scholarly journals GRA2 and ROP1 Recombinant Antigens as Potential Markers for Detection of Toxoplasma gondii-Specific Immunoglobulin G in Humans with Acute Toxoplasmosis

2009 ◽  
Vol 16 (4) ◽  
pp. 510-514 ◽  
Author(s):  
Lucyna Holec-Gąsior ◽  
Józef Kur ◽  
Elżbieta Hiszczyńska-Sawicka
Keyword(s):  
Toxoplasma Gondii ◽  
Recombinant Proteins ◽  
Immunoglobulin G ◽  
Expression System ◽  
Chronic Phase ◽  
Chronic Infections ◽  
Serum Samples ◽  
Past Infection ◽  
Recombinant Antigens ◽  
Acute Toxoplasmosis

ABSTRACT A goal of the current study was to evaluate serological applications of Toxoplasma gondii GRA2 and rhoptry protein 1 (ROP1) antigens. Soluble recombinant GRA2 and ROP1 antigens as fusion proteins containing six histidyl residues at the N and C terminals were obtained using an Escherichia coli expression system. Purification by one-step metal affinity chromatography allowed recovery of milligram amounts of pure recombinant proteins per liter of culture. The usefulness of these antigens for diagnosis of human infections was tested on 167 serum samples obtained during routine diagnostic tests. A panel of 37 serum samples from patients with acute toxoplasmosis was compared to a panel of 90 serum samples from individuals with past infection. The results indicated that both GRA2 and ROP1 recombinant antigens detected antibodies more frequently in samples from individuals with acute infections (100% and 94.6%, respectively) than in samples from individuals with chronic infections (22.5% and 15.5%, respectively). These results suggest that immunoglobulin G antibodies against GRA2 and ROP1 antigens are produced during the acute stage of toxoplasmosis but are uncommon in the chronic phase of the infection. Hence, these recombinant proteins can be used as specific molecular markers to differentiate between acute and chronic infections.

scholarly journals Use of MAG1 Recombinant Antigen for Diagnosis of Toxoplasma gondii Infection in Humans

2007 ◽  
Vol 14 (3) ◽  
pp. 220-225 ◽  
Author(s):  
Lucyna Holec ◽  
Elżbieta Hiszczyńska-Sawicka ◽  
Artur Gąsior ◽  
Anna Brillowska-Dąbrowska ◽  
Józef Kur
Keyword(s):  
Toxoplasma Gondii ◽  
Diagnostic Tests ◽  
Expression System ◽  
Recombinant Antigen ◽  
Acute Stage ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Acute Toxoplasmosis ◽  
Toxoplasma Gondii Infection ◽  
Potential Use

ABSTRACT This paper describes the cloning, purification, and serological applications of matrix antigen MAG1 of Toxoplasma gondii. The expression system used allows the production of a large amount of T. gondii recombinant protein, which was assessed for its potential use in an enzyme-linked immunosorbent assay (ELISA) for detection of T. gondii infection in humans. Serum samples from 117 patients with different stages of infection, along with 10 serum samples from seronegative patients obtained for routine diagnostic tests, were used. The results were compared with those of an ELISA that uses a native T. gondii antigen extract. The MAG1 antigen detected antibodies more frequently from the acute stage (97.3%) than from the chronic stage (7.5%) of toxoplasmosis. Hence, this antigen may be used as a tool for detection of T. gondii immunoglobulin G antibodies in persons with acute toxoplasmosis.

scholarly journals Evaluation of Recombinant Dense Granule Antigen 7 (GRA7) of Toxoplasma gondii for Detection of Immunoglobulin G Antibodies and Analysis of a Major Antigenic Domain

1999 ◽  
Vol 6 (1) ◽  
pp. 24-29 ◽  
Author(s):  
Dirk Jacobs ◽  
Martine Vercammen ◽  
Eric Saman
Keyword(s):  
Monoclonal Antibody ◽  
Toxoplasma Gondii ◽  
Immunoglobulin G ◽  
Chronic Phase ◽  
Dense Granule ◽  
Leader Peptide ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Human Sera ◽  
Antigenic Domain

ABSTRACT Dense granule protein 7 (GRA7) of Toxoplasma gondii was expressed in Escherichia coli as a fusion protein. The leader peptide contained a 25-amino-acid mouse tumor necrosis factor fragment and six histidyl residues. After purification by metal chelate affinity chromatography, the antigen was evaluated in an enzyme-linked immunosorbent assay for detection of immunoglobulin G (IgG). For two sets of IgG-positive human serum samples, obtained from routine screening, an overall sensitivity of 81% was obtained. For chronic-phase sera, the sensitivity of detection was 79%, but chronic-phase sera with low titers were more difficult to detect (65% sensitivity for sera with immunofluorescence titer of 1/64). When GRA7 was combined with Tg34AR (rhoptry protein 2 C-terminal fragment), the sensitivity rose to 96%. For a set of acute-phase serum samples tested on GRA7, the sensitivity of detection was 94%, and high-titer IgM-positive sera were detected at an especially high rate. In contrast, when Tg34AR was used, the sensitivity was only 85% for this latter set of serum samples. Three truncated GRA7 fragments containing the same leader peptide as that of recombinant GRA7 were produced. The shortest fragment (97 N-terminal amino acids) was not reactive with human sera or with a specific anti-GRA7 monoclonal antibody, while the two larger fragments were reactive. The most important antigenic domain of GRA7 for human sera was localized between residues 97 and 146. The epitope for the specific monoclonal antibody could be further narrowed down by the use of synthetic peptides, but this epitope is not recognized by sera from T. gondii-infected humans. These results indicate that GRA7 may be considered as an additional tool for studying the immune response to T. gondii.

scholarly journals Recombinant Antigens To Detect Toxoplasma gondii-Specific Immunoglobulin G and Immunoglobulin M in Human Sera by Enzyme Immunoassay

2000 ◽  
Vol 38 (3) ◽  
pp. 1144-1150 ◽  
Author(s):  
D. Aubert ◽  
G. T. Maine ◽  
I. Villena ◽  
J. C. Hunt ◽  
L. Howard ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Immunoglobulin G ◽  
Acute Infection ◽  
Relative Sensitivity ◽  
Immunoglobulin M ◽  
Recombinant Antigens ◽  
Disease Spectrum ◽  
Human Sera ◽  
Acute Toxoplasmosis ◽  
Sensitivity Specificity

We have evaluated the diagnostic utility of eleven Toxoplasma gondii recombinant antigens (P22 [SAG2], P24 [GRA1], P25, P28 [GRA2], P29 [GRA7], P30 [SAG1], P35, P41 [GRA4], P54 [ROP2], P66 [ROP1], and P68) in immunoglobulin G (IgG) and IgM recombinant enzyme-linked immunosorbent assays (Rec-ELISAs). Following an initial evaluation, six recombinant antigens (P29, P30, P35, P54, P66, and P68) were tested in the IgG and IgM Rec-ELISAs with four groups of samples which span the toxoplasmosis disease spectrum (negative, chronic infection, acute infection, and recent seroconversion). Our results suggest that the combination of P29, P30, and P35 in an IgG Rec-ELISA and the combination of P29, P35, and P66 in an IgM Rec-ELISA can replace the tachyzoite antigen in IgG and IgM serologic tests, respectively. The relative sensitivity, specificity, and agreement for the IgG P29-P30-P35 Rec-ELISA were 98.4, 95.7, and 97.2%, respectively. The resolved sensitivity, specificity, and agreement for the IgM P29-P35-P66 Rec-ELISA were 93.1, 95.0, and 94.5%, respectively. Relative to the tachyzoite-based immunocapture IgM assay, the IgM P29-P35-P66 Rec-ELISA detects fewer samples that contain IgG antibodies with elevated avidity from individuals with an acute toxoplasmosis.

scholarly journals Limited Value of Assays Using Detection of Immunoglobulin G Antibodies to the Two Recombinant Dense Granule Antigens, GRA1 and GRA6 Nt of Toxoplasma gondii, for Distinguishing between Acute and Chronic Infections in Pregnant Women

2004 ◽  
Vol 11 (6) ◽  
pp. 1016-1021 ◽  
Author(s):  
Josette Ferrandiz ◽  
Corinne Mercier ◽  
Martine Wallon ◽  
Stéphane Picot ◽  
Marie-France Cesbron-Delauw ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Pregnant Women ◽  
Immunoglobulin G ◽  
Dense Granule ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chronic Infections ◽  
Serum Samples ◽  
Positive Serology ◽  
Group 2 ◽  
Group 1

ABSTRACT An enzyme-linked immunosorbent assay (ELISA) using two recombinant antigens of Toxoplasma gondii (GRA1 and GRA6 Nt) was developed in order to differentiate between pregnant women with a serological profile of recently acquired infection and those with chronic infection. Both proteins were expressed in Escherichia coli as glutathione S-transferase fusion proteins. Thirty-two serum samples from subjects who presented seroconversion within 3 months before sampling (group 1; acute profile), 46 serum samples from women who had a positive serology at least 1 year before sampling (group 2; chronic profile), and 100 serum samples from pregnant women who were not infected by T. gondii (group 3) were examined for immunoglobulin G (IgG) reactivity. For both antigens, the specificity reached 98%. In both groups of infected patients, the overall sensitivity scored was 60% for GRA1 and 83% for GRA6 Nt. In group 1, 34% of sera reacted with GRA1 whereas 84% of sera reacted with GRA6 Nt; in group 2, however, sensitivities were 78.2 and 82.6%, respectively. Combination of the readings obtained with both antigens yielded a sensitivity of 91%. A serological follow-up of 10 women who seroconverted during pregnancy displayed three different serological patterns: (i) a GRA profile paralleling the IgG curve, as detected by the commercial kit, (ii) a GRA1 profile, or (iii) GRA1 and GRA6 Nt profiles remaining negative for at least 8 weeks after the reference test gave positive results. Taken together, these results suggest that neither GRA1 nor GRA6 Nt is sensitive enough to be used routinely to differentiate between acute and chronic toxoplasmic infections.

Usefulness of Toxoplasma gondii recombinant antigens (GRA1, GRA7 and SAG1) in an immunoglobulin G avidity test for the serodiagnosis of toxoplasmosis

2006 ◽  
Vol 100 (2) ◽  
pp. 333-337 ◽  
Author(s):  
H. Pietkiewicz ◽  
E. Hiszczyńska-Sawicka ◽  
J. Kur ◽  
E. Petersen ◽  
H. V. Nielsen ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Immunoglobulin G ◽  
Recombinant Antigens ◽  
Avidity Test

Cloning and Expression of Fasciola gigantica Cathepsin-B Recombinant Proteins

2020 ◽  
Author(s):  
O. K. Raina ◽  
Andleeb Aftab ◽  
Savita Bisen ◽  
Rohit Lall ◽  
Shobha Yadav ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Cathepsin B ◽  
Expression System ◽  
Cysteine Proteases ◽  
Scientific Data ◽  
Fasciola Gigantica ◽  
Cloning And Expression ◽  
Recombinant Antigens ◽  
Diagnostic Potential ◽  
Prokaryotic Expression System

Fasciola gigantica cathepsin (cysteine) proteases are potential diagnostic antigens for animal and human fasciolosis. These include cathepsin-L proteases that have been exploited in the diagnosis of animal fasciolosis. However, no scientific data on the diagnostic potential of F. gigantica cathepsin B proteases is available. Therefore, three recombinant antigens of F. gigantica viz. cathepsin (cat) B-1, cat B-2 and cat B-3 were expressed in prokaryotic expression system. The recombinant antigens were purified under denaturing conditions by Nickel affinity chromatography and an optimal level of the recombinant proteins was obtained. These recombinant proteins will further be evaluated for their potential in the early prepatent diagnosis of F. gigantica infection in domestic ruminants.

scholarly journals Toxoplasma gondii Recombinant Antigens in the Serodiagnosis of Toxoplasmosis in Domestic and Farm Animals

Animals ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 1245
Author(s):  
Bartłomiej Ferra ◽  
Lucyna Holec-Gąsior ◽  
Weronika Grąźlewska
Keyword(s):  
Toxoplasma Gondii ◽  
Dairy Products ◽  
Farm Animals ◽  
Antigenic Structure ◽  
High Price ◽  
Diagnostic Tools ◽  
Serum Samples ◽  
Recombinant Antigens ◽  
Scientific Disciplines ◽  
Undercooked Meat

Toxoplasmosis is caused by an intracellular protozoan, Toxoplasma gondii, and is a parasitic disease that occurs in all warm-blooded animals, including humans. Toxoplasmosis is one of the most common parasitic diseases of animals and results in reproductive losses. Toxoplasmosis in humans is usually caused by eating raw or undercooked meat or consuming dairy products containing the parasite. Diagnosis of toxoplasmosis is currently based on serological assays using native antigens to detect specific anti-T. gondii antibodies. Due to the high price, the available commercial agglutination assays are not suited to test a large number of animal serum samples. The recent development of proteomics elucidated the antigenic structure of T. gondii and enabled the development of various recombinant antigens that can be used in new, cheaper, and more effective diagnostic tools. Continuous development of scientific disciplines, such as molecular biology and genetic engineering, allows for the production of new recombinant antigens and provides the basis for new diagnostic tests for the detection of anti-T. gondii antibodies in animal serum samples.

scholarly journals Serodiagnosis of Recently Acquired Toxoplasma gondii Infection Using an Enzyme-Linked Immunosorbent Assay with a Combination of Recombinant Antigens

2000 ◽  
Vol 7 (5) ◽  
pp. 781-787 ◽  
Author(s):  
Shuli Li ◽  
Gina Galvan ◽  
Fausto G. Araujo ◽  
Yasuhiro Suzuki ◽  
Jack S. Remington ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Pregnant Women ◽  
Immunosorbent Assay ◽  
Immunoglobulin G ◽  
Enzyme Linked Immunosorbent Assay ◽  
Recombinant Antigens ◽  
Single Antigen ◽  
Toxoplasma Gondii Infection

ABSTRACT An enzyme-linked immunosorbent assay (ELISA) using four recombinant antigens of Toxoplasma gondii (rP22, rP25, rP29, and rP35) was used in an attempt to differentiate pregnant women with toxoplasma serologic profiles (TSPs) indicative of recently acquired infections (acute profile) from those with TSPs indicative of infections acquired in the distant past (chronic profile). In general, immunoglobulin G antibodies in sera from women with the acute profile reacted more strongly with the recombinant antigens than did those in sera from women with the chronic profile. However, reactivities differed significantly between antigens that reacted with a single serum and between sera that reacted with a single antigen. Because of these variations, we employed a combination of the four antigens in an ELISA (Comb-ELISA) and evaluated its ability to distinguish pregnant women with the acute profile from those with the chronic profile. Eighteen of 20 (90%) sera from acute-profile women were positive in the Comb-ELISA, whereas 69 of 70 (98.6%) sera from the chronic-profile women were negative. Thus, the Comb-ELISA may be useful for diagnosis of toxoplasmosis in pregnant women and for differentiation between recently acquired infections and infections acquired in the more distant past.

scholarly journals Molecular Characterization and Diagnostic Value ofTaenia solium Low-Molecular-Weight Antigen Genes

2000 ◽  
Vol 38 (12) ◽  
pp. 4439-4444 ◽  
Author(s):  
Yasuhito Sako ◽  
Minoru Nakao ◽  
Takashi Ikejima ◽  
Xian Zhi Piao ◽  
Kazuhiro Nakaya ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Expression System ◽  
Taenia Solium ◽  
Chimeric Protein ◽  
Immunoblot Analysis ◽  
Diagnostic Value ◽  
Enzyme Linked Immunosorbent Assay ◽  
Parasitic Infections ◽  
Low Molecular Weight ◽  
Serum Samples

Neurocysticercosis (NCC) caused by infection with the larvae ofTaenia solium is an important cause of neurological disease worldwide. In order to establish an enzyme-linked immunosorbent assay (ELISA) for this infection using recombinant proteins, we carried out molecular cloning and identified four candidates as diagnostic antigens (designated Ag1, Ag1V1, Ag2, and Ag2V1). Except for Ag2V1, these clones could encode a 7-kDa polypeptide, and Ag2V1 could encode a 10-kDa polypeptide. All of the clones were very similar. Except for Ag2V1, recombinant proteins were successfully expressed using anEscherichia coli expression system. Immunoblot analysis of NCC patient sera detected recombinant proteins, but because reactivity to recombinant Ag1 was too weak, Ag1 was not suitable as an immunodiagnostic antigen. So, Ag1V1 and Ag2 were chosen as ELISA antigens, and the Ag1V1/Ag2 chimeric protein was expressed. Of 49 serum samples from NCC patients confirmed to be seropositive by immunoblot analysis, 44 (89.7%) were positive by ELISA. No assays of serum samples from patients with other parasitic infections recognized the Ag1V1/Ag2 chimeric protein. The Ag1V1/Ag2 chimeric protein obtained in this study had a high value for differential immunodiagnosis.

scholarly journals IgG AVIDITY WESTERN BLOT USING Toxoplasma gondii rGRA-7 CLONED FROM NUCLEOTIDES 39-711 FOR SERODIAGNOSIS OF ACUTE TOXOPLASMOSIS

2013 ◽  
Vol 55 (2) ◽  
pp. 79-83 ◽  
Author(s):  
Poonam S. Deshpande ◽  
Dupadahalli Kotresha ◽  
Rahmah Noordin ◽  
Muhammad Hafiznur Yunus ◽  
Geita Saadatnia ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Western Blot ◽  
Chronic Infection ◽  
Acute Infection ◽  
Congenital Infection ◽  
Recombinant Antigen ◽  
Serum Samples ◽  
Serological Profile ◽  
Igg Avidity ◽  
Acute Toxoplasmosis

Toxoplasmosis is an important cause of congenital infection. The present study was performed to evaluate the usefulness of recombinant (r) GRA-7 cloned from nucleotides (n) 39-711 in discriminating between acute and chronic toxoplasmosis. First, commercial IgM, IgG and IgG avidity ELISAs were used to determine the serological profile of the sera. Serum samples were from 20 symptomatic patients with acute infection (low IgG avidity, IgM positive), 10 with chronic infection (high IgG avidity, IgM negative) and 10 with indeterminate IgG avidity (IgM positive) which were tested for IgG avidity status with an in-house developed IgG avidity Western blot using the rGRA-7 recombinant antigen. All 20 sera from cases of probable acute infection showed bands which either faded out completely or reduced significantly in intensity after treatment with 8 M urea, whereas the band intensities of the 10 serum samples from chronic cases remained the same. Of the 10 sera with indeterminate IgG avidity status, after treatment with 8 M urea the band intensities with six sera remained the same, two sera had completely faded bands and another two sera had significantly reduced band intensities. Discrimination between acute and chronic toxoplasmosis was successfully performed by the in-house IgG avidity Western blot.

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