• Biotechnology and Bioprocess Engineering:BBE
• /
• v.9 no.2
• /
• pp.127-131
• /
• 2004

A low-cost, simple strip reader system using a linear movement mechanism of CD-ROM deck has been developed to characterize a lateral flow membrane-based immunochromatographic assay. The test strip reader was assembled by a CD-ROM deck and home-made optical head especially designed for immunoassays. The optical head for detecting reflected light from the test strip surface consists of green light-emitting diode, large area silicon photodiode, and anodized aluminum mounting block providing a slit structure for cutting light from the LED. The stepping motor of the deck was operated in the full step mode, whose distance of each reading point is about 0.15mm. The performance of the strip reader was tested by analysis of HBV(hepatitis B virus) antigen test kit. This strip reader can be useful for inexpensive, disposable, and membrane-based assays that provide visual evidence of the presence of an analyte in a liquid sample.

• KSBB Journal
• /
• v.11 no.2
• /
• pp.140-150
• /
• 1996

To develop a home-version assay system for plasma lipoprotein cholesterol, variables that can control the assay performance were optimized. The system was constructcd by using two major components: nitrocellulose membrane strip with immobilized enzymes (cholesterol esterase, cholesterol oxidase, and horseradish peroxidase); and sample carrier solution containing non-ionic detergent (Triton X-100) and chromogen (3,3'-diaminobenzidine). Once a sample combined with the carrier was absorbed from the bottom of the strip, cholesterol was delivered by capillary action to the immobilized enzymes and a sequential reactions took place. In the final reaction, the chromogen was oxidized and then generated a color as signal that was proportional to the concentration of cholesterol. The signal intensity was enhanced by optimizing conditions for the immobilization of enzymes and the chemical composition of carriel. Under these conditions, a dose-response curve was obtained and revealed a high sensitivity enough to measure the cholesterol in blood.

• KSBB Journal
• /
• v.13 no.5
• /
• pp.577-584
• /
• 1998

A sensitive membrane strip assay for plasma lipoprotein cholesterol that can be performed without handling reagents has been investigated. We previously developed an assay system with immobilized enzymes (cholesterol esterase and cholesterol oxidase) on the surfaces of nitrocellulose membrane(1). In such a case, the amount of enzymes present on the membrane was limited by its surface area and, thus, the detection capability was relatively poor (> 50 mg/dL cholesterol). To overcome this problem, we devised a new system with non-immobilized enzymes by placing them within interstitial spaces of a celullose membrane pad in a dry state. Upon contact with sample medium, the enzymes were immediately dissolved and participated in the reactions with cholesterol in a liquid phase. We constructed a user-friendly system consisting of four membrane pads fro sample application, cholesterol decomposition, color development as signal, and medium absorption to invoke a continuous flow (sequential location from the bottom). A sample containing lipoproteins was added into the application pad by capillary action and transferred to the next pad for decomposition. The decomposition pad (namely, enzyme pad) contained a detergent (sodium cholate) for the destruction of lipoprotein particles, the two enzymes for cholesterol decomposition, and a chromogen (3,3'-diaminobenzidine). As a consequence of the enzyme reactions, hydrogen peroxide was produced, and then reacted in the presence of the chromogen with horseradish peroxidase immobilized on the signal generation pad. Finally, a colorimetric signal directly proportional to the cholesterol concentration was produced. The detection limit determined from this system under optimal conditions was at least 2 times lower than of the enzyme-immobilized system.

• Applied Chemistry for Engineering
• /
• v.25 no.5
• /
• pp.544-547
• /
• 2014

In this study, an enzyme-linked immunosorbent assay (ELISA) and immuno-chromatographic technique were combined to fabricate immuno-strip sensors for the detection of Legionella pneumophila. The immuno-strip sensor was manufactured with four different membranes. A nitrocellulose membrane was used to immobilize capture antibody and generate signals due to the high affinity to antibodies, and glass fiber membranes were used as a conjugate release pad and a sample application pad. A cellulose membrane was used as an absorption pad to induce sample flow by the capillarity. Colorimetric signals produced by sandwich immuno-reaction and enzyme reaction could be analyzed qualitatively and quantitatively within 30 min. Under the given experimental conditions, sensor signals with L. pneumophila samples were observed qualitatively by naked eyes and measured quantitatively in a range of $1.3{\times}10^3-1.3{\times}10^6CFU/mL$ with a digital camera and home-made image analysis software.

• Journal of Microbiology and Biotechnology
• /
• v.26 no.11
• /
• pp.1855-1862
• /
• 2016

Cronobacter sakazakii (C. sakazakii) is a foodborne pathogen, posing a high risk of disease to infants and immunocompromised individuals. In order to develop a quick, easy, and sensitive assay for detecting C. sakazakii, a rabbit anti-C. sakazakii immunoglobulin G (IgG) was developed using sonicated cell protein from C. sakazakii. The developed anti-C. sakazakii (IgG) was of good quality and purity, as well as species-specific. The developed rabbit anti-C. sakazakii IgG was attached to the surface of a sulforhodamine B-encapsulated liposome to form an immunoliposome. A test strip was then prepared by coating goat anti-rabbit IgG onto the control line and rabbit anti-C. sakazakii IgG onto the test line, respectively, of a plastic-backed nitrocellulose membrane. A purple color signal both on the test line and the control line indicated the presence of C. sakazakii in the sample, whereas purple color only on the control line indicated the absence of C. sakazakii in the sample. This immunochromatographic strip assay could produce results in 15 min with a limit of detection of $10^7CFU/ml$ in C. sakazakii culture. The immunochromatographic strip assay also showed very good specificity without cross-reactivity with other tested Cronobacter species. Based on these results, the developed immunochromatographic strip assay is efficient for the detection of C. sakazakii and has high potential for on-site detection.

• ALGAE
• /
• v.28 no.3
• /
• pp.289-296
• /
• 2013

For the quantitative detection of algal toxin, microcystin, a chemiluminescence immunochromatographic assay method was developed. The developed system consists of four parts, chemiluminescence assay strip (nitrocellulose membrane), horse radish peroxidase labeled microcystin monoclonal antibodies, chemiluminescence substrate (luminol and hydrogen peroxide), and luminometer. The performance of the chemiluminescence immunochromatographic assay system was compared with high performance liquid chromatography (HPLC) detection. The detection limit of chemiluminescence immunochromatographic assay system is several orders of magnitude lower than with HPLC. The chemiluminescence immunochromatography and HPLC results correlated very well with the correlation coefficient ($r^2$) of 0.979.

• Applied Chemistry for Engineering
• /
• v.22 no.5
• /
• pp.522-525
• /
• 2011

In this study, an established enzyme-linked immunosorbent assay and immuno-chromatography technique are combined to fabricate an immuno-strip sensor for the detection of S. aureus. The immuno-strip is manufactured by using four different functional membranes. The capture antibody is immobilized on the nitrocellulose membrane due to the high affinity and the capillary action through porous membranes induces a flow of sample. A colorimetric signal is appeared according to the enzyme reaction and is analyzed by the digital camera (qualitative analysis) and home-made image analysis software (quantitative analysis). Under the optimal conditions, samples with S. aureus in the range of $2.7{\times}10^4{\sim}2.7{\times}10^7CFU/mL$ can be detected by the colorimetric method within 30 min.

• Korean Journal of Veterinary Research
• /
• v.45 no.2
• /
• pp.191-197
• /
• 2005

An immunochromatographic (IC) strip for the rapid detection of Salmonella spp. in the enriched sample was developed. Affinity purified Salmonella polyclonal antibody was conjugated with 40 nm colloidal gold particles which were prepared by citrate method in our laboratory. The antigen-antibody-gold complex was captured by Salmonella antibody attached to test line of nitrocellulose membrane during the capillary migration of sample. Specificity of the IC strip was calculated to be 100% (12/12) and sensitivity was 97.6% (41/42) in the test with pure cultured bacteria. Salmonella was artificially inoculated into raw pork macerated with enrichment broth. And then it was 10-fold diluted from $5.2{\times}10^{8}CFU/ml$ to 5.2 CFU/ml. The IC strip could detect $5.2{\times}10^{6}CFU/ml$ before enrichment. However, the lowest limit of detection was 5.2 CFU/ml after overnight incubation. The results indicated that the IC assay was a rapid, economical and simple method with high specificity and sensitivity for the detection of Salmonella spp. without using any equipment.

• Journal of Microbiology and Biotechnology
• /
• v.19 no.1
• /
• pp.83-92
• /
• 2009

Individual immunochromatographic assays (ICG) for ochratoxin A (OTA) and zearalenone (ZEA) were optimized and used in the development of a one-step simultaneous immunochromatographic assay (OS-ICG) for the rapid multianalysis of two mycotoxins in corn samples. The nitrocellulose membrane of the OS-ICG was treated with OTA-bovine serum albumin (BSA), ZEA-ovalbumin (OVA), and anti-mouse IgG in the OTA test, ZEA test, and control zones, respectively. Monoclonal antibody-gold conjugates (OTA3 MAb-gold and ZEA2C5 MAb-gold) were sprayed onto the conjugate pad. The visual detection limits were 2.5 and 5 ng/ml for OTA and ZEA, respectively, and the results were obtained within 15 min after starting the analysis. An efficient, simple, and rapid extraction method using 30% MeOH/PBS was established and validated by analyzing the corn samples spiked with OTA/ZEA mixtures (0/0, 5/10, 10/20, and $20/30\;{\mu}g/kg$). The cut-off values of the OS-ICG for the spiked corn were 5 and $10\;{\mu}g/kg$ for OTA and ZEA, respectively. Natural corn samples were analyzed by OS-ICG, direct competitive enzyme-linked immunosorbent assay (DC-ELISA), and HPLC. Results of the OS-ICG were in good agreement with those obtained by DC-ELISA and HPLC. The developed OS-ICG offers a rapid, easy-to-use, and portable analytical system and can be used as a convenient qualitative tool for the on-site simultaneous determination of OTA and ZEA in cereals, food, and agricultural products in one analytical cycle.

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