The Development of Toxoplasma gondii Recombinant Trivalent Chimeric Proteins as an Alternative to Toxoplasma Lysate Antigen (TLA) in Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of Immunoglobulin G (IgG) in Small Ruminants.

This study presents an evaluation of seventeen newly produced recombinant trivalent chimeric proteins (containing the same immunodominant fragment of SAG1 and SAG2 of Toxoplasma gondii antigens, and an additional immunodominant fragment of one of the parasite antigens, such as AMA1, GRA1, GRA2, GRA5, GRA6, GRA7, GRA9, LDH2, MAG1, MIC1, MIC3, P35, and ROP1) as a potential alternative to the whole-cell tachyzoite lysate (TLA) used in the detection of infection in small ruminants. These recombinant proteins, obtained by genetic engineering and molecular biology methods, were tested for their reactivity with specific anti-Toxoplasma IgG antibodies contained in serum samples of small ruminants (192 samples of sheep serum and 95 samples of goat serum) using an enzyme-linked immunosorbent assay (ELISA). The reactivity of six recombinant trivalent chimeric proteins (SAG1-SAG2-GRA5, SAG1-SAG2-GRA9, SAG1-SAG2-MIC1, SAG1-SAG2-MIC3, SAG1-SAG2-P35, and SAG1-SAG2-ROP1) with IgG antibodies generated during T. gondii invasion was comparable to the sensitivity of TLA-based IgG ELISA (100%). The obtained results show a strong correlation with the results obtained for TLA. This suggests that these protein preparations may be a potential alternative to TLA used in commercial tests and could be used to develop a cheaper test for the detection of parasite infection in small ruminants.

In silico epitope prediction of Borrelia burgdorferi sensu lato antigens for the detection of specific antibodies.

Despite many years of research, serodiagnosis of Lyme disease still faces many obstacles. Difficulties arise mainly due to the low degree of amino acid sequence conservation of the most immunogenic antigens among B. burgdorferi s.l. genospecies, as well as differences in protein production depending on the environment in which the spirochete is located. Mapping B-cell epitopes located on antigens allows for a better understanding of antibody-pathogen interactions which is essential for the development of new and more effective diagnostic tools. In this study, in silico B-cell epitope mapping was performed to determine the theoretical diagnostic potential of selected B. burgdorferi s.l. proteins (BB0108, BB0126, BB0298, BB0689, BB0323, FliL, PstS, SecD, EF-Tu). Bioinformatics software predicted 35 conserved linear and 31 conformational epitopes with the degree of identity among B. burgdorferi s.l. of at least 85%, which may prove to be useful in the development of a new tool for the diagnosis of Lyme disease.

Antibody Cross-Reactivity in Serodiagnosis of Lyme Disease.

Lyme disease is a tick-borne disease caused by spirochetes belonging to the Borrelia burgdorferi sensu lato complex. The disease is characterized by a varied course; therefore, the basis for diagnosis is laboratory methods. Currently, a two-tiered serological test is recommended, using an ELISA as a screening test and a Western blot as a confirmatory test. This approach was introduced due to the relatively high number of false-positive results obtained when using an ELISA alone. However, even this approach has not entirely solved the problem of false-positive results caused by cross-reactive antibodies. Many highly immunogenic B. burgdorferi s.l. proteins are recognized nonspecifically by antibodies directed against other pathogens. This also applies to antigens, such as OspC, BmpA, VlsE, and FlaB, i.e., those commonly used in serodiagnostic assays. Cross-reactions can be caused by both bacterial (relapsing fever Borrelia, Treponema pallidum) and viral (Epstein-Baar virus, Cytomegalovirus) infections. Additionally, a rheumatoid factor has also been shown to nonspecifically recognize B. burgdorferi s.l. proteins, resulting in false-positive results. Therefore, it is necessary to carefully interpret the results of serodiagnostic tests so as to avoid overdiagnosis of Lyme disease, which causes unnecessary implementations of strong antibiotic therapies and delays in the correct diagnosis.

Epitope Mapping of BmpA and BBK32 Borrelia burgdorferi Sensu Stricto Antigens for the Design of Chimeric Proteins with Potential Diagnostic Value.

Lyme disease is a tick-borne zoonosis caused by Gram-negative bacteria belonging to the Borrelia burgdorferi sensu lato (s.l.) group. In this study, IgM- and IgG-specific linear epitopes of two B. burgdorferi sensu stricto (s.s.) antigens BmpA and BBK32 were mapped using a polypeptide array. Subsequently, two chimeric proteins BmpA-BBK32-M and BmpA-BBK32-G were designed to validate the construction of chimeras using the identified epitopes for the detection of IgM and IgG, respectively, by ELISA. IgG-ELISA based on the BmpA-BBK32-G antigen showed 71% sensitivity and 95% specificity, whereas a slightly lower diagnostic utility was obtained for IgM-ELISA based on BmpA-BBK32-M, where the sensitivity was also 71% but the specificity decreased to 89%. The reactivity of chimeric proteins with nondedicated antibodies was much lower. These results suggest that the identified epitopes may be useful in the design of new forms of antigens to increase the effectiveness of Lyme disease serodiagnosis. It has also been proven that appropriate selection of epitopes enables the construction of chimeric proteins exhibiting reactivity with a specific antibody isotype.

Detection of Toxoplasma gondii Infection in Small Ruminants: Old Problems, and Current Solutions.

Toxoplasmosis is a parasitic zoonosis of veterinary importance, with implications for public health. Toxoplasma gondii infection causes abortion or congenital disease in small ruminants. Moreover, the consumption of infected meat, cured meat products, or unpasteurized milk and dairy products can facilitate zoonotic transmission. Serological studies conducted in various European countries have shown the high seroprevalence of specific anti-T. gondii antibodies in sheep and goats related to the presence of oocysts in the environment, as well as climatic conditions. This article presents the current status of the detection possibilities for T. gondii infection in small ruminants and their milk. Serological testing is considered the most practical method for diagnosing toxoplasmosis; therefore, many studies have shown that recombinant antigens as single proteins, mixtures of various antigens, or chimeric proteins can be successfully used as an alternative to Toxoplasma lysate antigens (TLA). Several assays based on DNA amplification have been developed as alternative diagnostic methods, which are especially useful when serodiagnosis is not possible, e.g., the detection of intrauterine T. gondii infection when the fetus is not immunocompetent. These techniques employ multicopy sequences highly conserved among different strains of T. gondii in conventional, nested, competitive, and quantitative reverse transcriptase-PCR.

Evaluation of long-term immunity and protection against T. gondii after immunization with multivalent recombinant chimeric T. gondii proteins.

Toxoplasmosis caused by the opportunistic, cosmopolitan protozoan Toxoplasma gondii is one of the most common parasitoses in the world. Although it may prove dangerous or even fatal for immunocompromised individuals, immunoprophylaxis for humans is still nonexistent. Thus, the aim of the current work was to assess the ability of two immunogenic recombinant chimeric T. gondii proteins, SAG2-GRA1-ROP1 (SGR) and SAG1-MIC1-MAG1-GRA2 (SMMG), selected in previous experiments to induce long-lasting immunity when administered with a safe adjuvant. Thus, the determination of immunological parameters and parasite challenge were performed both two weeks after the last boost injection and 6 months postvaccination. Both experimental vaccines triggered specific humoral and cellular responses in immunized C3H/HeOuJ male mice, characterized by the production of specific IgG (IgG1/IgG2a) antibodies in vivo and the synthesis of key Th1/Th2 cytokines by Toxoplasma lysate antigen-stimulated splenocytes in vitro. Although the levels of specific antibodies and cytokine release were in most cases lower six months postimmunization, the protection rates conferred by the vaccination were comparable regardless of the time after the administration of the last vaccine dose. The results indicate that both preparations induce long-lasting immunity, which makes them attractive candidates for further research aimed at boosting their immunogenicity and immunoprotective capacity.

IgG Avidity Test as a Tool for Discrimination between Recent and Distant Toxoplasma gondii Infection-Current Status of Studies.

Toxoplasma gondii, an obligate intracellular protozoan parasite, is the causative agent of one of the most prevalent zoonoses worldwide. T. gondii infection is extremely important from a medical point of view, especially for pregnant women, newborns with congenital infections, and immunocompromised individuals. Thus, an accurate and proper diagnosis of this infection is essential. Among the available diagnostic tests, serology is commonly used. However, traditional serological techniques have certain limitations in evaluating the duration of T. gondii infection, which is problematic, especially for pregnant women. Avidity of T. gondii-specific IgG antibodies seems to be a significant tool for discrimination between recent and distant infections. This article describes the problem of diagnosis of T. gondii infection, with regard to IgG avidity tests. The IgG avidity test is a useful serological indicator of toxoplasmosis, which in many cases can confirm or exclude the active form of the disease. IgG antibodies produced in the recent primary T. gondii infection are of low avidity while IgG antibodies with high avidity are detected in the chronic phase of infection. Furthermore, this paper presents important topics of current research that concern the usage of parasite recombinant antigens that may improve the performance of IgG avidity tests.

Borrelia burgdorferi BmpA-BBK32 and BmpA-BBA64: New Recombinant Chimeric Proteins with Potential Diagnostic Value.

Currently, the diagnosis of Lyme disease is based mostly on two-tiered serologic testing. In the new generation of immunoenzymatic assays, antigens comprise whole-cell lysates of members of the Borrelia burgdorferi sensu lato (s.l.) species complex, with the addition of selected recombinant proteins. Due to the high diversity of members of the B. burgdorferi s.l. genospecies and the low degree of conservation among the amino acid sequences of their proteins, serodiagnostic methods currently in use are not sufficient for the correct diagnosis of borreliosis. Two divalent chimeric proteins (BmpA-BBK32 and BmpA-BBA64) were expressed in Escherichia coli. Following purification by one-step metal-affinity chromatography, preparations were obtained containing milligram levels of chimeric protein exhibiting electrophoretic purity in excess of 98%. Reactivity of the new chimeric proteins with specific human IgG antibodies was preliminarily determined by Western blot. For this purpose, 20 negative sera and 20 positive sera was used. The new chimeric proteins were highly reactive with IgG antibodies contained in the serum of patients suffering from borreliosis. Moreover, no immunoreactivity of chimeric proteins was observed with antibodies in the sera of healthy people. These promising results suggest that new chimeric proteins have the potential to discriminate between positive and negative sera.

The Immunogenic and Immunoprotective Activities of Recombinant Chimeric T. gondii Proteins Containing AMA1 Antigen Fragments.

Toxoplasmosis, one of the most common parasitoses worldwide, is potentially dangerous for individuals with a weakened immune system, but specific immunoprophylaxis intended for humans is still lacking. Thus, efforts have been made to create an efficient universal vaccine for both animals and humans to overcome the shortcomings of currently used treatment methods and protect all hosts against toxoplasmosis. The current work represents a relatively new approach to vaccine development based on recombinant chimeric Toxoplasma gondii antigens. In the present research, three tetravalent chimeric proteins containing different portions of the parasite's AMA1 antigen-AMA1domainI-SAG2-GRA1-ROP1L (ANSGR), AMA1domainsII,III-SAG2-GRA1-ROP1L (ACSGR) and AMA1fullprotein-SAG2-GRA1-ROP1L (AFSGR)-were tested for their immunogenic and immunoprotective capacities. All tested proteins were immunogenic, as evidenced by the triggering of specific humoral and cellular immune responses in vaccinated C3H/HeOuJ mice, defined by the production of specific IgG (IgG1/IgG2a) antibodies in vivo and synthesis of key Th1/Th2 cytokines by Toxoplasma lysate antigen-stimulated splenocytes in vitro. Although all tested preparations provided partial protection against chronic toxoplasmosis in immunized and T. gondii-challenged mice, the intensity of the generated immunoprotection depended on the fragment of the AMA1 antigen incorporated into the chimeric antigen's structure.

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

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.

Toxoplasma gondii Recombinant antigen AMA1: Diagnostic Utility of Protein Fragments for the Detection of IgG and IgM Antibodies.

Toxoplasma gondii is an important zoonotic protozoan that infects a wide variety of vertebrates as intermediate hosts. For this reason, the diagnosis of this disease is very important and requires continuous improvement. One possibility is to use recombinant antigens in serological tests. Apical membrane antigen 1 (AMA1), a protein located in specific secretory organelles (micronemes) of T. gondii, is very interesting in regard to its potential diagnostic utility. In the present study, we attempted to identify a fragment of the AMA1 protein with a high sensitivity and specificity for the serological diagnosis of human toxoplasmosis. The full-length AMA1 and two different fragments (AMA1N and AMA1C) were produced using an Escherichia coli expression system. After purification by metal affinity chromatography, recombinant proteins were tested for their utility as antigens in enzyme-linked immunosorbent assays (ELISAs) for the detection of IgG and IgM anti-T. gondii antibodies in human and mouse immune sera. Our data demonstrate that the full-length AMA1 recombinant antigen (corresponding to amino acid residues 67-569 of the native protein) has a better diagnostic potential than its N- or C-terminal fragments. This recombinant protein strongly interacts with specific anti-T. gondii IgG (99.4%) and IgM (80.0%) antibodies, and may be used for developing new tools for diagnostics of toxoplasmosis.

Toxoplasma gondii Tetravalent Chimeric Proteins as Novel Antigens for Detection of Specific Immunoglobulin G in Sera of Small Ruminants.

The detection of Toxoplasma gondii infection in small ruminants has important significance for public health and veterinary medicine. This study, for the first time, describes the reactivity of four tetravalent chimeric proteins (AMA1N-SAG2-GRA1-ROP1, AMA1C-SAG2-GRA1-ROP1, AMA1-SAG2-GRA1-ROP1, and SAG2-GRA1-ROP1-GRA2) containing immunodominant regions from the AMA1 (apical membrane antigen 1), SAG2 (surface antigen 2), GRA1 (dense granule antigen 1), GRA2 (dense granule antigen 2), and ROP1 (rhoptry antigen 1) with specific IgG antibodies from the sera of small ruminants with the use of an indirect enzyme-linked immunosorbent assay (ELISA). The reactivity of individual chimeric antigens was analyzed in relation to the results obtained in IgG ELISA based on a Toxoplasma lysate antigen (TLA). All chimeric proteins were characterized by high specificity (between 96.39% to 100%), whereas the sensitivity of the IgG ELISAs was variable (between 78.49% and 96.77%). The highest sensitivity was observed in the IgG ELISA test based on the AMA1-SAG2-GRA1-ROP1. These data demonstrate that this chimeric protein can be a promising serodiagnostic tool for T. gondii infection in small ruminants.

The Impact of the Antigenic Composition of Chimeric Proteins on Their Immunoprotective Activity against Chronic Toxoplasmosis in Mice.

Toxoplasmosis may pose a serious threat for individuals with weakened or undeveloped immune systems. However, to date, there is no specific immunoprophylaxis for humans. Thus, the aim of this study was to evaluate the immunogenicity of three trivalent-SAG2-GRA1-ROP1L (SGR), SAG1L-MIC1-MAG1 (SMM), and GRA1-GRA2-GRA6 (GGG)-and two tetravalent-SAG2-GRA1-ROP1-GRA2 (SGRG) and SAG1-MIC1-MAG1-GRA2 (SMMG)-chimeric T. gondii proteins, as well as their protective potential against chronic toxoplasmosis in laboratory mice. All three trivalent recombinant proteins possessed immunogenic properties, as defined by specific humoral and cellular responses in vaccinated mice characterized by the synthesis of specific IgG (IgG1/IgG2a) antibodies in vivo and the release of Th1/Th2 cytokines by stimulated splenocytes in vitro. Immunization with all three recombinant proteins provided partial protection against toxoplasmosis, although the protective capacity strongly depended on the individual antigenic composition of each preparation. The antigens providing the highest (86%) and lowest (45%) protection, SGR and SMM, respectively, were supplemented with GRA2 antigen fragment, to form the tetravalent chimeric proteins SGRG and SMMG. Further study revealed that the tetravalent preparations exhibited high immunogenic potential; however, the addition of another antigen to the recombinant protein structure had distinct effects on the protection generated, compared to that of the trivalent counterparts, depending on the antigen tested.

The development of an indirect ELISA for the detection of goose parvovirus antibodies using specific VP3 subunits as the coating antigen.

BACKGROUND: In Poland, the leader in goose production in Europe, goose parovirus infection, or Derzsy's disease (DD), must be reported to the veterinary administration due to the serious economic and epizootic threat to waterfowl production. Prophylactic treatment for DD includes attenuated live or inactivated vaccines. Moreover, the control of DD includes the monitoring of maternal derived antibody (MDA) levels in the offspring and antibody titers in the parent flock after vaccination. The aim of this study was to develop an ELISA for the detection of goose parvovirus (GPV) antibodies. RESULTS: Two recombinant protein fragments derived from VP3 (viral protein 3) GPV, namely VP3ep6 and VP3ep4-6 with a mass of 20.9 and 32.3 kDa, respectively, were produced using an Escherichia coli expression system. These proteins were purified by one-step nickel-affinity chromatography, which yielded protein preparations with a purity above 95%. These recombinant proteins were useful in the detection of serum anti-GPV antibodies, and this was confirmed by Western blotting. However, recombinant VP3ep4-6 protein showed a greater ability to correctly identify sera from infected geese. In the next stage of the project, a pool of 166 goose sera samples, previously examined by a virus neutralization test (VN), was tested. For further studies, one recombinant protein (VP3ep4-6) was selected for optimization of the test conditions. After optimization, the newly developed ELISA was compared to other serological tests, and demonstrated high sensitivity and specificity. CONCLUSION: In conclusion, the VP3ep4-6 ELISA method described here can be used for the detection of antibodies to GPV in serum.

The first study on the usefulness of recombinant tetravalent chimeric proteins containing fragments of SAG2, GRA1, ROP1 and AMA1 antigens in the detection of specific anti-Toxoplasma gondii antibodies in mouse and human sera.

This study presents an evaluation of four tetravalent recombinant chimeric proteins containing fragments of the Toxoplasma gondii antigens, SAG2, GRA1, ROP1 and AMA1, as potential replacements of a the soluble, whole-cell tachyzoite lysate (TLA) used in serological assays. Recombinant chimeric proteins (SAG2-GRA1-ROP1-AMA1N, AMA1N-SAG2-GRA1-ROP1, AMA1C-SAG2-GRA1-ROP1, and AMA1-SAG2-GRA1-ROP1) obtained by genetic engineering were tested for their reactivity with specific IgM and IgG antibodies from sera of experimentally infected mice and humans with T. gondii infection using an enzyme-linked immunosorbent assay (ELISA). In total 192 serum samples from patients with acquired T. gondii infection and 137 sera from seronegative individuals were examined. The reactivity of chimeric antigens with antibodies generated during T. gondii invasion was measured and compared to the results obtained in assays based on whole-cell Toxoplasma antigen. Chimeric proteins proved effective in differentiation between T. gondii-infected and uninfected individuals (100% sensitivity and specificity in the IgG ELISAs) which shows their potential usefulness as a replacements for TLA in standardized commercial tests for the serodiagnosis of toxoplasmosis. In addition, the chimeric proteins were tested for use in avidity determination. Obtained results were comparable to those of the corresponding commercial assays, suggesting the utility of these proteins for avidity assessment. Furthermore, this study demonstrated that the AMA1-SAG2-GRA1-ROP1 chimeric protein has the potential to distinguish specific antibodies from serum samples of individuals with the early and chronic phase of T. gondii infection.

Identification of evolutionary conserved DNA sequence and corresponding S21 ribosomal protein region for diagnostic purposes of all Borrelia spirochetes.

It is still under investigation, whether all Borrelia sp. causing Lyme borreliosis and other diseases are already identified and properly classified as human pathogens. For this reason, it is of great importance to develop a diagnostic ELISA test that detects all Borrelia sp. The aim of this study was to identify conserved DNA and protein regions present in all currently known Borrelia sp. In experimental studies 31 available Borrelia sp. genomes were aligned and screened for the presence of evolutionary conserved regions. As a result of bioinformatics analysis, one evolutionally conserved DNA region encoding a core fragment of the S21 ribosomal protein was identified. Both a couple of genus-specific PCR primers and the S21 protein B-cell epitope were designed for prospective diagnostic purposes.

A novel chemiluminescent immunoassay based on original acridinium ester labels as better solution for diagnosis of human toxoplasmosis than conventional ELISA test.

Toxoplasma gondii infection is one of the most common human zoonosis. Laboratory diagnosis of this disease is mainly based on the results of serological methods detecting specific antibodies in the patient's sera. In this study we aimed to evaluate the performance of a chemiluminescence immunoassay (CLIA) based on the use of a novel immunochemical reagent in the form of the conjugate of original acridinium label (AL) attached to secondary antibody (IgG-AL) and SAG2-GRA1-ROP1L chimeric antigen for T. gondii specific antibodies detection. The CLIA test was compared with conventional ELISA, which was based on the same recombinant antigen and differed only in terms of the detection methodology of immune complexes. The new CLIA assay proved to be more sensitive and better differentiated sera of patients with T. gondii infection from sera of healthy individuals, being a promising alternative to more labor, cost-demanding and less versatile ELISA as screening test in toxoplasmosis diagnostics.

A novel chemiluminescent immunoassay for detection of Toxoplasma gondii IgG in human sera.

This study describes Toxoplasma gondii IgG chemiluminescent immunoassay (CLIA) based on the use of a novel immunochemical reagents in the form of the conjugates of original acridinium ester (AE) labels attached to antibodies and SAG2-GRA1-ROP1L chimeric antigen and shows that this test is useful for diagnostic purposes.

First report of seroprevalence of Toxoplasma gondii infection in sheep in Pomerania, northern Poland.

INTRODUCTION AND OBJECTIVE: Toxoplasmosis is parasitic disease which has economic relevance for both veterinary and human medicine. In sheep, toxoplasmosis is a major cause of abortion and can thus cause reproductive problems. The current study aimed to determine the occurrence of anti-Toxoplasma gondii IgG antibodies in sheep from 13 districts of northern Poland and thereby obtain actual data about T. gondii seroprevalence in this population of animals. MATERIALS AND METHODS: Blood samples from 1,646 animals from 99 herds were collected, and an in-house enzyme-linked immunosorbent assay (ELISA) based on native Toxoplasma lysate antigen (TLA) was used for serological testing. The diagnostic sensitivity of diagnostic test used in this study was 98.6%, and specificity 94.9% for the group of 113 sheep sera (74 seropositive and 39 seronegative) previously characterized by using an commercial agglutination test. RESULTS: Antibodies against T. gondii were found in 921 (55.9%) of all tested animals. The percentage of infected sheep was the highest (67.6%) for older animals (>6 years), whereas for younger ones it was significantly lower (50.1% - 57.2% for 1-5-year-old animals, respectively). Furthermore, a higher percentage of seropositive animals was noted among males (63%) than females (55.5%). The results also showed that the size of the herd is not a factor which may affect the seroprevalence of T. gondii infection in the examined population of sheep. CONCLUSION: The results of this study indicate that T. gondii infection in sheep from region of northern Poland is relatively high, and consumption of ovine meat and milk can be regarded as a significant source of infection for humans.

A new Toxoplasma gondii chimeric antigen containing fragments of SAG2, GRA1, and ROP1 proteins-impact of immunodominant sequences size on its diagnostic usefulness.

This study presents the first evaluation of new Toxoplasma gondii recombinant chimeric antigens containing three immunodominant regions of SAG2, GRA1, and one of two ROP1 fragments differing in length for the serodiagnosis of human toxoplasmosis. The recombinant chimeric antigens SAG2-GRA1-ROP1L (with large fragment of ROP1, 85-396 amino acid residues) and SAG2-GRA1-ROP1S (with a small fragment of ROP1, 85-250 amino acid residues) were obtained as fusion proteins containing His6-tags at both ends using an Escherichia coli expression system. The diagnostic utility of these chimeric antigens was determined using the enzyme-linked immunosorbent assay (ELISA) for the detection of specific anti-T. gondii immunoglobulin G (IgG). The IgG ELISA results obtained for the chimeric antigens were compared to those obtained for the use of Toxoplasma lysate antigen (TLA) and for a mixture of recombinant antigens containing rSAG2, rGRA1, and rROP1. The sensitivity of the IgG ELISA was similar for the SAG2-GRA1-ROP1L chimeric antigen (100 %), the mixture of three proteins (99.4 %) and the TLA (97.1 %), whereas the sensitivity of IgG ELISA with the SAG2-GRA1-ROP1S chimeric antigen was definitely lower, reaching 88.4 %. In conclusion, this study shows that SAG2-GRA1-ROP1L chimeric antigen can be useful for serodiagnosis of human toxoplasmosis with the use of the IgG ELISA assay. Therefore, the importance of proper selection of protein fragments for the construction of chimeric antigen with the highest reactivity in ELISA test is demonstrated.