Immunoblotting detection methods

Figure 10.13. Comparison of immunoperoxidase (Lane A), peroxidase anti-peroxidase (Lane B) and biotin-streptavidin (Lane C) immunoblotting detection methods.15 [Reprinted, with permission, from K. E. Johansson, in Handbook of Immunoblotting of Proteins , O. J. Bjerrum and N. H. H. Heegaard, Eds., CRC Press, Boca Raton, FL, 1988. 1988 by CRC Press, Inc.]...

Fluorescamine, or 4-phenylspiro[furan-2(3//),l/-phthalan]-3,3,-dione, is used to introduce a fluorescent label on electroblotted proteins via reaction with free amines. Transferred proteins are visualized on blot transfer membranes with UV light. This stain can be very sensitive and can be used in conjunction with a second detection method such as immunoblotting (also see Basic Protocol 3). However, the protein is irreversibly modified because fluorescamine reacts with available amino groups (i.e., lysines and the protein N terminus if it was not previously blocked). 

Fluorescent labels are advantageous because they can be used not only for sequential detection methods on the same blot with minimal potential interference, but also for detection prior to protein extraction from the membrane. For example, after visualization of proteins with a fluorescent label, the blot can be photographed and specific bands marked with a pencil, either directly on the membrane or through a plastic bag. The latter method leaves a permanent indentation on the membrane. The blot can then be probed with antisera (i.e., immunoblotted unitbja). 

Matson, C.T. and Drewes, L.R. (2003) Immunoblot detection of brain vascular proteins. Methods in Molecular Medicine, 89. 479-487. 

Several diagnostic tests are available to detect acute HCV infection through detection of antibodies or viral target amplification. Antibody detection methods include enzyme immunoassay (EIA) and the recombinant immunoblot assay (RIBA). Specific antibodies to HCV by EIA are positive in only 50% to 70% of patients during the initial onset of symptoms, but 90% of patients have HCV antibodies after 3 months. A number of viral antigens are included in the current version of EIA, resulting in a 99% sensitivity and specificity for detection of HCV antibodies in immunocompetent patients. Patients with autoimmune disorders may have a false-positive EIA and no detectable HCV RNA, in which case the RIBA may be used as a supplemental test to rule out HCV. Viral target amplification techniques are used to detect HCV RNA either qualitatively or quantitatively. Qualitative tests are more sensitive with a detection limit of up to 100 copies/mL and should be reserved to determine spontaneous clearance of acute infection. Spontaneous clearance of HCV can occur in 50% of patients within 3 months of the acute onset of symptoms. ... 

A number of methods are used for the detection of antibody-antigen bindings. The most common methods used are radioisotopes, fluorophores, color changing enzymes, and metallic and magnetic beads. The advanced detection methods make use of chemiluminescent chemicals and immunoblotting and signal amplification. 

One example of the applications of the inactive endosialidase for polysialic acid detection is the staining of neuroblastoma cells (Fig. 7). The fusion protein with an inactivated endosialidase has also been used for the detection of polysialic acid in cells, bacteria, paraffin-embedded tissue sections, frozen sections, fluorescence counting, immunoblots, and flow cytometry [168, 203, 204]. The fusion protein represents a sensitive and specific detection method of polysialic acid. A further advantage is the convenience of use as a single-step reagent. A potential cross reaction concerns the rare E. coli K92 polysaccharide with the alternating a2,8/a2,9 sialic acid linkages. Such polymers have not been found in eukaryotic samples so far. 

Laboratory confirmation is vital to effective treatment of HSV, especially in individuals in whom a clinical diagnosis cannot be obtained. There are several methods by which a definitive diagnosis may be acquired, and these include virologic typing, serologic diagnosis, rapid point-of-care antigen detection, enzyme-linked immunosorbent assay (ELISA), immunoblot, and DNA polymerase chain reaction.27... 

The practicing clinician has a number of different tests available to aid in the evaluation of patients with suspected hepatitis C. These include measurement of alanine aminotransferase (ALT) levels, liver biopsy, serological tests (ELISA and recombinant immunoblot assay), and molecular methods for detection and quantitation of HCV RNA. 

The limitations of ELISA methods include the specificity of antibodies, the concentrations of primary antibody and antigen, and the type of reaction solution. Nonspecific binding of either of the antibodies to related antigens, unrelated proteins of other bacteria, or even the microtiter plate may lead to false positive reactions.49,52 57 Use of a monoclonal antibody may decrease crossreactivity with other antigens. For detection of low numbers of bacteria, as in drinking water, the sample may be filtered to concentrate the cells or cultured in a selective broth until it reaches the minimum detection limit for ELISA.49,58 Commercial test kits using dipsticks, immunoblots, and sandwich ELISA methods have been developed for the identification of pathogenic bacteria.58,59... 

A significant advantage of this method is that is provides quantitative information since the IEF technique actually separates the phospho- and dephospho- forms of the protein, the ratio of these species as detected by IEF/immunoblotting is a direct readout of the protein s phosphorylation status within the cell (assuming no selective loss of one species during isolation and analysis). 

Once transfectomas have been generated, they must be screened for the production of the desired fusion. The binding site provided by the expression of the VNP heavy chain in J558L cells and association of the heavy chain with the resident light chain means that the protein fusion can be captured on a solid phase by NP or NIP, and detected using commercially available antibody to mouse X chain and an appropriate enzyme conjugate, in a simple ELISA screening procedure. Similarly, purification can be achieved by affinity adsorption of the fusion onto an NP matrix. An immunoblot method is described here for characterization of selected transfectomas, which allows the mol-wt of the fusion product to be estimated. 

An example of the use of polyacrylamide gels in an ief system in combination with immunoblotting is given in Reference 40 where this method is used to detect low quantities of group specific component (GC) subtypes. Another example of p oly acrylamide in combination with ief is given in Reference 41 where the technique is used as a screening tool for inheritance of a certain polymorphic protein. 

Methods for the detection of anti-Ro and anti-La include traditional immunodiffusion, commercial ELISA kits, and methods such as immunoblotting and immunoprecipitation. 

Second, methods for the characterization of complex antisera are difficult. Antisera to E. coli protein mixtures have been developed with impressive spectra of reactivity using conventional immunization methods (6,22-23). An exact assessment of the spectrum of antibody reactivity is often limited, however, by the resolution of the analytical methods used. Counter immunoelectrophoresis is limited by the relatively low sensitivity of detection and resolution for complex mixtures of reacting species. One dimensional silver stained SDS-PAGE and immunoblotting provides sensitive detection limits but lacks resolution. Therefore, methods which have a high degree of resolution and sensitivity are required to best compare potential improvements in the production of antibodies to minor components in the mixture. 

The best method to determine the success of antibody production to the minor components was made by two dimensional SDS-PAGE and immunoblotting (24). A comparison of the antisera (day 112 antisera) from the three groups demonstrated that the cascade immunization antisera detected a number of minor components (Figure 4C, arrows) which were not observed with the conventional or passive antisera (Figure 4B and D). It was clear from these results that the cascade antisera was far superior in its spectrum of antibody reactivity and, in fact, was comparable or superior in detection of ECPs to silver stain (Figure 4A). Although silver stain appeared to have an improved detection of certain low MW or basic proteins,... 

The use of an immunoprecipitation method has been reported for detecting Hu, Ri, and Yo antibodies [165, 166], This method is highly specific and sensitive, as it can detect low levels of antibodies not demonstrated by immunohistochemistry or immunoblotting, and is useful in prevalence studies. However, low levels of antibodies are often not correlated with clinical disease and must be regarded with caution in patients without neurological symptoms. For instance, 25% of patients with SCLC have low levels of Hu antibodies [167]. There are also case reports of individuals with cancer and high antibody levels without PNS, even after prolonged observation [166, 168],... 

In immunoblotting procedures, a mixture of antigens is first separated by gel electrophoresis and subsequently transferred from the gel to a membrane, where it is detected by antibody. Comparative levels of reactivity with antibody can be assessed by incubation with serially diluted antibody preparations. A comprehensive review of the technique is available.44 Because the test is done with proteins that are at least partially degraded, relatively distant relationships can be detected.43 46 The method has been used to calculate SDI values for proteins of potyviruses.47... 

Morsky (1988) has described a method for detecting human lysozyme (with a sensitivity of S5 ng) in body fluids, by a procedure involving immunoblotting. 

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