Antibody-based detection methods

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. 

The exquisite dereplication ability of the mammalian immune systems to identify and remove unwanted xenobiotics has been utilized by some natural products chemists. Application of antibody-based detection methods for natural products have thus far been limited to those natural products that are of significant economic interest, either as agricultural contaminants or as potential pharmaceutical constituents. However, the development of more readily available microbially derived antibody fragments (58) and the advent of various methods for increasing their affinity (59) augur well for more widespread use of these technologies for dereplication in the future. Of the numerous reports of the use of this technique of antibody-based detection to identify natural products and related family members, two representative examples follow. 

Antibody-Based Detection Methods From Theory to Practice... 

ANTIBODY-BASED DETECTION METHODS FROM THEORY TO PRACTICE... 

Ricin can be detected in the blood or other bodly fluids of exposed animals using competitive radioimmunoassays or enzyme-linked immunosorbent assays (ELISA). These methods generally do not distinguish between active ricin molecules versus partially degraded or otherwise inactivated toxin. The postexposure time limit for accurate antibody-based detection of ricin in biological samples varies and depends on the route of exposure and the absorbed dose. In the laboratory, ELISA detects ricin in oro-nasal swabs of NHP exposed to ricin aerosol up to 24 h after exposure (Franz and Jaax, 1997). Likewise, ELISA detects ricin in selected tissues of laboratory rats up to 48 h after an i.m. challenge (Leith et al., 1988). 

Duan, L., Wang, Y., Li, S.S-C., Wan, Z., and Zhai, J. (2005) Rapid and simultaneous detection of human hepatitis B virus and hepatitis C virus antibodies based on a protein chip assay using nano-gold immunological amplification and silver staining method. BMC Infect. Dis. 5, 53. http //www.biomedcentral. com/1471-2334/5/53. 

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. 

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