Types of Assays

An assay varies in depth and complexity depending upon the crude oil type and its final use. The assay can be an inspection assay or comprehensive assay. There are various types of assays, which vary considerably in the amount of determined experimental information  

Assays that include yields and properties of those streams used as feed for catalytic reforming (naphtha) and catalytic cracking (gas oils) 

The physical and chemical properties of various heavy crude oils with API gravity ranging from 10° to 21° are presented in Table 1.9. It is clearly seen that the heavier the crude oil (lower API gravity), the higher the content of impurities (sulfur, nitrogen, carbon residue, asphaltenes, metals). Viscosity, which is commonly provided 

Physical and Chemical Properties of Heavy Crude Oils 

Total acid number (mg KOH/g) Total nitrogen (wppm) 

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This equation illustrates the components of a competitive protein binding assay system. That is, the reaction system contains both radioactive and non-radioactive free ligand (P and P) and both radioactive and non-radioactive protein bound ligand (P Q and PQ). This type of assay assumes that binding protein will have the same affinity for the labeled or non-labeled material that is being measured. Although this assumption is not always completely valid, it usually causes no problems of consequence with most radioassays or radioimmunoassays. 

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. 

These types of assay are rapid, taking approximately 2 hours, show good precision and are much more specific than microbiological assays. 

Discovery solubility assays can be subdivided depending on how the solubility point is determined. One type of assay measures compound coming out of solu-hon. The other type of assay measures compound in solution. Both methods have advantages and limitahons. 

When compounds are selected for preliminary PK studies, the identification of an appropriate dosing vehicle for iv studies requires solubility studies in various vehicles. Also, the study of thermodynamic solubility is useful as this more closely reflects the environment experienced by compounds on oral dosing. Higher throughput thermodynamic solubility assays have been introduced recently [23] so it will be possible to introduce this type of assay earlier in the discovery process. 

The initial velocity of reaction is defined by the slope of a linear plot of product (or substrate) concentration as a function of time (Chapter 2), and we have just discussed the importance of measuring enzymatic activity during this initial velocity phase of the reaction. The best measure of initial velocity is thus obtained by continuous measurement of product formation or substrate disappearance with time over a convenient portion of the intial velocity phase. However, continuous monitoring of assay signal is not always practical. Copeland (2000) has described three types of assay readouts for measuring reaction velocity continuous assays, discontinuous... 

Fig. 6.20. Types of assays to visualize lipid trafficking in membranes. (A) Self-quenching method. Here the self-quenching is released upon transfer to unlabeled acceptor membranes that are usually in large excess. (B) FRET assays. Here the donor membrane contains a transferable lipid (green) that is quenched by FRET to a non transferable acceptor lipid (red). Upon transfer to an unlabeled acceptor membrane the green-labeled lipid becomes unquenched. 

Measuring FRET by fluorescence lifetime imaging microscopy (FRET-FLIM) offers the ability to see beyond the resolution of the optical system ( 10-100 times that of modern far field microscopes [5]). FRET efficiency can be used as a proxy for molecular distance, thereby allowing the easy detection and somewhat more challenging quantification of molecular interactions. Although many types of assay exist, FRET-FLIM is a highly suitable technique that is capable of in situ measurements of molecular interactions and conformation in living and fixed cells. 

Preparation of a series of phycobiliprotein tandem dyes allows multiplexed analysis of different targets in a sample. In addition, since RPE can be excited by the argon-ion laser at 488 nm, a fluorescein-labeled probe can be used concurrently with RPE alone and RPE-tandem conjugates to create a multiplexed system of different fluorescent probes that can be used simultaneously. Table 9.3 shows the different combinations of dyes that can be used in this type of assay with RPE and APC. 

Dyed particles also are commonly used in diagnostic lateral flow tests (like the common home pregnancy test), as the colors can be seen with the eye without the need for special detectors. In this type of assay, antibodies or antigens are coupled to the dyed particles and a sample solution applied to the test strip carries them along within a membrane. The particles then are captured at points in the membrane that represent either a control or a positive sample result. Large numbers of color particles docking at these points within the membrane create the visual lines associated with these disposable tests. 

A comparison of the blank corrected values before and after conjugation should give an indication of the percent of peptide coupled. To be more quantitative, a standard curve must be run to focus in on the linear response range of the peptide-Ellman s reaction. Using cysteine as a representative sulfhydryl compound (similar in Ellman s response to a peptide having one free sulfhydryl), it is possible to obtain very accurate determinations of the amount which coupled to the activated carrier. Figure 19.20, discussed previously in this section, shows the results of this type of assay. 

Other molecules can be used in this type of assay approach. Hydrazide-modified (strept)avidin, lectins, biocytin, fluorescent probes and other detectable molecules can be used to detect specifically glycoconjugates in biological samples (Wilchek and Bayer, 1987). 

One of the most popular bioassay for interferons is termed the cytopathic effect inhibition assay . This assay is based upon the ability of many interferons to render animal cells resistant to viral attack. It entails incubation of the interferon preparation with cells sensitive to destruction by a specific virus. That virus is then subsequently added, and the percentage of cells that survive thereafter is proportional to the levels of interferon present in the assay sample. Viable cells can assimilate certain dyes, such as neutral red. Addition of the dye followed by spectrophotometric quantitation of the amount of dye assimilated can thus be used to quantitate percentage cell survival. This type of assay can be scaled down to run in a single well of a microtitre plate. This facilitates automated assay of large numbers of samples with relative ease. 

Biology and biochemistry laboratories perform three general types of assays ... 

This type of assay is known as direct method of iodimetry . 

As it has been discussed earlier, iodine cannot be used directly as an oxidizing agent in such type of assays, whereas the liberated iodine quantitatively produced by the oxidation of iodide with bromine (excess) may be assayed by titrating against sodium thiosulphate solution. 

A competitive ELISA assay for Lp(a) was recently described (Y4) in which the microtiter plate was coated with Lp(a) purified from a pool of donors. The method is simple and easy to perform, with satisfactory analytical parameters. A good stability and a reproducible coating of plates with the large Lp(a) lipoprotein is, however, critical in this type of assay. Wang et al. (W6) described an indirect sandwich assay for the measurement of Lp(a) in plasma and in dried blood spots, which can be applied to screening elevated Lp(a) levels in newborns (V3, V4). 

Baldo-Enzi (Bl) compared the results of five different commercial immunoassays (a two-site RIA, an endpoint nephelometric assay, and three different ELlSAs) for freshly drawn serum and for plasminogen-free fractions (obtained by one-step ultracentrifugation). He observed a variable influence of plasminogen on the results of the Lp(a) determination and found no comparability between the five types of assays. This was partly due to lack of standardization. The different assays were more comparable when the plasminogen-free fraction was used. 

If the product is an antibody, then it is essential to distinguish the immunoglobulin product, e.g., mouse IgG, from any media immunoglobulin components, e.g., bovine IgG. Lucas et al.16 developed an immunoassay to measure nanogram quantities of bovine IgG in the presence of a large excess of a structurally homologous protein, mouse MAb. The bovine IgG was a contaminant that copurified with the product from a protein A column. For the bovine IgG assay, whole IgG and protein A-purified IgG reacted differently in the assay. It is important to evaluate these types of assays for cross-reactivity. For other media components, such as chemicals or antibiotics, ELISA is probably not the most appropriate method due to the low immunogenicity of chemicals. Techniques such as HPLC would be better to detect these chemical components. 

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