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ELISA amplification

Several qualitative and quantitative immunochemical methods for CAP analysis in biological matrices of animal origin have been described [101,102, 104,105] (see Table 3). Van de Water et al. [ 102] described an ELISA that detected CAP in swine muscle tissue with an IC50 value of 3 ng mL1. This immunoassay was improved and subsequently optimized incorporating the streptavidin-biotin amplification system. There are also several commercially available test kits (see Table 4). RIDASCREEN is a competitive enzyme immunoassay for the quantitative analysis of CAP residues in milk, eggs, and meat in a microtiter plate. The measurement is made photometrically, obtaining a LOD of 100 ng L 1 in meat and eggs and 150 ng L 1 in milk. The test has been also applied to the analysis of tetracyclines. [Pg.212]

Both the ELISA and Western blot suffer from the problem that antibodies may not appear in an aposed individual s blood until months after the initial exposure. Methods for using PCR to screen blood samples for HIV are being developed, PCR amplification of the HIV provirai DNA 7-ovides the ability to detect HIV at earlier stages of infection, because the viral nucleic acid is r resent immediately upon exposure. It is used to detect HIV infection in newborns whose mothers are HIV positive. [Pg.107]

Enzyme linked coagulation assay for amplification of ELISA... [Pg.358]

If the major aim of the ELISA is to obtain quantification of substances present in extremely low concentrations there are a number of adaptations to the technique that can be used. Such techniques often use alkaline phosphatase enzyme systems, which can be used, for example, to lock into a circular redox cycle producing an end product such as red formazan which, is hugely amplified in comparison to standard amplification methods (4). Chemiluminescent amplified ELISA principles have also been shown to give very high... [Pg.118]

The use of europium chelates, with their unusually long fluorescence decay times, as labels for proteins and antibodies has provided techniques that are referred to as time-resolved fluoroimmunoassays (TRFIA). Fluorophores as labels for biomolecules will be the topic of Sect. 3. Nevertheless, TRFIAs always have to compete with ELISA (enzyme-linked immunosorbent assays) techniques, which are characterized by their great versatility and sensitivity through an enzyme-driven signal amplification. Numerous studies have been published over the past two decades which compare both analytical methods, e.g., with respect to the detection of influenza viruses or HIV-1 specific IgA antibodies [117,118]. Lanthanide luminescence detection is another new development, and Tb(III) complexes have been applied, for instance, as indicators for peroxidase-catalyzed dimerization products in ELISAs [119]. [Pg.71]

For all of these targets the versatility of enzyme-linked immunosorbent assays (ELISAs) qualifies this method as an almost universal detection platform, using the highly specific recognition potential of antibodies in conjugation with a detection enzyme and various signal-generating substrates [8, 9]. In contrast to the enormous amplification power of the PCR, these enzymatic methods normally have a detection limit of several millions of molecules. [Pg.240]

Fig. 1. Comparison of enzyme-linked immuno sorbent assay (ELISA, left) and immuno-polymerase chain reaction (IPCR, right). During ELISA, an antibody-enzyme conjugate is bound to the target antigen. The enzyme converts a substrate in solution to a detectable product. In IPCR, the antibody-enzyme conjugate is replaced by an antibody-DNA conjugate. The subsequent addition of a DNA polymerase enzyme (e.g., Taq), nucleotides and a specific primer pair uses the antibody-linked DNA marker sequence as a template for amplification of the DNA. The PCR product is finally detected as an indicator of the initial amount of antigen. Fig. 1. Comparison of enzyme-linked immuno sorbent assay (ELISA, left) and immuno-polymerase chain reaction (IPCR, right). During ELISA, an antibody-enzyme conjugate is bound to the target antigen. The enzyme converts a substrate in solution to a detectable product. In IPCR, the antibody-enzyme conjugate is replaced by an antibody-DNA conjugate. The subsequent addition of a DNA polymerase enzyme (e.g., Taq), nucleotides and a specific primer pair uses the antibody-linked DNA marker sequence as a template for amplification of the DNA. The PCR product is finally detected as an indicator of the initial amount of antigen.
Vascular endothelial growth factor 2000 C Factor 5 (IPCR 0.2 pg/ml fluorescence-ELISA 1 pg/ml) Sims et al. [49], real-time IPCR (see Section 2.2.3) detection of IPCR-amplificate was carried out online during PCR according to the TaqMan-principle, AbiPrism7700 sequence detector (Applied Biosystems)... [Pg.243]

In addition to successful linking of target antigen and DNA marker, as discussed in the previous chapter, the subsequent amplification of the DNA is the second key factor for efficient IPCR. Similar to many protocols developed for quantitative PCR [2], the DNA amplification product has to be converted into a detectable signal. Typically, a simple yes/no decision on the presence of the DNA marker is not sufficient, and a quantitative readout dependent on the antigen concentration is needed. Therefore, in many IPCR applications the cycle number in PCR-amplification is limited to the exponential phase of the amplification for example, 30 or fewer cycles [10, 24-26, 29, 31, 33, 37]. Alternatively, successful applications of 40 cycles were also reported [34-36, 38, 39, 41], underlining the relative flexibility of PCR conditions for the amplification step. The need for an optimized cycle number is only important for end point determinations such as gel electrophoresis (Section 2.2.1) or PCR-ELISA (Section 2.2.2). Recently, the... [Pg.258]

Eig. 5. Several endpoint detection methods were compared for the detection of immuno-polymerase chain reaction (IPCR) amplificate from a direct IPCR (Fig. 3A) of mouse-IgG. Although all IPCR/DNA-detection combinations were able to improve the detection limit of a comparable enzyme-linked immunosorbent assays (ELISA) of approximately 10 amol IgG in a 30-fL sample volume, several differences were observed in actual detection limit, and the linearity of the concentration/signal ratio dependent on the DNA quantification was applied. Best results were obtained for PCR-ELISA (see also Fig. 6) in combination with fluorescence- or chemiluminescence-generating substrates (b, c). With photometric substrates (d) or gel electrophoresis and subsequent spot densitometry (a), a 10-fold decrease in sensitivity was observed. In addition to the more sigmoid curve in gel electrophoresis, an enhanced overall error of 20% compared to 13% in PCR-ELISA was observed for two independent assays. The simple addition of a double-strand sensitive intercalation marker to the PCR-amplificate and measurement in a fluorescence spectrometer further decreased sensitivity (e) and appears therefore to be unsuited for IPCR amplificate quantification. (Figure modified according to references 37 and 65.)... [Pg.260]

PCR-ELISA [37]) were described. The immobilized hapten-labeled amplification product was subsequently detected by an antibody-enzyme conjugate similar to conventional ELISA. By choice of a chemiluminescence- or fluorescence-inducing substrate, the sensitivity of the IPCR essay is further enhanced [37]. A comparison of different detection methods is given schematically in Fig. 6 typical results are compared in Fig. 5. [Pg.261]

With these parameters, an optimized IPCR is comparable to conventional ELISA the additional PCR signal amplification has no negative effect on the performance of the immunoassay. The optimized IPCR protocol is therefore well suited for all clinical applications compatible with the widespread ELISA method. [Pg.270]

For the detection of CEA in human blood serum, as well as for the detection of prions (see also Section 3.4) Niemeyer et al. applied the one-step DDI-IPCR [60] described above (2.1.4). This method combines the advantages of coupling the antigen in solution, effective hybridization-directed immobilization, IPCR signal amplification, and real-time detection (2.2.3) into a very fast and robust protocol with a 1000-fold sensitivity increase over conventional ELISA. [Pg.272]

Bates, D. L. (1987) Enzyme amplification in diagnostics. Trends BkotedawL 5, 204-209 Johannsson, A. and Bates, D L (1988) Ampliflcation by second enzymes, in ELISA and Other Sobd Phase Immunoassays (Kemeny,D. M. andChallacombe,S.J.,eds ),John Wiley, NY, pp 85-106. [Pg.281]


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See also in sourсe #XX -- [ Pg.427 ]




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