scholarly journals Detection of Equine Antibodies to Babesia caballi by Recombinant B. caballi Rhoptry-Associated Protein 1 in a Competitive-Inhibition Enzyme-Linked Immunosorbent Assay

1999 ◽  
Vol 37 (7) ◽  
pp. 2285-2290 ◽  
Author(s):  
Lowell S. Kappmeyer ◽  
Lance E. Perryman ◽  
Stephen A. Hines ◽  
Timothy V. Baszler ◽  
Jonathan B. Katz ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Competitive Inhibition ◽  
Immunofluorescence Assay ◽  
Cloning And Expression ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Gene Encoding ◽  
Peptide Epitope ◽  
Babesia Caballi ◽  
Serum Dilution

A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was developed for detection of equine antibodies specific forBabesia caballi. The assay used recombinant B. caballi rhoptry-associated protein 1 (RAP-1) and monoclonal antibody (MAb) 79/17.18.5, which is reactive with a peptide epitope of a native 60-kDa B. caballi antigen. The gene encoding the recombinant antigen was sequenced, and database analysis revealed that the gene product is a rhoptry-associated protein. Cloning and expression of a truncated copy of the gene demonstrated that MAb 79/17.18.5 reacts with the C-terminal repeat region of the protein. The cELISA was used to evaluate 302 equine serum samples previously tested for antibodies to B. caballi by a standardized complement fixation test (CFT). The results of cELISA and CFT were 73% concordant. Seventy-two of the 77 serum samples with discordant results were CFT negative and cELISA positive. Further evaluation of the serum samples with discordant results by indirect immunofluorescence assay (IFA) demonstrated that at a serum dilution of 1:200, 48 of the CFT-negative and cELISA-positive serum samples contained antibodies reactive with B. caballi RAP-1. Four of five CFT-positive and cELISA-negative serum samples contained antibodies reactive withB. caballi when they were tested by IFA. These data indicate that following infection with B. caballi, horses consistently produce antibody to the RAP-1 epitope defined by MAb 79/17.18.5, and when used in the cELISA format, recombinant RAP-1 is a useful antigen for the serologic detection of anti-B. caballi antibodies.

scholarly journals Molecular Cloning of a Babesia caballi Gene Encoding the 134-Kilodalton Protein and Evaluation of Its Diagnostic Potential in an Enzyme-Linked Immunosorbent Assay

2004 ◽  
Vol 11 (1) ◽  
pp. 211-215 ◽  
Author(s):  
Yoh Tamaki ◽  
Haruyuki Hirata ◽  
Noriyuki Takabatake ◽  
Sabine Bork ◽  
Naoaki Yokoyama ◽  
...  
Keyword(s):  
Molecular Cloning ◽  
Immunosorbent Assay ◽  
Horse Serum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gene Encoding ◽  
Infected Horse ◽  
Babesia Caballi ◽  
Babesia Equi ◽  
Diagnostic Potential ◽  
Control Horse

ABSTRACT A Babesia caballi gene encoding the 134-kDa (BC134) protein was immunoscreened with B. caballi-infected horse serum. An enzyme-linked immunosorbent assay (ELISA) using recombinant BC134 protein could effectively differentiate B. caballi-infected horse sera from Babesia equi-infected or noninfected control horse sera. These results suggest that the recombinant BC134 protein is a potential diagnostic antigen in the detection of B. caballi infection.

scholarly journals Detection of Anaplasma antibodies in wildlife and domestic species in wildlife-livestock interface areas of Kenya by major surface protein 5 competitive inhibition enzyme-linked immunosorbent assay

2008 ◽  
Vol 75 (3) ◽  
Author(s):  
J.J.N. Ngeranwa ◽  
S.P. Shompole ◽  
E.H. Venter ◽  
A. Wambugu ◽  
J.E. Crafford ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Competitive Inhibition ◽  
Surface Membrane ◽  
Clinical Signs ◽  
Surface Protein ◽  
Domestic Animal ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Domestic Species ◽  
Major Surface

The seroprevalence of Anaplasma antibodies in wildlife (eland, blue wildebeest, kongoni, impala, Thomson's gazelle, Grant's gazelle, giraffe and plains zebra) and domestic animal (cattle, sheep and goat) populations was studied in wildlife / livestock interface areas of Kenya. Serum samples were analyzed by competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA), using a recombinant antigen (MSP-5) from Anaplasma marginale surface membrane. A monoclonal antibody, FC-16, was used as the primary antibody, while anti-mouse conjugated to horseradish peroxidase was used as the secondary antibody. The results indicate a high seroprevalence in both wildlife and livestock populations, in contrast to earlier reports from Kenya, which indicated a low seroprevalence. The differences are attributed to the accurate analytical method used (CI-ELISA), as compared with agglutination techniques, clinical signs and microscopy employed by the earlier workers.

scholarly journals  Toxoplasma gondii and Neospora caninum antibodies in goats in the Czech Republic

2012 ◽  
Vol 57 (No. 3) ◽  
pp. 111-114 ◽  
Author(s):  
E. Bartova ◽  
K. Sedlak
Keyword(s):  
Czech Republic ◽  
Toxoplasma Gondii ◽  
Immunosorbent Assay ◽  
Competitive Inhibition ◽  
Neospora Caninum ◽  
Fluorescent Antibody ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Antibodies ◽  
Serum Samples ◽  
The Czech Republic

Toxoplasma gondii is zoonotic protozoan parasite that causes infections in many vertebrate species. The present study determined the seroprevalence of T. gondii and N. caninum in goats from the Czech Republic. Serum samples were collected from 251 healthy adult goats in the Czech Republic during the years 2006 to 2009. Sera samples were tested for serum antibodies to Toxoplasma gondii by an enzyme-linked immunosorbent assay with cut off equal to or higher than 50% S/P. The same samples were tested for serum antibodies to Neospora caninum by a competitive-inhibition enzyme-linked immunosorbent assay with cut off equal to or higher than 30% inhibition; positive sera were confirmed by an indirect fluorescent antibody test with cut-off titre equal to or higher than 40. Sera positive in both tests were marked as positive. In total, 166 (66%) and 15 (6%) goat sera reacted positively for T. gondii and N. caninum antibodies, respectively. All sera positive for N. caninum antibodies were simultaneously positive for T. gondii antibodies. This is the first detection of N. caninum antibodies in goats in the Czech Republic. Our findings indicate that goats in the Czech Republic are frequently exposed to T. gondii, but less frequently to N. caninum.  

scholarly journals An enzyme-linked immunosorbent assay for the detection of mouse polyomavirus-specific antibodies in laboratory mice

1994 ◽  
Vol 28 (3) ◽  
pp. 257-261 ◽  
Author(s):  
H. W. J. Broeders ◽  
J. Groen ◽  
G. van Steenis ◽  
A. D. M. E. Osterhaus
Keyword(s):  
Immunosorbent Assay ◽  
Haemagglutination Inhibition ◽  
Immunofluorescence Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Pathogen Free ◽  
Laboratory Mice ◽  
Serum Samples ◽  
Laboratory Rodents ◽  
Antibody Titres ◽  
Specific Pathogen

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection and quantification of IgM and IgG serum antibodies to mouse polyomavirus (MPV). To evaluate the potential of this ELISA for the screening of laboratory rodents, serum samples from specific pathogen free (SPF) BALB/c RIVM mice, collected after experimental intraperitoneal infection with MPV, were tested by this assay. The results were compared with those obtained from the same sera in an immunofluorescence assay (IFA) and a haemagglutination inhibition assay (HIA). The ELISA proved to be the most sensitive of the 3 assays, allowing the detection of seropositive animals within 7 days post-infection and giving antibody titres that were about 4 to 8 times higher than those found in the IFA and HIA respectively. More than 5000 serum samples from non-infected specific pathogen free laboratory mice and 90 from 10 SPF N:NIH/RIVM mice experimentally infected with K-papovavirus, were negative in this assay, thus confirming the specificity of the ELISA

scholarly journals Comparison of Enzyme-Linked Immunosorbent Assay, Western Blotting, Microagglutination, Indirect Immunofluorescence Assay, and Flow Cytometry for Serological Diagnosis of Tularemia

2004 ◽  
Vol 11 (6) ◽  
pp. 1008-1015 ◽  
Author(s):  
Mustafa Porsch-Özcürümez ◽  
Nele Kischel ◽  
Heidi Priebe ◽  
Wolf Splettstösser ◽  
Ernst-Jürgen Finke ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Western Blotting ◽  
Francisella Tularensis ◽  
Immunosorbent Assay ◽  
Indirect Immunofluorescence ◽  
Immunofluorescence Assay ◽  
Indirect Immunofluorescence Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Diagnostic Sensitivity And Specificity

ABSTRACT The serodiagnostic efficiencies of five different approaches to detecting antibodies (immunoglobulins G, A, and M) developed in clinically proven infections with Francisella tularensis have been assessed. Fifty serum samples from patients suffering from tularemia during an outbreak in Sweden were compared with samples from 50 healthy blood donors (controls) by using an enzyme-linked immunosorbent assay (ELISA), microagglutination (MA), Western blotting (WB), an indirect immunofluorescence assay (IIFA), and flow cytometry (FC). ELISA, WB, and FC were based on the use of preparations of lipopolysaccharides (LPS) of the live vaccine strain of Francisella tularensis subsp. holarctica (ATCC 29684) as a capture antigen. Whole methanol-fixed bacteria were used for IIFA and MA. Optimized protocols yielded a diagnostic sensitivity and specificity of 100% for WB, MA, and FC, 98% for ELISA, and 93% for IIFA. A total of 6,632 serum samples from individuals between the ages of 18 and 79 years, representatively recruited from all regions of Germany, were screened to estimate and confirm the positive predictive value (PVpos) of the ELISA. Serum samples from 15 (0.226%) individuals tested positive for F. tularensis-specific antibodies by ELISA and confirmatory WB. The resulting prevalence-dependent PVpos of 10.2% and specificity of 98.1% were consistent with our findings for tularemia patients and controls. We conclude that the combined usage of a screening ELISA and a confirmatory WB based on LPS as a common antigen, as well as the MA, is a suitable serodiagnostic tool, while the quality of the IIFA is hampered by subjective variations of the results. FC is a promising new approach that might be improved further in terms of multiplex analyses or high-throughput applications.

scholarly journals A Peptide-Based Enzyme-Linked Immunosorbent Assay for Detecting Antibodies Against Avian Infectious Bronchitis Virus

2021 ◽  
Vol 7 ◽  
Author(s):  
Liping Yin ◽  
Qi Wu ◽  
Zhixian Lin ◽  
Kun Qian ◽  
Hongxia Shao ◽  
...  
Keyword(s):  
Antibody Response ◽  
Immunosorbent Assay ◽  
Infectious Bronchitis Virus ◽  
Immunofluorescence Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Specific Antibody Response ◽  
Infectious Bronchitis ◽  
Conserved Sequence ◽  
Sensitivity Specificity

Infectious bronchitis virus (IBV) causes substantial loss to the poultry industry despite extensive vaccination. Assessing the antibody response is important for the development and evaluation of effective vaccines. We have developed an enzyme-linked immunosorbent assay (ELISA) for the detection of IBV-specific antibodies, using a synthetic peptide based on a conserved sequence in the IBV spike protein. This peptide-based ELISA (pELISA) specifically detects antibodies to different genotypes of IBV but not antibodies against other common chicken viruses. This assay could detect IBV-specific antibody response on as early as day 7 postinfection. In the testing with field serum samples collected from chickens administered with IBV vaccines, the sensitivity, specificity, and accuracy of pELISA were 98.30, 94.12, and 98.8%, respectively, relative to indirect immunofluorescence assay. Our data demonstrate that the pELISA is of value for the detection of IBV antibody and the evaluation of IBV vaccines.

scholarly journals Use of Protein AG in an Enzyme-Linked Immunosorbent Assay for Screening for Antibodies against Parapoxvirus in Wild Animals in Japan

1999 ◽  
Vol 6 (3) ◽  
pp. 388-391 ◽  
Author(s):  
Yasuo Inoshima ◽  
Shinya Shimizu ◽  
Nobuyuki Minamoto ◽  
Katsuya Hirai ◽  
Hiroshi Sentsui
Keyword(s):  
Immunosorbent Assay ◽  
Japanese Monkey ◽  
Cervus Nippon ◽  
Black Bear ◽  
Immunofluorescence Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Nyctereutes Procyonoides ◽  
Wild Animals ◽  
Serum Samples ◽  
Myocastor Coypus

ABSTRACT Using protein AG in an enzyme-linked immunosorbent assay (ELISA), we tried to detect antibodies against parapoxvirus in 9 species of wild animals in Japan: the Japanese badger (Meles meles anakuma), Japanese black bear (Ursus thibetanus japonicus), Japanese deer (Cervus nippon centralis), Japanese monkey (Macaca fuscata), Japanese raccoon dog (Nyctereutes procyonoides viverrinus), Japanese serow (Capricornis crispus), Japanese wild boar (Sus scrofa leucomystax), masked palm civet (Paguma larvata), and nutria (Myocastor coypus). A total of 272 serum samples were collected over the period from 1984 to 1995 and were tested by the protein AG-ELISA, the agar gel immunodiffusion test, and an indirect immunofluorescence assay. The protein AG-ELISA was effective in a serological survey for parapoxvirus in wild animals, and antibodies were detected only in Japanese serows. A total of 24 of 66 (36.4%) Japanese serows reacted positively, and they were found in almost all prefectures in all years tested. These results suggest that epizootic cycles of parapoxvirus exist widely in Japanese serows and that they could be reservoirs for the virus in the field in Japan. Moreover, it is probable that they might carry the virus to domestic animals such as cattle, sheep, and goats.

scholarly journals Development of a sensitive competitive enzyme-linked immunosorbent assay for serodiagnosis of Burkholderia mallei, a Tier 1 select agent

2021 ◽  
Vol 15 (12) ◽  
pp. e0010007
Author(s):  
Ulrich Wernery ◽  
Elaine Chan ◽  
Rekha Raghavan ◽  
Jade L. L. Teng ◽  
Ginu Syriac ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
High Sensitivity ◽  
Microtiter Plate ◽  
Enzyme Linked Immunosorbent Assay ◽  
Burkholderia Mallei ◽  
Serum Samples ◽  
Serum Dilution ◽  
Cutoff Value ◽  
Tier 1 ◽  
Serious Disease

Glanders is a highly contagious and potentially serious disease caused by Burkholderia mallei, a Tier 1 select agent. In this study, we raised a monoclonal antibody (mAb) against the lipopolysaccharide (LPS) of B. mallei and developed a competitive enzyme-linked immunosorbent assay (cELISA) for B. mallei infection. Using the titrated optimal conditions of B. mallei-LPS (2 ng) for microtiter plate coating, sample serum dilution at 1:20 and 3.5 ng/μL anti-LPS mAb B5, the cutoff value of the cELISA was determined using serum samples from 136 glanders-free seronegative horses in Hong Kong. All calculated percentage inhibition (PI) values from these seronegative samples were below 39.6% inhibition (1.5 standard deviations above mean PI) and was used as the cutoff value. The diagnostic sensitivity of the developed LPS-based cELISA was first evaluated using sera from donkeys and mice inoculated with B. mallei. An increasing trend of PI values above the defined cELISA cutoff observed in the donkey and mouse sera suggested positive detection of anti-LPS antibodies. The sensitivity and specificity of the LPS-based cELISA was further evaluated using 31 serologically positive horse sera from glanders outbreaks in Bahrain and Kuwait, of which 30 were tested positive by the cELISA; and 21 seronegative horse sera and 20 seronegative donkey sera from Dubai, of which all were tested negative by the cELISA. A cELISA with high sensitivity (97.2%) and specificity (100%) for the detection of B. mallei antibodies in different animals was developed.

scholarly journals Cloning and Expression of a 48-KilodaltonBabesia caballi Merozoite Rhoptry Protein and Potential Use of the Recombinant Antigen in an Enzyme-Linked Immunosorbent Assay

1999 ◽  
Vol 37 (11) ◽  
pp. 3475-3480 ◽  
Author(s):  
Hiromi Ikadai ◽  
Xuenan Xuan ◽  
Ikuo Igarashi ◽  
Shigeyasu Tanaka ◽  
Takumi Kanemaru ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Cloning And Expression ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cdna Expression Library ◽  
Expression Library ◽  
Infected Horse ◽  
Babesia Caballi ◽  
Reading Frame ◽  
Highly Sensitive ◽  
Rhoptry Protein

A cDNA expression library prepared from Babesia caballimerozoite mRNA was screened with a monoclonal antibody BC11D against the rhoptry protein of B. caballi merozoite. A cDNA encoding a 48-kDa protein of B. caballi was cloned and designated BC48. The complete nucleotide sequence of the BC48 gene had 1,828 bp and was shown to contain no intron. Southern blotting analysis indicated that the BC48 gene contained more than two copies in theB. caballi genome. Computer analysis suggested that this sequence contained an open reading frame of 1,374 bp with a coding capacity of approximately 52 kDa. The recombinant protein expressed by the vaccinia virus vector in horse cells had an apparent molecular mass of 48 kDa, which was the same as that of the native B. caballi 48-kDa protein. Moreover, recombinant proteins expressed by the pGEX4T expression vector in Escherichia coli as glutathione S-transferase fusion proteins were used for antigen in an enzyme-linked immunosorbent assay (ELISA). The ELISA was able to differentiate very clearly between B. caballi-infected horse sera and B. equi-infected horse sera or noninfected normal horse sera. These results suggest that this simple and highly sensitive test might be applicable to the detection of B. caballi-infected horses in the field.

scholarly journals Use of Peptide-Based Enzyme-Linked Immunosorbent Assay followed by Immunofluorescence Assay To Document Ehrlichia chaffeensis as a Cause of Febrile Illness in Nicaragua

2016 ◽  
Vol 54 (6) ◽  
pp. 1581-1585 ◽  
Author(s):  
Ijeuru Chikeka ◽  
Armando J. Matute ◽  
J. Stephen Dumler ◽  
Christopher W. Woods ◽  
Orlando Mayorga ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Large Scale ◽  
Febrile Illness ◽  
The United States ◽  
Immunofluorescence Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Ehrlichia Chaffeensis ◽  
Acute Febrile Illness ◽  
Serum Samples ◽  
Content Type

Ehrlichia chaffeensis, the etiologic agent of human monocytic ehrlichiosis (HME), has been extensively studied as a cause of acute febrile illness and an emerging tick-borne zoonosis in the United States. Limited data suggest its presence in other regions, including Central and South America but not Nicaragua to date. Diagnosis ofE. chaffeensisinfection by indirect immunofluorescence assay (IFA) is the reference standard due to its presumed high sensitivity and specificity, but IFA is impractical, variably reproducible, and cumbersome for large epidemiologic studies and for clinical diagnosis in resource-poor regions. We evaluated a high-throughput, objective peptide-based enzyme-linked immunosorbent assay (ELISA) for use alone or in combination with IFA. We found that it performed best as a screening test (sensitivity, 100%; specificity, 84%) to reduce the proportion of serum samples that were required by the more cumbersome and subjective IFA testing to <20%. Using a two-step diagnostic approach (IFA is performed if the ELISA is positive), we identifiedE. chaffeensisor a serologically and antigenically similar organism as a heretofore unrecognized cause of acute febrile illness in humans in Nicaragua and demonstrated the utility of the peptide ELISA as a screening tool for large-scale clinical studies.

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