Immunocapture PCR

Key Words Immunocapture PCR reverse transcription potato Solanum tuberosum virus. 

Sharman, M., Thomas, J. E., and Dietzgen, R. G. (2000) Development of a multiplex immunocapture PCR with colourimetric detection for viruses of banana. J. Virol. Methods 89, 75-88. 

One advantage of this hybrid method is that the antibodies used in the immunocapture step do not need to provide the ultimate level of specificity required for the assay. For example, a genus-specific capture antibody may be used to provide the template for a species-specific PCR assay. In fact, each step of the technique can be tailored to provide the required level of discrimination. 

Immunocapture-polymerase chain reaction (IC-PCR) is a synthesis of two commonly used diagnostic tools. This method exploits the high-affinity binding of antibodies to provide a facile method of purification, usually from a complex matrix, supplying the substrate for PCR detection. PCR exponentially amplifies a deoxyribonucleic acid (DNA) template in a temperature-dependent fashion by the annealing of oligonucleotide primers, enzymatic extension of bound primers by a heat-stable polymerase, followed by denaturation of... 

Fig. 1. IC-RT-PCR of potato latent virus. Agarose gel analysis of IC-RT-PCR assays from infected potato plants, immunocaptured using a PVS° polyclonal antibody, which also had affinity to potato latent virus (10). Lane M 100-bp molecular weight standard (SuperLadder-Low ABgene) lane 1, negative control (water) lane 2, positive control (potato virus S) and lane 3, potato latent virus.

Nolasco, G., de Bias, C., Torres, V., and Ponz, F. (1993) A method combining immunocapture and PCR amplification in a microtiter plate for the detection of plant viruses and subviral pathogens. J. Virol. Methods 45, 201-218. 

Kokko, H. I., Kivineva, M., and Karenlampi, S. O. (1996) Single-step immunocapture RT-PCR in the detection of raspberry bushy dwarf virus. Biotechniques 20, 842-846. 

Jacobi, V., Bachand, G. D., Hamelin, R. C., and Castello J. D. (1998) Development of a multiplex immunocapture RT-PCR assay for detection and differentiation of tomato and tobacco mosaic tobamo-viruses. J. Virol. Methods 74, 167-178. 

Helguera, P. R., Taborda, R., Docampo, D. M., and Ducasse, D. A. (2001) Immunocapture reverse transcription-polymerase chain reaction combined with nested PCR greatly increases the detection of Prunus necrotic ring spot virus in the peach. J. Virol. Methods 95, 93-100. 

Ptacek, J., Skopek, J., Dedic, P., and Matousek, J. (2002) Immunocapture RT-PCR probing of potato virus Y isolates. Acta Virol. 46, 63-68. 

Immunoassays for nucleic acids usually employ a solid phase on which the analyte is immobilized either before or after hybridization. Two general configurations, immunocapture or hybridization capture (enzyme-linked oligonucleotide-sorbent assay or ELOSA), are possible (Figure 1). Careful design and optimization of each component can yield a high-performance assay. Typical components for a microwell PCR immunoassay are provided in Table 1. 

Figure 1 Immune complexes formed in typical PCR immunoassay. The two general configurations for DNA immunoassay that are shown differ in the means of capture. Analyte DNA is assumed to have been amplified with a 5 -biotinylated primer. In the immunocapture approach, biotin provides the means for capture onto an avidin-coated multiwell plate. In the hybridization approach, the analyte is captured by hybridization to an immobilized probe followed by immunoenzymatic detection with an avidin-alkaline phosphatase conjugate.

Immunocapture Immunocapture uses a solid phase coated with avidin or streptavidin to capture nucleic acids containing biotin. Biotin is introduced into the nucleic acids by the use of a biotinylated de-oxyribonucleoside triphosphate (dNTP) or a 5 -bio-tinylated primer during PCR amplification. Haptens such as digoxygenin or fluorescein are also widely used in combination with monoclonal antibodies. 

Table 5 General procedure for PCR immunocapture assay Denature the nucleic acid analyte...

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