Section 19-5 Immunoassays

After screening for toxicity, identification and/or quantification assays may need to be carried out if the screening method is not specific for the cyanobacterial toxin(s) under investigation. Suitable assays for these purposes include the physicochemical assays, HPLC, MS, and CE, and to some extent the immunoassays and protein phosphatase inhibition assays summarized in Section 2. 

Many of the coagulation factors measured by global coagulation tests have limited stability, and the time and temperature of storage of sample will affect their measurements. Concepts of analyte stability and half-life in plasma extend to markers measured by immunoassay. Markers of platelet activation are affected by artifactual activation in vitro upon collection of the blood specimen. This section will highlight some of the nonanalytical variables that, if uncontrolled, can lead to spurious results and thus affect the interpretation of laboratory data. 

Figure 22.18 Biotinylated liposomes may be used in immunoassay systems to enhance the signal for detection or measurement of specific analytes. The liposome components may be constructed to include fluorescent molecules to facilitate detection of antigens within tissue sections.

The following sections briefly describe the principal enzymes used for conjugation with other protein molecules, particularly in the design of ELISA and other immunoassay systems. 

Protocols for the activation of enzyme molecules with SMCC (or sulfo-SMCC) can be found in Chapter 20, Section 1.1. Conjugates formed using this method usually result in high-activity complexes giving excellent sensitivity for use in immunoassays or other applications. 

Today s personalized medicine requires analysis of a large number of biological samples in a short period on the day they are collected from patients so that a proper informed dose adjustment can be made before subsequent dosing. The high-throughput analytical procedures developed to meet this demand are reviewed in subsequent sections covering immunoassays, HPLC alone and combined with tandem mass spectrometry detection (HPLC-MS/MS), and ultra-performance liquid chromatography with MS/MS detection (UPLC-MS/MS). 

Consider one small molecule, phenylalanine. It is an essential amino acid in our diet and is important in protein synthesis (a component of protein), as well as a precursor to tyrosine and neurotransmitters. Phenylalanine is one of several amino acids that are measured in a variety of clinical methods, which include immunoassay, fluorometry, high performance liquid chromatography (HPLC see Section 4.1.2) and most recently MS/MS (see Chapter 3). Historically, screening labs utilized immunoassays or fluorimetric analysis. Diagnostic metabolic labs used the amino acid analyzer, which was a form of HPLC. Most recently, the tandem mass spectrometer has been used extensively in screening labs to analyze amino acids or in diagnostic labs as a universal detector for GC and LC techniques. Why did MS/MS replace older technological systems The answer to this question lies in the power of mass spectrometer. 

The enzyme immunoassay procedure [57] discussed in section 5.6.1.5 for the determination of polychlorobiphenyls in soils has also been applied to sediments. 

The standard urine immunoassay for detection of cocaine (23a) abuse during the gestation period of newborn babies was frequently found to yield negative results in cases where positive results were shown by extraction of meconium with a solvent, followed by HPLC. The drug and metabolites such as norcocaine (23b) and cocaethyline (23c) were detected140. See Section IV.C for an alternative analysis of cocaine. 

Fluorescence immunoassays require labeling of antibodies, antigens, or both. The structure and some of the properties of antibodies that are important to the construction of immunoassays are briefly discussed first in this section, followed by a general discussion of probes and some of their characteristics. 

The fluorescence lifetime can be measured by time-resolved methods after excitation of the fluorophore with a light pulse of brief duration. The lifetime is then measured as the elapsed time for the fluorescence emission intensity to decay to 1/e of the initial intensity. Commonly used fluorophores have lifetimes of a few nanoseconds, whereas the longer-lived chelates of europium(III) and terbium(III) have lifetimes of about 10-1000 /tsec (Table 14.1). Chapter 10 (this volume) describes the advantages of phase-modulation fluorometers for sensing applications, as a method to measure the fluorescence lifetime. Phase-modulation immunoassays have been reported (see Section 14.5.4.3.), and they are in fact based on lifetime changes. 

Immunoassays based on phase-modulation spectroscopy have been implemented by two distinctly different approaches. Phase-resolved immunoassays rely on fluorescence intensity measurements, in which the emission of one fluorescent species in a mixture is suppressed, and the remainder is quantitated. Phase fluorescence immunoassays utilize measurements of the phase angle and modulation, which change in response to fluorescence lifetime changes. Common aspects of the theory and instrumentation are discussed in this section, followed by individual discussions of the different approaches. 

When drying these sections, it is necessary to allow them to dry at room temperature. The use of a warming plate or oven while the sections are still wet can result in the loss of some antigens proportional to the drying temperature. Once the slides are dry though, they must be heated in an oven prior to the immunoassay. There is no adverse effect associated with heating dried sections. 

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