• Title/Summary/Keyword: i-ELISA

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A Highly Sensitive Indirect Enzyme-Linked Immunosorbent Assay (ELISA) Based on a Monoclonal Antibody Specific to Thermal Stable-Soluble Protein in Pork Fat for the Rapid Detection of Pork Fat Adulterated in Heat-Processed Beef Meatballs

  • Sol-A Kim;Jeong-Eun Lee;Dong-Hyun Kim;Song-min Lee;Hee-Kyeong Yang;Won-Bo Shim
    • Food Science of Animal Resources
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    • v.43 no.6
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    • pp.989-1001
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    • 2023
  • Processed foods containing pork fat tissue to improve flavor and gain economic benefit may cause severe issues for Muslims, Jews, and vegetarians. This study aimed to develop an indirect enzyme-linked immunosorbent assay (iELISA) based on a monoclonal antibody specific to thermal stable-soluble protein in pork fat tissue and apply it to detect pork fat tissue in heat-processed (autoclave, steam, roast, and fry) beef meatballs. To develop a sensitive iELISA, the optimal sample pre-cooking time, coating conditions, primary and secondary dilution time, and various buffer systems were tested. The change in the iELISA sensitivity with different 96-well microtiter microplates was confirmed. The detection limit of iELISA performed with an appropriate microplate was 0.015% (w/w) pork fat in raw and heat-treated beef. No cross-reactions to other meats or fats were shown. These results mean that the iELISA can be used as an analytical method to detect trace amounts of pork fat mixed in beef.

Development of indirect ELISA for the detection of canine adenovirus type 2 antibodies in dog sera

  • Yang, Dong-Kun;Kim, Ha-Hyun;Lee, Siu;Oh, Dongryul;Yoo, Jae Young;Hyun, Bang-Hun
    • Journal of Veterinary Science
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    • v.21 no.4
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    • pp.63.1-63.9
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    • 2020
  • Background: Canine adenovirus type 2 (CAV-2) induces infectious laryngotracheitis in members of the family Canidae, including dogs. To date, no ELISA kits specific for CAV-2 antibody have been commercialized for dogs in Korea. Objectives: We aimed to develop new indirect enzyme-linked immunosorbent assay (I-ELISA) to perform rapid, accurate serological surveys of CAV-2 in dog serum samples. Methods: In total, 165 serum samples were collected from dogs residing in Chungbuk and Gyeongbuk provinces between 2016 and 2018. The Korean CAV-2, named the APQA1701-40P strain, was propagated in Madin-Darby canine kidney cells and purified in an anion-exchange chromatography column for use as an antigen for I-ELISA. The virus-neutralizing antibody titers of CAV-2 in the dog sera were measured by virus neutralization (VN) test. Results: We compared the results obtained between the VN and new I-ELISA tests. The sensitivity, specificity, and accuracy of new I-ELISA were 98.6%, 86.4% and 97.0% compared with VN test, respectively. New I-ELISA was significantly correlated with VN (r = 0.91). Conclusions: These results indicate that new I-ELISA is useful for sero-surveillance of CAV-2 in dog serum.

Purification of antigenic proteins of Paragonimus westermani and their applicability to experimental cat paragonimiasis (폐(肺)디스토마(Paragonimus westermani) 감염(感染) 고양이 혈청(血淸)에 대(對)한 ELISA 항체가(抗體價)의 의의(意義))

  • Choi, Won-Young;Yoo, Jae-Eul;Nam, Ho-Woo;Choi, Hyung-Rak
    • Parasites, Hosts and Diseases
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    • v.24 no.2
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    • pp.177-186
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    • 1986
  • This study was designed to evaluate the partially purified antigens which were fractionated from crude extract of Paragonimus westermani and to monitor the enzyme-linked immunosorbent assay (ELISA) in experimental cat paragonimiasis during the course of infection as well as before and after chemotherapy. Crude extract of 6-month-old adult P. westermani was fractionated to 5 antigens by successive applications of ammonium sulfate precipitation, ion exchange chromatography and gel filtration. And the cats, 10 in each group, were infected with 60, 30, 15, and 5 metacercariae, then the half of each group was treated with praziquantel 2 times in one day of 100mg per kilogram of weight on 150 days after the infection. Sera were collected every 10 days. ELISA was performed with the concentration of $2{\mu}g/ml$ antigen, 100 times diluted sera and 1,000 times diluted alkaline phosphatase conjugated anti-cat IgG. The results were as follows: 1. Absorbance by ELISA with proteins precipitated by differential concentration of ammonium sulfate was the highest at $51{\sim}65%$ precipitate (PA2), followed by $0{\sim}50%$ precipitate (PAl), $66{\sim}80%$ precipitate (PA3), and $81{\sim}90%$ precipitate (PA4). Unprecipitated protein over 90% ammonium sulfate (PA5) showed the lowest antigenicity. 2. Fractionation of PA1, PA2, and PA3 through the DEAE-cellulose column did not differentiate the antigenic proteins. 3. By passing through the Sephadex G-200 column, PA1 and PA2 were fractionated to high molecular weight proteins and those of low molecular weight which showed high absorbance by ELISA (PA1-I, II and PA2-I, II). But PA3 was shown to have a fraction of high molecular weight proteins (PA3-I) which showed high antigenicity. 4. SDS-polyacrylamide gel electrophoresis of PA1-I, P A1-II, PA2-I, PA2-II, PA3-I, and crude extract was performed. Fraction PA1-I was composed of proteins which had the molecular weight of 270 kilodaltons(KD) to 196 KD; of them 220KD protein was major band. Fraction PA2-I was composed of $255{\sim}225\;KD$, and PA3-I, $255{\sim}240\;KD$, respectively. Fraction PA1-II and fraction PA2-II consisted of 30 KD proteins. 5. Absorbance by ELISA began to increase within $10{\sim}20$ days after the infection and reached the highest on $140{\sim}180$ days, then made plateau thereafter. 6. Absorbance by ELISA decreased after praziquantel treatment. In 60 metacercariae infection group, the absorbance had been decreasing, but remained within the positive range during observation period, while those of 30, 15, and 5 metacercariae infection groups turned to negative range. 7. Fraction PA1-II showed the highest antigenicity in ELISA, then fraction PA2-I, fraction PA1-I, fraction PA2-II, fraction PA3-I and crude extract followed. In early phase of infection, the absorbance of fraction PA1-II showed more rapid increase than those of the other fractions and it came to positive range at $20{\sim}30$ days after infection.

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Improvement of indirect enzyme-linked immunosorbent assay for detection of Japanese encephalitis virus antibodies in swine sera

  • Yang, Dong-Kun;Kim, Ha-Hyun;Jo, Hyun-Ye;Lee, Seung Heon;Jang, Sang-Ho;Lee, Sang-Oh;Choi, Sung-Suk;Cho, In-Soo
    • Korean Journal of Veterinary Research
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    • v.57 no.1
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    • pp.31-36
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    • 2017
  • Japanese encephalitis (JE) is an important zoonosis caused by the mosquito-transmitted JE virus (JEV), which is a causative agent of reproductive failure in pregnant sows. Detection of JEV antibodies in swine is performed by hemagglutination inhibition (HI), virus neutralization (VN), and the plaque reduction neutralization test (PRNT). The most stringent PRNT is the 90% endpoint PRNT ($PRNT_{90}$). These conventional assays are difficult to carry out in diagnostic laboratories with insufficient instruments or cell culture systems. An alternative assay that is easily conducted and time efficient is required. In this study, we improved the indirect enzyme-linked immunosorbent assay (I-ELISA) with clarified antigen for the detection of JEV antibodies. The I-ELISA results obtained from 175 swine serum samples were compared with HI, VN, and $PRNT_{90}$ results. The sensitivity of I-ELISA was 91.8%, 95.0%, and 94.7% compared with HI, VN, and $PRNT_{90}$ results, respectively. The specificity of I-ELISA was 92.2%, 94.7%, and 94.7% compared with HI, VN, and $PRNT_{90}$ results, respectively. Moreover, the I-ELISA results were significantly correlated with the HI (r = 0.93), VN (r = 0.95), and $PRNT_{90}$ (r = 0.92) results. These results suggest that the improved I-ELISA is useful for serosurveillance of JEV in swine.

Development of an ELISA for the Organophosphorus Insecticide Chlorpyrifos

  • Cho, Young Ae;Lee, Hye-Sung;Park, Eun-Yeong;Lee, Yong-Tae;Hammock, Bruce D.;Ahn, Ki-Chang;Lee, Jae-Koo
    • Bulletin of the Korean Chemical Society
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    • v.23 no.3
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    • pp.481-487
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    • 2002
  • A selective enzyme-linked immunosorbent assay (ELISA) for the insecticide chlorpyrifos was developed. Four haptens for chlorpyrifos were synthesized and two of them were used as immunogens after coupling to keyhole limpet hemocyanin by two differe nt approaches. Rabbits were immunized with either of them and the sera were screened against 4 haptens coupled to ovalbumin (OVA). Using the sera of highest specificity, an antigencoated ELISA was developed, which shows an I50 of 160 ppb with a detection limit of 10 ppb. An antibody-coated ELISA was also developed, which shows an $I_{50}$ of 20 ppb with a detection limit of 0.1 ppb. The antibodies showed negligible cross-reactivity with other organophosphorus pesticides except for insecticides chlorpyrifos-methyl and bromophos-ethyl, which makes these assays suitable for the selective detection of chlorpyrifos.

Development of an ELISA for the Organophosphorus Insecticide Isofenphos

  • Park, Han-Jin;Park, Won-Chul;Jung, Tae-Owan;Rha, Choon-Sup;Lee, Yong-Tae
    • Bulletin of the Korean Chemical Society
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    • v.23 no.4
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    • pp.599-603
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    • 2002
  • A selective enzyme-linked immunosorbent assay (ELISA) for the insecticide isofenphos was developed. Three different analogues (haptens) of isofenphos were synthesized and were coupled to carrier proteins through the pesticide thiophosphate group t o use as immunogens or coating antigens. Rabbits were immunized with one of the haptens coupled to BSA for production of polyclonal antibodies and the sera were screened against each of the other two haptens coupled to ovalbumin (OVA). Using the sera of highest specificity, an antigen-coated ELISA was developed, which showed an I50 of 96 ng/mL with the detection limit of 2 ng/mL. The antibodies showed negligible cross-reactivity with other organophosphorus pesticides and the phenol metabolite of isofenphos, which makes the developed assay suitable for the selective detection of isofenphos. An antibody-coated ELISA was also developed, which showed an I50 of 580 ng/mL with a detection limit of 70 ng/mL.

Determination of urinary cortisol : creatinine ratios by sequential thin layer chromatography and ELISA in dogs (개에서 thin layer chromatography 박층크로마토그라피 및 ELISA를 이용한 요중의 cortisol : creatinine비 측정)

  • Sohn, Dae-ho;Na, Ki-jeong;Oh, Tae-ho;Lee, Hye-sook;Han, Hong-ryul
    • Korean Journal of Veterinary Research
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    • v.39 no.5
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    • pp.1006-1012
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    • 1999
  • This study was conducted to evaluate the ELISA kit for measuring the level of cortisol in the urine. The CV of within-run variation and day to day variation were 0.4~2.8 and 1.8~5.7, respectively. The minimum limitation of measurement was 1ng/ml. The cross reaction was high ($CR_{50}(%)=11.4{\sim}43.2$) in prednisolone, 11-deoxycortisol, 21-deoxycortisol and predinosone. There was low and no cross reaction in other steroid. To develop the ELISA kit we measured the cortisol level in diluted urine with PBS (procedure I), extracted urine with methylene chloride (procedure II) and extracted methylene chloride-extracted urine from thin-layer chromatography (procedure III). The CV value of procedure I, II, III was 9.4~28.3%, 7.2~8.9% and 2.5~5.7%, respectively. There was significant difference between procedure I with II, and pro-cedure I with III(p < 0.01), but no difference between procedure II with III significantly(p < 0.01). The mean UCCR of urine collected through am 8 to 10 was $9.5{\pm}7.6$(0.14~28.0) in 12-month-old dog(n = 47). In this study we can measure the cortisol level in extracted urine with methylene chloride and sequential thin-layer chromatography accurately using ELISA kit.

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Diagnosis of canine brucellosis using recombinant ribosomal protein L7/L12 (재조합 리보솜 단백질 L7/L12을 이용한 개 브루셀라병의 진단)

  • Lee, Hyang-Keun;Kim, Jong-Wan;Ha, Yun-Mi;Her, Moon;Kim, Ji-Yeon;Lee, Kichan;Kang, Sung-Il;Jung, Suk-Chan
    • Korean Journal of Veterinary Research
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    • v.52 no.1
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    • pp.25-31
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    • 2012
  • Brucella (B.) canis is mainly transmitted by direct or indirect contact with aborted fetuses and placenta. It's also known to be able to infect human, which likely results in providing veterinarians and companion animal owners for infectious risk. To develop diagnostic ELISA, we cloned and expressed rp1L gene of B. canis, which encodes the ribosomal protein L7/L12. Using this purified recombinant protein, indirect-ELISA (iELISA) was evaluated using 78 positive and 44 negative sera. The sensitivity and the specificity of iELISA were 94% and 89%, respectively. The results indicated that indirect-ELISA using recombinant ribosomal protein L7/L12 may be useful for diagnosis of canine brucellosis.

Evaluation on diagnostic efficiency of the standard tube agglutination test for bovine brucellosis (소 브루셀라병 표준시험관응집반응법 진단효율 평가)

  • Sung, So-Ra;Kim, Ji-Yeon;Her, Moon;Lee, Kichan;Gu, Jeong-Hui;Kang, Sung-Il;Lee, Hyang-Keun;Kim, Suk-Mi;Jung, Suk-Chan
    • Korean Journal of Veterinary Service
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    • v.35 no.4
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    • pp.269-273
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    • 2012
  • A confirmatory serological test, the standard tube agglutination test (STAT) is evaluated for the diagnostic efficiency in brucellosis Korea. A total of 345 bovine samples were collected from regional veterinary branch under national brucellosis monitoring program from January 2010 to June 2012 in Korea. These samples were diagnosed as suspected serum and brucellosis positive by the Rose Bengal test (RBT) and the STAT, respectively. The STAT was compared and evaluated with three serological test such as the indirect-enzyme linked immunosorbent assay (I-ELISA), competitive-enzyme linked immunosorbent assay (C-ELISA) and fluorescence polarisation assay (FPA) prescribed for international trade by OIE. Among the 345 bovine serum samples, 302 (87.5%) were diagnosed as positive in the STAT, while 215 (62.3%), 223 (64.6%) and 194 (56.2%) serum samples were diagnosed as positive for brucellosis in the I-ELISA, C-ELISA and FPA, respectively. The STAT showed quite high positive results as compared with three prescribed tests of OIE. FPA, I-ELISA and C-ELISA have shown 60.6%, 64.9% and 67.2% correlation, respectively as compared to the STAT. However correlations of three prescribed tests ranged high 84.1~97.7%. Especially, correlation between I-ELISA and C-ELISA is quite high, 97.7%. These results suggest that the STAT has shown many false-positive reactions. Therefore, additional serological test, such as ELISAs and FPA, would be necessary to adopt as a confirmatory test in the national surveillance program of bovine brucellosis in Korea.

Studio sulla letteratura femminista in Italia in Elsa Morante (이탈리아 페미니즘 문학연구 -엘사 모란테를 중심으로-)

  • Kim, Hyo Jung
    • Lettere Italiane
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    • no.37
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    • pp.31-72
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    • 2012
  • In questa tesi ho focalizzanto sui due romanzi di Elsa Morante: Menzogna e Sortilegio e L'isola di Arturo, riferendo i fenomeni generali delle narratrici italiane. Le scrittrici si sono limitate a scegliere i temi e i sentimenti dei personaggi nell' ambiente sociale in cui si prevalgono i valori della famiglia patriarcale. Quasi tutti i personaggi dei romanzi di Morante sono figurati dall'esperienza propria della sua vita. Specialmente la protagonista della Menzogna e sortilegio, Elisa si identifica della Morante. La fantasia di Elisa ci esprime il sogno non realizzato dei suoi genitori. Arturo è preso dal velo fantastico di Maia, vivendo felicemente nell'isola di Procida. Questo mondo illusorio è quello psicologico creato dall'autrice e fa raggiunre alla vita totale dei suoi abitanti. In questi due romanzi ho messo in rilievo il mondo immaginario e letterario di Elsa Morante: nel primo romanzo, quello creato e ricordato da Elisa, nel secondo, quello infantile ricordato dalle memorie di Arturo. In questo mondo irrazionale i nostri personaggi possono trovare riposo dimenticando il dolore reale e ottenendo la totalità esistenziale, di cui parla Georg Luckás nel La teoria del romanzo.