Research BriefA double antibody sandwich enzyme-linked immunosorbent assay for detection of secreted antigen 1 of Babesia microti using hamster model
Graphical abstract
Highlights
► A sandwich ELISA targeting secreted antigen 1 of Babesia microti (BmSA1) was developed. ► The sandwich ELISA detected circulating BmSA1 earlier than antibody detection by indirect ELISA. ► The kinetics of circulating BmSA1 coincided with the profile of parasitemia. ► This assay was more sensitive than the microscopic examination of Giemsa-stained blood smears. ► BmSA1 could be a potential marker for surveillance of human babesiosis.
Introduction
Babesia microti is a tick-transmitted intraerythrocytic protozoon of the genus Babesia. The parasite has recently emerged as lethal opportunistic pathogens in immunocompromised patients (Homer et al., 2000, Vannier and Krause, 2009). Human babesiosis is caused by some babesial species that have distinct geographic distributions on the basis of the presence of their hosts (Vannier and Krause, 2009). B. microti has initially been identified to be prevalent in diverse regions of the United States (Scholtens et al., 1968, Krause et al., 2003) and recently emerged in different parts of the world including Europe (Herwaldt et al., 2003, Hildebrandt et al., 2007) and Asia (Shih et al., 1997, Wei et al., 2001). The infection usually causes asymptomatic presentation to flu-like symptoms with fever, chills, sweat, headache, fatigue, and anemia (Homer et al., 2000, Gubernot et al., 2009a). Severe or fatal illness mainly occurs in elderly, splenectomized and immunocompromised people (Rosner et al., 1984, Krause et al., 2008, Gubernot et al., 2009b). In human, B. microti is known to be mainly transmitted in nature by Ixodes scapularis tick. However, a large number of infections have been documented to be transmitted via blood transfusion (Saito-Ito et al., 2000, Leiby, 2006, Gubernot et al., 2009b). The lack of specific symptoms in asymptomatic individuals exhibiting low parasitemia can be a potential threat for blood transfusion (Krause et al., 1998). Therefore, effective diagnostic tests are urgently needed for detection of the parasite carriers and differentiation of the infection stage.
Microscopic examination of Giemsa-stained blood smear has been considered as the gold standard for Babesia diagnosis, but it is problematic for early and chronic stages of infection when the parasitemia is very low. Alternatively, polymerase chain reaction (PCR) has been used for detecting B. microti infection yielding high sensitivity and specificity (Persing et al., 1992). However, this method requires expensive laboratory equipments and well-trained personnel, thus these problems have limited the application of the assay. Inoculation of susceptible animals is reliable, but not practical for routine diagnosis (Kjemtrup and Conrad, 2000). On the other hand, serological methods, including immunofluorescent antibody test (IFAT), indirect enzyme-linked immunosorbent assay (iELISA), and immunochromatographic test (ICT) have been developed for antibody detection (Krause et al., 1994, Homer et al., 2003, Luo et al., 2011). However, these tests are incapable of detecting the infection at an early stage. Furthermore, antibody level may only indicate past exposure to the parasite rather than current infection and even potential disease. This is because the antibody level can remain for a long time, even after the parasites have been eliminated (Gubernot et al., 2009a). Secreted antigens or circulating antigens that are released into plasma by the parasites could be potential targets for antigen detection necessary for defining the current infection status in the host. Indeed, double antibody sandwich ELISA (DAS-ELISA) for detecting circulating antigens has been successfully applied for serological diagnosis of many protozoan infections (Montealegre et al., 1987, De Arruda et al., 2004, Dondorp et al., 2005, Chen et al., 2008). We believe that a combined iELISA and DAS-ELISA approaches could provide powerful tools for accurate diagnosis of B. microti infection. Therefore, we have attempted to develop a DAS-ELISA based on the antibodies to the recombinant B. microti secreted antigen 1 (rBmSA1). Our data might provide a new avenue for diagnosis of Babesia infection in human and animals.
Section snippets
Parasites and experimental animals
B. microti human Gray strain (US type, American Type Culture Collection, Catalog No. 30221) was maintained in specific pathogen-free (SPF) Golden Syrian hamsters (Clea, Japan) by intraperitoneal injection with 107 B. microti-infected erythrocytes. Infection was monitored by microscopic examination of Giemsa-stained blood smears. Blood was collected during different stages of infection, and then the serum fraction was separated by centrifuging the collected blood at 3500g for 30 min, and then
Development of DAS- ELISA
Circulating BmSA1 was previously identified by immunoscreening of a B. microti cDNA expression library using antisera generated using plasma of hamsters infected with B. microti (Luo et al., 2011). In order to confirm the secretory nature of BmSA1, Western blotting was carried out using sera of B. microti-infected hamster and SPF hamster probed with rabbit anti-rBmSA1 serum. As shown in Fig. 1, authentic BmSA1 was detected in serum of B. microti-infected hamster and the molecular weight was
Discussion
Diagnosis of human babesiosis can be achieved either directly by detecting parasites by microscopy and PCR, or indirectly by detecting the antibody to the parasitic pathogen using IFAT and iELISA (Hunfeld et al., 2008). However, these tests are not always sensitive and specific enough to detect the parasitic infection and to differentiate the stage of infection. Thus, an alternative diagnostic strategy is extremely desirable for the detection of this disease. During the parasite infection, a
Acknowledgment
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
References (33)
- et al.
Toxoplasma gondii: detection of MIC10 antigen in sera of experimentally infected mice
Experimental Parasitology
(2008) - et al.
Babesia gibsoni: serodiagnosis of infection in dogs by an enzyme-linked immunosorbent assay with recombinant BgTRAP
Experimental Parasitology
(2008) - et al.
Babesiosis: recent insights into an ancient disease
International Journal for Parasitology
(2008) - et al.
Human babesiosis: an emerging tick-borne disease
International Journal for Parasitology
(2000) - et al.
Identification and characterization of a novel secreted antigen 1 of Babesia microti and evaluation of its potential use in enzyme-linked immunosorbent assay and immunochromatographic test
Parasitology International
(2011) - et al.
Babesiosis in splenectomized adults. Review of 22 reported cases
The American Journal of Medicine
(1984) - et al.
Spherical body protein 4 is a new serological antigen for global detection of Babesia bovis infection in cattle
Clinical and Vaccine Immunology
(2011) - et al.
Evaluation of a rapid ELISA technique for detection of circulating antigens of Toxoplasma gondii
Microbiology and Immunology
(2008) - et al.
Quantitative determination of sporozoites and circumsporozoite antigen in mosquitoes infected with Plasmodium falciparum or P. Vivax
Annals Tropical Medicine and Parasitology
(2004) - et al.
Estimation of the total parasite biomass in acute falciparum malaria from plasma PfHRP2
PLoS Medicine
(2005)
Detection of circulating excretory secretory antigens in human fascioliasis by sandwich enzyme-linked immunosorbent assay
Journal of Clinical Microbiology
Babesia infection through blood transfusions: reports received by the US Food and Drug Administration, 1997–2007
Clinical Infectious Diseases
Transfusion-transmitted babesiosis in the United States: summary of a workshop
Transfusion
Molecular characterization of a non-Babesia divergens organism causing zoonotic babesiosis in Europe
Emerging Infectious Diseases
First confirmed autochthonous case of human Babesia microti infection in Europe
European Journal of Clinical Microbiology and Infectious Diseases
Babesiosis
Clinical Microbiology Reviews
Cited by (22)
Development of double-antibody sandwich ELISA for rapidly quantitative detection of antigen concentration in inactivated SCRV vaccine
2020, AquacultureCitation Excerpt :Vaccine quality control is vital to the SCRV vaccine production. Double antibody sandwich ELISA was higher sensitivity and specificity than indirect ELISA, which can accurately quantify antigens with simple operation (Hutchings and Ferris, 2006; Li et al., 2014; Luo et al., 2012; Ten Haaf et al., 2017). In this study, we established an optimized DAS-ELISA method based on the capture antibody 4H8 and detector antibody 4E12 for testing SCRV vaccine antigen content.
An optimized double-antibody sandwich ELISA for quantitative detection of WSSV in artificially infected crayfish
2018, Journal of Virological MethodsCitation Excerpt :Fig. 3). Double antibody sandwich ELISA has higher sensitivity and specificity than indirect ELISA (Hutchings and Ferris, 2006; Luo et al., 2012; Li et al., 2014), and can accurately quantify antigens with easy experimental operation (Fæste and Plassen, 2008; Tang et al., 2010; Haaf et al., 2017). In the present work, an optimized DAS-ELISA technique was established for WSSV detection at protein level, which used the rabbit antisera against WSSV and mixed MAbs against WSSV as the capture and detection antibodies, respectively.
Development of droplet digital PCR for the detection of Babesia microti and Babesia duncani
2015, Experimental ParasitologyCitation Excerpt :In addition, microscopic diagnosis is difficult in patients with very low parasitemia during early or chronic stages of infection. Serological methods such as immunofluorescent antibody test and enzyme-linked immunosorbent assay have been developed for detection of B. microti infection (Homer et al., 2003; Krause et al., 1994; Luo et al., 2011). As molecular assays have become more commonplace in the diagnosis of parasitic infections, both conventional and real-time polymerase chain reaction (PCR) have been developed for detection of B. microti in human samples (Bloch et al., 2013; Chan et al., 2013; Persing et al., 1992; Rollend et al., 2013; Teal et al., 2012).
Babesia Species
2014, Mandell, Douglas, and Bennett's Principles and Practice of Infectious DiseasesDevelopment and evaluation of serodiagnostic assays with recombinant BgSA1 of Babesia gibsoni
2014, Veterinary ParasitologyCitation Excerpt :Chequerboard titration was carried out to optimize the concentration of antigen (0.5 μg/well), serum samples (100 μl of 1:100 dilution), goat anti-dog HRP conjugated secondary antibody (100 μl of 1:5000 dilution, Bethyl, USA) and substrate solution (100 μl of OPD, Amresco, USA). ELISA was performed on serum/plasma samples collected from 75 dogs suspected for B. gibsoni infection following the method described by Luo et al. (2012). Absorbance was recorded using microplate reader (SPECTRA MAX M5, USA) at 492 nm.