Immunoblot assays

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. 

In an immunoblot assay (Fig. 5-28c), proteins that have been separated by gel electrophoresis are transferred electrophoretically to a nitrocellulose membrane. The membrane is blocked (as described above for ELISA), then treated successively with primary... 

Ikegaki, N. and Kennett R. H. (1989) Glutaraldehyde fixation of the primary antibody-antigen complex on nitrocellulose paper increases the overall sensitivity of immunoblot assay. J. Immunol. Methods 30, 205-210. 

RIBA Recombinant immunoblot assay tRNA Transfer RNA... 

A six-year-old child had anaphylaxis 30 minutes after a fifth dose of DT vaccine (22). Skin tests, in vitro determination of specific IgE antibodies, and immunoblotting assays showed that the IgE response was directed against tetanus and diphtheria toxoids. Cross-reactivity between the two toxoids was not demonstrated, indicating the presence of co-existing but non-cross-reacting IgE and IgG antibodies. 

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. ... 

The luciferase reporter assay captures the effect of test compounds on downstream signaling events (i.e., activation of NFAT and AP-1). However, such effects could also be due to off-target activity and not (solely) inhibition of the PTP of interest. If the direct substrate(s) of the target PTP is known, and phospho-specific antibodies for this/these substrate(s) are available, immunoblot assays can be employed that directly probe the phosphorylation levels of the substrate(s). If no direct substrate of the target PTP has been... 

PBMC are a mixed cell population, only 40 % of which constitute T cells. While with flow cytometry, effects of inhibitors on T cells (or even subsets of T cells) can be separated and recorded, PBMC cannot be used to verify such effects in immunoblot assays, as tyrosine-phosphorylated proteins from non-T cells may mask the effect of the inhibitor. Therefore, primary human T cells are purified by negative selection from freshly isolated PBMC see Subheading 3.7) using the StemSep Human T Cell Enrichment Kit. Follow the selection protocol step-by-step according to the vendor s instructions. Expect to recover >1x10 T cells from 7X 10 purified PBMC (from 50 mL buffy coat). 

Cells are now ready for immunoblot assays. Proceed as described in Subheadings 3.12 and 3.5, respectively. Selective PTP inhibitors are expected to have a similar effect on phosphorylation levels of substrate proteins as observed in cells in which the PTP has been acutely eliminated by RNAi. However, the combination of knockdown and small-molecule inhibition should not result in greater effects than observed for each alone. 

A false positive has a more direct economical impact on the manufacturer. A false positive due to a specific reaction may result in significant losses because manufactured products, potentially unsafe for allergic persons, are hardly released to the market. Similarly, false positives by nonspecific reactions may lead to economical loses due to unnecessary recalls, or additional tedious procedures to ensure product safety. To verify doubtful positive results, immunoblotting assay can be utilized as a confirmatory test. 

Figure 2.21 Immunoblot assay demonstrating the degree of phosphorylation of the BCR-ABL substrate CRKL in individual patients. (Reproduced from Druker, B.J., et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in a chronic myeloid leukemia. N. Engl. J. Med. 2001, 344, 1031-1037, copyright 2001, Massachusetts Medical Society. All rights reserved.)...

The nuclear gene mutant viridis-zb has been shown to lack completely PSI activity (8) and the associated EPR signals (9). In the polypeptide pattern of the mutant the Chl - protein 1 and the PSI subunits II and III (18.3 kda and 15.2 kda respectively) are below the level of detection. Subunits II and III are, however, present in small amounts as shown by immunoblotting assay using monoclonal antibodies (10). But the Chl - apoprotein 1 of viridis - zb is not detectable. 

Other Methods SDS-PAGE was performed according to Laemmli (17). The immunoblot assay was performed according to the protocol from Bio-Rad Laboratories using anti-C. reinhardtii periplasmic CA antibody as the primary antibody. In vitro translations were performed using the rabbit reticulocyte lysate system from Stratagene. Protein and chlorophyll concentrations were determined spectrophotometrically. 

Detection of CFi subunits. CFi subunits were separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose paper essentially as described by Towbin et al. (1979). Specific proteins were detected by the immunoblot assay described by BioRad Laboratories, USA. 

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