Reverse Enzyme-Linked Immunosorbent Assay Using Monoclonal Antibodies against SAG1-Related Sequence, SAG2A, and p97 Antigens from<i>Toxoplasma gondii</i>To Detect Specific Immunoglobulin G (IgG), IgM, and IgA Antibodies in Human Sera

Carvalho, Fernando R.; Silva, Deise A. O.; Cunha-Júnior, Jair P.; Souza, Maria Aparecida de; Oliveira, Taísa Carrijo de; Béla, Samantha Ribeiro; Faria, Gabriele Garcias de; Lopes, Carolina; Mineo, José Roberto

doi:10.1128/cvi.00069-08

Cited by 26 publications

(19 citation statements)

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“…Since fewer clones are screened in colony blotting, antibodies to major antigens must be abundant in the libraries. Indeed, SAG1 is the most abundant surface molecule of T. gondii (17), and serum antibodies to this molecule have been detected in patients with toxoplasmosis (6,12,19,28). Therefore, the results of the present study show that screening by colony blotting is effective for libraries constructed from immune patients.…”

Section: Discussionmentioning

confidence: 99%

Generation of a Neutralizing Human Monoclonal Antibody Fab Fragment to Surface Antigen 1 ofToxoplasma gondiiTachyzoites

Feng

Ohnishi

et al. 2011

Infect Immun

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A combinatorial immunoglobulin gene library was constructed from lymphocytes in peripheral blood of a patient with toxoplasmosis and screened for production of human monoclonal antibody Fab fragments to recombinant surface antigen 1 (SAG1) of Toxoplasma gondii. Two Fab clones, Tox203 and Tox1403, which consisted of a common heavy chain and different light chains, showed positive staining on the entire surface of tachyzoites in confocal microscopy. Sequence analysis of the heavy-chain gene revealed that the closest germ line V segments were VH3-23. The germ line D segment was D1-7, and the closest germ line J segment was JH4. In the light-chain genes, the closest germ line V segment was V1-17 with the J1 or J4 segments. The dissociation constants of these Fab fragments with recombinant SAG1 were 3.09 ؋ 10 ؊9 M for Tox203 and 2.01 ؋ 10 ؊8 M for Tox1403, indicating that the affinity of Tox203 was 7 times higher than that of Tox1403. Preincubation of T. gondii tachyzoites with Tox203 significantly inhibited their attachment to cultured MDBK cells. Passive immunization of mice with Tox203 also significantly reduced mortality after challenge with T. gondii tachyzoites. This is the first report of bacterial expression of human monoclonal antibody Fab fragments to SAG1 of T. gondii. These results also demonstrate that human Fab fragments to SAG1 might be applicable for immunoprophylaxis of toxoplasmosis.Toxoplasma gondii is an obligate intracellular parasite in the phylum Apicomplexa and infects a variety of warm-blooded domestic and wild animals worldwide. It is an important foodborne parasite transmitted primarily from animals to humans through meat, as well as through oocysts shed by cats into the environment (25). Infection in humans is usually asymptomatic in immunocompetent hosts. However, primary infection during pregnancy can result in severe neonatal malformations and ocular complications in the fetus. In immunocompromised hosts such as patients with human immunodeficiency virus/ AIDS, reactivation of the latent infection results in symptomatic diseases such as toxoplasmic encephalitis (14,22). Transmission of T. gondii by organ transplantation from a seropositive donor to a seronegative recipient is also an important potential cause of disease in heart, heart-lung, kidney, liver, and liver-pancreas transplant patients (25).The surface of T. gondii is the first component to contact the host cells. The T. gondii surface is coated by closely related antigens that belong to the surface antigen 1 (SAG1)-related sequences (SRS) superfamily (13, 16). SAG1 is the most abundant and immunogenic of these antigens and is important for the process of invasion. Treatment of T. gondii with mouse monoclonal or rabbit polyclonal antibodies to SAG1 inhibits parasite attachment to host cells (24). Fab fragments derived from a mouse monoclonal antibody also showed dose-dependent inhibition of parasite attachment (23). Therefore, human monoclonal antibodies to SAG1 may be applicable for prevention of transmission and reactivation ...

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Section: Discussionmentioning

confidence: 99%

Generation of a Neutralizing Human Monoclonal Antibody Fab Fragment to Surface Antigen 1 ofToxoplasma gondiiTachyzoites

Feng

Ohnishi

et al. 2011

Infect Immun

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“…Capture ELISA for IgM and IgA antibodies to T. gondii were performed in house as previously described [21]. Briefly, highbinding microtiter plates (Costar-Corning Incorporated, New York, USA) were coated overnight at 4 • C with goat anti-human IgM or anti-human IgA antibodies (10 g/mL) (Sigma) diluted in 0.06 M carbonate buffer (pH 9.6).…”

Section: Capture Elisa For Detection Of Igm and Iga Antibodies To T mentioning

confidence: 99%

Analysis of IgG subclasses (IgG1 and IgG3) to recombinant SAG2A protein from Toxoplasma gondii in sequential serum samples from patients with toxoplasmosis

et al. 2012

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The kinetics of the humoral immune response was evaluated using the recombinant SAG2A protein comparatively to soluble Toxoplasma antigen (STAg) by ELISA in sequential serum samples of patients with toxoplasmosis up to 12 months of illness onset. The follow up of IgM and IgA levels to STAg showed a gradual decrease, with the majority of patients (88%) seropositive for IgM up to 12 months of infection, whereas IgA seropositivity was relatively low (78%) compared to IgM (100%) in the first 3 months of infection. The follow up of IgG and IgG1 antibodies showed a similar increasing profile for both SAG2A and STAg, with slightly higher seropositivity for STAg. The kinetics of IgG3 to STAg was similar to that of IgG1, contrasting with the kinetics of IgG3 to SAG2A that showed high levels up to 6 months of infection, with continuous decreasing over the time. Higher IgG3 seropositivity to SAG2A than STAg was also observed in the initial phases of infection. A higher IgG3/IgG1 ratio for SAG2A than STAg was detected in the first 3 months of infection, with decreasing profile over the time. The associations of IgG3/IgG1 ratio>1.0 with positive IgM or IgA antibodies were predominantly found in the first 3 months of infection, whereas associations of IgG3/IgG1 ratio<1.0 with positive IgM or negative IgA antibodies were mostly observed from 3 to 12 months of infection. In conclusion, our results demonstrate a differential kinetics of IgG3 antibodies to SAG2A and STAg in patients with toxoplasmosis up to 12 months of infection. Also, the IgG3/IgG1 ratio to SAG2A in association with classical serological markers of acute phase could be potential tools to distinguish early acute from convalescent phases of Toxoplasma gondii infection.

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“…Chronic infection is similar in humans and animals and results in lifelong immune protection against reinfection [6]; specific immunity against T. gondii infection involves both cellular [7] and humoral [8] mechanisms. Studies evaluating immune responses to toxoplasmosis vaccines based on recombinant proteins or attenuated or mutant parasites have reported varying levels of vaccine efficacy [9].…”

Section: Introductionmentioning

confidence: 98%

Humoral responses and immune protection in mice immunized with irradiated T. gondii tachyzoites and challenged with three genetically distinct strains of T. gondii

et al. 2011

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Toxoplasma gondii is an obligate intracellular parasite that infects a variety of mammals and birds. T. gondii also causes human toxoplasmosis; although toxoplasmosis is generally a benign disease, ocular, congenital or reactivated disease is associated with high numbers of disabled people. Infection occurs orally through the ingestion of meat containing cysts or by the intake of food or water contaminated with oocysts. Although the immune system responds to acute infection and mediates the clearance of tachyzoites, parasite cysts persist for the lifetime of the host in tissues such as the eye, muscle, and CNS. However, T. gondii RH strain tachyzoites irradiated with 255Gy do not cause residual infection and induce the same immunity as a natural infection. To assess the humoral response in BALB/c and C57BL/6J mice immunized with irradiated tachyzoites either by oral gavage (p.o.) or intraperitoneal (i.p.) injection, we analyzed total and high-affinity IgG and IgA antibodies in the serum. High levels of antigen-specific IgG were detected in the serum of parenterally immunized mice, with lower levels in mice immunized via the oral route. However, most serum antibodies exhibited low affinity for antigen in both mice strain. We also found antigen specific IgA antibodies in the stools of the mice, especially in orally immunized BALB/c mice. Examination of bone marrow and spleen cells demonstrated that both groups of immunized mice clearly produced specific IgG, at levels comparable to chronic infection, suggesting the generation of IgG specific memory. Next, we challenged i.p. or p.o. immunized mice with cysts from ME49, VEG or P strains of T. gondii. Oral immunization resulted in partial protection as compared to challenged naive mice; these findings were more evident in highly pathogenic ME49 strain challenge. Additionally, we found that while mucosal IgA was important for protection against infection, antigen-specific IgG antibodies were involved with protection against disease and disease pathogenesis. Most antigen responsive cells in culture produced specific high-affinity IgG after immunization, diverse of the findings in serum IgG or from cells after infection, which produced low proportion of high-avidity IgG.

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Reverse Enzyme-Linked Immunosorbent Assay Using Monoclonal Antibodies against SAG1-Related Sequence, SAG2A, and p97 Antigens fromToxoplasma gondiiTo Detect Specific Immunoglobulin G (IgG), IgM, and IgA Antibodies in Human Sera

Cited by 26 publications

References 31 publications

Generation of a Neutralizing Human Monoclonal Antibody Fab Fragment to Surface Antigen 1 ofToxoplasma gondiiTachyzoites

Generation of a Neutralizing Human Monoclonal Antibody Fab Fragment to Surface Antigen 1 ofToxoplasma gondiiTachyzoites

Analysis of IgG subclasses (IgG1 and IgG3) to recombinant SAG2A protein from Toxoplasma gondii in sequential serum samples from patients with toxoplasmosis

Humoral responses and immune protection in mice immunized with irradiated T. gondii tachyzoites and challenged with three genetically distinct strains of T. gondii

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