Full text of DOI:10.1111/j.1348-0421.2002.tb02703.x

Microbiol. Immunol., 46(5), 327―332, 2002
Development of Antigen Detection ELISA for the
Diagnosis of Brugian and Bancroftian Filariasis
Using Antibodies to Recombinant Filarial
Antigens Bm-SXP-1 and Wb-SXP-1
ꢀ1
ꢀ2
Pattabhiraman Lalitha , Devarajan Eswaran , Muniratnam Gnanasekar,
ꢀ3
ꢀ4
Kakuturu Venkata Nagaraja Rao , Rangarajan Badri Narayanan, Alan Scott ,
ꢀ5
Thomas Nutman , and Perumal Kaliraj*
Centre for Biotechnology, Anna University, Chennai 600 025, India
Received November 5, 2001; in revised form, February 4, 2002. Accepted February 12, 2002
Abstract: Antibodies specific to recombinant filarial antigens Wb-SXP-1 and Bm-SXP-1 have been used to
develop a sandwich ELISA for the detection of circulating filarial antigen (CFA) in sera from patients with
lymphatic filariasis caused by Wuchereria bancrofti of Brugia malayi. In patients with W. bancrofti infections,
a high proportion of microfilaria (mf) positive (MF) and low proportions of patients with chronic pathol-
ogy (CP) and endemic normals (EN) showed the presence of CFA. Similarly in patients with brugian infec-
tions a high proportion of mf positive individuals contained CFA while none of the patients with chronic
pathology or endemic normals showed the presence of CFA. Sera from patients with other parasitic
infections (OPI) like O. volvulus, Loa loa, Ascaris lumbricoides and from individuals residing in areas non-
endemic to filariasis did not exhibit any reactivity. This assay shows promise for the detection of microfi-
laremic infections in lymphatic filariasis and its usefulness as a diagnostic tool especially in B. malayi infec-
tions, needs to be further evaluated.
Key words: Filariasis, Wuchereria bancrofti, Brugia malayi, ELISA, Recombinant antigen
The lymphatic dwelling filariae Wuchereria bancrofti
and Brugia malayi are estimated to infect approximate-
ly 119 million people worldwide (8). Affected individ-
uals may suffer from clinical manifestations such as
elephantiasis, hydrocoele, and adenolymphangitis or
may have clinically silent manifestations of infection
that are associated with subtle abnormalities of the lym-
phatic system. A diagnostic technique based on direct
demonstration of the parasite or parasite products such as
circulating filarial antigen (CFA) is the only definite
way to detect infection in humans.
Various groups of researchers have developed diag-
nostic tests that detect and quantify CFA (1, 3
―
5, 10, 13,
15, 18) in individuals harbouring Wuchereria bancrofti.
At present, monoclonal antibody based assays in an
ELISA (9) and a rapid-format card test (16, 17) are
being employed globally for the identification of infec-
tion with W. bancrofti. In brugian filariasis no test is
available to detect CFA and night blood smear to identify
microfilaria (mf) positive (MF) individuals is the only
reliable method for diagnosis.
*Address correspondence to Dr. Perumal Kaliraj, Centre for
Biotechnology, Anna University, Chennai 600 025, India. Fax/Tel:
91―44―2350299. E-mail: pkaliraj@annauniv.edu
Thus, there is a need to develop immunodiagnostic
tests that identifies CFA in B. malayi infections. In the
Present addresses: ꢀ1School of Health Sciences, PPSK, USM,
Kelantan, Malaysia. ꢀ2Department of Bioimmunotherapy, MD
Anderson Cancer Center, University of Texas, Houston, U.S.A.
ꢀ3Biomedical Engineering Department, Northwest University,
Evanston, Illinois 60208, U.S.A. ꢀ4Department of Molecular
Microbiology and Immunology, Johns Hopkins University, Bal-
timore, Maryland, U.S.A. ꢀ5Helminth Immunology Division,
Laboratory of Parasitic Diseases, National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Bethesda,
Maryland 20892, U.S.A.
Abbreviations: BmA, soluble extract of adult Brugia malayi;
Bm-SXP-1, Brugia malayi derived SXP; CFA, circulating filari-
al antigen; CP, patients with chronic pathology; EN, asymptomatic
amicrofilaremic individuals; IMAC, immobilized metal affinity
chromatography; MF, asymptomatic microfilaremic patients; mf,
microfilaria; NEN, individuals from areas non-endemic to filari-
asis; OPI, other parasitic infections; Wb-SXP-1, Wuchereria
bancrofti derived SXP.
327

328
P. LALITHA ET AL
present study we have developed an ELISA for the
detection of CFA in lymphatic filarial infections caused
by both W. bancrofti or B. malayi using antibodies to the
recombinant filarial antigens Bm-SXP-1 and Wb-SXP-
1 (11).
crofti or B. malayi infections, and the sera were Og4C3
negative.
Development of antibodies to recombinant Bm-SXP-
1 and Wb-SXP-1. We have previously reported the
cloning and characterization of the SXP protein from B.
malayi adult worm cDNA library and its orthologue
from Wuchereria bancrofti L3 cDNA library (11). The
full-length Bm-SXP-1 and Wb-SXP-1 cDNAs were
cloned into pRSET B and expressed as N-terminal six-
histidine-tagged fusion proteins. The recombinant pro-
teins were purified on immobilized metal affinity chro-
matography (IMAC).
The purified recombinant Brugia malayi SXP-1 (Bm-
SXP-1) and Wuchereria bancrofti SXP-1 (Wb-SXP-1)
were used for raising antibodies in mice and rabbits.
A group of five outbred male BALB/c mice were immu-
nized intraperitoneally with 2 µg of Bm-SXP-1 or Wb-
SXP-1 in complete Freunds adjuvant (CFA) followed by
three intramuscular booster doses of similar concentra-
tions in incomplete Freunds adjuvant (IFA) at two-week
intervals. Antibodies to Bm-SXP-1 or Wb-SXP-1 were
raised in rabbits by immunizing with 5 µg of each protein
in CFA followed by three booster doses of similar con-
centrations in IFA by intramuscular injections at two-
week intervals. Sera were collected from mice and rab-
bits after two weeks of each immunization. The
immunoreactivity of these antibodies was tested against
the purified Bm-SXP-1 and Wb-SXP-1 by Western blot
and the antibody titers were determined by ELISA. The
antisera were adsorbed with E. coli proteins to remove
cross reactivity. The rabbit and mouse monospecific
antisera against Bm-SXP-1 reacted only with the recom-
binant protein of 23 kDa and not with the vector protein.
Normal mouse and normal rabbit serum did not show any
Materials and Methods
Study population. Serum samples from a variety of
patients were used (Table 1). The serum samples from
W. bancrofti infected individuals were collected from
an area endemic for bancroftian filariasis in and around
Chennai, India. The microfilarial status of the individuals
was assessed by membrane filtration of 1 ml of
heparinized venous blood collected between 9.00 PM and
2.00 AM using 3 µm polycarbonate filters (Nucleopore,
Pleasanton, Calif., U.S.A.) followed by visualization
under a light microscope. Sera were collected after
informed consent from 34 microfilaremic patients (MF),
31 with chronic lymphatic disease (CP), and 23 micro-
filariae negative healthy individuals residing in the
endemic areas (endemic normals [EN]). The serum
samples from Brugia malayi infected individuals were
obtained from patients residing in areas endemic to Bru-
gian filariasis such as Kerala, India and in Kelantan,
Malaysia (supplied by Dr. N. Rahmah, Universiti Sains
Malaysia, Malaysia) consisting of 30 with MF, 13 with
CP, and 10 uninfected individuals residing in endemic
areas (EN). Non-endemic normal (NEN) sera were
obtained from healthy US blood bank donors who had
never traveled outside the United States (n＝19). Five
sera from patients with other parasitic infections such as
O. volvulus, Loa loa and Ascaris lumbricoides were
also included. These patients had no history of W. ban-
Table 1. Details of patients selected in the present study
Category
No. samples
mf＋
Og4C3＋
Source
W. bancrofti
Centre for Biotechnology,
Anna University, Chennai, India
MF
CP
EN
34
31
33
34
0
0
34
0
0
B. malayi
MF
CP
Dr. V. Kumaraswami,
30
13
10
30
0
0
0
0
0
Tuberculosis Research Centre, Chennai,
India and Dr. N. Rahmah, Universiti
Sains Malaysia, Malaysia
EN
Non-endemic
normal (NEN)
19
0
0
Dr. T.B. Nutman, Laboratory
of Parasitic Diseases, NIH, U.S.A.
Other parasitic
infections (OPI)
Onchocerca volvulus
Loa loa
Dr. T.B. Nutman, Laboratory
of Parasitic Diseases, NIH, U.S.A.
5
5
5
0
0
0
0
0
0
Ascaris lumbricoides

329
CIRCULATING ANTIGEN IN FILARIASIS
reactivity with Bm-SXP-1. Similar findings were manufacturer.
observed with Wb-SXP-1 using rabbit and mouse anti-
sera.
Statistical analysis. Data were analysed by Kruskal-
Wallis one-way analysis of variance. Mann-Whitney
U test was employed to identify the higher reactivity
group. P-values were adjusted for multiple compar-
isons by using Bonferroni’s correction. For paired values,
Sign test was used. In the present study P＜0.05 was
considered statistically significant.
Detection of circulating filarial antigen by ELISA
using antibodies against recombinant Bm-SXP-1 and
Wb-SXP-1. The optimal dilutions of mouse and rabbit
antisera, patient’s serum, and goat anti-rabbit IgG alka-
line phosphatase conjugates were determined by chequer
board titration. For the Bm-SXP-1 based antigen assay,
flat bottom 96-well microtiter plates (Immunolon 4,
Dynatech Laboratories, Inc., Alexandria, Va., U.S.A.)
were coated with 100 µl of mouse anti-Bm-SXP-1 anti-
body diluted in carbonate buffer pH 9.6 (1:1,500) and
incubated overnight at 4 C. The plates were washed in
phosphate buffered saline (PBS) containing 0.05% Tween
20 (Sigma) and blocked with PBS containing 3% bovine
serum albumin (BSA) for 2 hr at 37 C. After further
washes, sera samples (1:100) treated with glycine (0.15
M, pH 2.0) and neutralized with equal volume of Tris (0.1
M, pH 9.0) was added (6) to the wells in duplicates and
the plates were incubated at 37 C for 2 hr. The plates
were washed as before and incubated at 37 C for 1 hr
with rabbit anti-Bm-SXP-1 antibody (1:1,000). After
washing the plates, biotin labeled goat anti-rabbit IgG
was added and incubated at 37 C for 1 hr. The plates
were further washed and incubated with Streptavidin
conjugated with alkaline phosphatase (1:10,000) for 1 hr
at 37 C. After washing as before, substrate solution
was added (1 mg/ml p-nitro phenyl phosphate in sub-
Results
Development of an ELISA with Antibodies to Bm-SXP-1
and Wb-SXP-1 to Quantitate Circulating Filarial Anti-
gens (CFA)
The rabbit and mouse antiserum against Bm-SXP-1
reacted only with the recombinant protein of 23 kDa
and not with the vector protein. Normal mouse and
normal rabbit serum did not show any reactivity with
Bm-SXP-1. Similar findings were observed with Wb-
SXP-1 using rabbit and mouse antisera. The specificity
of the rabbit and mouse antiserum against Bm-SXP-1 or
Wb-SXP-1 were tested for the reactivity with different
filarial recombinant proteins by Western blot and the
results were negative. Preliminary experiments were
carried out with mouse and rabbit anti-SXP-1 antibodies
to determine the choice of antisera to be used as the
capture antibody and the secondary antibody for the
standardization of ELISA. It was observed that the best
results were obtained with mouse anti-Bm-SXP-1/Wb-
SXP-1 antisera as the capture antibody and the rabbit
antisera as the secondary antibody.
strate buffer) (Sigma Chemicals, U.S.A.). After 10
―
15
min the reaction was stopped by addition of 3 M NaOH.
Absorbance was measured at 405 nm in a micro plate
ELISA reader (BIO-TEK EL311sx, Winooski, Vermont,
U.S.A.) and means were taken of the duplicate read-
ings.
The Wb-SXP-1 based antigen assay was performed as
described above with the following dilutions: mouse
anti-Wb-SXP-1 1:2,000, patient’s serum 1:4 and goat
anti-rabbit IgG alkaline phosphatase conjugate 1:1,000.
The sensitivity of the ELISA was determined using nor-
mal human serum spiked with different concentrations of
purified Bm-SXP-1/Wb-SXP-1 antigen, and a standard
graph was constructed for quantitating the antigen con-
centration (ng) in the filarial sera.
Circulating filarial antigen detection using mono-
clonal antibody based Og4C3 commercial kit. Circu-
lating filarial antigen was also quantitated in the sera
using a commercially available mAb based Og4C3
ELISA kit (Tropical Biotechnology, Townsville, Aus-
tralia). The antigen units were extrapolated from a stan-
dard curve prepared from standards provided in the kit.
Samples with OD values greater than 80 antigen units
were considered to be positive as recommended by the
Detection of CFA in Bancroftian Filarial Sera
The results of ELISA with anti-Bm-SXP-1 antibodies
using sera from patients with W. bancrofti infection is
given in Fig. 1A. Kruskal-Wallis one-way ANOVA
showed that CFA among the groups were highly signif-
icant (P＜0.001). Individual comparisons were made
between the groups to identify the higher mean Bm-
SXP-1 category using Mann-Whitney U test. MF group
had significantly higher antigen units (P＜0.001). Sim-
ilarly MF group exhibited higher antigen units (P＜
0.001) when Wb-SXP-1 antibodies were used (Fig. 2A).
Antigen concentration of 10 ng and above in the sam-
ples were considered positive for Bm-SXP-1 and Wb-
SXP-1 assay. Eighty-eight percent (30/34) MF, 22%
(7/31) CP and 26% (6/23) EN were positive for Bm-
SXP-1 CFA (Fig. 1A) and 95% (20/21) MF, 10% (2/20)
CP and 3% (1/33) EN were positive for Wb-SXP-1 CFA
(Fig. 2A).
Using the above criteria for the positivity, paired com-
parison with Wb-SXP-1 antigen and Bm-SXP-1 antigen
of the same MF (n＝21) did not show any significant dif-

330
P. LALITHA ET AL
Fig. 1. Circulating filarial antigen levels in individual sera of
patients with bancroftian filariasis as determined by Bm-SXP-1
(A) and Og4C3 assay (B). The horizontal line represents the
mean antigen concentration (ng) or antigen units＋3SD of a
group of NEN individuals. Each dot represents corresponding anti-
gen concentration in ng or antigen units of individual patients. MF,
microfilaremics; CP, individuals with chronic pathology; EN,
endemic normals; NEN, non-endemic normals; OPI, other para-
sitic infections.
Fig. 2. Circulating filarial antigen levels in individual sera of
patients with brugian filariasis as determined by Bm-SXP-1 (A)
and Og4C3 assay (B). The horizontal line represents the mean
antigen concentration (ng) or antigen units＋3SD of a group of
NEN individuals. Each dot represents corresponding antigen
concentration in ng or antigen units of individual patients. MF,
microfilaremics; CP, individuals with chronic pathology; EN,
endemic normals; NEN, non-endemic normals; OPI, other para-
sitic infections.
ference (P＞0.05). All the 21 MF samples showed pos-
itivity for Bm-SXP-1 CFA and only one did not show
positivity for Wb-SXP-1 CFA. All the bancroftian MF
sera tested (34/34) were positive for circulating filarial
antigen by Og4C3 ELISA while none of the CP, EN
and NEN reacted in the assay (Fig. 1B).
the sera from NEN and patients with other parasitic
infections were positive in either of the assays (Figs. 1, 2
and 3).
Discussion
Detection of CFA in Brugian Filarial Sera
Antibodies raised against purified recombinant filari-
al proteins Wb-SXP-1 and Bm-SXP-1 have been used to
develop a sandwich ELISA to diagnose microfilaria
positive individuals. When compared to ELISA devel-
oped using Bm-SXP-1 antibody, the ELISA with Wb-
SXP-1 antibody identified apparently a high proportion
of MF’s with bancroftian filariasis.
Monoclonal antibody based ELISA (Og4C3) has been
used by our laboratory and in those of others to diagnose
filarial infecton (2) by quantitating circulating filarial
Circulating antigen levels in patients with brugian
filariasis using Bm-SXP-1 antibodies were quantitated by
ELISA as described above. MF patients exhibited high-
est levels of the circulating antigens 25/30 (83.3%) com-
pared to CP [0% (0/13)] and EN [10% (1/10)] (Fig. 3A).
Similar results were obtained using antibodies to Wb-
SXP-1 {MF [12/15 (80%)], CP [0/10 (0%)] and EN
[0/10 (0%)]} (Fig. 2B). All the brugian sera tested were
negative for CFA by Og4C3 ELISA (Fig. 3B). None of

331
CIRCULATING ANTIGEN IN FILARIASIS
group tested positive for SXP-1.
The possible explanations for the above findings could
be (a) SXP-1 is a somatic antigen and released by mul-
tiple stages of filarial worms, (b) Og4C3 is a mono-
clonal antibody based ELISA while in the present study
polyclonal antibodies have been employed. These anti-
bodies may bind to cross reactive antigens present in the
serum samples of CP and EN and may be false positive.
(c) It is possible that some of the CP and EN individuals
could be infected at the time of sample collection though
it is difficult to demonstrate microfilaria positivity in
them. (d) Since the CP and EN individuals are in an
endemic area they may be exposed to infective bites of
mosquitoes and thus may trigger a positive response
for SXP-1. The assay developed in the present study
appears specific since sera from patients with other par-
asitic infections (OPI) and sera from non-endemic nor-
mals (NEN) were negative for circulating SXP-1. NEN
as known were not exposed to infective mosquito bites
and hence do not carry the infection. It is possible that
patients with OPI have SXP-1 but present in a concen-
tration range not detectable by the ELISA developed in
this study. Experiments are in progress to evaluate this
assay in normal individuals in areas endemic to ban-
croftian filariasis to examine the question of false posi-
tivity.
In the present study, all the MF patients with B.
malayi infection were microfilaria positive (mf) (Table
1). Sera from MF patients with brugian filariasis were all
negative by Og4C3 assay while a high percentage of
them were circulating Bm-SXP-1 and Wb-SXP-1 antigen
positive. None of the CP and EN from endemic area of
brugian infection contained the circulating antigen. The
negative response in Og4C3 assay indicates that the MF
patients in question are harbouring only B. malayi and
not W. bancrofti in fection. Thus the test appears to be
specific and identifies a high percentage (80%) of micro-
filariae positive individuals harbouring B. malayi infec-
tion. The remaining 20% were not positive by this
assay. The possible explanation for this could be that the
levels of SXP antigen present in these samples could be
very low and hence not detectable by this assay. Though
there are certain limitations, this assay might be very use-
ful for the epidemiological survey of filarial infections.
In summary, an ELISA developed with antibodies to
recombinant filarial proteins Bm-SXP-1 and Wb-SXP-1
has been successfully used to quantitate circulating fila-
rial antigens in sera from bancroftian and brugian infec-
tions. This will be helpful for the sero-epidemiological
studies in endemic areas of Brugia malayi and mixed
infections.
Fig. 3. Circulating filarial antigen levels in individual sera of
patients with bancroftian filariasis (A) and brugian filariasis (B)
as determined by Wb-SXP-1 assay. The horizontal line represents
the mean antigen concentration (ng) or antigen units＋3SD of a
group of NEN individuals. Each dot represents corresponding anti-
gen concentration in ng or antigen units of individual patients. MF,
microfilaremics; CP, individuals with chronic pathology; EN,
endemic normals; NEN, non-endemic normals; OPI, other para-
sitic infections.
antigen. We have shown that all MFs were positive by
Og4C3 test while CP, EN and NEN were negative (7).
The results of the present study using antibody to Wb-
SXP-1 supports the findings of Og4C3 assay though
the sample size used for the evaluation was rather small.
Lymphoproliferative and cytokine response studies
on various clinical categories of filarial patients have
shown that MF individuals have low Th1 response to
filarial specific antigens compared to CP and EN indi-
viduals where enhanced Th1 activity is observed (12, 14).
Thus MF individuals were not able to clear the antigen
and hence were positive in the antigen assay. Since CP
and EN have high levels of cell mediated immune
response due to increased T cell activation and elevated
levels of gamma interferon, there is increased macro-
phage activation and the elimination of the antigen. In
spite of this, some of the individuals in the CP and EN
We sincerely thank Prof. Kunthala Jayaraman for initiating

332
P. LALITHA ET AL
research in filariasis at our Centre. P. Lalitha and D. Eswaran each
contributed equally to this work. P. Lalitha and M. Gnanasekar
each received a senior research fellowship award from the Coun-
cil for Scientific and Industrial Research, New Delhi, India. D.
Eswaran received a senior research fellowship award from ICMR,
New Delhi, India. This work was supported in part by PL-480
project sponsored by USIF, Filarial Genome Project supported by
WHO and grants from AICTE, New Delhi, India. We thank the
Department of Public Health and Preventive Medicine, Govt.
of Tamil Nadu, India for providing us with the blood samples. The
help rendered by Dr. V. Kumaraswamy, Tuberculosis Research
Centre, Chennai, and Dr. N. Rahmah, Universiti Sains Malaysia,
Kelantan, Malaysia for providing us the brugian filarial patients
serum is gratefully acknowledged.
8) Michael, E., Bundy, D.A., and Grenfell, B.T. 1996. Reassess-
ing the global prevalence and distribution of lymphatic fila-
riasis. Parasitology 4: 409―428.
9) More, S.J., and Copeman, D.B. 1990. A highly specific and
sensitive monoclonal antibody-based ELISA for the detection
of circulating antigen in bancroftian filariasis. Trop. Med. Par-
asitol. 41: 403―406.
10) Ramzy, R.M., Gad, A.M., Faris, R., and Weil, G.J. 1991.
Evaluation of a monoclonal antibody based antigen assay for
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J. Trop. Med. Hygiene 44: 691―695.
11) Rao, K.V.N., Eswaran, D., Ravi, V., Gnanasekar, M.,
Narayanan, R.B., Kaliraj, P., Jayaraman, K., Marson, A.,
Raghavan, N., and Scott, A.L. 2000. The Wuchereria ban-
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