Development of Double Antibody Sandwich ELISA for Detection of Duck or Goose Flavivirus

In order to establish double antibody sandwich enzyme-linked immunosorbent assay （DAS-ELISA） for detection of duck or goose flavivirus, polyclonal antibody against the flavivirus strain JS804 in geese and monoclonal antibody against the E protein of flavivirus strain JS804 in geese were used as the capture antibody and detection antibody, respectively. The optimal dilution of the capture antibody and detecting antibody capable of detecting the flavivirus strain JS804 in geese were 1：3 200 and 1：160 in the check-board titration, respectively. The reaction time of sample was 1 h, and the optimal working dilution of HRP-labeled goat-anti-mouse IgG was 1：10 000. The positive standard value was 0.247 （OD450.m）. The geese flavivirus could be detected at a minimal concentration of 1.875 μg mL^-1. The ELISA had no cross-reaction with Newcastle disease virus （NDV）, Avian influenza virus （AIV）, Infectious bronchitis virus （IBV）, Infectious bursal disease virus （IBDV）, Duck hepatitis virus （DHV）, and Gosling plague virus （GPV）. Twenty clinical samples were detected by the DAS-ELISA and RT-PCR respectively, with the agreement rate of 75%. The results revealed that the DAS-ELISA possessed favorable specificity and higher sensitivity, indicating a suitable method for rapid detection of the duck or goose flavivirus.

ELISA of polyclonal antibody to fish MT and study on heavy metal tolerance in fish

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Development and optimization of a double antibody sandwich ELISA for the detection of goose T cell surface CD8α molecule

CD8, a glycoprotein on the surface of T cells, is involved in the defense against viral infection and plays significant roles in antigen presentation and in the antiviral immune response. CD8 is composed of two chains. Of these, the CD8α chain was chosen for the detection because it involved in both the CD8αα homodimer and the CD8αβ heterodimer. Here, we established a double antibody sandwich enzyme-linked immunosorbent assay（DAS-ELISA） for specific detection of goose CD8α（go CD8α）. The results showed that the optimal coated antibody and antigen dilutions were 1：50（the antibody titer was 1：12 800） and 1：32（0.3 ng m L^–1）, respectively, while the optimal capture antibody and horseradish peroxidase（HRP）-labelled goat anti-rabbit Ig G dilutions were 1：50（the antibody titer was 1：51 200） and 1：4 000（the antibody titer was 1：5 000）, respectively. The optimal blocking buffer was 5% bovine serum albumin（BSA）. The best incubating condition was overnight at 4℃, the best blocking time was 120 min and the best anti-capture antibody working time was 150 min. In addition, the minimum dose detectable by DAS-ELISA was 5×10^–3 ng m L^–1. Most importantly, go CD8α expression levels in goose spleen mononuclear cells（MNCs） post-Goose parvoviruse（GPV） infection were found to be significantly up-regulated using the DAS-ELISA method, which was consistent with previous results obtained using real-time quantitative PCR. In conclusion, the DAS-ELISA method reported here is a novel, specific technique for the clinical detection of go CD8α.

Effect of Attenuated Highly Pathogenic Pig Reproductive and Respiratory Syndrome(HP-PRRS) TJM-F92 Strain Vaccine on Immune Antibody Levels against Classical Swine Fever(CSF) and Foot-and-Mouth Disease(FMD)

Effects of attenuated highly pathogenic pig reproductive and respiratory syndrome（HP-PRRS）TJM-F92 strain vaccine on immune antibody level against classical swine fever（CSF）and foot-and-mouth disease（FMD）were studied from October 8 to November 12 in 2014,in order to optimize vaccination program of CSF,HP-PRRS and FMD and to provide scientific guidance for animal disease control and prevention work.The results showed that attenuated HP-PRRS（TJMF92 strain）vaccine had no significant effect on immune antibody level of hog cholera lapinized virus（HCLV,ST passage cell vaccine）attenuated vaccine and FMD-O inactivated vaccines（OZK/93 strain）,and single or combined use of three vaccines received good immunization effects.

Comparative evaluation of a rapid diagnostic test,an antibody ELISA,and a pLDH ELISA in detecting asymptomatic malaria parasitaemia in blood donors in Buea,Cameroon

Background:In malaria endemic areas,infected blood donors serve as a source of infection to blood recipients,which may adversely affect their prognosis.This necessitates the proper screening of blood to be used for transfusion in these areas.The purpose of this study was to determine the prevalence of malaria parasitaemia in blood donors in Buea,Cameroon,and to evaluate the performance of a rapid diagnostic test(RDT),a malaria antibody enzyme-linked immunosorbent assay(ELISA),and a Plasmodium lactate dehydrogenase(pLDH)ELISA in the detection of asymptomatic malaria parasitaemia in the target population.Methods:In a prospective study conducted between September 2015 and June 2016,1240 potential blood donors were enrolled.The donors were screened for malaria parasites using Giemsa microscopy(GM)and a RDT.A sub-sample of 184 samples,comprising 88 positive and 96 negative samples,were selected for the evaluation of the pLDH ELISA and the antibody ELISA.The chi-square test and correlation analysis were performed as part of the statistical analyses.The statistical significance cut-off was set at P<0.05.Results:The prevalence of malaria parasitaemia in this study was found to be 8.1%(95%CI:6.6-9.7).The prevalence was not observed to be dependent on the age or sex of the participants.The RDT had a sensitivity(88.0%),specificity(99.1%),and negative predictive value(99.0%)higher than the ELISAs.The performance of the pLDH ELISA,which demonstrated the highest positive predictive value(91.6%),was generally comparable to the RDT.The sensitivity was lowest with the antibody ELISA(69.9%),which also demonstrated the highest false positive and false negative rates.The detection threshold for the pLDH(three parasites/μl)was lower compared to the RDT(50-60 parasites/μl).Non-significant positive correlations were observed between the parasite density and the pLDH titers and malaria antibody titers.Conclusions:Overall,the RDT and the pLDH ELISA demonstrated a perfectly correlated agreement with GM,meanwhile the antibody ELISA demonstrated a substantially correlated agreement with GM.The pLDH is therefore recommended for mass screening of blood(to detect malaria parasitaemia)for transfusions in the study area.However,where this is not feasible,an RDT will suffice.

Establishment of enzyme-linked immunosorbent assay for beef and lamb contents in cooked meat

In this study,an enzyme 1linked immunosorbent assay(ELISA)was established to detect beef and 1amb components,and its performance was tested.Double-antibody sandwich ELISA was adopted and determined a coating concentration of capture antibody 3G5 of 1:4000,a working concentration of enzyme-labeled antibody 2E7-horseradish peroxidase(HRP)of 1:1000,a sample incubation time of 60 min and a detection antibody reaction time of 60 min.The specificity,sensitivity,repeatability and stability of this assay were detemmined.The limit of detection for beef and 1amb skeleta1 muscle troponin I was 45 mg/kg,the inter-assay and intra-assay recovery rates ranged from 80.4%to 115.7%,the coefficients of variation were below 13.6%,and the cIoss reaction rates of the tissue components of chicken,duck and fish were below 13.4%.The sandwich ELISA method established in this study is stable and has high accuracy.The test results were consistent with the polymerase chain reaction(PCR)method at 50 and 100 g/kg-Therefore,this ELISA method can be used to quantitatively detect beef and 1amb components in meat products.

Development of a quantitative ELISA kit for human secreted CD306/LAIR-2 and its application

AIM: A quantitative ELISA kit for the detection of human secreted CD306/LAIR-2 was developed using monoclonal and polyclonal antibodies which were raised against a highly purified recombinant human secreted CD306/LAIR-2. METHODS: Anti-human secreted CD306/LAIR-2 monoclonal and polyclonal antibodies were raised by immunizing mouse or rabbit with recombinant human secreted CD306/LAIR-2. The monoclonal antibody was purified by protein G affinity, whereas the polyclonal antibody was purified by protein A affinity. The best match pair of antibodies were found and used to develop a double antibody sandwich ELISA kit for the detection of human secreted CD306/LAIR-2 in human samples. RESULTS: A human secreted CD306/LAIR-2 ELISA kit was formulated with highly purified recombinant human secreted CD306/LAIR-2, highly specific monoclonal and polyclonal antibodies. This kit realized the quantitative measurement of recombinant human CD306/LAIR-2 and natural CD306/LAIR-2 in human serum samples. CONCLUSIONS: The developed human secreted CD306/LAIR-2 ELISA kit is a reliable quantitation immunoassay kit.