Antibody detection methods

Fig. 6 An illustration of antibody detection method using an antigen-antibody capture configuration.

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. ... 

Antibody detection methods can be used to test expression conditions in batch/dia-lysis mode, and Coomassie blue-stained SDS-PAGE is often sufficient for an analysis of dialysis mode reactions. However, the simplest method for the rapid screen-... 

Applications for Highly-Sensitive Detection Method of Small Molecules by the Supramolecular Complexes between Antibodies... 

Here, we describe the design and preparation of antibody supramolecular complexes and their application to a highly sensitive detection method. The complex formation between antibodies (IgG) and multivalent antigens is investigated. When an antibody solution is mixed with divalent antigen, a linear or cyclic supramolecule forms [26-29]. With trivalent antigens, the antibody forms network structures. These supramolecular formations are utilized for the ampH-fication of detection signals on the biosensor techniques. 

Scheme 6. A highly sensitive detection method using an antibody specific for IgG (Fc) and gold nanoparticle...

The anti-DNA antibody has been used as a marker molecule of Systemic Lupus Erthematosus (SLE) which is a severe autoimmune disease. Enzyme immunoassay is the most reliable, widely used method of assay however, the electrochemical detection method reported here should be interesting for the purpose of a rapid and convenient diagnostic tool of SLE. 

The increasing attention directed to the adverse effects of variation in sample preparation upon the quality of IHC staining of FFPE tissues has served to reinforce the importance of determining the optimal AR method for each antibody/detection system/antigen to achieve optimal retrieval and optimal staining of tissues that may have been processed and stored in different and unknown ways (see Chapter 5 for details). Practically, in considering... 

Vassallo J, Pinto GA, Alvarenga JM, et al. Comparison of immunoexpression of 2 antibodies for estrogen receptors (1D5 and 6F11) in breast carcinomas using different antigen retrieval and detection methods. Appl. Immunohistochem. Mol. Morphol. 2004 12 177-182. 

Optical imaging methods for hypoxia are not yet applicable in vivo but immunohistochemical antibody detection of nitroimi-dazoles (e.g. pimonidazole) has been used extensively for in vitro work and biopsy tissue staining (89). 

The performance of a biotreatment system ultimately depends on optimization of the activity of microbes and the ability to control the process parameters of the treatment system [157]. In this respect, the ability to monitor gene copy numbers and gene expression is highly useful for real time optimization of the efficiency of a biotreatment system. Advanced molecular techniques as well as low cost methods (e.g., antibody detection of enzymes based on color reaction strips fluorescence i.e., GFP marked organisms with UV light detection) can also be applied to monitor the microbial community structure, persistence of the added bacteria, and their interactions with indigenous populations. 

Antibody-based detection methods include immuno-cytochemistry, which gives qualitative data but has very good spatial resolution. Radioimmunoassays provide a quantitative measure of release or content. One of the major limitations of all antibody-based methods is the potential for cross-reactivity among the many peptides. For example, some of the most sensitive gastrin antisera also detect CCK, since the peptides share a common COOH-terminal tetrapeptide sequence. Methods for detection of the mRNAs encoding neuropeptides include Northern blots, which provide quantitative data and information on splice variants, but lack fine anatomical resolution. The more commonly used polymerase chain reaction, which can be quantitative but often is used in a more qualitative manner, provides great sensitivity. Alternatively, in situ hybridization preserves anatomical relationships and can be used to obtain both qualitative and quantitative data. 

In summary, these are the clinically relevant questions about the immunogenicity of rDNA species-specific proteins will antibody be induced in the recipient that will neutralize the therapeutic effect or lead to immune complex disease What is the class (e.g., IgG or IgE) and specificity (i.e., reactivity against specific protein or contaminant) of the antibody induced The former antibody type could potentially neutralize the product and produce immune complex disease, while the latter could result in an anaphylaxis response. It is possible that the antibody induced is of insignificant health consequence, and its presence is known only because of improvements made in the sensitivity of detection methods with the introduction of the enzyme-linked immunosorbent (ELISA) assay. 

The cross-reaction of secondary species-specific antibodies with primary antibodies from the same species is obviously avoided by direct (one antibody layer) methods. The direct method offers an easy way for simultaneous labeling of a pair or more antigens, even when using primary antibodies from the same species. Recently, a direct technique with primary antibodies that are covalently labeled by different fluorophores was described for a simultaneous detection of up to seven... 

Another antibody-based method is immunoligand assay (ILA) technology. The Threshold system (Molecular Devices, Sunnyvale, CA) shortens assay development and assay turnaround time.11 It adapts a sensitive detection system originally... 

For many years, due to the availability and low cost of radioisotope-labeled secondary antibodies, radioactive detection was the method of choice in Western blotting. Newer methods that are less hazardous and easier to use, while maintaining comparable sensitivity, have been developed. Today, Western blotting detection methods can be light-based, (chemiluminescence, bioluminescence, chemifluorescence, and fluorescence), radioactivity-based, or color-based. It is important to note that the detection sensitivity depends on the affinity of the primary antibody for the antigen and on the affinity of the secondary antibody for the primary antibody and can therefore vary considerably from one protein sample to another and from one antibody batch to another. 

---