ELISA Subject

Biological techniques, e.g., immunoassays, are among the most sensitive analytical methods, but are limited by the availability of the specific antisera and are subject to cross-reactivity. Huang et al. [36] employed an enzyme-linked immunosorbent assay (ELISA) for determination of estradiol, its conjugates, and ethynylestradiol in wastewaster treatment plant effluents (see Table 4). The reported limit of detection (LOD) of 0.1 ng L 1 reflects the sen-... 

Fig. 7. Frequency distribution of plasma Lp(a) concentrations (a) and apo(a) alleles (b) in three populations. Plasma Lp(a) levels were measured in 381 subjects by the sandwich ELISA technique apo(a) allele size (number of K-4 repeats) was estimated using pulsed-field gel electrophoresis and genomic blotting. [With permission of Gaw et at. (G11).]...

The ELISA can be used for identification and quantitation of the protein product (biopharmaceutical) of interest throughout the development, production, and manufacturing process. For example, in the initial development phase, ELISAs can aid in the selection of the best cell line. In the early manufacturing steps, it can be used to identify the appropriate product-containing pools or fractions in process to be subjected to further purification. Because of the selectivity of ELISA, it is a suitable tool to select out the protein of interest from complex protein mixtures, such as cell culture fermentation media or product pools in early steps of protein recovery as well as downstream processing. Even complex mixtures do not require much sample preparation. It is important to determine... 

ELISA is a method that is simple to perform. Ferris and Fischer28 evaluated the performance of subjects with no formal laboratory training or experience (sixth and seventh graders), and this sample of analysts were able to perform the ELISA with success. 

Patients and blood donors are routinely screened for exposure to HIV by means of ElISA and Western blot assays of blood samples (F uie 1-7-15). The assays are designed to detect antibodies to HIV in the blood of the test subject The ELISA is used as the primary screening assay because it is very sensitive. Because the reference interval for the test is set to include everyone with antibodies to HIV, it also gives false positives and thus has a rather low positive predictive value, especially in low-risk populations. The Western blot (or immunoblot) is used as the confirmatory test for HIV exposure. In the Western blot technique, specific HIV proteins are separated by gel electrophoresis and blotted to a filter. The filter is incubated with the test sample. If the sample contains antibodies to HIV, they will bind to the proteins on the filter. The filter is next washed and incubated with a labeled goat anti-human IgG to visualize any bound human antibodies. The Western blot is highly specific. The combination of an ELISA and Western blot has a positive predictive value of greater than 99%,... 

In this chapter, we will survey the kinds of solid supports (substrates) and surface chemistries currently used in the creation of nucleic acid and protein microarrays. Which are the best supports and methods of attachment for nucleic acids or proteins Does it make sense to use the same attachment chemistry or substrate format for these biomolecules In order to begin to understand these kinds of questions, it is important to briefly review how such biomolecules were attached in the past to other solid supports such as affinity chromatography media, membranes, and enzyme-linked immxm-osorbent assay (ELISA) microtiter plates. However, the microarray substrate does not share certain unique properties and metrics with its predecessors. Principal among these are printing, spot morphology, and image analysis they are the subjects of subsequent chapters. 

The source and quality of the lymphocytes and the antibody titer of the desired antibodies within an individual will influence the difficulty of producing antibodies from a library derived from that individual. The sources of lymphocytes that have been used successfully to produce human antibodies of the same specificity as the subject s serum antibodies have been peripheral blood lymphocytes from leukophoresis (20), thyroid lymphocytes (18,21), and lymphocytes from lymph nodes draining the site of the immune response (22) The serum reactivity of the donor should be analyzed in as much detail as possible in order to clone Ig isotypes and subclasses responsible for the response of interest. Subclass-specific antisera can be used in antigen-specific ELISA to achieve this. 

In the example of a-human atrial natriuretic peptide (ANP), found at increased plasma levels in patients with heart failure, Numata et al. [70] demonstrated how IPCR sensitivity accelerated conventional assay procedures. For individual treatment of the cardiac patients, a prompt detection of atrial distension by the presence of the ANP marker would be desirable. Common ANP tests, however, take 2-3 days for the quantification of plasma by radiometric or ELISA techniques. With sandwich IPCR, the assay time could be shortened to 5 hours. A good correlation between IPCR and radiometric detection was maintained, combined with an additional improvement of the detection limit to 2 ng/L ANP. The average level of ANP in plasma for 25 patients with heart failure was found to be 117 100 ng/L, significantly higher than the typical level of 20 14 ng/L for healthy subjects. 

A standard double-sandwich enzyme-linked immunosorbent assay (ELISA) or Western blot analysis is used. As the concentration of factor is very low in normal plasma (approximately 0.1 mg/1 for factor VIII), it is necessary to subject plasma samples to cryoprecipitation in order to concentrate the sample, prior to Western blot analysis. The cryoprecipitation protocol described by Bi et al. is as follows (Bi et al., 1996 Sarkar et al., 2000 Mah et al., 2003). Plasma samples are collected as described above for the Coatest assay. Plasma is then frozen at -80 °C overnight. Frozen samples are then subject to centrifugation at 7000 x g for 20 min at 4°C. The precipitate is washed with... 

Improved quality of autoantigen preparation (human recombinant antigen vs guinea pig liver enzyme) in ELISAs led to a diagnostic accuracy comparable to indirect immunofluorescent detection of endomysium antibodies [115, 117, 118, 120, 122-127]. These advances in ELISA technology have led to a progressive replacement of the more labor-intensive and -subjective immunofluorescent techniques. 

The main advantage of the ELISA over Western blotting is its ability to analyze many samples at one time. In addition, modifications to the general ELISA protocol can be used to quantify the exact concentration of antigen in an unknown solution, the concentration of a specific antibody in an unknown solution, the dissociation constant for a particular antigen-antibody interaction, or the relative affinity of two different antibodies for the same antigen. An example of an indirect ELISA, and its use in the determination of the concentration of an antigen in an unknown solution, is the subject of Experiment 17. 

The hairy roots were cultured in MS liquid medium and the adventitious roots were cultured in MS liquid medium containing 0.5 mg/1 lAA at 25°C in the dark. After 4 weeks, the fresh hairy and adventitious roots (2.5 cm in length) were thoroughly washed with water and divided into 5 parts (each 0.5 cm) from the distal to the proximal segment along the root axis. Each segment was subjected to ELISA to determine lAA and scopolamine content. Extraction and determination of lAA were carried... 

For the ELISA, 70% ethanol extracts of dried plant samples were diluted with an appropriate volume of C-PBS and subject to the ELISA. 

The alkaloid contents of transformed and non-transformed calli were analyzed by an ELISA and an HPLC. The crude extract can be immediately subjected to the ELISA analysis as described above (sections 3.1, 3.4) using morphine-specific monoclonal antibody [145]. The morphine-specific antibody used has a high affinity for morphine, codeine, ethylmorphine, dihydromorphine, and dihydrocodeine, less reactivity with dihydromorphinone, dihydrocodeinone, and norcodeine, but almost no reactivity with naloxone and naltrexone [145]. 

A number of assays for the hydrolase-type of Hyals were developed in the last decade that facilitated their characterization. These include microtiter-based ELISA-like assays, in which a highly specific HA-binding protein substitutes for the antibody component [186]. Biotinylated HA bound to microtiter plates is subjected to Hyal activity, and the remaining HA quantified by an avidin-enzyme color reaction [107]. HA substrate gel zymography procedures were also formulated that facilitated additional studies of these enzymes [187]. 

Agazzi et al. (2004) prepared products, spiked with bovine brain tissue at concentrations of 0.0%, 0.5%, 1.0%, and 2.0% that were subjected to three differing heat treatments (none, 80 °C for 20 min, and 120 °C for 20 min), coded them, and sent samples of each product to each of 19 laboratories for analysis by the R-ELISA method. They reported that all samples with 0% CNS tissue were correctly identified as negative for presence of CNS tissue and that, at the 0.5% level of CNS tissue... 

Fig. 10. Glycated Cu,Zn-SOD in streptozotocin-induced diabetic rat lens. Pooled lenses from normal and streptozotocin-induced diabetic rats [9 weeks after intravenous injection of streptozotocin (STZ)] were homogenized, and Cu,Zn-SOD was extracted by chloroform/ethanol and subjected to a boronate affinity column. Upper panel, normal rat lens lower panel, diabetic rat lens. The SOD activity and immunoreactive Cu,Zn-SOD were measured using the xanthine/xanthine oxidase method and ELISA, respectively.

Fig. 13. Effect of serum dilution on the ELISA. The sera from three different individuals were diluted 8- to 128-fold, and aliquots of the dilutions were subjected to ELISA.

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