Antibody Detection Techniques

PPL used Highest concentration used for skin test Patients challenged with PN Patients experiencing reactions % Authors 

Clinical form of reaction Immediate-type (20-30 min) skin tests with benzylpenicillin and/or PPL Epicutaneous test with benzylpenicillin 200000 U/ml (24-48 h reaction) Pos. Neg.  

Human skin-sensitizing antibodies from penicillin-sensitive patients can be transferred to normal individuals (Waldo and Tyson 1949 Coleman and Siegel 1956 Redmond and Levine 1967), monkeys (Kunz et al. 1966,1967 Assem and Schild 1968), or experimental rodents (Sonntag and Marcus 1963 Palomeque et al. 1965), although in the last case it is likely that IgG antibodies are detected, not the relevant IgE antibodies. The technique is interesting in experimental studies and its results appear roughly parallel to those of skin tests, but it is too cumbersome to be of practical use. 

Additional serological techniques, such as complement consumption tests, radioimmunoprecipitation (Gleich and Stankievic 1969), fluorescence polarization (Dandliker et al. 1965), and variations of sandwich hemagglutination techniques (e.g., red-cell-linked antigen-antiglobulin reaction Kraft et al. 1976), have been used in the detection of antipenicillin antibodies, but all appear to have been supplanted by modern RIA and ELISA techniques. 

The lymphocyte transformation test, first proposed as a morphological assessment of lymphoblast production in culture upon incubation with the antigenic drug (Heitmann and Kuwert 1966 Halpern et al. 1967), was soon used in its biochemical version, based on measurement of DNA synthesis and [ HJthymidine uptake (Rose et al. 1966 Vischer 1966 Evans 1967 Fellner et al. 1967 a Assem and Vickers 1972 Sarkany and Gaylarde 1978 Warrington and Tse 1979). Al- 

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Figure 7.8 Some examples of antibody detection techniques, (a) Direct labelling of the primary antibody, (b) indirect using labelled secondary antibody, (c) indirect using biotinylated secondary antibody and labelled streptavidin.

Most sample components analyzed with electrophoretic techniques are invisible to the naked eye. Thus methods have been developed to visualize and quantify separated compounds. These techniques most commonly involve chemically fixing and then staining the compounds in the gel. Other detection techniques can sometimes yield more information, such as detection using antibodies to specific compounds, which gives positive identification of a sample component either by immunoelectrophoretic or blotting techniques, or enhanced detection by combining two different electrophoresis methods in two-dimensional electrophoretic techniques. 

Conductometric transducers, as the oldest electrochemical devices, seem not to enjoy wide applications due to their poor selectivity. For example, Yagiuda et al. proposed a conductometric immunosensor for the determination of methamphetamine (MA) in urine [89], The decrease in the conductivity between a pair of platinum electrodes might result from the direct attachment of MA onto the anti-MA antibodies immobilized on the electrode surface. The system was claimed to be a useful detection technique of MA in comparison with a gas chromatography-mass spectrometry method. 

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. 

However, luminescence-based detection techniques often require a high number of steps. Consider ELISA as an example. As a first step, the sample is introduced into a 96-well plate an antibody targeting the antigen of interest has been immobilized to the wells of the plate. After a rinse, the wells contain the antibody and any bound antigen. However, although the antigen has been isolated, the protocol is nowhere near completion. The remaining steps include another antibody (different from the first) to form a sandwich assay, a secondary antibody with an enzymatic label, and a substrate that is luminescent when activated by the enzyme. Finally, the sample is analyzed by relatively expensive detection optics to determine the amount of analyte that was captured in the assay. The steps are illustrated in Fig. 14.1a. 

Western blot and immunohistochemical detection are the two most widely used techniques to confirm the expression of a given P450 isoform in isolated brain regions or thin slices. Antibodies detect actual protein,... 

Very few immunosensors are commercially available. The commercial immunosensors are either the detector or bioanalyzer types. The PZ 106 immunosensor from Universal Sensors Inc. (New Orleans, LA) has been used as a detector to measure antibody-antigen reaction. Ohmicron (Newtown, PA) developed a series of pesticide immuno-bioanalyzers that have been used in field tests. Pharmacia Biosensor USA (Piscataway, NJ) recently introduced BIAcore immunodetection system. A combination of a unique flow injection device and surface plasmon resonance (SPR) detection technique provides a real time analysis. A carboxylmethyldextran layer added to plasmon generating gold film is a hydrophobic, activatable, and flexible polymer that provides high antibody and low non-specific bindings. System demonstration at the Institute of Food Technologists (IFT) 1994 meeting in Atlanta drew attention of food scientists. It should easily be adapted for food protein characterization. 

Because MIB-1 monoclonal antibody is used extensively to determine the cell proliferation index, its applications are discussed below. This antibody detects the nuclear antigen Ki-67 expressed in proliferating cells but not in resting cells. The antibody reacts with the nuclei of cells in mid-Gj (first gap), S (DNA synthesis), G2 (second gap), and M (mitosis) phases, but not in the G0 or quiescent phases. The use of MIB-1 antibody is one of the simplest and most reliable labeling techniques for assessing the rate of proliferation of a neoplastic cell population. Thus, the antibody can be used to assess the growth fraction (i.e., the number of cells in cell cycle) of normal, reactive, and neoplastic tissues. 

The processing sequence can be varied with complete freedom in order to accomplish different purposes in an assay (i.e., for either RIA or ELISA for either antigen or antibody detection by either the direct or the indirect techniques). As an example, the following sequence of steps is appropriate for ELISA detection of human antibody to a specific viral antigen, by the indirect (labeled second antibody) technique (Fig. 5A shows diagrammatically how this can be accomplished). 

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