INTRODUCTION
Lymphatic filariasis is a mosquito-borne disease and a major public health problem affecting nearly 120 million people across the world caused by Wuchereria bancrofti, Brugia malayi, and Brugia timori. In India alone, about 554.2 million people are reported to be at the risk of infection, with 27 million actually carrying Mf in blood and another 21 million suffering from clinical (CL) manifestations like hydrocele, lymphedema, and elephantiasis.1-3 The World Health Organization has launched the Global Programme for the Elimination of Lymphatic Filariasis (GPELF) and the target of the Global Programme is to eliminate lymphatic filariasis by the year 2020 through mass drug administration and alleviation of disability.4,5
The diagnosis and monitoring of filariasis has conventionally relied upon microscopic detection of Mf in peripheral blood, which is unreliable and lacks sensitivity. Further, the limitations associated with CL diagnosis, such as inconsistent symptoms and atypical manifestations seen in occult cases in the absence of Mf call for the need to develop suitable specific and rapid immunodiagnostic assays for control programs, particularly GPELF, and for mass surveys of community for filarial prevalence.6-8
Immunodiagnosis based on parasite antigen detection are considered to be very useful for the detection of active infection in filariasis. Extensive studies have been conducted to identify and isolate the filarial antigens of immunodiagnostic use.9 Though native antigens like Mf excretory—secretory antigens or purified somatic antigens have been shown to be quite useful for detecting circulating filarial antigen in bancroftian filariasis, one of the major problems is in the large-scale preparation of these antigens with consistent quality for bulk preparation of commercial diagnostic kits.10
Recently, by immunoscreening W. bancrofti complementary deoxyribonucleic acid libraries, novel recombinant filarial antigens rWbL2 have been identified to be reactive with different groups of filarial cases (Anandharaman et al. 2009 and Siva Prasad et al., unpublished observations). The WbL2 is a W. bancrofti L3 larval protein of the SXP/RAL-2 family protein.11 After specific expression of this WbL2 gene in the subventral pharyngeal gland, this protein secretion will occur.
In the light of the above facts, the present study was done to generate monospecific polyclonal antibodies to recombinant filarial antigen rWbL2 and comparatively evaluate their diagnostic utility to detect circulating filarial antigens in infected cases.
MATERIALS AND METHODS
Human Sera
Blood samples were collected from Mf individuals and patients with CL filariasis in Sevagram and surrounding villages in Maharashtra State, India, which are endemic for nocturnally periodic W. bancrofti. The presence of Mfs was checked by examining fresh night blood (collected between 9.00 pm and 2.00 am) preparations. After informed consent, sera were collected from 25 Mf patients and 25 CL filariasis individuals manifesting hydrocele, lymphedema, and elephantiasis. Blood samples were also collected from 25 healthy individuals (EN—endemic normals), who had lived in a filarial endemic region for over 5 years and had no history of filariasis and from 15 students volunteers arriving at the Mahatma Gandhi Institute of Medical Sciences in Sevagram (at the time of their arrival) from places like Chandigarh, Kashmir, etc., where there is no filariasis (NEN—nonendemic normal). Sera were separated and stored at −20°C with sodium azide as preservative.
Expression and Purification of Recombinant Filarial Antigens rWbL2
The recombinant gene constructs pRSET-B WbL2 were maintained in Escherichia coli host TOP10F, i.e., deficient in T7 polymerase and, hence, the proteins were not expressed. For expression studies, the gene constructs pRSET-B WbL2 was transformed into BL21(DE3)pLysS, which contain a chromosomal copy of T7 ribonucleic acid polymerase, under the control of UV5 promoter and, hence, used for the expression of genes cloned under T7 promoter.12 Cloned genes were induced with gratuitous inducers, such as isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 1 mM. Purification of recombinant proteins was done by immobilized metal affinity chromatography (IMAC).
Generation of Monospecific Polyclonal Antibodies to Recombinant WbL2 Antigens in BALB/c Mice and Rabbits
The BALB/c mice and rabbits were bred conventionally at our institutional animal house, which is registered with the Committee for the Purpose and Control of Supervision on Experimental Animals.
Immunization Schedule
Around 6 to 8-week-old BALB/c mice and rabbits of 1 year old were used for immunization with rWbL2 following standard immunization protocol as described by Catty and Raykundalia.13
Five BALB/c mice in each group were immunized intraperitoneally with rWbL2. The immunization schedule consisted of an initial dose of 15 μg antigen in 100 μL of 0.05 phosphate-buffered saline (PBS) emulsified in an equal volume of Freund's complete adjuvant. This was followed by three more booster doses of rWbL2 (15 μg each) emulsified in Freund's incomplete adjuvant at intervals of 7 days.
Two rabbits in each group were immunized with rWbL2 antigen following the schedule given below using Freund's complete (200 μg subcutaneous for first dose) or in incomplete adjuvants (for subsequent doses) as described in the protocol of Pocono Rabbit Farm and Laboratory Inc, Canadensis, Pennsylvania.
Sera were collected from mice/rabbits, prior to first dose and 3 days after the final dose of immunization schedule, from ear marginal veins of rabbit and from tail of BALB/c mice. Sera were separated and antibody titers were checked. The immunoreactivity of these antibodies (mouse and rabbit anti-rWbL2 antibodies) was tested against the purified WbL2 by Western blot, and the antibody titers were determined by ELISA.
Preparation of IgG Fraction of Human Filarial Serum Immunoglobulin
Human filarial serum immunoglobulin was prepared from 20 mL of pooled CL filarial serum by 33% saturation with ammonium sulfate followed by diethylaminoethyl cellulose ion exchange (Whatman DE52) chromatography as described by Reddy et al.14
Detection of WbL2 or Its Equivalent Antigen in Circulation by Double Antibody Sandwich ELISA using Antibodies against Recombinant WbL2
Double antibody sandwich ELISA was employed using different combinations of FSIgG or mouse anti-rWbL2 polyclonal antibodies as capture antibodies, and rabbit antibodies against rWbL2 as the probing antibodies to detect filarial antigen in sera.15
Procedure
The wells in polystyrene microtiter plates (NUNC, Denmark) were coated with 100 μL of capture antibodies (FSIgG at 60 ng/100 μL/well or mouse anti-rWbL2 antibodies at 1:1,000 dilution) in 0.06 M carbonate buffer, pH 9.6 at 4°C overnight. The wells were washed one time with PBS Tween 20 (PBS/T) and then blocked with 100 μL of 2% bovine serum albumin at 37°C for 1 hour. The wells were washed again three times with 0.01 M PBS having 0.05% Tween 20 at 1 minute interval and incubated each well with 100 μL of optimally diluted sera (1:10) at 37°C for 1 hour. The wells were again washed three times at 1 minute intervals and further incubated with optimally diluted 1:10,000 probing antibody (rabbit anti-WbL2 antibody in PBS/T) at 37°C for 1 hour. After five washings with PBS/T, the wells were incubated with 100 μL of optimally diluted (1:25,000) goat antirabbit horseradish peroxidase conjugate for 45 minutes. Following final washing, each well was incubated with 100 μL of substrate consisting of tetramethylbenzidine/H2O2 substrate as 1 in 20 dilutions in double distilled water for 15 minutes. Reaction was stopped by adding 50 μL of 2N H2SO4. The results were read at 450 nm using ELISA reader. Sera samples showing absorbance value equal to or higher than the mean + 2 standard deviation absorbance of NEN sera were considered as positive.
Statistical Analysis
Filarial antigen levels in different filarial endemic groups (25 in each group, i.e., Mf, CL, EN, and 15 in NEN) of filarial patients were analyzed using Statistical Package for the Social Sciences version 16.0 software and the tests of significance were tested at p < 0.01 and p < 0.05 by independent t-test.
RESULTS
Generation of Monospecific Polyclonal Antibodies to Recombinant Filarial Antigen WbL2
The gene constructs of pRSET-B WbL2 were transformed into E. coli BL21(DE3)pLysS cells and the cloned genes were induced with 1 mM IPTG. The rWbL2 antigens were expressed as hexahistidine tagged proteins with molecular weights of 23 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Fig. 1). The same was confirmed by Western blotting using anti-His antibody as probe (Fig. 2). In the first part of the study, rWbL2 were expressed and used to immunize both rabbits and BALB/c mice to raise antibodies. Immunization of BALB/c mice and rabbits with rWbL2 yielded high antibody levels. The immunoreactivity of these antibodies [rabbit and mouse specific anti rWbL2 antibodies] was tested against the purified rWbL2 by western blotting technique. The rabbit and mouse monospecific antisera against rWbL2 reacted only with the recombinant protein of 23 kDa [ rWbL2 protein] and not with the vector protein (Fig. 1).
Detection of Filarial Antigen WbL2 or Its Equivalent Antigen in Circulation by Sandwich ELISA with Antibodies to WbL2
Preliminary experiments were carried out with mouse and rabbit anti-WbL2 antibodies to determine the choice of antibodies to be used as the capture antibody and the secondary antibodies as probing antibodies for standardization of ELISA. It was observed that the best results were obtained with mouse anti-WbL2 antibodies and FSIgG as the capture antibody and the rabbit antibodies as probing antibody.
By using mouse anti-WbL2 polyclonal antibody as capture antibody in double antibody sandwich ELISA, the mean filarial WbL2 or its equivalent antigen levels in sera of CL individuals were significantly higher than the mean filarial antigen levels in NENs (p < 0.05; Graphs 1 and 2). A total of 10 of 25 (40%) microfilaremic and 14 of 25 (56%) CL filarial sera were positive for filarial WbL2 antigen by using mouse anti-WbL2 polyclonal antibody as capture antibody (Tables 1 and 2). Nine of the 25 endemic sera were found positive for filarial WbL2 antigen. None of the NEN sera was found positive for WbL2 antigen.
Graph 1
Scatter gram showing filarial WbL2 or its equivalent antigen levels in circulation in sera of different groups using mouse anti-rWbL2 antibody as capture antibody. Single dot indicates absorbance value in individual serum sample and solid line indicates mean value in each group. Mf vs NEN (p > 0.05); CL vs NEN (p < 0.05); EN vs NEN (p > 0.05)
Graph 2
Levels of filarial WbL2 or its equivalent antigen in circulation in sera of different groups using mouse anti-rWbL2 antibody as capture antibody. Bars represent mean ± standard error of mean absorbance values in each group. Mf vs NEN (p > 0.05); CL vs NEN (p < 0.05); EN vs NEN (p > 0.05)
Table 1
Detection of filarial WbL2 or its equivalent antigen using WbL2 antigen
Table 2
WbL2 or its equivalent circulating antigen detection using mouse anti-WbL2 as a capture antibody
By using FSIgG as capture antibody in double antibody sandwich ELISA, the mean filarial WbL2 or its equivalent antigen levels in sera of microfilariemic and CL individuals were significantly higher than the mean filarial antigen levels in NENs (p < 0.01; Graphs 3 and 4). A total of 15 of 25 (60%) microfilariemic and 18 of 25 (72%) CL filarial sera were positive for filarial WbL2 or its equivalent antigen using FSIgG as capture antibody (Tables 1 and 3). One of the 25 endemic sera was found positive and none of the 15 NEN sera were positive.
Graph 3
Scatter gram showing filarial WbL2 or its equivalent antigen levels in circulation in sera of different groups using FSIgG as capture antibody. Single dot indicates absorbance value in individual serum sample and solid line indicates mean value in each group Mf vs NEN (p < 0.01); CL vs NEN (p < 0.01); EN vs NEN (p > 0.05)
Graph 4
Levels of filarial WbL2 or its equivalent antigen in circulation in sera of different groups of human filarial sera using FSIgG as capture antibody. Bars represent mean ± standard error of mean absorbance values in each group. Mf vs NEN (p < 0.01); CL vs NEN (p < 0.01); EN vs NEN (p > 0.05)
Table 3
The WbL2 or its equivalent circulating antigen detection using FSIgG as a capture antibody
Table 4
Performance characters of filarial antigen assay using rWbL2 antigen
DISCUSSION
In the present study, rabbit and mouse antibodies raised against rWbL2 along with IgG of human Cl filarial serum (FSIgG) were explored in different combinations to standardize double antibody sandwich ELISA to detect circulating filarial antigen in sera of different filarial groups. The WbL2 is a W. bancrofti L3 larval protein of the SXP/RAL-2 family protein. After specific expression of this WbL2 gene in the subventral pharyngeal gland, protein secretion will occur.11 The rWbL2 antigens were expressed as hexahistidine tagged proteins with molecular weights of 23 kDa in SDS-PAGE (Fig. 1). The same was confirmed by Western blotting using anti-His antibody as probe (Fig. 2). There is a similarity of WbL2 proteins with the other proteins of SXP/RAL-2 family, such as Ancylostoma caninum (Ac-16), Ascaris suum (As14), Acanthocheilonema viteae (Av-RAL-2), Setaria digitata (Sd-SXP-1), Brugia malayi (Bm-SXP-1), and Onchocerca volvulus (Ov17 and P36991). This suggests that WbL2 may be having similar properties with these nematode-specific proteins.16 Some of the above proteins have been identified as a target vaccine candidate for protection from nematode infection upon immunization. One of the previous studies explored the antigenic properties of the SXP/RAL-2 family protein, and protective immune response elicited by these protein family against filarial and nematode infection.17
In our present study, the filarial antigen assay was standardized to detect WbL2 or equivalent antigen using FSIgG and mouse anti-WbL2 antibodies as capture antibodies. Both the antigen assays (based on FSIgG and mice anti-WbL2 antibodies) gave 100% specificity. However, using mouse antibodies, WbL2, or equivalent antigen could be detected only with 40% sensitivity (for microfilaremic cases). This assay also showed 56% of 25 CL filarial sera as positive (Table 4).
The best results for filarial antigen detection were obtained when the assay was configured to detect WbL2 or equivalent antigen using FSIgG as capture antibody. The assay detected filarial antigen with 60% sensitivity and 100% specificity (Table 4). The positive and negative predictive values were 100 and 60% respectively. Another positive aspect of this assay was that it detected 72% of CL filaria as positive, suggesting the presence of active filarial infection in these CL cases.
Immunological response studies on different CL categories of filarial patients have shown that Mf individuals have low Th1 response to filarial-specific antigens compared with CL filariasis cases and EN. Thus, more Mf individuals should be positive because of low clearance of antigen in antigen-based assay. In CL filariasis cases, there is more antigen clearance due to macrophage activation and increased Th1 response.18 Despite this fact, more CL cases tested positive for WbL2 antigens. The fact that the circulating WbL2 or equivalent filarial antigen could be detected in these CL cases and ENs even though they were negative for Mf in night blood smear suggests the presence of adult worms. This implies the presence of active infection in these cases. It is possible that some CL and EN group cases are from endemic areas, and they may be exposed to fresh infective bites of mosquitoes. In our study, the positivity for filarial antigen using the above assays varied from 4 to 36% in EN group. The EN is a very heterogeneous group in filaria-endemic areas, consisting of some which are truly negative and some with prepatent or unisexual infections.
The assay developed in this study has 100% specificity since sera from NEN were negative for circulating WbL2 antigen. The NEN groups of individuals were not exposed to infective mosquito bites and, hence, they are not filaria-infection carriers. In our study, all NEN individuals were negative for WbL2 antigens in circulation.
In the present study, all the Mf cases with W. bancrofti infection were Mf positive. These assays were able to detect 40 to 60% microfilaremic individuals. Possible explanation for this low detection in Mf staged individuals could be that the level of WbL2 antigens in these samples could be very low and, hence, not detectable by this assay.
Hamilton et al19 explored the rabbit antibodies raised against B. malayi adult worm antigens. These are useful to detect filarial antigen in W. bancrofti microfilaremic cases. In the earlier report from this laboratory, polyclonal antibodies raised against B. malayi adult soluble antigens were shown to be useful for the detection of filarial antigen in 90 to 93% active microfilaremic infection and 30% CL filarial sera.20 Two commercially available kits employ monoclonal antibodies directed against animal filarial parasites, such as by ICT diagnostics (AMRAD, Australia) and the Trop Bio Test. The ICT filarial antigen card test (Binax) was devised by Weil et al21 by applying rapid immuno-chromatographic technique using specific polyclonal antibodies and AD 12.1 monoclonal antibodies attached to colloidal gold. Another monoclonal antibody raised against O. gibsoni adult antigen (Og4C3) by More and Copeman22 was found to be quite sensitive and specific to detect filarial antigen in W. bancrofti-infected cases. These kits are highly sensitive in detecting mf carriers in different geographical areas.23 However, these tests show poor sensitivity in detecting amicrofilaremic individuals with pathology and adult worm burdens.24
The IgG fraction of FSIgG has been used earlier in sandwich ELISA for the detection of filarial antigen in microfilaremic sera.14 Malhotra and Harinath25 used FSIgG and W. bancrofti mf ES antigen—penicillinase in microtiter plate inhibition ELISA and detected Mf ES antigen in 75% of microfilaremic sera, 28% of EN sera, and none of the NEN sera.
Lalitha et al26 have developed sandwich ELISA by using antibodies against recombinant antigens, BmSXP-1 and WbSXP-1, which have a detection of high proportion of Mf-positive cases with bancroftian filariasis in WbSXP-1 than BmSXP-1 assays.27
In the present study, the specific antibodies raised against novel recombinant antigens rWbL2 could be explored to develop suitable filarial antigen assays. It was possible to come out with a filarial antigen assay that could detect WbL2 or its equivalent antigen with 60% sensitivity and 100% specificity. However, comparison of these two sandwich assays with rWbL2, for detection of circulating WbL2 or its equivalent antigen, using mouse anti-WbL2 antibodies or FSIgG as capture antibodies showed the latter to have superior performance characters with 60% sensitivity and 100% specificity to detect and monitor active filarial infection. Possible explanation for this low detection by using mouse anti-WbL2 as capture antibodies in Mf-staged individuals could be due to levels of WbL2 antigens in these samples being very low and, hence, not detectable by the assay. Sandwich assay with rWbL2 [for detection of circulating WbL2 or its equivalent antigen] using FSIgG as capture antibodies thus proved to have potential as a useful diagnostic and monitoring tool in the elimination program.
Hence, immunomonitoring along with CL examination of filarial-specific WbL2 antigen in circulation could be helpful in determining the appropriate period of DEC treatment for CL relief and cure, which are necessary for proper implementation of GPELF and to make world free of lymphatic filariasis.28
Though there are certain limitations, this assay for WbL2 or its equivalent antigen detection might be useful for epidemiological survey and detection of filariasis cases. In the present study, filarial-specific WbL2 or their equivalent antigen detections by using FSIgG as capture antibodies have been shown to be good immunological markers than detection of rWbL2 or their equivalent antigens using mouse anti-WbL2 antibodies.