• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.2
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• pp.112-117
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• 2004

The performance of an immuno-analytical system can be assessed in terms of its analytical sensitivity, i.e., the detection limit of an analyte, which is determined by the amount of analyte molecules bound to the capture antibody that has been immobilized onto a solid surface. To increase the number of the binding complexes, we have investigated a site-directed immobilization of an antibody that has the ability to resolve a current problem associated with a random arrangement of the insolubilized immunoglobulin. The binding molecules were chemically reduced to produce thiol groups that were limited at the hinge region, and then, the reduced products were coupled to biotin. This biotinylated antibody was bound to a streptavidin-coated surface via the streptavidin-biotin reaction. This method can control the orientation of the antibody molecules present on a solid surface and also can significantly reduce the possibility of steric hindrance in the antigen-antibody reactions. In a two-site immunoassay, the introduction of the site-directly immobilized antibody as the capture enhanced the sensitivity of analyte detection approximately 10 times compared to that of the antibody randomly coupled to biotin. Such a novel approach would offer a protocol of antibody immobilization in order for the possibility of constructing a high performance immunochip.

• KSBB Journal
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• v.18 no.3
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• pp.190-196
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• 2003

An analytical system detecting DNA particularly utilizing a concept of membrane strip chromatography initially applied to home-version tests for, such as, pregnancy and ovulation has been developed. We have chosen S. typhimurium as model analyte among food-contaminating microorganisms that occurred in high frequencies, and invA gene, as a detection target, specific to Salmonella species. This gene was able to be amplified by PCR under optimal conditions employing newly designed primers in our laboratory. The PCR product was specifically measured via hybridization between the analyte and a DNA probe, which was a totally different feature from the conventional gel electrophoresis detecting the products based only on the molecular size. It is notable thar the DNA probe sequence was specially designed such that no separation of excess primers present after PCR was required. This was immobilized on a nitrocellulose (NC) membrane via streptavidin-biotin linkage minimizing a steric effect when the hybridization with the amplified DNA took place. The analyrical system detected the microorganism in a concentration of minimum $10^3$ cfu/mL (i.e., 10 cells per system), estimated from the standard curve, 20 to 40 minutes after adding the sample. This sneitivity was approximately 10 times higher than that of gel electrophoresis as an analytical tool conventionally used. Furthermore, the assay was able to be run at room temperature, which would ofter an extra advantage to users.