• 한국미생물·생명공학회지
• /
• 제46권3호
• /
• pp.269-276
• /
• 2018

Rabies virus (RABV)-specific antibodies in animals and humans are measured using standard methods such as fluorescent antibody virus neutralization (FAVN) tests and rapid fluorescent focus inhibition tests, which are based on cell culture systems. An alternative assay that is safe and easy to perform is required for rapid sero-surveillance following mass vaccination of animals. Two purified monoclonal antibodies (4G36 and B2H17) against RABV were selected as capture and detection antibodies, respectively. A genetically modified RABV, the ERAGS strain, was propagated and concentrated by polyethylene glycol precipitation. Optimal conditions for the RABV antigen, antibodies, and serum dilution for a blocking enzymelinked immune sorbent assay (B-ELISA) were established. We evaluated the sensitivity, specificity, and accuracy of the B-ELISA using serum samples from 138 dogs, 71 raccoon dogs, and 25 cats. The B-ELISA showed a diagnostic sensitivity of 95.8-96.3%, specificity of 91.3-100%, and accuracy of 96.0-97.2% compared to the FAVN test. These results suggest that the B-ELISA is useful for sero-surveillance of RABV in dogs, raccoon dogs, and cats.

• Journal of Veterinary Science
• /
• 제23권5호
• /
• pp.32.1-32.11
• /
• 2022

Background: Classical swine fever (CSF) is a severe infectious disease of pigs that causes significant economic losses to the swine industry. Objectives: This study developed a solid-phase blocking enzyme-linked immunosorbent assay (spbELISA) method for the specific detection of antibodies against the CSF virus (CSFV) in porcine serum samples. Methods: A spbELISA method was developed based on the recombinant E2 expressed in Escherichia coli. The specificity of this established spbELISA method was evaluated using reference serum samples positive for antibodies against other common infectious diseases. The stability and sensitivity were evaluated using an accelerated thermostability test. Results: The spbELISA successfully detected the antibody levels in swine vaccinated with the C-strain of CSFV. In addition, the detection ability of spbELISA for CSFV antibodies was compared with that of other commercial ELISA kits and validated using an indirect immunofluorescence assay. The results suggested that the spbELISA provides an alternative, stable, and rapid serological detection method suitable for the large-scale screening of CSFV serum antibodies. Conclusions: The spbELISA has practical applications in assessing the vaccination status of large pig herds.

• 한국수산과학회지
• /
• 제50권5호
• /
• pp.547-552
• /
• 2017

The viral hemorrhagic septicemia virus (VHSV) has an extensive host range, and infects farmed and wild fish inhabiting both freshwater and marine ecosystems. Enzyme-linked immunosorbent assay (ELISA) is highly useful in diagnosing viral hemorrhagic septicemia. However, ELISA shows high, non-specific background reaction with fish antibodies. In this study, we optimized the antigen and antibody concentrations used for detecting specific antibodies in VHSV-infected olive flounder to reduce non-specific binding, and improve the sensitivity of ELISA. The results suggested that OD (optical Density) values were valid when ELISA was performed with $0.1{\mu}g/well$ of virus, involving blocking with blocking buffer (Roth, Roti-Block), 1:300-1:600 dilution with flounder antisera, and 1:1000 dilution with anti-flounder IgM and HRP-conjugated goat anti-mouse IgG for detecting the VHSV antibody in flounder sera. Furthermore, 11 different VHSV strains isolated in Korea from 2012 to 2016 were used to infect the fish. The results showed no correlation between viral pathogenicity and antibody production. This research is a basic study on the application of antibody detection in the diagnosis of viral hemorrhagic septicemia in the olive flounder.

• 한국동물위생학회지
• /
• 제15권1호
• /
• pp.32-45
• /
• 1992

In order to establish and enzyme-linked immunosorbent assay to ILTV, field virus strain of ILTV was propagated in chorioallantoic membrane of the embryonated eggs. purified and used as antigen. The antisera selected from the field samples and immunized chickens based on serum neutralization test were used as the standard positive and negative sera in all tests. It was found that optimal antigen concentration was $2{\mu}g$ of protein per well and a 1 : 100 dilution of standard serum showed low background optical density with negative serum and high P/N values of positive sera. A 1 : 500 dilution of the rabbit anti-chicken IgG peroxidase conjugate produced a high P/N values and thirty minutes was chosen as suitable time to read the optical density of the enzyme substrate reaction and optical density was consistent during the 16 hours after stopper was treated. When coated antigen was kept on microplate for varying time up to 16 hours at $4^{\circ}C$ or $37^{\circ}C,$ no significant difference was observed between the treatment. The coated antigen could be kept without change of antigenicity for at least one month at $-70^{\circ}C,\; -20^{\circ}C,\; 4^{\circ}C$ and room temperature. When blocking buffer contanining bovine serum albumin was mixed directly with conjugate and serum at 10% level induced higher P/N values compared to blocking antigen coated microplate with the same blocking buffer. The coefficience of correlation between ELISA and SN test was 0.577. When antibody response of chickens, vaccinated with ILTV, was examined by ELISA and SN test, antibody rising and decay pattern between the two test was similar until 11 weeks of age. However 12 weeks onward antibody titer checked on by SN test was slightly lower than that tested by ELISA.

• 한국임상수의학회지
• /
• 제27권6호
• /
• pp.704-712
• /
• 2010

Porcine circovirus-2 (PCV2) 는 돼지에서 여러 형태의 질병과 증후군의 발생과 관련이 되어있어 현재는 PCV-associated diseases (PCVAD)로 총괄적으로 분류된다. PCVAD에 의한 높은 경제적 손실 때문에 많은 양돈장들이 PCV2의 감염을 확인하기 위하여 혈청을 검사하고 있다. 하지만, 기존의 혈액채취법은 비용이 높고 많은 인력이 소요되므로 큰 규모의 병원체 검사에는 어려움이 있었다. 이에 본 연구에서는 혈액채취법을 이용한 돈군의 PCV2 검사법에 대한 대체방법으로 돈방 단위의 구강액채취법의 유용성을 실제 농장에서 평가하였다. 세 곳의 다른 양돈 농장들에서 각각 6개의 25두 규모의 돈방들을 선정하여 생후 3, 5, 8, 12, 16주에 돈방 마다 하나의 구강액과 5개의 혈청을 채취하였다. 모든 시료들은 real-time PCR을 이용하여 PCV2 DNA를 검사하였고 IgG 또는 IgA 간접형광항체 검사법및 세 가지의 ELISA 검사법 (blocking ELISA, indirect ELISA, and IgG/IgM sandwich ELISA)을 이용하여 PCV2에 대한 항체를 검사하였다. 구강액에서 PCV2 DNA는 8주까지는 간헐적으로 검출이 되다가 16주에는 모든 돈방에서 검출이 되었으며, 모체유래 PCV2 특이 IgG는 3주부터 검출이 되었고 모든 농장에서 5-8주까지 지속이 되었다. 16주에는 한 농장 (Site 1)의 모든 돈방에서 감염에 의한 IgG와 IgA가 검출되었다. 혈청에서의 PCV2 DNA와 PCV2 항체의 검출은 구강액에서의 검출과 높은 상관관계를 보였다. 따라서 구강액은 돈군의 PCV2 감염을 모니터링 하기 위해 혈청대신 사용할 수 있는 저비용, 고효율의 시료가 될 수 있을 것으로 사료된다.

• 한국동물위생학회지
• /
• 제32권4호
• /
• pp.293-298
• /
• 2009

Porcine respiratory coronavirus (PRCV) is antigenically related to transmissible gastroenteritis virus (TGEV). Differential serological diagnosis between PRCV and TGEV infection is not possible with the classical sero-neutralization test. Infection with PRCV or TGEV induces antibodies which neutralize both viruses to the same titer. However, the enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) can differentiate between PRCV and TGEV infection. This study was carried out to investigate the prevalence of PRCV infection of swine in Gyeongnam province. A total of 391 serum samples from 37 herds in Gyeongnam were examined for antibody to PRCV using blocking ELISA. All serum samples were collected from 130- to 150-day-old pigs between August and December 2006. By ELISA, 182 out of 391 sera tested (46.5%) and 29 out of 37 sample herds (78.4%) were positive against PRCV. Our data suggested that seropositive herds for PRCV are distributed diffusely throughout Gyeongnam. The PCR methods were established to diagnose PRCV spike protein (S) gene. PCR were conducted to identify the PRCV genome against 150 pigs in PRCV antibody positive herds.

• 한국식품위생안전성학회지
• /
• 제13권4호
• /
• pp.419-424
• /
• 1998

Zearalenone 검출을 위하여 Z-M-26 hybridoma cell을 mouse의 복강에 투여한 후 생산, 정제한 항체와 합성한 Zearalenone-oxime-OVA conjugate를 이용하여 ELISA법을 확립하였다. Carbonyl buffer로 희석한 Zearalenone-oxime-OVA conjugate를 4$^{\circ}C$에서 하룻밤 coating 하고 1% BSA용액으로 하룻밤 blocking한 다음, 1,000배 희석한 항체를 Zearalenone 또는 시료와 혼합하여 하룻밤 반응시키는 것이 효과적이었다. 또한 2차 항체와 기질용액의 반응시간은 각각 1시간, 30분이 적당하였고, 발색된 반응액 450nm에서 측정하였다. 이 분석법의 결과 0.1~100 ppb의 Zearalenone이 측정 가능하였으며, 본 실험에서 확립한 indirect competitive ELISA법은 농산물 중 Zearalenone 분석에 효과적으로 활용할 수 있으리라 생각된다.

• 농약과학회지
• /
• 제5권2호
• /
• pp.1-12
• /
• 2001

다클론항체를 이용하여 환경 시료중 유기인계 살충제 acephate를 분석할 수 있는 경합적 간접효소면역측정법(competitive indirect ELISA)을 개발하기 위하여 분석물과 화학구조가 유사하며 hexanoic acid를 지닌 3종의 hapten을 합성하였다. 이들 hapten을 active ester 방법으로 담체단백질인 keyhole limpet hemocyanin과 접합시켜 면역원으로 사용하있고, bovine serum albumin과 접합시켜 homologous 혹은 heterologous ELISA를 위한 코팅항원으로 사용하였다. 생산된 항체들과 코팅항원을 최종선발하여 acephate 잔류분석을 위한 ELISA를 위하여 최적화하였다. 단백질의 농도가 $1{\mu}g/mL$인 코팅항원 (hapten-3-BSA)과 다클론항체 #8377를 16000배로 희석한 heterologous ELISA에서 최적화된 acephate의 $IC_{50}$ 값은 110 ng/mL이었고, 분석범위와 최소검출한계는 각각 $10{\sim}1000$과 4 ng/mL 이었다. Acephate의 주분해산물이고 살충제로 사용되는 methamidophos을 위시한 몇몇 유기인계 살충제의 항체에 대한 교차반응은 모두 0.02%이하였다. 이와 같은 결과는 본 ELISA가 농산물 및 환경시료중의 acephate 잔류분석을 위하여 편리한 방법이 될 수 있음을 시사한다.

• Journal of Veterinary Science
• /
• 제23권4호
• /
• pp.55.1-55.12
• /
• 2022

Background: ASF was first reported in Kenya in 1910 in 1921. In China, ASF spread to 31 provinces including Henan and Jiangsu within six months after it was first reported on August 3, 2018. The epidemic almost affected the whole China, causing direct economic losses of tens of billions of yuan. Cause great loss to our pig industry. As ELISA is cheap and easy to operate, OIE regards it as the preferred serological method for ASF detection. P54 protein has good antigenicity and is an ideal antigen for detection. Objective: To identify a conservative site in the African swine fever virus (ASFV) p54 protein and perform a Cloth-enzyme-linked immunosorbent assay (ELISA) for detecting the ASFV antibody in order to reduce risks posed by using the live virus in diagnostic assays. Method: We used bioinformatics methods to predict the antigen epitope of the ASFV p54 protein in combination with the antigenic index and artificially synthesized the predicted antigen epitope peptides. Using ASFV-positive serum and specific monoclonal antibodies (mAbs), we performed indirect ELISA and blocking ELISA to verify the immunological properties of the predicted epitope polypeptide. Results: The results of our prediction revealed that the possible antigen epitope regions were A23-29, A36-45, A72-94, A114-120, A124-130, and A137-150. The indirect ELISA showed that the peptides A23-29, A36-45, A72-94, A114-120, and A137-150 have good antigenicity. Moreover, the A36-45 polypeptide can react specifically with the mAb secreted by hybridoma cells, and its binding site contains a minimum number of essential amino acids in the sequence 37DIQFINPY44. Conclusions: Our study confirmed a conservative antigenic site in the ASFV p54 protein and its amino acid sequence. A competitive ELISA method for detecting ASFV antibodies was established based on recombinant p54 and matching mAb. Moreover, testing the protein sequence alignment verified that the method can theoretically detect antibodies produced by pigs affected by nearly all ASFVs worldwide.

• Natural Product Sciences
• /
• 제10권5호
• /
• pp.211-216
• /
• 2004

Citrus unshiu (Rutaceae) has long been known as an anti-inflammatory and anti-allergic agent. In the present study, the inhibitory effect of CUWE (Citus unshiu water extract) on the production of $TNF-{\alpha}$ and tryptase was examined. In addition, a possible mechanism for the inhibition of trypsin-stimulated human leukemic mast cell-1 (HMC- 1 ) activation was determined. To do so, $TNF-{\alpha}$ production from the HMC-1 cells that were stimulated by trypsin (100 nM) in the presence or absence of CUWE $(10,\;100,\;and\;100\;{\mu}g/ml)$ was measured by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-PCR. The tryptase production was evaluated by reverse transcription-PCR. Extracellular signal-regulated kinase (ERK) activation was analyzed by Western blot. Trypsin activity was measured by using Bz-DL-Arg-p-nitroanilide (BAPNA) as substrate. Results showed that the CUWE inhibited production of both $TNF-{\alpha}$ and tryptase from the trypsin-stimulated HMC-1 in a dose-dependent manner. The CUWE a1so inhibited the ERK phosphorylation and trysin activity. These results indicate that the CUWE had an inhibitory effect on $TNF-{\alpha}$ and the tryptase productions by blocking the ERK phosphorylation and trypsin activity.