scholarly journals Development of a Novel Double Antibody Sandwich ELISA for Quantitative Detection of Porcine Deltacoronavirus Antigen

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2403
Author(s):  
Wei Wang ◽  
Jizong Li ◽  
Baochao Fan ◽  
Xuehan Zhang ◽  
Rongli Guo ◽  
...  
Keyword(s):  
Sandwich Elisa ◽  
Virus Titer ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
N Protein ◽  
Double Antibody ◽  
Rabbit Polyclonal Antibody ◽  
Inactivated Vaccines ◽  
Das Elisa

Porcine deltacoronavirus (PDCoV) can cause diarrhea and dehydration in newborn piglets. Here, we developed a double antibody sandwich quantitative enzyme-linked immunosorbent assay (DAS-ELISA) for detection of PDCoV by using a specific monoclonal antibody against the PDCoV N protein and an anti-PDCoV rabbit polyclonal antibody. Using DAS-ELISA, the detection limit of recombinant PDCoV N protein and virus titer were approximately 0.5 ng/mL and 103.0 TCID50/mL, respectively. A total of 59 intestinal and 205 fecal samples were screened for the presence of PDCoV by using DAS-ELISA and reverse transcriptase real-time PCR (RT-qPCR). The coincidence rate of the DAS-ELISA and RT-qPCR was 89.8%. DAS-ELISA had a sensitivity of 80.8% and specificity of 95.6%. More importantly, the DAS-ELISA could detect the antigen of PDCoV inactivated virus, and the viral antigen concentrations remained unchanged in the inactivated virus. These results suggest that DAS-ELISA could be used for antigen detection of clinical samples and inactivated vaccines. It is a novel method for detecting PDCoV infections and evaluating the PDCoV vaccine.

scholarly journals The Use of Chicken Igy in a Double Antibody Sandwich Elisa for the Quantification of Melittin in Bee Venom and Bee Venom Melittin Content in Cosmetics

2015 ◽  
Vol 59 (1) ◽  
pp. 97-107
Author(s):  
Lindsey Y. K. Suh ◽  
Tayabaa Kartoon ◽  
Naiyana Gujral ◽  
Youngmee Yoon ◽  
Joo Won Suh ◽  
...  
Keyword(s):  
Sandwich Elisa ◽  
Competitive Elisa ◽  
Bee Venom ◽  
Raw Material ◽  
Enzyme Linked Immunosorbent Assay ◽  
Western Blot Assay ◽  
Detection Systems ◽  
Indirect Competitive Elisa ◽  
Double Antibody ◽  
Das Elisa

Abstract Two enzyme-linked immunosorbent assay (ELISA) - based detection systems: indirect competitive ELISA and biotinylated double antibody sandwich ELISA (DAS-ELISA) were developed to determine the melittin concentration in honeybee (Apis mellifera) venom and the melittin concentration in cosmetics which contain bee venom. The indirect competitive ELISA employed chicken anti-melittin IgY. The biotinylated DAS-ELISA employed anti-melittin monoclonal antibody (MAb) and biotinylated anti-melittin IgY. To produce anti-melittin IgY; Sigma melittin was emulsified with Freund‘s incomplete adjuvant and immunised to Leghorn laying chickens intramuscularly at four different sites (50 μg/mL, 0.25 mL per site) of the breast muscles. After 5 to 8 weeks of the immunisation, anti-melittin IgY was extracted and analysed by ELISA. The anti-melittin IgY antibody produced was highly specific to melittin and did not cross-react with other bee venom proteins, as examined by ELISA and a western-blot assay. Indirect competitive ELISA demonstrated a higher range of melittin detection (2.5 to 80 μg/mL). Double antibody sandwich ELISA using MAb as the capture antibody and biotinylated polyclonal IgY as the detection antibody, provided a lower range of detection (2.5 - 40 ng/mL), which has a 1000 times higher sensitivity than that of indirect competitive ELISA. Therefore, indirect competitive ELISA is a useful tool to measure the concentration of melittin in bee venom as a raw material. Biotinylated DAS-ELISA, on the other hand, is more suitable for nanoscale quantification of melittin in commercial products.

scholarly journals A Double-Antibody Sandwich ELISA for Sensitive and Specific Detection of Swine Fibrinogen-Like Protein 1

2021 ◽  
Vol 12 ◽  
Author(s):  
Xin Zhang ◽  
Haipeng Zhu ◽  
Xu Zheng ◽  
Yunjie Jiao ◽  
Lulu Ning ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Sandwich Elisa ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Detection ◽  
Serum Samples ◽  
Double Antibody ◽  
Programmed Death ◽  
Prokaryotic Expression Vector ◽  
Das Elisa

Fibrinogen-like protein 1 (FGL1), a member of the fibrinogen family, is a specific hepatocyte mitogen. Recently, it has been reported that FGL1 is the main inhibitory ligand of lymphocyte activating gene 3 (LAG3). Furthermore, the FGL1-LAG3 pathway has a synergistic effect with programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) pathway and is regarded as a promising immunotherapeutic target. However, swine FGL1 (sFGL1) has not been characterized and its detection method is lacking. In the study, the sFGL1 gene was amplified from the liver tissue of swine and then inserted into a prokaryotic expression vector, pQE-30. The recombinant plasmid pQE30-sFGL1 was transformed into JM109 competent cells. The recombinant sFGL1 was induced expression by isopropyl-β-d-thiogalactoside (IPTG) and the purified sFGL1 was used as an antigen to produce mouse monoclonal antibody (mAb) and rabbit polyclonal antibody (pAb). After identification, a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for sensitive and specific detection of sFGL1 was developed. Swine FGL1 in samples was captured by anti‐sFGL1 mAb followed by detection with anti‐sFGL1 rabbit pAb and HRP-conjugated goat anti-rabbit IgG. The limit of detection of the developed sFLG1-DAS-ELISA is 35 pg/ml with recombinant sFLG1. Besides, it does not show cross‐reactivity with the control protein. Then serum samples of PRRSV-negative and -positive pigs were tested with the established DAS-ELISA and calculated according to the equation of y=0.0735x+0.0737. The results showed that PRRSV infection enhanced the serum FGL1 levels significantly. Our research provides a platform for the research on the functional roles of swine FGL1.

Development of double-antibody sandwich ELISA for rapidly quantitative detection of antigen concentration in inactivated SCRV vaccine

Aquaculture ◽  
2020 ◽  
Vol 520 ◽  
pp. 734671 ◽  
Author(s):  
Yinjie Niu ◽  
Peng Zhang ◽  
Luyao Wang ◽  
Ningqiu Li ◽  
Qiang Lin ◽  
...  
Keyword(s):  
Sandwich Elisa ◽  
Quantitative Detection ◽  
Antigen Concentration ◽  
Double Antibody

An optimized double-antibody sandwich ELISA for quantitative detection of WSSV in artificially infected crayfish

2018 ◽  
Vol 251 ◽  
pp. 133-138 ◽  
Author(s):  
Xiaoqian Tang ◽  
Qianrong Liang ◽  
Lushan Liu ◽  
Xiuzhen Sheng ◽  
Jing Xing ◽  
...  
Keyword(s):  
Sandwich Elisa ◽  
Quantitative Detection ◽  
Double Antibody

Sandwich ELISA for Detection of Pig Meat in Raw Beef Using Antisera to Muscle Soluble Proteins

1988 ◽  
Vol 51 (10) ◽  
pp. 790-798 ◽  
Author(s):  
ROSARIO MARTÍN ◽  
JUAN I. AZCONA ◽  
CARMEN CASAS ◽  
PABLO E. HERNÁNDEZ ◽  
BERNABÉ SANZ
Keyword(s):  
Horseradish Peroxidase ◽  
Immunosorbent Assay ◽  
Peroxidase Activity ◽  
Sandwich Elisa ◽  
Soluble Proteins ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Antibodies ◽  
Double Antibody ◽  
Colored Product ◽  
Pig Meat

A double-antibody sandwich ELISA (enzyme-linked immunosorbent assay) has been successfully developed for the detection of defined amounts of pig meat (1–50%) in raw beef. Antibodies against pig sarcoplasmic extracts were produced in rabbits. Pig-specific antibodies were affinity purified by removing antibodies which crossreacted with horse, chicken or beef extracts followed by immunoadsorption and elution from a pig-extract column. The ELISA involved capturing antigens in sarcoplasmic extracts with pig specific antibodies immobilized on 96-well plates, detecting bound antigen with pig specific, horseradish peroxidase-labeling antibody, and measuring peroxidase activity by the conversion of a clear substrate to a colored product.

Comparison of Real-Time Polymerase Chain Reaction and Enzyme-Linked Immunosorbent Assay for Sensitive and Quantitative Detection of Hazelnuts in Nut Pastes

2018 ◽  
Vol 101 (6) ◽  
pp. 1864-1867 ◽  
Author(s):  
Ľubica Piknová ◽  
Veronika Janská ◽  
Tomáš Kuchta ◽  
Peter Siekel
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Sandwich Elisa ◽  
Pcr Analysis ◽  
Quantitative Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chain Reaction ◽  
Wide Range ◽  
Order Of Magnitude ◽  
Polymerase Chain

Abstract Background: Hazelnuts, being a frequent agent of allergenic reactions, need to be detected in food products. Thus, it is necessary to develop and further investigate appropriate methods for detection. Objective: The aim of the study was to compare the analysis of nut pastes (peanut paste spiked with different amounts of hazelnut paste) as a model of contamination of confectionery. Methods: Real-time PCR and sandwich ELISA (RidaScreen Hazelnut Fast Kit) were used. Results: For real-time PCR, LOQ of 2 mg/kg and a quantification range from 2 to 10 000 mg/kg were determined. For ELISA, LOQ of 1 mg/kg and a quantification range from 1 to 100 mg/kg were determined. Conclusions: The comparison shows that the methods had comparable sensitivity with LOQs in the same order of magnitude. Although ELISA was slightly more sensitive, it required dilution of samples at higher concentrations of the analyte because of its narrow quantification range. Results of this study suggest that real-time PCR and ELISA are both suitable methods for the analysis of nut pastes over a wide range of concentrations. Achieved results could be useful for control as well as for technological purposes. Highlights: Real-time PCR analysis of peanut paste spiked with different amounts of hazelnut paste as a model is proposed. Sandwich ELISA analysis of peanut paste spiked with different amounts of hazelnut paste as a model is proposed. The analytical parameters of real-time PCR and ELISA methods are compared.

scholarly journals Changes in the Concentration of an Allexivirus During the Crop Cycle of Two Garlic Cultivars

Plant Disease ◽  
2006 ◽  
Vol 90 (10) ◽  
pp. 1293-1296 ◽  
Author(s):  
Eva E. Cafrune ◽  
Mónica Balzarini ◽  
Vilma C. Conci
Keyword(s):  
Virus Titer ◽  
Virus Detection ◽  
Coat Protein Sequence ◽  
Enzyme Linked Immunosorbent Assay ◽  
Virus Concentration ◽  
Accession Number ◽  
Crop Cycle ◽  
Garlic Virus ◽  
Sampling Times ◽  
Das Elisa

Garlic can be infected by a number of viruses, including allexiviruses. The coat protein sequence of an Allexivirus was detected in Argentina and deposited in the EMBL database as Garlic mite-borne filamentous virus (accession number X98991); it has high homology with Garlic virus A (GarV-A). For reliable virus detection, plants should be sampled when virus titer is high to reduce the risk of identifying infected plants as healthy. The objective of this study was to describe fluctuations in the concentration of this Argentine isolate of GarV-A in two garlic cultivars, Morado-INTA and Nieve-INTA, throughout the crop cycle using the double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Over a 2-year period, for both cultivars, virus concentration was assessed in samples from the tips section of the youngest leaves of GarV-A-infected plants, and from basal sections of both dormant and devernalized cloves of stored bulbs of Morado-INTA. The concentration of GarV-A varied during the crop cycle, but peaked at the beginning and again at the end of the crop cycle. Virus concentration was slightly higher in devernalized cloves compared with dormant cloves of Morado-INTA. No correlation between virus concentration and mean air temperature was observed. The results of this study recommend sampling times at the beginning of the crop cycle at 64 to 81 days after planting, and towards the end of the crop cycle to evaluate for the presence of GarV-A by DAS-ELISA.

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