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Development and evaluation of an enzyme-linked immunosorbent assay based on recombinant TgSRS2 for serodiagnosis of Toxoplasma gondii infection in cats.

ABSTRACT: An enzyme-linked immunosorbent assay (ELISA) based on recombinant SAG1-related sequence 2 of Toxoplasma gondii (rTgSRS2) was developed to detect toxoplasmosis in cats. The specificity and sensitivity of rTgSRS2 ELISA were confirmed using a series of serum samples from T. gondii-experimentally infected mice. A total of 76 field samples from cats were examined by the developed ELISA. The rTgSRS2 ELISA showed a good diagnostic performance characterized by high concordance (88.16) and kappa value (0.76) with latex agglutination test (LAT). The sensitivity and specificity of the test were 92.68% and 82.86%, respectively. These results suggest that the ELISA based on rTgSRS2 could be a useful tool for serodiagnosis of T. gondii infection in cats.

SUBMITTER: Gao Y

PROVIDER: S-EPMC7719894 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Development and evaluation of an enzyme-linked immunosorbent assay based on recombinant TgSRS2 for serodiagnosis of Toxoplasma gondii infection in cats.

Gao Yang Y Guo Huanping H Moumouni Paul Franck Adjou PFA Liu Mingming M Li Jixu J Efstratiou Artemis A Galon Eloiza May EM Ybanez Rochelle Haidee RH Masatani Tatsunori T Soma Takehisa T Xuan Xuenan X

The Journal of veterinary medical science 20201019 11

An enzyme-linked immunosorbent assay (ELISA) based on recombinant SAG1-related sequence 2 of Toxoplasma gondii (rTgSRS2) was developed to detect toxoplasmosis in cats. The specificity and sensitivity of rTgSRS2 ELISA were confirmed using a series of serum samples from T. gondii-experimentally infected mice. A total of 76 field samples from cats were examined by the developed ELISA. The rTgSRS2 ELISA showed a good diagnostic performance characterized by high concordance (88.16) and kappa value (0 ...[more]

PMID: 33071252

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Project description:Background and aimToxoplasma gondii is recognized as a zoonosis causing toxoplasmosis in animals globally. Cat is a definitive host of T. gondii and sheds oocyst through feces, which can infect human beings and animals through contaminated food ingestion. A precise diagnostic test is essential to prevent T. gondii infection in both humans and animals. This study aimed to develop and evaluate the pETite-dense granule antigen 7(GRA7)-based indirect enzyme-linked immunosorbent assay (ELISA) to detect T. gondii infection in cats.Materials and methodsT. gondii-GRA7 was cloned and expressed in the Expresso®small ubiquitin-related modifier (SUMO) T7 Cloning and Expression System. The recombinant pETite-GRA7 was purified using HisTrap affinity chromatography and confirmed using Western blot analysis. The recombinant protein was used to develop and evaluate the indirect ELISA for T. gondii infection detection. In total, 200 cat sera were tested using pETite-GRA7-based indirect ELISA and indirect fluorescent antibody test (IFAT). The statistical analysis based on Kappa value, sensitivity, specificity, positive predictive value, negative predictive value, χ 2 test, and receiver operating characteristic (ROC) curve was used to evaluate the performance of the test.ResultsA 606 bp GRA7 polymerase chain reaction (PCR) product was obtained from T. gondii RH strain genomic DNA. The gene was cloned into the pETite™ vector and transformed to HI-Control Escherichia coli BL21 (DE3) for protein expression. Approximately 35 kDa of recombinant pETite-GRA7 was observed and Western blot analysis showed positive bands against anti-6-His antibody and positive-T. gondii cat serum. A sample of 0.5 μg/mL of pETite-GRA7 was subjected to indirect ELISA to detect T. gondii infection in the cat sera. The results showed sensitivity and specificity of pETite-GRA7-based indirect ELISA at 72% and 96%, respectively. An acceptable diagnostic performance was characterized by high concordant results (94%) and substantial agreement (Kappa value=0.65) with IFAT. The seroprevalence levels of ELISA and IFAT were 10% and 9%, respectively, and were not significantly (p>0.05) different. The expected performance of ELISA at different cutoff points using the ROC curve analysis revealed 89% sensitivity and 92% specificity at the cutoff value of 0.146, with a high overall assay accuracy (area under the curve=0.94).ConclusionIn this study, the pETite™ vector, N-terminal 6xHis SUMO fusion tag, was used to improve the solubility and expression level of GRA7. The recombinant pETite-GRA7 showed enhanced protein solubility and purification without special condition requirements. This pETite-GRA7-based indirect ELISA showed high concordant results and substantial agreement with IFAT. ELISA revealed an acceptable sensitivity and specificity. These initial data obtained from cats' sera demonstrated that pETite-GRA7-based indirect ELISA could be a useful method for local serological diagnosis of T. gondii infection in cats in Thailand.

Project description:The outer surface protein C (OspC) and the internal 14-kDa flagellin fragment of strain GeHo of Borrelia burgdorferi sensu stricto were expressed as recombinant proteins in Escherichia coli and were purified for use in an immunoglobulin M (IgM) enzyme-linked immunosorbent assay (OspC-14-kDa antigen ELISA). No hint at disturbing protein-protein interferences, which might influence the availability of immunoreactive epitopes, was found when the recombinant antigens were combined in the ELISA. The recombinant OspC-14-kDa antigen ELISA was compared to a commercial IgM ELISA that used a detergent cell extract from Borrelia afzelii PKo as the antigen. According to the manufacturer's information, the cell extract contains, in addition to other antigens, the following diagnostically relevant antigens: the 100-kDa (synonyms, 93- and 83-kDa antigens), 41-kDa, OspA, OspC, and 17-kDa antigens. The specificity was adjusted to 95% on the basis of data for 154 healthy controls. On testing of 104 serum samples from patients with erythema migrans (EM), the sensitivity of the recombinant ELISA (46%) for IgM antibodies was similar to that of the commercial ELISA (45%). However, when 42 serum samples from patients with polyclonal B-cell stimulation due to an Epstein-Barr virus infection were tested, false-positive reactions were significantly less frequent in the recombinant ELISA (10%) than in the whole-cell-extract ELISA (23%). OspC displays sequence heterogeneity of up to 40% according to the genomospecies. However, when the reactions of serum specimens from controls and EM patients with OspC from representative strains of B. burgdorferi sensu stricto (strain GeHo) and B. afzelii (strain PKo) were compared in an ELISA, almost no differences in specificity and sensitivity were seen. This demonstrates that the sera predominantly recognize the common epitopes of OspC tested in this study. In conclusion, we suggest that the OspC-14-kDa antigens ELISA is a suitable test for the detection of an IgM response in early Lyme disease.

Project description:BackgroundSchistosomiasis is a huge threat to human and animal health. Apart from bovines, goats play an important role in the transmission of schistosomiasis in some endemic areas of China. An accessible, quality-assured goat schistosomiasis diagnostic technique is needed. Recently, our laboratory identified two recombinant diagnostic antigens, SjPGM and SjRAD23 via an immuno-proteomic method. The application of these two recombinant antigens to develop a higher sensitivity and specificity technique for the sheep schistosomiasis diagnosis is urgently needed.MethodsEpitopes of SjPGM and SjRAD23 were predicted and three polypeptides, two from SjRAD23 and one from SjPGM, were selected. Recombinant plasmids containing two to three DNA sequences encoding predicted polypeptides or large hydrophilic region of Sj23 (LHD-Sj23) were constructed and expressed. Eight recombinant schistosome antigens including four multi-epitope proteins and four recombinant single-molecule antigens as well as SEA, were assessed by ELISA in 91 sera from schistosome-infected goats, 44 sera from non-infected goats, 37 sera from Orientobilharzia-infected goats, and 12 from Haemonchus contortus-infected goats.ResultsELISA tests showed that three multi-epitope proteins had higher sensitivity than the four single-molecule antigens (rSjRAD23, rSjPGM, rBSjRAD23-1, rBSj23) and the multi-epitope protein rBSjPGM-BSjRAD23-1-BSj23 had the highest sensitivity (97.8 %, 89/91) and maintained good specificity (100 %, 44/44) as well as low cross-reactivity with haemonchosis (8.33 %, 3/12) and orientobilharziasis (13.51 %, 5/37) in the diagnosis of goat schistosomiasis. In contrast, when SEA was applied as a diagnosis antigen, it had 100 % (91/91) sensitivity, 75 % (33/44) specificity, 25 and 83.78 % cross-reactivity with haemonchosis (3/12) and orientobilharziasis (31/37), respectively.ConclusionsThe application of recombinant multi-epitope proteins may increase the sensitivity of diagnosis technique and retain high specificity of single-molecule antigens for schistosomiasis, and the recombinant antigen rBSjPGM-BSjRAD23-1-BSj23 has the potential to be used as a diagnosis antigen for goat schistosomiasis.

Project description:A portion of a cDNA encoding a 35-kDa antigen from Toxoplasma gondii was cloned into the CKS expression vector and expressed in Escherichia coli. By using the enzyme-linked immunosorbent assay (ELISA), the recombinant protein (rP35 antigen) was examined for reactivity with immunoglobulin G (IgG) antibodies in the sera of pregnant women. Of these women, 41 had a toxoplasma serologic profile suggestive of recently acquired T. gondii infection (Sabin-Feldman dye test [DT] titers from 1:256 to 1:32,000, positive IgM ELISA titers from 2.3 to 9.7, positive IgA ELISA from 1 to >28, and acute patterns in the differential agglutination [AC/HS] test) (group I), and 50 women had a toxoplasma serologic profile suggestive of infection acquired in the distant past (low DT titers from 1:16 to 1:512, negative IgM ELISA titers from 0 to 0.8, and chronic patterns in the AC/HS test) (group II). The classification of acute or chronic profile was based on the individual's clinical history as well as the combination of the results of the toxoplasma serological profile. An additional group (group III) was composed of sera from 50 women who were seronegative for T. gondii antibodies in the DT. The results revealed that whereas 85.3% of women in group I had IgG antibodies that reacted with the rP35 antigen, only 8% of women in group II had IgG antibodies that reacted with the same antigen. In immunoblots, the rP35 antigen was recognized by IgG antibodies in a pool of sera from individuals with a toxoplasma serologic profile compatible with acute infection but not in a pool of sera from individuals with a serologic profile characteristic of a chronic infection. These results reveal that IgG antibodies against the P35 antigen are produced during the acute stage of the infection but are uncommon in the latent or chronic phase of the infection. Thus, the rP35 antigen may be a useful serologic marker to differentiate between recently acquired infection and that acquired in the more distant past.

Dataset Information

Development and evaluation of an enzyme-linked immunosorbent assay based on recombinant TgSRS2 for serodiagnosis of Toxoplasma gondii infection in cats.

Publications

Development and evaluation of an enzyme-linked immunosorbent assay based on recombinant TgSRS2 for serodiagnosis of Toxoplasma gondii infection in cats.

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