GOOD assay

The GOOD assay is a single tube procedure. The five reaction steps are done without transferring the samples to new reaction vials. Reagents are simply added to the reaction and the samples either placed in an incubator or thermocycler. This makes it amenable for automation. 

The main benefit of the GOOD assay is that the introduction of sensitivity enhancing chemistry circumvents the need to purify and concentrate the products prior to MALDI analysis, making it very economical for SNP genotyping. 

The GOOD assay with negative ion-mode detection is easily accessible. The required primers can be obtained from most manufacturers of ohgonucleotides. The (jOOD assay with positive ion-mode detection, on the other hand, gives options for further derivatisations of the product oligomer. 

For the GOOD assay in the positive ion mode version, amino-modified phosphoramidites A , and are integrated into the extension 

The ability to multiplex reactions is of great importance for reducing the cost per SNP genotype and analysis time. Multiplexing at the PCR level and of the primer extension reaction is currently under investigation. The quality of the enzymatic reactions in a multiplex assay depends on the SNPs that have to be combined. The surrounding DNA sequences have an profound influence on the quality of the PCR reaction. 

---

Immunoassays employ monoclonal or polyclonal antibody preparations (Chapter 13) to detect and quantify the product (Box 7.1). The specificity of antibody-antigen interaction ensures good assay precision. The use of conjugated radiolabels (RIA) or enzymes (EIA) to allow detection of antigen-antibody binding renders such assays very sensitive. Furthermore, when compared with... 

To find a good assay for the desired pharmaceutical target is the critical step for successful HTS. Let us take a protein as an example for a target. Only substances that bind to protein can have physiological activity they provide a hit. Substances that do not bind fail the test and are no longer considered for this application. 

Two important characteristics of any assay method are its accuracy and its reproducibility. Accuracy is how close an estimate is expected to be in relation to the true value for the specimen. Reproducibility relates to how repeated estimates of the same specimen vary about their average value. Both accuracy and reproducibility are usually defined for a given concentration and may differ between low and high specimen concentrations. In a good assay method, these differences should be inconsequential across the working range of the method. 

Performing a system suitability test each day provides a good assay reference. 

Immunoassays are very versatile, and if one could select but a single method, it could be the method of choice. Fortunately we have a variety of techniques available and a good analyst should know when to apply them. Table I provides some general rules for determining how difficult an immunoassay will be. The terms used are relative and possibly other dimensions to the table could be the laboratory s experience with immunoassay and the problems faced. This table does not indicate that good assays cannot be developed for hard compounds it just Indicates that the expense, skill and time required may be greater for those compounds. For instance we have developed successful immunoassays for some lipophilic, small, unstable, volatile compounds. However, such compounds would be a poor choice to use for one s first venture into immunoassay development. 

Several assays have been patented for mutation detection these are mainly designed for single-nucleotide polymorphism (SNP) analysis and use MALDI-TOF spectrometry (Invader , Sequazyme-PinPoint assay, MassARRAY , GOOD assay) all of which use PCR amplification, or require a high DNA concentration in the sample (Invader ). 

In FP assays, the change of the polarization AP (highest minus lowest signal) is important. A AP of > 150 mP is a robust assay and 100 mP is considered as a good assay, but below 100 mP the z factor is usually not acceptable. 

Good assay reproducibility and recovery were observed for neat (undiluted) normal human serum and serum from rheumatoid arthritis patients for quantitation of a fully human anti-TNF-a monoclonal antibody [86]. It has also been reported that excess therapeutic antibodies present in serum were tolerated better in an assay for the detection of antitherapeutic antibodies based on the MSD ECL device [91]. Furthermore, the ECL procedure has been reported to be stable over a broad range of magnetic bead concentrations, probe concentrations, and hybridization conditions for a nucleic acid binding assay, making these assays more versatile and easier to transfer from one laboratory to another [88]. 

SNP genotyping by MALDI-TOF MS takes advantage of mass differences between allele-specific primer extension products. At present, three related assays are used, the PROBE— primer oligo base extension assay, which was further developed to the MassExtend assay by SEQUENOM— the PinPoint, and the GOOD assay (8). A representative scheme is depicted in Fig. 1. 

As the third step of the GOOD assay is a primer extension with a specifically tailored set of a-S-dNTPs and a-S-ddNTPs, dNTPs of the PCR have to be removed. This is done enzymatically by degradation of the dNTPs in the second step by shrimp alkaline phosphatase (SAP). 

Figure 2. Flowchart of the GOOD assay. SAP stands for shrimp alkaline phosphatase digestion, PE for primer extension, PDEfor phosphodiesterase. Alkyl for alkylation and MS...

Figure 4. Principle of the GOOD assay. CT synibolises the charge-tag, N" a DNA base and N a quartemary ammonium.

---