Dot-blot assay

One of the first practical applications for these fluorescent labelled heparins was to examine the heparin binding behavior of different proteins and peptides under study in our laboratories. To this end we used a modification of the dot-blot assay described by Hirose and colleagues (13). F-D labelled heparin (-1 fluorescein/heparin) was radiolabelled with 125Iodine using iodobeads, to a specific activity of approximately 0.5 x 106 cpm/pg. Solutions of proteins with known heparin-binding capacities were dotted on nitrocellulose paper. A series of replicates... 

The fluorescent labelling of heparin with F-D by this technique did not observably alter the biologic activity of the heparin as regards to its binding to antithrombin and catalysis of antithrombin s neutralization of activated coagulation factors. F-D labelled heparins also bound to other known heparin-binding proteins in a saturable and reversible manner, as demonstrated by the dot-blot assay technique (Figure 6). 

Figure 6. 125I-F-D Heparin dot blot assay. See text for methods and abbreviations. Upper panel incubation with 125I-F-D labelled porcine mucosal heparin alone, Lower panel the same conditions, but a 100-fold excess of unlabelled heparin has been added to the labelled heparin.

Quantification of the DNA isolated from the product involves concurrent inclusion in the dot blot assay of a set of spots, containing known quantities of DNA, and being derived from the producer cell. After autoradiography, the intensity of the test spot is compared with the standards. 

An alternative assay format entails the use of virus-specific DNA probes. These can be used to screen the biopharmaceutical product for the presence of viral DNA. The assay strategy is similar to the dot blot assays used to detect host-cell-derived DNA contaminants, as discussed earlier. 

Heinicke, E. Kumar, U. Munoz, D. G. Quantitative dot-blot assay for proteins using enhanced chemiluminescence. J. Immunol. Methods 1992,152(2), 227-236. 

A number of methods are available for analyzing tumor HER-2 status. The selection of the method depends on the target molecule to be detected. The target molecules are DNA mRNA, and protein (Fig. 12.2). HER-2 gene amplification can be detected by Southern blot (Press et al., 1994), slot blot (Naber et al., 1990), and dot blot assays (Descotes et al 1993), fluorescence in situ hybridization (FISH) (Persons et al., 1997), in situ hybridization (ISH) on isolated nuclei or tissue sections (Smith et al., 1994), and polymerase chain reaction (Gramlich et al., 1994). Assays to determine mRNA oveiexpiession include Northern blot (Slamon et al., 1989), Western blot (Press et al., 1994), slot blot (Naber et al., 1990) and ISH (Naber et al., 1990). Methods to assess HER-2/mcm protein product overexpression... 

Dot blot assay to determine the titer of rAA Vphysical particles and the particle to infectivity ratio... 

A dot blot assay by cuprolinic blue precipitation has been described by Jortikka et al. (1993), which is restricted to serum free conditions. 

The initial emphasis in analytical biotechnology was on broad safety concerns that translated into detection of host-cell components such as DNA, endotoxins, Escherichia colt proteins, and retroviral contamination.2 The detection of these impurities requires development of high-sensitivity assays that are based primarily on antibodies [e.g., enzyme-linked immunosorbent assay (ELISA) for E. coli proteins) or radioactivity (e.g., dot-blot assays for DNA detection). New developments are focused on low-sensitivity detection, characterization, and removal of undesirable target sequence variants. Bioseparations play an important role even after a product has been isolated and shown to contain a low level of contaminants for initiation of clinical studies. The focus shifts to achievement of a reproducible, large-scale manufacturing process. At this stage, analytical methods provide essential informa-... 

GiUes PN, Wu DJ, Foster CB, Dillon PJ> Channock SJ. Single nucleotide polymorphic discrimination by an electronic dot blot assay on semi-conductor microchips. Nature Biotechnol 1999 17 365-70. 

The amplicons prepared are subsequently analyzed by a variety of methods. They can be hybridized to oligonucleotide probes in reverse or forward dot-blot assays. The panel of probes is chosen to cover critical polymorphic positions in the HLA gene tested. The pattern of reactivity of the panel can then be analyzed to assign allele identities. ... 

There are currently several methods for analysis of the amplified target DNA. For HIV-1, liquid hybridization with radioactively labeled probes is used (K12). Tests for HLA genes and sickle cell anemia utilize the reverse dot-blot format with a nylon membrane (S3). Each clinical research format has a well-characterized detection method defining the optimum probe concentration, the hybridization times and temperatures, as well as the concentrations of indicator reagents. Table 5 describes the optima and tolerances of a nonradioactive dot-blot assay that uses biotinylated probes and detection by a chemiluminescent substrate and a strepta-vidin-HRP conjugate. 

Infectious Diseases DNA hybridization assays that employ dioxetane substrates for alkaline phosphatase were first briefly reported in 1988, viz., herpes simplex 1 vims (B21) and Chlamydia trachomatis (Cl 7). These two assays were followed by an assay for hepatitis B vims core antigen, HBVc, (B18, B26). Thus, 10 copies of HBVc-containing plasmid DNA were detected in 30 min in a dot-blot assay, whereas a colorimetric test required 10 copies in the same time (B18, B26). Optimization allowed 10 -10 copies to be detected in 2 hr. An in situ hybridization assay for HSV-1 detected its presence after only 5 min (B22). A more detailed account of the Chlamydia assay describes how probes were prepared against two sites on an endogenous 7.4-kb plasmid and how, in a 5-hr assay. 

Smith, C., et al. (1989). Sodium Dodecyl Sulfate Enhancement of Quantitative Immunoenzyme Dot-blot Assays on Nitrocellulose, /Ina/. Biochem. Ill 212-219. 

It is advisable to check the incorporation of labeled nucleotides to the probe, especially since good batches of probes can be stored frozen for long-term use. Scintillation counting for radionucleotides (4J or standard dot-blot assays (SJ for digoxygenin or biotin incorporation can be employed, and incorporation of... 

Sorensen, M. S., Duch, M., Paludan, K., Jorgensen, P., and Pedersen, F. S. (1992) Measurement of hygromycin B phosphotransferase activity in crude mammalian cell extracts by a simple dot-blot assay. Gene 112, 257-260. 

C. Dot-blot assay (Guttenberger et ai, 1991) Ammonium sulfate for biochemical purposes (Merck), benzoxanthene yellow (Hoechst 2495, Calbiochem), cellulose acetate membranes (SM 12000, Sartorius), glycine and SDS (Serva). 

The dot-blot assay is a versatile tool its different modifications enable one to cope with almost every potentially interfering substance. In the following description the steps for all modifications are included. 

Pro The dot-blot assay combines high sensitivity, an extended range of linearity (20 ng to 20 fig), and high tolerance to potentially interfering substances. The sample is not used up during assay. Hence, it may be reprobed for immunological tests or detection of glycoproteins (Neuhoff et al., 1981). 

Extraction or precipitation steps to eliminate interfering substances should be carefully controlled for complete recovery of protein (Lowry et al., 1951). The more demanding dot-blot assay frequently is a good alternative because of a considerable gain of convenience and accuracy with respect to a simplified sample preparation. 

In the case of photometers/fluorometers operating with filters (usually microplate readers) the correct wavelength may not be available. Instead, a similar wavelength may be employed Lowry assay 500-800 nm, Bradford assay 540-620 nm, dot-blot assay 380-450 nm (excitation), 450-520 nm (emission). In the case of fluorometry allow for a sufficient wavelength interval between excitation and emission (consult the operating instructions of your instrument). 

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