Setting up an assay

Alternatively, the whole reaction may be carried out in solution (figure 4B) and the antibody-antigen complexes subsequently precipitated from solution The separation out of solution of antibody-antigen complexes can be achieved 

Step 1 coat first antibody onto solid support wash away unbound antibody. 

Step 2 add labelled (fixed amount) and unlabelled antigen, which compete for first antibody. Wash away unbound antigen. Detect activity of bound labelled antigen. 

Step 1 mix labelled antigen with standard/sample unlabelled antigen. 

Allow labelled and unlabelled antigen to compete for antibody binding sites. 

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Abstract Neuroscientists may wish to quantify an enzyme activity for one of many reasons. In order to do so, the researcher must be able to set up an assay appropriately, and this requires some understanding of the kinetic behavior of the enzyme toward the substrate used. Furthermore, such an understanding is vital if the inhibitory effects of a drug are to be assessed appropriately. This chapter outlines key principles that must be adhered to, and describes basic approaches by which rather complex kinetic data might be obtained, in order that enzyme kinetics and inhibitor kinetics might be studied successfully by the nonexpert. 

Set up an assay tube with varying concentrations of the substrate. 

In setting up an assay, some thought should be given to the incubation volume, since the type of injector may necessitate larger volumes than might be available. 

Dr Mark Murcko of Vertex Pharmaceuticals provides an example of how this might work for a real-world project In the area of kinases or ion channels or GPCRs we ve already worked on other members of the family, he notes. We already have some know-how about how to set up an assay in the field. We may already have useful compounds—not drug candidates, perhaps—but pretty decent starting points. Let s say we read a paper in Nature that suggests that kinase X is a great target for cancer. But it s speculative. 

Since liver (biopsy) material is not very convenient for diagnosis we have now set up an assay allowing PhyH activity measurement in skin fibroblast homogenates. This assay is ba on a gas chromatography / mass spectrometry (GC-MS) detection method (Jansen et al, manuscript in preparation). Using this assay we have measured PhyH activ-... 

One issue related to supporting a metabolic stability assay with HPLC/MS/MS is the need to set up an MS/MS method for each compound. While it may only take 10 min to infuse a compound solution and find the corresponding precursor and product ions (along with minimal optimization of the collision energy), the processes of MS/MS development would require 4 hr per day if one wanted to assay 25 compounds per day. MS vendors have responded to this need by providing software tools that can perform the MS/MS method development step in an automated fashion. Chovan et al.68 described the use of the Automaton software package supplied by PE Sciex (Toronto, Canada) as a tool for the automated MS/MS method development for a series of compounds. The Automaton software was able to select the correct precursor and product ions for the various compounds and optimize the collision energy used for the MS/MS assays of each compound. They found that the Automaton software provided similar sensitivity to methods that would have been developed by manual MS/MS procedures. Chovan et al. also reported that the MS/MS method development for 25 compounds could be performed in about an hour with the Automaton software and required minimal human intervention. 

Introduction. Our aim is to obtain the physicochemical background in order to be able to set up an absolute viscosimetric method for the assay of endocellulases in enzyme mixtures, using a buffered HEC substrate and giving the definition of enzymic activities in katals, according to the recommendations of the International Union of Biochemistry. 

This chapter presents a strategy for the design of an HPLC assay system. Section 4.1, Setting up the Assay, focuses on the enzymatic reaction and the steps leading to the development of the assay. These steps are previewed in Table 4.1. Section 4.2, The Use of HPLC to Establish Optimal Conditions for the Enzymatic Reaction, discusses the procedure for monitoring the activity of an enzyme with the HPLC method and the use of HPLC assays to determine the parameters required for obtaining optimal activity. 

Setting up an HPLC assay for activities on intact cells requires only one major change since the reaction mixture will contain cells, the samples for HPLC analysis cannot be injected directly onto the analytical column thus the reactions must be terminated and any precipitated proteins removed (see Chapter 4). Termination can be accomplished by centrifugation at a low speed or by filtration (see Fig. 4.9B). However, care should be taken to avoid any cell breakage, particularly if the product of the primary or even secondary reactions can also be found as a naturally occurring intracellular component. 

One measure of success is anecdotal At more than one scientific meeting, graduate students and colleagues have told me how pleased they were with the book. By following the directions provided, they have been able to set up HPLC assays, and they have found the book most useful as a reference source. A more quantitative measure would be the number of investigators who rely on HPLC to monitor enzymatic activities. If we use publications as a measure, we find that the 1987 edition reported HPLC assays of 62 activities. In contrast, the 1998 edition cites 169 activities involving assay by HPLC. Thus, 11 years brought an almost threefold increase in the number of activities assayed by HPLC. 

The first consideration when setting up an AlphaScreen assay is the choice of an assay format In most cases, the decision will depend on the interaction partners under investigation and on the biological tools available for their detection. The interaction partners can be coupled to the beads directly via reductive amination of reactive aldehyde groups, similar to the immobilization on a Biacore sensor chip (see above). The usefulness of this approach is limited by the reaction conditions, which may not be appropriate for maintaining the biologically active conformation of the biomolecule. Therefore the biomolecule of interest is usually not coupled to the beads directly, but instead captured via an antibody, also preventing steric hindrance. While not strictly necessary, it is often convenient to use a biotinylated molecule which can be captured by streptavidin-coated donor beads. 

In the so-called direct assay format, a biotinylated component binds to an antibody directly coupled to acceptor beads or to a protein captured by this antibody (Figure 8.4A). In the indirect assay format, the antibody used for capturing the biomolecule is in turn bound by a secondary antibody or by Protein A conjugated to the acceptor beads. In principle, any method capable of capturing the interaction partners to donor and acceptor beads, respectively, is suitable for setting up an AlphaScreen assay. 

Assume 06-MeG content to be 6% of total cpm and set up ATase assay using up to 10-fold excess of ATase (see Subheading 3.5.). The plateau level will give an accurate measure of 06-MeG content (calculate from specific activity... 

It is essential that test-kits are used for the intended purpose in a specific matrix. Thus test-kits can limit the applications in which the technology can be applied. In some cases, it will be necessary to set up an in-house assay format for which it will be necessary to buy the separate individual immunoreagents, such as antibodies, enzyme-tracer or analyte-derivative protein conjugates. In some cases this is difficult because the quality and supply are not always guaranteed. A recent list of antibody manufacturers, suppliers and services is given by Blow, 2007. 

For the reductive cleavage of SAM into methionine and the deoxyadenosyl radical (DOA ), an electron has to be transferred from a reduced [Fe-S] cluster. The reducing system consisting of NADPH, flavodoxine, and flavodoxine reductase has been identified in E. This allowed to set up an in vitro assay, which besides the... 

Prior to P450 reaction phenotyping or inhibition experiments, it is important to determine enzyme kinetic parameters such as Km and Umax for the formation of selected metabolites that are subjected to quantitative analysis by LC-MS. For example, -warfarin is catalyzed by CYP2C9 to a specific metabolite, 7-hydroxy-5 -warfarin (Fig. 15.13). Thus, a CYP2C9 inhibition assay is developed based on the reaction. In the assay, Y-warfarin is incubated with HLM in the presence of a test compound, followed by quantification of 7-hydroxy-5 -warfarin by LC—MS (Zhang et ah, 2001). To set up this assay in our lab, enzyme kinetics for the formation of 7-hydroxy-iS-warfarin in HLM was determined. In this experiment, warfarin was incubated at concentrations from 0 to 250 >M with HLM at optimized conditions. Rates of 7-hydroxy-S -warfarin formation at various substrate concentrations were determined as shown in Figure 15.13a, from which Km and Umax values were calculated. The warfarin assay represented an analytical challenge since the turnover of warfarin in the HLM system was extremely low. To be able to quantitatively determine low concentrations of 7-hydroxy-5 -warfarin in the incubations, a very sensitive LC—MS method that used MRM with a 4000 QTRAP has been developed (Fig. 15.13a). 

An additional major problem for any laboratory desiring to set up this assay is the lack of easily available positive controls. The only positive controls that are currently available are the blood or bronchoalveolar cells from subjects known to be positive. Because there are very few such persons, the availability of this material is limited. Therefore it is recommended that samples for beryllium proliferation testing only be sent to a laboratory with experience in beryllium testing. Since it is important that the cells arrive alive and in good condition, direct contact with the laboratory should be made before a sample is sent. A list of laboratories that are currently performing a beryllium proliferation assay can be obtained from the Medical Department at Brush Wellman, Inc., 17876 St. Clair Avenue, Cleveland, Ohio 44110-2697, USA. 

Assay of Enzymes In body fluids, enzyme levels aie measured to help in diagnosis and for monitoiing treatment of disease. Some enzymes or isoenzymes are predominant only in a particular tissue. When such tissues are damaged because of a disease, these enzymes or isoenzymes are Hberated and there is an increase in the level of the enzyme in the semm. Enzyme levels are deterrnined by the kinetic methods described, ie, the assays are set up so that the enzyme concentration is rate-limiting. The continuous flow analyzers, introduced in the early 1960s, solved the problem of the high workload of clinical laboratories. In this method, reaction velocity is measured rapidly the change in absorbance may be very small, but within the capabiUty of advanced kinetic analyzers. 

Assays are usually set up according to the instructions provided with the reagents. These directions are usually very detailed and should be referred to in establishing an assay. However, the following illustrates the most common type of assay and may be helpful in explaining some of the various nomenclature used in the field of competitive protein binding assays. 

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