Abstract
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.
Highlights
Toxoplasmosis is a widespread disease caused by the ubiquitous obligate intracellular parasite Toxoplasma gondii, which infects a wide range of hosts, including humans [1]
The aim of this study was to improve the performance of the IgM, IgG and IgG avidity enzymelinked immunosorbent assay (ELISA) using generation recombinant chimeric proteins and to demonstrate the diagnostic utility of four T. gondii recombinant tetravalent chimeric proteins (SAG2-GRA1-ROP1-AMA1N, AMA1N-SAG2-GRA1ROP1, AMA1C-SAG2-GRA1-ROP1, and AMA1-SAG2-GRA1-ROP1), composed of combination of four well-characterized antigens, including dense granule antigen (GRA1), rhoptry antigen (ROP1), surface antigen (SAG2), and different regions of apical membrane antigen (AMA1)
SAG2-GRA1-ROP1-AMA1N, AMA1N-SAG2-GRA1-ROP1, AMA1C-SAG2-GRA1-ROP1, and AMA1-SAG2-GRA1-ROP1 were expressed as insoluble proteins with a calculated molecular mass between 96.96–128.42 kDa (Table 2), and were purified by one-step metal affinity chromatography
Summary
Toxoplasmosis is a widespread disease caused by the ubiquitous obligate intracellular parasite Toxoplasma gondii, which infects a wide range of hosts, including humans [1]. From the medical point of view, the reliable recognition of T. gondii invasion is very important in pregnant women, where the significant risk of tachyzoite transmission via the placenta to the fetus, can lead to miscarriages or cause neonatal malformations, neurological damage, and blindness in newborns [2,3]. In light of many recent studies, it should be emphasized that the diagnosis of long-acquired T. gondii infection is important and may explain the biological basis of some behavioral disorders and neurological diseases. It is assumed that the chronic parasite infection can be associated inter alia with the occurrence of seizures, slow response time, memory loss, increased risk of bipolar disorder, schizophrenia and depression, and even suicide rates [5,6,7,8,9,10,11]
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