Detection system EnVision

Figure 8. Transplanted kidney stained for C4d using (a) an ultrasensitive polymer detection system (EnVision) and (b) standard strepavidin-biotin peroxidase system. There was clear staining of peritubular capillaries in (b) compared to the polymer system where the stain extended into the intertubular interstitium masking the capillaries.

Residue chemists would be most interested in comparable confidence bands or confidence bandwidths. These values become a performance characteristic for any detection system. The values indicate the precision of not only the prepared standards but also the precision of the overall operating detection system. It is ultimately envisioned that a given system of a separation column in a chromatograph with a certain type of detector should give bands of standardized values. If a chemist finds he has not met... 

In contrast to the EPOS system, a modification of the highly sensitive two-step immunohistochemical EnVision system allows the detection of a broad spectrum of antigens in frozen sections in less than 13 min (Kammerer et al., 2001). In this study 38 out of 45 antibodies tested showed specific staining. In fact, the modified EnVision procedure allows the use of any suitable primary antibody, preferably monoclonal antibodies. Like the EPOS system, EnVision employs a dextran polymer coupled to horseradish peroxidase molecules for detection. No attempt was made to block endogenous peroxidase, nor was any antigen retrieval pretreatment used. Because of the very short incubation durations, a humid chamber is not required to avoid evaporation of immunoreagents. 

Antimouse Ig-s polymer-conjugate, EnVision+ detection system (Dako K4001). Incubation 30 min at room temperature. 

HIER treatment efficiently exposes endogenous biotin. This effect can lead to high background staining in tissues rich in mitochondria (e.g. the epithelia of kidney, liver, and gastrointestinal tissues). For this reason, we recommend the use of one of the newer nonbiotin-based polymer conjugate detection systems such as EnVision+ (Dako). 

Despite being relatively new technology, aptamers have a tremendous potential and can be envisioned to rival antibodies and other traditional recognition elements for molecular detection and recognition, due to their inherent affinity, selectivity, and ease of synthesis. In addition, the combination of aptasensors with electrochemical detection methods has the added advantage of further cost reduction and miniaturization of such systems. 

An alternative to the avidin-biotin technology, the EnVision +System (Dako) detection method, is recommended for universal use in diagnostic and research studies. It is based on enhanced polymer methodology. In comparison with APAAP, PAP, ChcmMatc , CSA, LABC, and SABC methods, the En Vision +System yields optimal detection (Sabattini et al., 1998). Its sensitivity is at least as good as that of Strept ABC techniques, and its use completely eliminates the problem of endogenous biotin. 

Rapid immunohistochemical study of frozen sections is necessary for intraoperative diagnosis in some cases. Rapid immunostaining is also helpful in confirming or excluding tumor clearance in resection margins or in detecting micrometastases in sentinel lymph nodes in breast cancer patients. Two methods to immunostain frozen sections are the enhanced polymer one-step staining (EPOS) system and the EnVision system both systems are detailed later. 

EPOS, which contained primary antibodies, the EnVision system contained secondary antibodies with anti-mouse Ig and anti-rabbit Ig specificity. This universal reagent could be used to detect any tissue-bound primary antibody of mouse or rabbit origin. The utility of this method opened the door to a new family of polymer-based immunohistochemical methods. The sensitivity of these methods compared to LSAB and ABC methods was comparable or even slightly greater in most cases (7). With the latest development of EnVision FLEX+ the sensitivity has been improved even further. However, because of the large molecular size of the polymer conjugates, accessibility to certain epitopes was restricted, presumably due to steric hindrance, in a minority of cases. 

As envisioned previously, ESI response is concentration-dependent. A linear response versus concentration is up to the maximum concentration of about 10 M. When analyte concentration exceeds this limit, the ESI response levels off. This is because ESI intensity is proportional to the surface concentration of an ion. At about lO M, the droplet surface is completely saturated and higher concentration will not increase the total number of surface charges available for ion formation [25]. This will impact the high concentration end of quantitation using LC/ESl-MS. For the low concentration end, the detection limit depends on the sensitivity of LC/MS system, including efficient ion transfer/detection and removal of chemical noise in the system. 

Advantages of microfluidics for bioanalysis could potentially include lower costs for laboratory reagents and equipment and the avoidance of system carryover. Perhaps the biggest advantage will be in the introduction of novel formats for innovation that are not readily envisioned at the present time. For instance, the solution to improved ionization or parallel detection may lie in nanotechnology. As nanotechnology is reduced to practice, we may witness a dramatic shift in how future bioanalysis is conducted in drug discovery. 

Within the next 5-10 years the use of ISE type devices for continuous monitoring during surgical procedures and at the bedside of critically ill patients should become commonplace. While there are still problems to overcome, recent experiments with animals have already demonstrated that such measurements are feasible. It is probable that biomedical instrument manufocturers will produce both extracorporeal (e.g., the Miles Biostator already mentioned) and catheter-type systems for continuous detection of blood electrolyte levels. The use of telemetry for monitoring ISE potentials will undoubtedly play a major role in the development of the catheter devices. Ideally, one can envision, in the near future, operating nrams equipped with in vivo or extracorporeal devices and video screens which continuously display the patient s electroijrte levels just as blood pressure and heart rates are currently monitored. 

The sensitivity and selectivity of gas chromatography make it a strong fimdamen-tal technology for addressing DARPA s needs in the area of CWA detection. No other spectrometric analyzer offers to separate the components of a mixture and then make small amounts of them available for further identification and quantification. However, current GC systems are unacceptably large and slow for the envisioned applications. 

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