Immuno polymerase chain reaction

4 IMMUNO-POLYMERASE CHAIN REACTION 13.4.1 History and Principle of IPCR Assays 

The immuno-polymerase chain reaction (IPCR) technique was first described by Sano et al. as a highly sensitive method for antigen detection [56]. In this method, the enzyme label used in ELISA is substituted with a specific DNA molecule as a marker and the DNA marker is amplified by PCR to increase assay sensitivity. The production of specific PCR products indicates the presence of the antigen of 

FIGURE 13.2 Principle of IPCR assay. Source Reprinted with kind permission of Elsevier from Ref. [58], 

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Fluit, A. C., Torensma, R., Visser, M. J., Aarsman, C. J., Poppelier, M. J., Keller, B. H., Klapwijk, P., and Verhoef, J. (1993). Detection of Listeria monocytogenes in cheese with the magnetic immuno-polymerase chain reaction assay. Appl. Environ. Microbiol. 59, 1289-1293. 

Introduction Immuno-Polymerase Chain Reaction—Principle of the Method. . 239... 

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.

Overview of Immuno-Polymerase Chain Reaction (IPCR) Applications... 

Fig. 3. Comparison of different enzyme-linked immuno sorbent assay (ELISA) methods adapted for immuno-polymerase chain reaction (IPCR). Dependent on the purification grade of the sample to be analyzed and the availability of specific and functionalized antibodies, several typical ELISA protocols were adapted to IPCR. In the direct approach (A), the pure antigen is immobilized to the microplate surface and subsequently detected by a labeled specific antibody. If no labeled antibody is available (e.g., because of unpurified ascites fluid containing the antibody or loss in activity following labeling), a standardized labeled secondary species-specific antibody is used for detection of the primary antigen-specific antibody (B). For the detection of the antigen from matrices such as serum, plasma, tissue homogenate, and so on, a capture antibody immobilized to the microplate surface was used either in a direct (C) or indirect (D) sandwich approach, with the latter one additionally including a secondary species-specific detection antibody. For different methods of coupling antibody and DNA, abbreviated by in this figure, compare Fig. 2. Note that protein A chimeras (Fig. 2A) are not compatible with capture antibodies (Fig. 3C, D).

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.)... 

Fig. 9. Correlation between Aviscumine dose levels and Cmax concentrations in plasma as quantified by immuno-polymerase chain reaction in the sample taken 1 hour after treatment. For the application of 10 ng/kg-2400 ng/kg, only one patient was monitored for each dose (P l-P 9, respectively). Starting with 3200 ng/kg, the calibration curve was adapted and cohorts ranging between 4 and 10 individuals were treated with the same dose (e.g., P 10-P 13 all received a treatment with 3200 ng/kg). The figure shows average concentrations for these patients. Note the linear ratio between the given dose and the concentration found in plasma. (Figure adapted from 73 and 87.)...

Fig. 10. Different techniques for competitive immuno-polymerase chain reaction of small-molecule compounds. (A) Using biotinylated haptens and a DNA-streptavidin nanocircle conjugate [105], hapten-DNA conjugates were synthesized and used for a competitive assay in a sample containing free hapten and capture antibody-coated surfaces [92]. (B) Hapten-coated microplates were simultaneously incubated with a sample containing free hapten and a hapten-specific antibody. Following competitive coupling, the immobilized antibody was subsequently detected by a species-specific antibody-DNA conjugate [93, 94].

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Immuno-polymerase chain reaction detects the presence of an antigen by PCR amplification of a single-stranded DNA bound (directly or indirectly) to an antibody Dissociation constant (3-Mercaptopropyl)-trimethoxysilane N-Hydroxysuccinimide... 

Lubelli, C., Chatgilialoglu, A., Bolognesi, A., Strocchi, P., Colombatti, M. and Stirpe, F. (2006) Detection of ricin and other ribosome-inactivating proteins by an immuno-polymerase chain reaction assay. Anal Biochem, 355, 102-109. 

Barletta, J. (2006) Applications of real time immuno polymerase chain reaction (rt PCR) for the rapid diagnoses of viral antigens and pathologic proteins. Molecular Aspects of Medicine, 21, 224 253. 

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