scholarly journals Development of a double monoclonal antibody–based sandwich enzyme-linked immunosorbent assay for detecting canine distemper virus

2020 ◽  
Vol 104 (24) ◽  
pp. 10725-10735
Author(s):  
Yuan Zhang ◽  
Gang Xu ◽  
Lu Zhang ◽  
Jiakai Zhao ◽  
Pinpin Ji ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Immunosorbent Assay ◽  
Colloidal Gold ◽  
Canine Distemper Virus ◽  
Sandwich Elisa ◽  
Enzyme Linked Immunosorbent Assay ◽  
Test Results ◽  
Canine Distemper ◽  
Serum Samples ◽  
Ideal Method

Abstract Canine distemper virus (CDV) infection causes mass mortality in diverse carnivore species. For effective virus surveillance, rapid and sensitive assays are needed to detect CDV in field samples. In this study, after BABL/c mice were immunized with recombinant CDV-fusion (F) protein, monoclonal antibodies (mAbs) against recombinant CDV-F protein (designated 1A5, 1A6, and 7D5) were produced using traditional hybridoma cell technology. Next, capture antibody (1A6, 800 ng/well) and horseradish peroxidase (HRP)–conjugated detection antibody (HRP-7D5, 1:100, 500 ng/well) were used in a double monoclonal antibody–based sandwich enzyme-linked immunosorbent assay (ELISA) for CDV detection after optimization of both mAb amounts per well using a checkerboard titration test. Based on sandwich ELISA test results for 120 known CDV-negative samples, the cutoff value for a positive result was set to an OD450 nm value ≥ 0.196. As compared with test results obtained from commercial immune colloidal gold test strips, the low limits of detection for the two assays were revealed to be 100 TCID50 per 100 μL. In addition, the sandwich ELISA agreed 100% and 96.4% with commercial immune colloidal gold test strips when testing serum and stool samples. The sandwich ELISA assay provided statistically similar CDV detection. Thus, the sandwich ELISA developed here to detect CDV in fecal and serum samples provided good sensitivity, high specificity, and good reproducibility and should serve as an ideal method for large-scale surveillance of CDV infections in carnivores. Key points • Three CDV mAbs that recognized different epitopes and bound to virion were generated. • The sandwich ELISA based mAbs to detect CDV in fecal and serum samples was developed. • The sandwich ELISA is an ideal method for detecting CDV infections in the field.

Sandwich enzyme immunoassay of osteocalcin in serum with use of an antibody against human osteocalcin

1993 ◽  
Vol 39 (6) ◽  
pp. 942-947 ◽  
Author(s):  
D A Monaghan ◽  
M J Power ◽  
P F Fottrell
Keyword(s):  
Monoclonal Antibody ◽  
Enzyme Immunoassay ◽  
Solid Phase ◽  
Sandwich Elisa ◽  
Normal Subjects ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Standard Curve ◽  
Microtiter Plates ◽  
Human Osteocalcin

Abstract We have developed and thoroughly validated a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) on microtiter plates for osteocalcin in human serum with use of an antibody raised against human osteocalcin. We used a monoclonal antibody against bovine osteocalcin as the capture antibody; the second antibody was a polyclonal antibody against human osteocalcin. The amount of bound second antibody was determined with use of swine anti-rabbit antibody labeled with horseradish peroxidase. We demonstrated independence of volume and determined the recovery of added standard and within- and between-assay precision. The minimal detection limit for osteocalcin was between 1.0 and 1.5 micrograms/L and the midpoint of the standard curve ranged from 14 to 17 micrograms/L. The intraassay CV was < or = 8% in the range 2.7-52 micrograms/L; the interassay CV was usually < or = 15% in the same range. Analytical recovery of human osteocalcin standard added to serum samples was consistently > 90%. Values for osteocalcin measured in serum from 44 normal subjects were similar to those obtained with a competitive enzyme immunoassay (EIA) that used a monoclonal antibody against bovine osteocalcin. There was a good correlation between the two assays [r2 = 0.877, slope and intercept (+/- SE) = 0.88(+/- 0.051) and 0.316(+/- 0.523), respectively]. The range and mean (+/- SD) for the sandwich ELISA and the competitive EIA were 1.7-18.1 micrograms/L [8.7(+/- 4.4) micrograms/L] and 1.9-22.8 micrograms/L [9.1(+/- 4.4) micrograms/L], respectively.

scholarly journals Antibodies against canine distemper virus, parvovirus and Ehrlichia spp. in wild captive carnivores in midwestern Brazil

2018 ◽  
Vol 38 (8) ◽  
pp. 1681-1684
Author(s):  
Isis I.G.G. Taques ◽  
Thaís O. Morgado ◽  
Ísis A. Braga ◽  
Regina C.R. Paz ◽  
Sandra H.R. Corrêa ◽  
...  
Keyword(s):  
Canine Distemper Virus ◽  
Clinical History ◽  
Neutralization Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Canine Distemper ◽  
Serum Samples ◽  
Virus Neutralization Assay ◽  
Hemagglutination Inhibition Test ◽  
History Of ◽  
Indirect Immunofluorescent

ABSTRACT: The occurrence of antibodies against canine distemper virus (CDV), parvovirus and Ehrlichia spp. in wild captive carnivores was evaluated in a zoological park in midwestern Brazil. Serum samples were collected between 2007 and 2014 from 45 carnivores. Antibodies were evaluated by virus neutralization assay for CDV, hemagglutination inhibition test for parvovirus, indirect immunofluorescent and Enzyme-linked immunosorbent assay for Ehrlichia spp. Antibodies against CDV and parvovirus were detected in 75% of Canidae and Felidae. Procyonidae were negative for CDV, although one Mustelidae was positive. TwoCanidae presented antibodies reactive to E. canis antigens. The high antibodies rates to CDV and parvovirus suggest the contact with both pathogens, however since no clinical history of disease are registered in the Zoo-UFMT, we can presume that carnivores have responded satisfactorily against the antigens. The low serological rates observed against Ehrlichia spp. may be resulted to the low occurrence of ticks among carnivores.

scholarly journals Serological Survey of Canine Distemper Virus Infection Using Enzyme-Linked Immunosorbent Assay.

1995 ◽  
Vol 57 (4) ◽  
pp. 761-763 ◽  
Author(s):  
Tsuyoshi GEMMA ◽  
Naoko MIYASHITA ◽  
Yeon-Sil SHIN ◽  
Masatsugu OKITA ◽  
Takeshi MORI ◽  
...  
Keyword(s):  
Virus Infection ◽  
Immunosorbent Assay ◽  
Canine Distemper Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serological Survey ◽  
Canine Distemper

scholarly journals Quantitation of Canine Distemper Virus and Antibodies by Enzyme-Linked Immunosorbent Assays Using Protein A and Monoclonal Antibody Capture

1989 ◽  
Vol 1 (2) ◽  
pp. 110-115 ◽  
Author(s):  
Leon N. D. Potgieter ◽  
Peace A. Ajidagba
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Cell Cultures ◽  
Canine Distemper Virus ◽  
Solid Phase ◽  
Sandwich Elisa ◽  
Protein A ◽  
Canine Distemper ◽  
Clinical Specimens ◽  
Immunosorbent Assays

Monoclonal antibodies produced from 19 cloned hybridomas were selected for this study. Specific canine distemper virus (CDV) antibodies in medium from cloned hybridomas were detected by direct enzyme-linked immunosorbent assays (ELISA) and by indirect immunofluorescence. Three different sandwich ELISA systems were developed either to detect CDV in cell cultures and clinical specimens or to detect specific antibody in canine sera. Protein A and monoclonal antibodies attached in sequence to a solid phase constituted the capture system in the assays. Viral antigens were detected by sandwiching extracts of clinical specimens (or infected cell cultures), monoclonal antibody, and peroxidase-labeled protein A in sequence onto the capture layer. In 1 procedure, biotin-labeled antibody and peroxidase-labeled avidin were used as the last 2 layers in the assay. The CDV antibodies in dog sera were quantitated in a similar manner, but the sequential sandwiching levels consisted of partially purified CDV, serum specimen, and peroxidase-labeled protein A, respectively. The procedures were specific and highly sensitive.

scholarly journals Development and Application of a Double-Antigen Sandwich Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Porcine Circovirus 2

2012 ◽  
Vol 19 (9) ◽  
pp. 1480-1486 ◽  
Author(s):  
Meng Ge ◽  
Wei Luo ◽  
Daliang Jiang ◽  
Runcheng Li ◽  
Wenwei Zhao ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Immunosorbent Assay ◽  
Sandwich Elisa ◽  
Test Procedure ◽  
Porcine Circovirus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Swine Industry ◽  
Elisa Kit ◽  
Porcine Circovirus 2

ABSTRACTA double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) is described for detection of porcine circovirus 2 (PCV2) antibodies using the well-characterized recombinant PCV2 capsid protein. In a comparative test of 394 pig sera against an indirect immunofluorescence (IIF) test and a commercial ELISA kit (also based on the recombinant PCV2 capsid protein), the results showed that the diagnostic sensitivity, specificity, and accuracy of the assay were, respectively, 90.61, 94.02, and 91.62% compared with IIF and 94.38, 95.28, and 94.67% compared with the commercial ELISA kit. Assay of 12 PCV-free pigs over a 5-week period produced only PCV2-negative titers by all 3 methods. These results and the seroprofiles of 4 pig farms obtained by both the commercial ELISA kit and the double-antigen sandwich ELISA indicate that the sandwich ELISA is a reliable method for detection of antibodies to PCV2. Additionally, the method described here permits the use of undiluted test serum samples simultaneously loaded with horseradish peroxidase (HRP)-conjugated antigen into the test well, and the complete test procedure can be performed in less than 90 min. This double-antigen sandwich ELISA should be a useful tool to aid swine industry professionals in deciding the intervention strategies for the control of PCV2-associated diseases.

Sign in / Sign up

Export Citation Format

Share Document