessed. These assessments were performed using blood obtained from male Sprague-Dawley rats exposed to 0, 20 or 40mg/kg/day N-ethyl-N-nitrosourea via oral gavage for three consecutive days. Blood was collected from these rats on multiple occasions from Day 29 to Day 71 and samples were analysed for Pig-a mutation using the rat MutaFlow kit. Frequencies of reticulocytes (RET), mutant phenotype erythrocytes (RBC(CD59-)) and mutant phenotype RET (RET(CD59-)) were determined. Overall, the proportion of RET and frequencies of RBC(CD59-) and of RET(CD59-) were consistent throughout the different assessments. The assay demonstrated acceptable intra-run and inter-run variability with coefficients of variation of ≤4.8 and 20.6%, respectively. The method was shown to be independent of the analyst performing the assay and unaffected by small changes in assay conditions. Comparable results were obtained from freshly collected samples and samples refrigerated for up to 4 days. Although technically challenging, the rat Pig-a gene mutation assay using a high-throughput automated method was shown to be reliable. The different assay parameters evaluated during the conduct of this study yielded acceptable results. Thus, the method was considered suitable for use in GLP toxicology studies.

Characterization of Human Flavin-Containing Monooxygenase (FMO) 3 and FMO5 Expressed as Maltose-Binding Protein Fusions

The flavin-containing monooxygenase (FMO) family of enzymes oxygenates nucleophilic xenobiotics and endogenous substances. Human FMO3 and FMO5 are the predominant FMO forms in adult liver. These enzymes are naturally membrane-bound, and recombinant proteins are commercially available as microsomal preparations from insect cells (i.e., Supersome FMO). As an alternative, FMO3 has previously been expressed as a soluble protein, through use of an N-terminal maltose-binding protein (MBP) fusion. In the current study, MBP fusions of both human FMO3 and FMO5 were prepared to >90% purity in the presence of detergent and characterized for biochemical and kinetic parameters, and the parameters were compared with those of Supersome FMO samples. Although MBP-FMO enzymes afforded lower rates of turnover than the corresponding Supersome FMOs, both types of FMO showed identical substrate dependencies and similar responses to changes in assay conditions. Of interest, the FMO3 enzymes showed a 2-fold activation of k(cat)/K(m) in the presence of Triton X-100. Oligomeric analysis of MBP-FMO3 also showed disassociation from a high-order oligomeric form to a monomeric status in the presence of Triton X-100. This report serves as the first direct comparison between Supersome FMOs and the corresponding MBP fusions and the first report of a detergent-based activation of k(cat)/K(m) that corresponds to changes in oligomerization.

IMPORTANCE OF pH ON ANTIBROWNING ACTIVITY OF OXALIC ACID

The importance of maintaining stable pH when studying antibrowning activity of oxalic acid in a catechol-polyphenol oxidase model system was demonstrated. Enzyme inhibition in the reaction medium showing a decrease in pH caused by the addition of increasing oxalic acid concentrations was compared with the inhibition of the same oxalic acid concentrations at a stable pH of 5.0. Relative antibrowning activity of oxalic acid was significantly lower at stable pH conditions than at variable pH conditions. The main reason for this was because of the very slight alterations around the pH used for the experiments, which were sufficient to induce significant differences in the catalytic activity of the enzyme. Oxalic acid at pH 5.0 was found to be a noncompetitive inhibitor of the mixed-type with an inhibitor constant (Ki) of 0.8 mM. PRACTICAL APPLICATIONS Fruits and vegetables are prone to polyphenol oxidase (PPO)-catalyzed browning reactions during handling and processing, and the food industry is interested in methods to prevent browning for minimally processed crops. Although not an approved food additive, oxalic acid is a common component of several foods from plant origin. There are few reports on the inhibition of PPO by oxalic acid in reaction model systems. The importance of maintaining a stable pH when studying the antibrowning activity of oxalic acid in a catechol-PPO model system was demonstrated. Small additions of oxalic acid into the assay system induced significant pH variation. Minute changes in the degree of acidity severely affected PPO activity and resulted in significant errors in measuring inhibition. Identifying how environmental changes in assay conditions affect activity and inhibition are important in interpreting how this enzyme is controlled on foods by inhibitors.

Detection of influenza virus resistance to neuraminidase inhibitors by an enzyme inhibition assay

We previously characterized influenza viruses whose selection in the presence of neuraminidase (NA) inhibitors resulted in a substituted residue (position 119, 152, 274, or 292) in the NA active center. To identify the most favorable conditions for detecting NA inhibitor-resistant viruses we compared the results of four modifications of the NA inhibition assay utilizing a fluorogenic substrate. The IC 50 values were highly dependent upon assay conditions, and most mutant enzymes were more sensitive to changes in assay conditions (e.g. addition of PO 4 3−, Ca 2+, DMSO, or EDTA) than wild-type enzymes or a mutant NA with an Arg292 → Lys substitution. Although the levels of resistance to zanamivir, oseltamivir carboxylate, and BCX-1812 (RWJ-270201) for each mutant varied among assays, mutants with substitutions at framework residues 119 or 274 exhibited sensitivity to at least one inhibitor. Viruses with substitutions at catalytic residues 152 or 292 were resistant to each inhibitor in all assays. Monitoring resistance in a clinical setting will require a panel of resistant viruses to ensure that assay conditions are favorable for detecting variants with substituted residues in the NA active center. Because variants selected in the presence of one NA inhibitor could be variably resistant to other inhibitors, all three inhibitors should be used in drug susceptibility testing.

Ornithine decarboxylase assay in human colorectal mucosa. Methodologic issues of importance to quality control.

Ornithine decarboxylase may be a useful biomarker for risk of neoplasia in colorectal tissues. Investigators have reported enzyme activities varying by as much as 10- to 20-fold using variations of the usual 14CO2 release assay. We have examined the effect of different methodologic factors on calculated ornithine decarboxylase activity. Major effects on the assay result (greater than 20% change) were produced by: (1) use of Tris vs. phosphate buffer, the former yielding 1.5- to 4-fold greater activity; (2) protein content of the reaction mixture with significant error if less than 50 micrograms; (3) use of alpha-difluoro-methylornithine-inhibited blank versus buffer-only blank. Other changes in assay conditions, including addition of sucrose, detergent, protease inhibitors, specific activity of 14C-ornithine, the nature of the trapping agent used, and incorporation of a sonication step, did not have a significant effect on ODC quantification (less than or equal to 20%). Thus, seemingly minor variations in assay conditions can greatly affect the results, which may provide a partial explanation for the variability of ODC activities reported in the literature. Strict quality control measures are mandatory in the interpretation of clinical observations utilizing this marker as an endpoint.

Lung cytochrome P450-dependent benzyloxyphenoxazone debenzylase and ethoxyphenoxazone deethylase activities in total microsomal and isolated alveolar type II cells: Responses to changes in assay conditions with special reference to non-linear dependence at low enzyme concentration

1. 1. Responses of cytochrome P450-dependent ethoxyphenoxazone deethylase (EtOPx'ase) and benzyloxyphenoxazone debenzylase (BzOPx'ase) activities to changes in assay conditions were measured in total microsomal and isolated alveolar type II (t 11) cells from rats pretreated with β-naphthoflavone. 2. 2. Whereas microsomal EtOPx'ase activity was unaffected by storage at −80°C for up to 4 months, BzOPx'ase activity began to decline after only 1 month. 3. 3. The microsomal and type II activities were unaffected by changes in pH (7.2–8.0) or salt content. 4. 4. The type II activities increased after sonication 2.3–2.7-fold or in the presence of 10 μM dicumarol 1.7–1.9-fold. 5. 5. Type II BzOPx'ase was sensitive to metyrapone (MP) whereas EtOPx'ase was sensitive to α-naphthoflavone (ANF). I 50, values for the tu activities were calculated as: 0.63 μM—MP (BzOPx'ase), 80 μM—MP (EtOPx'ase), 0.024 μM—ANF (EtOPx'ase). At the highest concentration of ANF (10 μM), 50% inhibition t 11, BzOPx'ase was not observed. The results were similar to those obtained with the total lung microsomal fraction. 6. 6. Microsomal and t 11 BzOPx'ase activities exhibited non-linear dependence at low enzyme concentration. Linearity was restored by 0.5 mM dimyristoylphosphatidylcholine.

Mechanisms of aminoacyl-tRNA synthetases: a critical consideration of recent results.

During the last 10 years intensive and detailed studies on mechanisms and specificities of aminoacyl-tRNA synthetases have been carried out. Physical measurements, chemical modification of substrates, site-directed mutagenesis, and determination of kinetic parameters in misacylation reactions with noncognate amino acids have provided extensive knowledge which is now considered critically for its consistency. A common picture emerges: (1) The enzymes work with different catalytic cycles, kinetic constants, and specificities under different assay conditions. (2) Chemical modifications of substrates can have comparable influence on catalysis as can changes in assay conditions. (3) All enzymes show a specificity for the 2'- or 3'-position of the tRNA. (4) Hydrolytic proofreading is achieved in a pre- and a posttransfer process. In most cases pretransfer proofreading is the main step; posttransfer proofreading is often marginal. (5) Initial discrimination of substrates takes place in a two-step binding process. For some investigated enzymes, initial discrimination factors were found to depend on hydrophobic interaction and hydrogen bonds. (6) The overall recognition of amino acids is achieved in a process of at least four steps. At present, only a rough overall picture of aminoacyl-tRNA synthetase action can be given.

Lymphokine regulation of macrophage effector activities.

Our concept of the regulation of macrophage activation is ever expanding and contracting. In regard to the number of LK that regulate macrophages killing activities, we have entered a new phase. In the beginning there was one macrophage activation factor, MIF; then there were many macrophage activation factors, most uncharacterized and bearing a variety of names. Then came IFN, a genetically cloned single reagent that induced destruction of virtually every target assessed; all activities of macrophages were assumed to be regulated by IFN. Once again, however, the LK universe is expanding: the number of single, cloned reagents that induce macrophage killing activities is amazing. With just two targets, a fibrosarcoma cell and an intracellular amastigote of L. major, we can identify 5 different macrophage activation factors, four of which are cloned and sequenced. As more recombinant reagents become available, the story of macrophage activation is likely to become even more complex. It is fascinating not only that certain of the LK are capable of inducing single effector reactions in the absence of effects on other effector activities, but also that at least one effector reaction requires the cooperation of several molecularly distinct LK. The complexity of LK activation factors that regulate a single effector reaction in vitro is compounded by the complexity in effector cell populations. For example, inflammatory macrophages exposed to LK kill the fibrosarcoma tumor target 5 to 10-fold better than an equal number of resident peritoneal macrophages. In contrast, LK treated resident macrophages eliminate intracellular amastigotes of leishmania far more efficiently than inflammatory cells. Thus, changes in cell populations dramatically affect the capacity to demonstrate a single effector reaction. Further, simple changes in assay conditions also determine whether an effector reaction can be observed in vitro. And superimposed upon all these layers of complexity is the target itself. The mechanisms a macrophages uses to block the replication of a virus may be totally ineffective in the destruction of a multicellular helminth, such as Schistosoma mansoni. And there is no reason to suspect that the extracellular destruction of a tumor target occurs by the same means that the macrophage uses to kill an intracytoplasmic bacterium, such as a rickettsia.(ABSTRACT TRUNCATED AT 400 WORDS)

Investigations into the association of retroviruses with ovine lymphoma

To determine whether retroviruses are associated with sporadic ovine lymphoma, suspension cell cultures of four lymphomas and one control lymph node were labelled with tritiated uridine. Following differential and sucrose density gradient ultracentrifugation of their media a peak of radioactivity was found at a density of 1.15 to 1.18 g ml-1 in preparations from the cell cultures of two lymphomas and the normal lymph node. Sedimentation velocity centrifugation of the sodium dodecyl sulphate-treated radiolabelled material found an approximate sedimentation value of 7S. The assay for RNA directed DNA polymerase in ultracentrifuged pellets of media from cultures of 15 lymphomas and one control lymph node found activity in material from four lymphomas and the control node cultures. However, little variation in incorporation kinetics occurred with changes in assay conditions and activities were not associated with particles of density 1.15 g ml-1. It was concluded that the detected activities were not retroviral in origin.

Delayed induction by phenobarbital of udp-glucuronyltransferase activity towards bilirubin in fetal liver.

Phenobarbital pretreatment of pregnant mice (ASH/TO strain) gave rise to approximately equal concentrations of phenobarbital in both maternal and fetal liver. This pretreatment resulted in increased UDP-glucuronyltransferase (GT) activity towards bilirubin in maternal and neonatal liver in fetal liver on days 19 and 20 but in livers from earlier (15-18 day) fetuses GT either was not significantly increased or remained undetectable. Fetal liver is thus not competent to respond to phenobarbital by increasing its GT activity, until just before birth. This pattern persisted through changes in assay conditions and is contrasted with that occuring in embryos free from maternal influence. GT from adult and neontal liver is activated by 0.2 per cent digitonin; in fetal liver this response also does not appear until day 19.