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 Antibodies to Babesia equi in Horses by a Latex Agglutination Test Using Recombinant EMA-1

2001 ◽  
Vol 8 (3) ◽  
pp. 645-646 ◽  
Author(s):  
Xuenan Xuan ◽  
Ikuo Igarashi ◽  
Tetsuya Tanaka ◽  
Shinya Fukumoto ◽  
Hideyuki Nagasawa ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Screening Method ◽  
Latex Agglutination ◽  
Agglutination Test ◽  
Routine Screening ◽  
Latex Agglutination Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Babesia Caballi ◽  
Babesia Equi

ABSTRACT A latex agglutination test (LAT) using recombinant equi merozoite antigen 1 (EMA-1) for the detection of antibodies to Babesia equi was developed. The LAT was able to differentiate very clearly between sera from B. equi-infected horses and sera from Babesia caballi-infected horses or from normal horses. The LAT results were identical to those of a previously developed enzyme-linked immunosorbent assay. These results indicate that LAT using recombinant EMA-1 might be very useful as a routine screening method for the diagnosis of B. equi infection.

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 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 Cloning of a Novel Babesia equi Gene Encoding a 158-Kilodalton Protein Useful for Serological Diagnosis

2005 ◽  
Vol 12 (2) ◽  
pp. 334-338 ◽  
Author(s):  
Haruyuki Hirata ◽  
Naoaki Yokoyama ◽  
Xuenan Xuan ◽  
Kozo Fujisaki ◽  
Naoyoshi Suzuki ◽  
...  
Keyword(s):  
Gene Product ◽  
Fluorescent Antibody ◽  
Horse Serum ◽  
Antibody Test ◽  
Immune Serum ◽  
Phage Library ◽  
Enzyme Linked Immunosorbent Assay ◽  
Infected Horse ◽  
Reading Frame ◽  
Babesia Equi

ABSTRACT In this study, we characterized a Babesia equi Be158 gene obtained by immunoscreening a B. equi cDNA expression phage library with B. equi-infected horse serum. The Be158 gene consists of an open reading frame of 3,510 nucleotides. The recombinant Be158 gene product was produced in Escherichia coli and used for the immunization of mice. In Western blot analysis, mouse immune serum against the Be158 gene product recognized 75- and 158-kDa proteins from the lysate of B. equi-infected erythrocytes. In an indirect fluorescent-antibody test with the mouse immune serum, the Be158 antigen appeared in the cytoplasm of Maltese cross-forming parasites (which consist of four merozoites) and was located mainly in the extraerythrocytic merozoite body. When the recombinant Be158 gene product was used in an enzyme-linked immunosorbent assay as a serological antigen, it was found to react to B. equi-infected horse sera, indicating that the Be158 gene product is useful as a serologically diagnostic antigen for B. equi infection.

scholarly journals Development of an Enzyme-Linked Immunosorbent Assay for the Detection of Antibody to Epizootic Hemorrhagic Disease of Deer Virus

2000 ◽  
Vol 12 (2) ◽  
pp. 142-145 ◽  
Author(s):  
James O. Mecham ◽  
Michael M. Jochim
Keyword(s):  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Hemorrhagic Disease ◽  
Structural Protein ◽  
Serum Samples ◽  
Epizootic Hemorrhagic Disease ◽  
Diagnostic Potential ◽  
The Us ◽  
Serotype 1 ◽  
Infected Cells

An enzyme-linked immunosorbent assay has been developed to detect antibodies to epizootic hemorrhagic disease of deer virus (EHDV). The assay incorporates a monoclonal antibody to EHDV serotype 2 (EHDV-2) that demonstrates specificity for the viral structural protein, VP7. The assay was evaluated with sequential sera collected from cattle experimentally infected with EHDV serotype 1 (EHDV-1) and EHDV-2, as well as the four serotypes of bluetongue virus (BTV), BTV-10, BTV-11, BTV-13, and BTV-17, that currently circulate in the US. A competitive and a blocking format as well as the use of antigen produced from both EHDV-1-and EHDV-2-infected cells were evaluated. The assay was able to detect specific antibody as early as 7 days after infection and could differentiate animals experimentally infected with EHDV from those experimentally infected with BTV. The diagnostic potential of this assay was demonstrated with field-collected serum samples from cattle, deer, and buffalo.

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.

Denatured virion protein 1 of equine rhinitis B virus 1 contains authentic B-cell epitopes recognised in an enzyme-linked immunosorbent assay—Short communication

2008 ◽  
Vol 56 (2) ◽  
pp. 265-270 ◽  
Author(s):  
Gernot Kriegshäuser ◽  
Anne Cullinane ◽  
Ernst Kuechler ◽  
Timothy Skern
Keyword(s):  
Immunosorbent Assay ◽  
Metal Chelate ◽  
Horse Serum ◽  
Laboratory Diagnosis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Upper Respiratory Tract ◽  
Virion Protein ◽  
Current Standard ◽  
B Virus ◽  
High Level

Equine rhinitis B virus 1 (ERBV1), genus Erbovirus, family Picornaviridae , is a pathogen of horses which causes clinical and subclinical infection of the upper respiratory tract in horses. The virus is widespread in European horse populations and the current standard method for the detection of antibody against ERBV1 is by virus neutralisation (VN). VN tests, however, are labour-intensive and time-consuming, require tissue culture facilities, and generally do not provide same-day results. In this study, a protocol for the high-level expression and purification of recombinant virion protein 1 (rVP1) was established using metal-chelate affinity chromatography under denaturing condition. When used as a coating antigen in a prototype enzyme-linked immunosorbent assay (ELISA), denatured rVP1 was recognised by ERBV1 antibody present in horse serum. This finding suggests that denatured rVP1 is a promising candidate for the development of an ELISA to be used in the routine laboratory diagnosis of ERBV1 infection in horses.

Evaluation of immunoglobulin G4 subclass antibody in a peptide-based enzyme-linked immunosorbent assay for the serodiagnosis of human fascioliasis

Parasitology ◽  
2007 ◽  
Vol 134 (14) ◽  
pp. 2021-2026 ◽  
Author(s):  
C. TANTRAWATPAN ◽  
W. MALEEWONG ◽  
C. WONGKHAM ◽  
S. WONGKHAM ◽  
P. M. INTAPAN ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Large Scale ◽  
Laboratory Diagnosis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Parasitic Infections ◽  
Immunoglobulin G4 ◽  
Predictive Values ◽  
Diagnostic Potential ◽  
Human Fascioliasis ◽  
Specific Igg4

SUMMARYTo improve the diagnosis of human fascioliasis caused byFasciola gigantica, we developed a peptide-based enzyme-linked immunosorbent assay (peptide-based ELISA) based on the detection of specific IgG4 subclass antibody. Two identified B-cell epitopes ofF. giganticacathepsin L1 were synthesized as single synthetic peptides, acetyl-DKIDWRESGYVTELKDQGNC-carboxamide (peptide L) and acetyl-DKIDWRESGYVTEVKDQGNC-carboxamide (peptide V), and their diagnostic potential was evaluated. The sera of 25 patients infected withF. gigantica, 212 patients with other parasitic infections, 32 cholangiocarcinoma patients and 57 healthy controls were analysed. The sensitivity, specificity, accuracy, and positive and negative predictive values of this assay were the same with both peptides at 100%, 99·7%, 99·7%, 96·2% and 100%, respectively. These highly sensitive and specific peptide-based ELISAs for the detection of specific IgG4 antibody could be useful for laboratory diagnosis of human fascioliasis in future large-scale surveys throughout Southeast Asia where this disease is prevalent.

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