scholarly journals A Highly Sensitive and Subspecies-Specific Surface Antigen Enzyme- Linked Immunosorbent Assay for Diagnosis of Johne's Disease

2006 ◽  
Vol 13 (8) ◽  
pp. 837-844 ◽  
Author(s):  
Shigetoshi Eda ◽  
John P. Bannantine ◽  
W. R. Waters ◽  
Yasuyuki Mori ◽  
Robert H. Whitlock ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Surface Antigens ◽  
Johne's Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Diagnostic Specificity ◽  
Serum Samples ◽  
Low Level ◽  
Specificity And Sensitivity ◽  
Highly Sensitive ◽  
High Level

ABSTRACT Johne's disease (JD), or paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis, is one of the most widespread and economically important diseases of livestock and wild ruminants worldwide. Control of JD could be accomplished by diagnosis and good animal husbandry, but this is currently not feasible because commercially available diagnostic tests have low sensitivity levels and are incapable of diagnosing prepatent infections. In this study, a highly sensitive and subspecies-specific enzyme-linked immunosorbent assay was developed for the diagnosis of JD by using antigens extracted from the surface of M. avium subsp. paratuberculosis. Nine different chemicals and various intervals of agitation by vortex were evaluated for their ability to extract the surface antigens. Various quantities of surface antigens per well in a 96-well microtiter plate were also tested. The greatest differences in distinguishing between JD-positive and JD-negative serum samples by ethanol vortex enzyme-linked immunosorbent assay (EVELISA) were obtained with surface antigens dislodged from 50 μg/well of bacilli treated with 80% ethanol followed by a 30-second interval of agitation by vortex. The diagnostic specificity and sensitivity of the EVELISA were 97.4% and 100%, respectively. EVELISA plates that had been vacuum-sealed and then tested 7 weeks later (the longest interval tested) had diagnostic specificity and sensitivity rates of 96.9 and 100%, respectively. In a comparative study involving serum samples from 64 fecal culture-positive cattle, the EVELISA identified 96.6% of the low-level fecal shedders and 100% of the midlevel and high-level shedders, whereas the Biocor ELISA detected 13.7% of the low-level shedders, 25% of the mid-level shedders, and 96.2% of the high-level shedders. Thus, the EVELISA was substantially superior to the Biocor ELISA, especially in detecting low-level and midlevel shedders. The EVELISA may form the basis for a highly sensitive and subspecies-specific test for the diagnosis of JD.

Development and preliminary application of gE enzyme-linked immunosorbent assay for detection of the antibody to gE protein ofPseudorabies virusin pigs

2005 ◽  
Vol 2 (2) ◽  
pp. 79-83 ◽  
Author(s):  
Tang Yong ◽  
Chen Huan-Chun ◽  
Qin Ya-Li ◽  
He Qi-Gai ◽  
Jin Mei-Lli ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Pseudorabies Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Diagnostic Specificity ◽  
Serum Samples ◽  
Agreement Rate ◽  
Glycoprotein E ◽  
Recombinant Glycoprotein ◽  
Specificity And Sensitivity ◽  
Preliminary Application

AbstractTo differentiate pigs infected withPseudorabies virus(PrV) from pigs vaccinated with gE-PrV, a glycoprotein E enzyme-linked immunosorbent assay (gE-ELISA) based on recombinant glycoprotein E (gE) (which was expressed byEscherichia coli, purified, denatured and renatured) was developed. By testing 115 serum samples, the diagnostic specificity and sensitivity of the developed gE-ELISA were evaluated to be 94.5% and 96.7%, respectively. Five serum samples were tested with plates from five lots, and the results had a coefficient of variation of less than 10%, showing good reproducibility of gE-ELISA. This gE-ELISA was compared with a commercial blocking ELISA by testing 356 serum samples. The agreement rate of the two assays was 92.13% (328/356). These results suggested that the gE-ELISA developed in our laboratory could be used in differentiating PrV-infected and gE-PrV-vaccinated pigs.

scholarly journals An enzyme-linked immunosorbent assay for the detection of IgG antibodies against Babesia equi in horses

2004 ◽  
Vol 34 (5) ◽  
pp. 1525-1529 ◽  
Author(s):  
Cristiane Divan Baldani ◽  
Rosangela Zacarias Machado ◽  
Paulo de Tarso Landgraf Botteon ◽  
Felipe Santoro Takakura ◽  
Carlos Luiz Massard
Keyword(s):  
Immunosorbent Assay ◽  
Serological Test ◽  
Epidemiological Studies ◽  
Sao Paulo ◽  
Enzyme Linked Immunosorbent Assay ◽  
São Paulo ◽  
Igg Antibodies ◽  
Serum Samples ◽  
Babesia Equi ◽  
Specificity And Sensitivity

A crude antigenic preparation of Babesia equi was used to develop and establish the suitability of an enzyme-linked immunosorbent assay (ELISA) for the detection of parasite carriers. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkboard titrations. The specificity and sensitivity of the ELISA were 100 %. A total of 90 serum samples were taken from horses from the Northeast region of São Paulo State and examined for diagnosis of equine B. equi infection by ELISA. Approximately 75% (n=67) of all the horses tested were found serologically positive for B. equi. These results suggest that the ELISA described may prove to be an appropriate serological test for epidemiological studies on B. equi infections in the field and that equine piroplasmosis is a cause for serious concern in the State of São Paulo, Brazil.

scholarly journals Detection of Francisella tularensis-Specific Antibodies in Patients with Tularemia by a Novel Competitive Enzyme-Linked Immunosorbent Assay

2012 ◽  
Vol 20 (1) ◽  
pp. 9-16 ◽  
Author(s):  
Neekun Sharma ◽  
Akitoyo Hotta ◽  
Yoshie Yamamoto ◽  
Osamu Fujita ◽  
Akihiko Uda ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Francisella Tularensis ◽  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
The Novel ◽  
Serum Samples ◽  
Content Type ◽  
Microagglutination Test ◽  
Highly Sensitive ◽  
High Degree

ABSTRACTA novel competitive enzyme-linked immunosorbent assay (cELISA) was developed and evaluated for detection of antibodies againstFrancisella tularensisin humans. The assay is based on the ability of serum antibodies to inhibit the binding of monoclonal antibodies (MAbs) directed againstF. tularensislipopolysaccharide antigens. The assay was evaluated using serum samples of tularemia patients, inactivatedF. tularensis-immunized rabbits, andF. tularensis-infected mice. Antibodies againstF. tularensiswere successfully detected in serum samples of tularemia patients as well as the immunized and infected animals. The cELISA method was compared to indirect ELISA (iELISA) and the commonly used microagglutination test (MA) using serum samples of 19 tularemia patients and 50 healthy individuals. The sensitivity and specificity of cELISA were 93.9 and 96.1%, respectively, in comparison to the iELISA. MA was less sensitive than cELISA with a sensitivity and specificity of only 81.8 and 98.0%, respectively. A high degree of correlation (R2= 0.8226) was observed between cELISA and iELISA results. The novel cELISA developed in this study appears to be highly sensitive and specific for serodiagnosis of human tularemia. The potential of the MAb-based cELISA to be used in both human and animal samples emphasizes its usefulness for serological survey of tularemia among multiple animal species.

Expression of sheep pathogen Babesia sp. Xinjiang rhoptry-associated protein 1 and evaluation of its diagnostic potential by enzyme-linked immunosorbent assay

Parasitology ◽  
2016 ◽  
Vol 143 (14) ◽  
pp. 1990-1999 ◽  
Author(s):  
QINGLI NIU ◽  
ZHIJIE LIU ◽  
JIFEI YANG ◽  
PEIFA YU ◽  
YUPING PAN ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Post Infection ◽  
Small Ruminants ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Diagnostic Potential ◽  
Positive Rate ◽  
Ct Antigen ◽  
High Level ◽  
Potential Use

SUMMARYOvine babesiosis is one of the most important tick-borne haemoparasitic diseases of small ruminants. The ovine parasite Babesia sp. Xinjiang is widespread in China. In this study, recombinant full-length XJrRAP-1aα2 (rhoptry-associated protein 1aα2) and C-terminal XJrRAP-1aα2 CT of Babesia sp. Xinjiang were expressed and used to evaluate their diagnostic potential for Babesia sp. Xinjiang infections by indirect enzyme-linked immunosorbent assay (ELISA). Purified XJrRAP-1aα2 was tested for reactivity with sera from animals experimentally infected with Babesia sp. Xinjiang and other haemoparasites using Western blotting and ELISA. The results showed no cross-reactivities between XJrRAP-1aα2 CT and sera from animals infected by other pathogens. High level of antibodies against RAP-1a usually lasted 10 weeks post-infection (wpi). A total of 3690 serum samples from small ruminants in 23 provinces located in 59 different regions of China were tested by ELISA. The results indicated that the average positive rate was 30·43%, and the infections were found in all of the investigated provinces. This is the first report on the expression and potential use of a recombinant XJrRAP-1aα2 CT antigen for the development of serological assays for the diagnosis of ovine babesiosis, caused by Babesia sp. Xinjiang.

scholarly journals Rapid Competitive Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Peste des Petits Ruminants Virus

2005 ◽  
Vol 12 (4) ◽  
pp. 542-547 ◽  
Author(s):  
Kang-Seuk Choi ◽  
Jin-Ju Nah ◽  
Young-Joon Ko ◽  
Shien-Young Kang ◽  
Nam-In Jo
Keyword(s):  
Immunosorbent Assay ◽  
Small Ruminants ◽  
Viral Disease ◽  
Turnaround Time ◽  
Enzyme Linked Immunosorbent Assay ◽  
Peste Des Petits Ruminants ◽  
Rinderpest Virus ◽  
Serum Samples ◽  
Specificity And Sensitivity ◽  
Relative Specificity

ABSTRACT Peste des petits ruminants (PPR) is a contagious viral disease of small ruminants that is of economic importance in Africa, the Middle East, and Asia. We developed a rapid competitive enzyme-linked immunosorbent assay (rapid c-ELISA) for the diagnosis and surveillance of PPR. This assay detects PPR virus (PPRV) antibodies in serum samples by quantifying the amount of monoclonal antibody (MAb) P-3H12 after 30 min of incubation of a serum-MAb conjugate mixture on plates coated with a PPRV recombinant nucleocapsid protein (rPPRV-N). We tested 249 PPRV-positive serum samples and 733 PPRV-negative serum samples from field ruminants. The threshold of percent inhibition (PI) was determined to be <50 on the basis of the mean PI plus 3 standard deviations for sera from PPRV-negative ruminants. The relative specificity and sensitivity of the rapid c-ELISA were 98.5% (722 of 733 serum samples) and 93.4% (234 of 249 serum samples), respectively. The rapid c-ELISA sensitively detected PPRV antibodies in hyperimmune sera (virus neutralization test [VNT] titer, >512), even at dilutions ≥512 in normal goat serum, and as early as 6 to 13 days postinfection from 12 goats, each of which was infected with one of the four PPRV lineages. Hyperimmune sera from animals experimentally vaccinated with rinderpest virus gave positive results by the rapid c-ELISA when the rinderpest virus VNT titers were >512, although the rapid c-ELISA titers were very low (2 to 16). However, the rapid c-ELISA was negative when the rinderpest virus VNT titer was ≤128. The rapid c-ELISA developed in the present work provides a short turnaround time and could be a useful tool for the diagnosis of PPR and screening for PPRV in the field.

scholarly journals An enzyme-linked immunosorbent assay (ELISA) for the detection of IgM antibodies against Leishmania chagasi in dogs

2009 ◽  
Vol 29 (2) ◽  
pp. 120-124 ◽  
Author(s):  
Débora Carvalho ◽  
Trícia M.F.S. Oliveira ◽  
Cristiane D. Baldani ◽  
Rosangela Z. Machado
Keyword(s):  
Visceral Leishmaniasis ◽  
Immunosorbent Assay ◽  
Fluorescent Antibody ◽  
Domestic Dog ◽  
Enzyme Linked Immunosorbent Assay ◽  
Leishmania Chagasi ◽  
Serum Samples ◽  
Igm Antibodies ◽  
Specificity And Sensitivity ◽  
Belo Horizonte

Visceral leishmaniasis is an emergent zoonosis with an increasing number of new cases in Brazil where the domestic dog is an important parasite reservoir in the infectious cycle of Leishmania chagasi. An enzyme-linked immunosorbent assay (ELISA), based upon the use of a total soluble antigenic preparation of L. chagasi, was adapted for the detection of IgM antibodies in the serum of infected dogs. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkboard titrations. The specificity and sensitivity of the ELISA were 100 %. A total of 110 serum samples were taken from dogs in Belo Horizonte, Minas Gerais, Brazil, and examined for anti-L. chagasi IgM antibody by ELISA and indirect fluorescent antibody test (IFAT). About 25% (n=27) of all the dogs tested were found serologically positive for L. chagasi by IFAT, while 89.09% (n=98) were seropositive by ELISA. The results obtained by ELISA and IFAT were significantly different (P<0.01). The combined use of ELISA and IFAT is recommended in order to enable veterinary services to more efficiently detect canine visceral leishmaniasis.

scholarly journals Lipopolysaccharide-Based Enzyme-Linked Immunosorbent Assay for Experimental Use in Detection of Antibodies to Lawsonia intracellularis in Pigs

2005 ◽  
Vol 12 (6) ◽  
pp. 693-699 ◽  
Author(s):  
J. J. Kroll ◽  
M. A. Eichmeyer ◽  
M. L. Schaeffer ◽  
S. McOrist ◽  
D. L. Harris ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Indirect Elisa ◽  
Experimental Studies ◽  
Antibody Test ◽  
Controlled Study ◽  
Future Research ◽  
Enzyme Linked Immunosorbent Assay ◽  
Lawsonia Intracellularis ◽  
Serum Samples ◽  
Specificity And Sensitivity

ABSTRACT An enzyme-linked immunosorbent assay (ELISA) for Lawsonia intracellularis was developed and compared with a whole-cell antigen-based immunofluorescence antibody test (IFAT). The antigen-containing lipopolysaccharide (LPS) was derived from Percoll gradient purified cultures of L. intracellularis by using a modification of the Westphal hot phenol procedure. The antigen was bound directly to polystyrene 96-well microtiter plates, and the assay was performed in an indirect ELISA format. Specificity and sensitivity values based on 80 known positive and 80 known negative serum samples from controlled experimental trials were 93.7% and 88.7%, respectively. Serological results from a controlled L. intracellularis challenge exposure study confirmed the high specificity and sensitivity of this assay (100% and 99.5%, respectively). Comparisons between the LPS ELISA and the IFAT in detecting anti-Lawsonia antibodies in this controlled study revealed significantly more LPS ELISA-positive pigs than IFAT-positive pigs on days 21, 28, 35, and 42 (P = 0.003, 0.030, 0.002, and 0.006, respectively). This indirect ELISA (LPS ELISA) test is an improved method of detecting antibodies in pigs soon after exposure to L. intracellularis, regardless of isolate type (vaccine or wild type) in experimental studies. The LPS ELISA may be used as a tool to support future research trials on vaccine efficacy and to further understand the immune response induced by L. intracellularis.

scholarly journals Development of a Highly Sensitive and Specific Enzyme-Linked Immunosorbent Assay Based on Recombinant Matrix Protein for Detection of Avian Pneumovirus Antibodies

2000 ◽  
Vol 38 (11) ◽  
pp. 4010-4014 ◽  
Author(s):  
Baldev R. Gulati ◽  
Kjerstin T. Cameron ◽  
Bruce S. Seal ◽  
Sagar M. Goyal ◽  
David A. Halvorson ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Matrix Protein ◽  
Clinical Signs ◽  
M Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Specific Test ◽  
Avian Pneumovirus ◽  
Highly Sensitive ◽  
The Matrix

The matrix (M) protein of avian pneumovirus (APV) was evaluated for its antigenicity and reliability in an enzyme-linked immunosorbent assay (ELISA) for diagnosis of APV infection, a newly emergent disease of turkeys in United States. Sera from APV-infected turkeys consistently contained antibodies to a 30-kDa protein (M protein). An ELISA based on recombinant M protein generated in Escherichia coli was compared with the routine APV ELISA that utilizes inactivated virus as antigen. Of 34 experimentally infected turkeys, 33 (97.1%) were positive by M protein ELISA whereas only 18 (52.9%) were positive by routine APV ELISA 28 days after infection. None of the serum samples from 41 uninfected experimental turkeys were positive by M protein ELISA. Of 184 field sera from turkey flocks suspected of having APV infection, 133 (72.3%) were positive by M protein ELISA whereas only 99 (53.8%) were positive by routine APV ELISA. Twelve serum samples, which were negative by M protein ELISA but positive by routine APV ELISA, were not reactive with either recombinant M protein or denatured purified APV proteins by Western analysis. This indicates that the samples had given false-positive results by routine APV ELISA. The M protein ELISA was over six times more sensitive than virus isolation (11.5%) in detecting infections from samples obtained from birds showing clinical signs of APV infection. Taken together, these results show that ELISA based on recombinant M protein is a highly sensitive and specific test for detecting antibodies to APV.

scholarly journals Comparative Performance of a Novel Herpes Simplex Virus Type 2-Specific Enzyme-Linked Immunosorbent Assay Using a Targeted Chain Oligopeptide, Peptide 55

2009 ◽  
Vol 16 (6) ◽  
pp. 931-934 ◽  
Author(s):  
A. M. Al-Sulaiman ◽  
P. J. Vallely ◽  
P. E. Klapper
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Igg Antibody ◽  
Comparative Performance ◽  
Simplex Virus ◽  
High Level

ABSTRACT Herpes simplex virus (HSV) glycoprotein G (gG2) has been used as the basis of many serological assays for the detection of HSV type 2 (HSV-2)-specific antibodies. In the present study, an enzyme-linked immunosorbent assay (ELISA), the Pathozyme Viro HSV-2 immunoglobulin G (IgG) ELISA (Omega Diagnostics, Alva, United Kingdom), based on an immunodominant epitope of gG2 presented in a branched-chain format (peptide 55), was compared with two commercially available gG2-specific assays, the Bioelisa HSV-2 IgG assay (Biokit, S.A., Barcelona, Spain) and the HerpesSelect HSV-2 IgG assay (Focus Diagnostics, Cypress, CA). A panel of 218 well-characterized serum samples was tested. Thirty-one samples were determined to be HSV-2 IgG antibody positive and 164 samples were determined to be negative with all three kits. The levels of concordance between the tests were 95.9% between the Omega and HerpeSelect assays, 90.8% between the Omega and Bioelisa assays, and 94.5% between the HerpeSelect and Bioelisa assays. Twenty-three samples gave discordant results. Western blot results showed that of these, the results for 77% were correctly identified by the Omega assay, the results for 68% were correctly identified by the HerpeSelect assay, and the results for 13.6% were correctly identified by the Bioelisa assay. Although there was a high level of agreement between the results obtained by the three assays and no false-positive results were detected by any of the three kits, confirmation of the results for samples with discordant results by Western blotting suggested that the peptide 55-based Omega assay is the most sensitive and specific assay among the assays evaluated.

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