• Journal of the Institute of Electronics Engineers of Korea SC
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• v.48 no.4
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• pp.102-107
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• 2011

In this paper, a microfluidic array biochip for simultaneously detecting multiple antigen-antibody bindings was designed and implemented. The biochip has the single channel in which microreaction chambers are serially connected, and the antibody-coated microbeads are packed in each microreaction chamber. In addition, the weir structure was fabricated in the microchannel using the gray-scale photolithography in order to trap the microbeads in the microreaction chamber. Three kinds of antibodies were chosen, and the antibodies were immobilized onto the microbeads by the streptavidin-biotin conjugation. In the experiment, as the fluorescence-labeled antigens were injected into the microchannel, the antigen-antibody bindings were completed in 10 minutes. When the solution with multiple antigens was injected into the microchannel, it was observed that the fluorescence intensity increased in only the corresponding microreaction chambers with few non-specific binding. The microfluidic array biochip implemented in this study provides, even with the consumption of tiny amount of sample and fast reaction time to simultaneously detect multiple immunoreactions.

• Korean Journal of Veterinary Research
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• v.37 no.3
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• pp.583-593
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• 1997

Indirect fluorescent antibody test(IFAT) and enzyme-linked imuunosorbent assay (IgG-ELISA) as serological diagnostic tools were conducted to evaluate the usefulness for diagnosis of canine babesiosis infected with Babesia gibsoni in domestic various dog breeds, american pit bullterrier, military shepherd, and mongrel dogs. The results obtained from this study were abstracted as follows. The nonionic detergent Triton X-100 and absorbent bio-bead $SM_2$ were useful reagents for the preparation of pure merozoite antigen of B gibsoni to be used in ELISA. The optimum reaction in ELISA was shown when the protein concentration of ELISA antigen was measured as 625ng/ml and the conjugate concentration was diluted into 1/6000 fold. The average OD value of ELISA in sera determined with negative responses in IFAT was measured as $0.255{\pm}0.051$(490nm) and the cut - off value of OD was determined as 0.399(490nm). The serum antibodies in both of IFAT and ELISA were detected on one week after artificially infected with B gibsoni and these high antibody titers, 512X in IFAT and 1024X in ELISA, were long lasted until 15 weeks after infection. The reproducibility of reaction and stability of the antigen absorbed microtitration polystyrene plate preserved in $4^{\circ}C$ refrigerator and $-20^{\circ}C$ freezer, respectively could be lasted until 135 days after storage. The positive rates in IFAT by dog breeds were shown 8.1%(60/744 heads) in mongrel dogs, 81.3%(78/96 heads) in american pit bullterrier and 15.6%(15/96 heads) in military shepherd, while the positive rate in ELISA shown 17.6%(131/744 heads) in mongrel dogs, 83.3%(80/96 heads) in american pit bullterrier and 36.5%(35/96 heads) in military shepherd, respiectively. In the total of 936 heads surveyed with IFAT and ELISA the positive rates in IFAT and ELISA were 16.4%(153/936 heads) and 26.3%(246/936 heads), respectivily. Agreement of reactions between IFAT and ELISA was shown 82.4% in 936 dog sera. The specificity and sensitivity of ELISA reaction were 83.5% and 76.5%, respectively. From the conclusion obtained in this study it was evaluated that IFAT and ELISA were useful as highly specific, sensitive and stable serelogical tools for the diagnosis of canine babesiosis in Korea.

• Korean Journal of Veterinary Research
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• v.31 no.2
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• pp.209-215
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• 1991

Serological survey were conducted on the leptospiral antibody in domestic animals which were fed in the three rural village occurred human leptospirosis. Names of three villages are Shinnam-li, Shinjeop-li and Jinai-li which are located in near the northeastern part of Yeoju town in Kyunggi province. Total 66 serum samples were collected from the domestic animals in which 12 dairy cows, 10 Korean native cattle, 12 pigs and 32 dogs were included. Leptospiral antibody were detected with 4 different serovars of leptospira living antigens, such as Leptospira icterohaemorrhagiae, L. pomona, L. canicola and L. tarassovi by microscopic agglutination test for each serum sample. The results are obtained as follow. 1. All 66 sera collected from the domestic animals at three villages showed negative reaction with 4 different serovars of leptospiral antigen. 2. Only one serum sample taken from a dairy cow in Shinjeop-li showed a weak positive reaction with Leptospira tarassovi. It is suggest that this positive case is not infected with L. tarassovi, but with vaccination. 3. It is indicated that all domestic animals which wen, fed in the villages occured human leptospirosis were not infected with above 4 different serovars of leptospira at least.

• Korean Journal of Veterinary Research
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• v.13 no.1
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• pp.39-46
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• 1973

Cultural method, dark field microscopy & fluorescent antibody technique were compared for their sensitivity of the detection of leptospires from experimentally infected mice. Two groups of mice were infected with L. icterohemorrhagiae (M20) and L. australis (Ballico), and the infected blood, urine and a number of organs were subjected to the bacterial isolation. The results obtained were summarized as follows: 1. L. icterohemorrhagiae (M20) and L. australis (Ballico) in blood, urine and various tissues of experimentally infected mice were detected with a negrigible non specificity, by the fluorescent antibody technique. 2. The fluorescent antibody technique, as applied to detection of leptospires in blood, urine and various infected tissue, proved to be better than cultural method and dark-field microscopy. 3. Early detection of leptospires by fluorescent antibody technique were possible in blood at 2 days after inoculation, whereas detection of organisms in liver, spleen, lung and kidney were observed later. By means of fluorescent antibody technique, the detection of leptospires in kidney and urine was possible up to 34 days postinoculation, whereas those in other parts were impossible. 4. Fluorescent antibody reaction of leptospires were highly specific to homologous antigen rather than to heterologous one. 5. Fluorescent antibody technique may be of value in the application for the demonstration of leptospira from clinical specimens.

• Journal of Food Hygiene and Safety
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• v.13 no.3
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• pp.206-212
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• 1998

An improved antibody-coated sensor system based on quartz crystal microbalance was developed for the detection of Salmonella spp. An antibody against Salmonella common structural antigen was immobilized onto one gold electrode of the piezoelectric quartz crystal surface by various immobilization procedures. The best results in sensitivity and stability were obtained with the thin layers of protein A and 3,3'-dithiopropionimidate.2HCI(DTBP), a homobifunctional thiol-cleavable crosslinker. After the addition of a S. typhimurium suspension into a reaction cell with 0.1 M sodium phosphate buffer, pH 7.2, the resonant frequency owing to S. typhimurium adsorption decreased conspicuously. The antibody-immobilized crystals prepared by the gold-protein A complex formation and DTBP thiolation showed the frequency shifts of 80 and 283 Hz, respectively. The time required for maximum frequency shift was about 30~60 min. The antibody-coated crystal could be reused for 6~8 consecutive assays.

• Korean Journal of Food Science and Technology
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• v.24 no.3
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• pp.204-208
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• 1992

This study was carried out to develop a practical enzyme-linked immunosorbent assay(ELISA) for the determination of soy protein in processed meat products as a preliminary study. The titer of antiserum raised in rabbit by injection of SDS-treated whole buffer extract(WBE) from isolates soy protein(ISP) was above 1:10,000 in indirect ELISA. When the SDS concentration was higher than 0.03% the antibody-antigen reaction was inhibited significantly. However, the antibody-antigen reaction inhibition was not observed when the SDS concentration was less than 0.02%. The antibodies used in this experiment also reacted with renatured antigen after removing SDS by dialysis, though not better than with SDS-denatured antigen(immunogen). The calibration curve with $100\;{\mu}g/100\;ml$ of sensitivity was obtained in indirect competitive ELISA.

• Bulletin of the Korean Chemical Society
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• v.18 no.1
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• pp.44-50
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• 1997

A method of dual-signal generation from two different enzymes was developed and utilized to simultaneously perform dual immunoassays in a single microwell. Two enzymes selected as tracers were horseradish peroxidase (HRP) and β-galactosidase (GAL). 3, 3', 5, 5'-Tetramethylbenzidine (TMB) and chlorophenolred-β-galactopyranoside (CPRG) as chromogenic substrates for the respective enzyme were used. Although the two enzymes showed their maximum activities at distinct pH conditions (pH 5.1 for HRP and 7.5 for GAL), the enzyme reactions were able to be concurrently carried out at pH 5.75 in a dual-substrate solution without signal loss. This performance was achieved by increasing TMB concentration two-fold, introducing potassium salt as activator of GAL reaction, and extending total reaction time 50%. The signal generation method was then used for dual-enzyme immunoassays to detect antibodies with co-immobilized Hepatitis C virus antigens (core and NS5) and a Hepatitis B virus antigen (PreS(2)) in a microwell. Dose-response curves of the assays revealed cooperativity between different antigen-antibody complex formation, which suggested that dual immunoassays can only be used for qualitative screening tests unless the antigens immobilized were spatially separated.

• Journal of agricultural medicine and community health
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• v.16 no.1
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• pp.61-69
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• 1991

Serodiagnosis of Clonorchis sinensis infections will probably be a first choice tool for screening of clonorchiasis in a future because of increasing difficulties in collection and examination of stools. The sensitive test such as ELISA can he used effectively. However there are some limitations in serological diagnosis for the detection of serum antibody. One of the major problems is the non-specificity of the antigens which produce cross reaction with other helminthic infection sera. To solve this problem. many investigators have tried to purify the antigens used. In this study, we determined the antigenic profile of the crude saline extract antigen of C. sinensis at early developmental stage based on SDS-PAGE and immunoblotting techniques for the purpose of understanding the nature of C. sinensis worm antigen The following results were obtained : 1) The SDS-PAGE showed many protein hands ranging from 10Kd to 91Kd relative molecular weight. Among them, 66, 46, 40, 33, 27, 24, 16, 14 and 10Kd bands were observed as a principle bands. The protein components of C. sinensis changed chronologically during their early developmental period. 44Kd band was stained unclearly in antigen of 2 weeks worm, but changed to concentrated state in antigen of 5 weeks worm. 35Kd band was found in antigen of 2 weeks worm, however this band was disappeared in antigen of 5 weeks worm. 22Kd band also lost its staining property gradually. 2) In spite of differences in antigenic profile, there was no differences in the data obtained by microplate ELISA using each antigen preparation. Absorbance value began to rise in between 2 to 3 weeks after infection. 3) By EITB. serum antibody recognized major protein bands with molecular weight of 91, 85, 63, 46, 40, 33, 24, 14 and 10Kd hand respectively. Among them 66, 33, 17 and 14Kd bands were observed as non-specific band because they reacted even in normal control sera. Generally, gradual increase of positive reactions were observed as the infection period of C. sinensis was prolonged. In other hand, the reaction of 10Kd hand did not occurred when 26th week sera was tested. 4) The positive reactions using antigens of 2 weeks worm, especially on 40 and 24Kd bands, were most strong and sharply demarcated compared to those of 3~5 weeks worm antigen.

• Korean Journal of Veterinary Service
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• v.17 no.2
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• pp.95-113
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• 1994

To establish an effective diagnostic measure for detection of the antibodies against Actinobacillus pleuropneumoniae, the methods for tube agglutination test (TAT), plate agglutination test (PAT), micro-agglutination test(MAT) and agar-gel immunodiffusion test(ID) were improved and standarized, and the comparative studies were carried out. The results obtained through the experiments were summarized as follows. 1. The rabbit hyperimmune sera to reference serotypes 1 to 6 were cross-tested with TAT, PAT, MAT and ID. In the homologous systems, the range of antibody titers in TAT was 80 to 640, showing the cross-reaction in serotypes 3, 4, 5 and 6. The range of antibody titers in PAT was 4 to 64, showing the cross-reaction in serotypes 3, 4, 5 and 6. In ID, the range of antigen titers was 8 to 32, and cross-reaction was observed in serotype 5. 2. The optimal concentration of antigen in PAT and MAT were 100mg /ml and 1.25mg /ml respectively. The most sensitive reaction in MAT was observed in 52$^{\circ}C$ for 18hrs. 3. In ID, the most promising antigen and the buffer for agar-gel were EDTA-treated antigen and 0.05M tris buffer (pH 7.2), respectively. 4. By the tests for 200 swine sera, it was found that the frequency of positive reaction were 203 in TAT, 240 in PAT and 163 in ID. 5. When compared the titers of TAT with those of MAT for 200 swine sera, MAT showed the higher titer than TAT being increased by relative correlation. Int was found that the titer for positive readings were 20 in TAT and 40 in MAT. 6. when compared the results of ID with those of TAT for 200 swine sera, all sera with TAT titer under 10 were negative in ID. Of the sera with TAT titer 20 and 40, 55.1% nd 91.8% were positive in ID, respectively. All sera with TAT titer above 80 were positive in ID. In comparison of ID and MAT, all sera with MAT titer under 20 were negative in ID. Of the sera with MAT titer 40 and 80, 24.7% and 93.9% were positive in ID, respectively. All sera with MAT titer over 160 showed positive in ID. 7. In conclusion, the established MAT showed high sensitivity but low specificity, wherease ID revealed low sensitivity but high specificity.

• The Transactions of The Korean Institute of Electrical Engineers
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• v.57 no.1
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• pp.137-141
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• 2008

Biochip, which implements bioanalytical process on a tiny surface, is one of candidates for medical diagnosis, drug screening, and molecular sensing. In general, a type of biochip based on microfluidics is composed of microcomponents including microchannel, pump, and valve, which require complicated processes. In this study, gray-scale photolithography(GSPL) was applied to fabricate a biochip with multiple layers. A mould for casting PDMS(polydimethylsiloxane) channel, was fabricated using GSPL. A gray-photomask was prepared by printing gray patterns on a high-quality glossy paper followed by photoreducing by 10:1 onto the photo-film. The formation of multiple layers was studied according to the change of gray level of pattern and the developing time. A biochip composed of a weir(multiple layer structure) and a reaction chamber in a single microchannel was fabricated in a glass plate. Finally, we investigated the application of biochip to antigen-antibody reaction by packing the microbead coated with antibody.