91
CHAPTER 3
RESULTS
In an earlier study, rWbSXP-1 was identified as a suitable immuno-
diagnostic candidate (Rao et al 2000). The rapid antibody flow-through kit
was developed to suit field conditions using finger prick blood samples
collected on filter paper (Basker et al 2004). The large scale expression and
purification of rWbSXP-1 was optimized in in the salt inducible recombinant
E.coli GJ1158. The „MF-Signal: rapid kit format‟ was originally developed
under the memorandum of understanding between Centre for Biotechnology
Anna University, Chennai and SPAN Diagnostics Ltd., Surat, Gujarat, India.
In the present work, to enhance the diagnostic efficiency of antigen,
WbSXP-1 protein was also expressed in a eukaryotic expression system,
namely baculovirus expression system. The antigen based immuno-dignostic
assay was developed for the detection of circulating WbSXP-1 antigen in
human lymphatic Filariasis with monoclonal and polyclonal antibody raised
against WbSXP-1 protein expressed in E.coli GJ1158. The present work was
based on the following objectives:
Cloning, expression, purification and characterization of
WbSXP-1 in Baculovirus expression system to enhance the
diagnostic efficiency of antigen for antibody detection assay.
Expression and purification of rWbSXP-1 in E.coli towards
development and characterization of monoclonal antibodies for
92
the development of prototype antigen based immuno-
diagnostics for Human Lymphatic Filariasis
3.1 RECOMBINANT FILARIAL CLONE pRSETB:WbSXP-1
USED IN PRESENT STUDY
An orthologue of BmSXP-1 was identified from W. bancrofti L3
cDNA library using BmSXP-1 specific DNA primers as a probe (Rao et al
2000). The identified WbSXP-1 gene (Accession no. AF098861) was cloned
in EcoRI site of pRSETB vector and was expressed in E.coli BL21 to derive
the diagnostic antigen rWbSXP-1 (Rao et al 2000). The identification of
WbSXP-1 set a platform for the development of a specific diagnostic method
to detect both Brugian and Bancroftian filariasis. In a previous study,
development of rWbSXP-1 specific ELISA with predominantly IgG4
antibodies and a detection method for circulating filarial antigen in
bancroftian and brugian filariasis using monospecific polyclonal antibodies
were demonstrated (Lalitha et al 2002). Further, the rapid flow-through
antibody assay kit developed using the WbSXP-1 antigen underwent national
(Basker et al 2004) and global evaluation (Lammie et al 2004). The antibody
test kit underwent field trials and showed 91 % sensitivity and 100 %
specificity (Basker et al 2004, Lammie et al 2004). The technology transfer
for production, quality control and application of recombinant antigen
rWbSXP-1 was done as per the MoU between CBT, Anna University and
Span Diagnostics Ltd.
The objective of this research work is to develop monoclonal
antibodies against recombinant antigen WbSXP-1 for the detection of
circulating SXP-1 antigen in both bancroftian as well as brugian filariasis. In
order to improve the detection of antigen in the diagnostic kit, Wbsxp-1 was
cloned and expressed in the Baculovirus expression system which is used for
93
expression of eukaryotic proteins for post-translational modifications,
improved processing and targeting.
The results of the research work are herewith presented.
3.2 CONFIRMATION OF RECOMBINANT CLONE
pRSETB:WbSXP-1 IN E.coli GJ1158
3.2.1 PCR Analysis of E.coli GJ1158 Transformants
Transformation of pRSETB: WbSXP-1 plasmid into E.coli GJ1158
host was carried out using CaCl2 method. The transformation efficiency was
estimated to be an average 45 colonies per microgram of plasmid. Plasmid
extraction was done from E.coli GJ1158 transformant and PCR of the
extracted pRSETB:WbSXP-1 was carried out using WbSXP-1-specific
forward and reverses primers. The amplification profile of the gene showed
~600 bp, thereby confirming the SXP gene insert (Figure 3.1).
Figure 3.1 Characterization of pRSETB:WbSXP-1 by PCR
Agarose gel (1%) electrophoresis pattern of PCR products amplified from
E.coli GJ1158 transformant using SXP specific primers. Lane1: 100 bp
ladder, Lane 2: Negative control Lanes 3: pRSETB:WbSXP-1
~ 600 bp
1 2 3
500bp
1 kb
94
3.2.2 Expression and Purification of Recombinant pRSETB:WbSXP-
1 in E.coli GJ1158
The large scale expression and purification of rWbSXP-1 in
osmotically (NaCl) induced E.coli (GJ1158) was optimized in our lab by S.
Janardhan et al (2007). Briefly, The expression of the recombinant pRSETB:
WbSXP-1 was done in E.coli GJ1158 hosts. The recombinant WbSXP-1 was
expressed as a 26 KDa fusion protein with 6X histidine tag. Expression study
in E.coli GJ1158 was carried out and the protein profile of the batch course is
shown in Figure 3.2. In GJ1158 the T7 RNA Polymerase is under the control
of ProUp promoter which is highly osmoresponsive. Consequently, there was
low leaky expression of rWbSXP-1 observed without the addition of inducer
(NaCl). The induction was performed using 250 mM NaCl for 3 h and an
expression of rWbSXP-1 was clearly observed.
Figure 3.2 Expression Profile of recombinant WbSXP-1 in osmotically
(NaCl) induced E.coli (GJ1158)
Total protein extracts from recombinant pRSETB:WbSXP-1 and control
were solubilised in 1 X SSB, separated by electrophoresis on 12% gel.
Lane 1: Molecular weight marker, Lanes 2 and 3: Uninduced culture,
Lanes 4: 1st hour induced culture, Lanes 5: 2
nd hour induced culture, Lanes
6: 3rd hour induced culture
97
66
45
30
20.1
14.4
WbSXP-1
(26kDa)
1 2 3 4 5 6 kDa
95
The purification of rWbSXP-1 was carried out with GJ1158 and the
protein was purified from the soluble fraction using 5 ml bed volume IMAC
column. The purification of the recombinant WbSXP-1 protein in soluble
form (6xHis–tag fusion protein) was successfully done in IMAC column,
result is shown in Figure 3.3. The binding and elution buffer used for native
isolation of proteins was the combination of 50 mM TRIS-Sodium phosphate,
10 mM imidazole and 0.4 M NaCl. The pH of the binding buffer was
optimized to be 8.0 and that of the elution buffer at 6.0. Elution gradient was
with 0.5 M stock of Imidazole and rWbSXP-1 eluted at 150-300 mM Imidazole.
Figure 3.3 SDS-PAGE analysis of recombination protein WbSXP-1
purification using IMAC
GJ1158 host cells containing WbSXP-1 were sonicated and the soluble
protein fraction was used in purification. Total cell lysate of induced and
the purified protein were resolved on 12% gel under reducing and
denaturing condition. Lane 1: Molecular weight marker, Lane 2: Induced
culture, Lane 3: sonicated culture supernatant, Lane 4: flow-through
supernatant, Lane 5: flow-through wash (early), Lane 6: flow-through
wash (late), Lane 7: flow-through 50 mM imidazole, Lane 8: 100 mM
imidazole, Lane 9: 150 mM imidazole, 10: 200 mM imidazole, Lane 11:
250 mM imidazole, Lane 12: 300 mM imidazole, Lane 13: 500 mM
imidazole
kDa 1 2 3 4 5 6 7 8 9 10 11 12 13
97
66
45
30
20.1
14.
WbSXP-1
(26kDa)
96
3.3 CHARACTERIZATION OF SXP GENE IN BACULOVIRUS
SYSTEM
3.3.1 Sub-Cloning of SXP in Baculovirus Expression System
The SXP gene from pRSETBSXP-1 was amplified from SXP
forward and reverses primes with BamH1 and EcoR1 restriction sites and
subcloned in pFastBac1 donor vector. The SXP-1 protein contains signal
peptide at amino-terminal region with possible cleavage site (Figure 3.4). The
SXP gene with signal sequence was cloned to Baculovirus expression system
(Figure 3.5).
MKYFIFLSIGLIAGALAQREAQIPQSDIPPFLSGAPNHVVKQFFDLLRA
DESKTDPQTEADIEAFMRRLGGVYQARFEQFKQEMKKQFAQYDKVH
QAALSRFSPAARQADARMSAIAESKQLTVKQKTEQIKAIMDSLSESVR
KEILEGFNSKFCVISVLLNVTIKIFSKWRKNHMRQKSNK
Figure 3.4 Wuchereria bancrofti SXP-1 amino acid sequence
(gi|3832519|gb|AF098861.1| SXP)
The signal peptide is present at amino-terminal region of SXP-1. The SXP-
1 protein was expressed in Baculovirus expression system along with signal
peptide. Signal peptide is in boldface and cleavage site is shown by arrow
mark.
Cleavage Site
97
Figure 3.5 Cloning strategy of SXP gene into Baculovirus system
SXP gene was subcloned in pFastBac1 donor vector with SXP native signal
sequence and expressed in Baculovirus expression system.
A faster approach for generating a recombinant baculovirus
(Luckow et al 1993) uses site-specific transposition with Tn7 to insert foreign
genes into bacmid DNA propagated in E. coli. The gene of interest is cloned
into a pFastBac vector, and the DH10BAC competent cells are transformed
with recombinant plasmid which contain the bacmid with a mini-attTn7 target
site and the helper plasmid. The mini-Tn7 element on the pFastBac plasmid
can transpose to the mini-attTn7 target site on the bacmid in the presence of
transposition proteins provided by the helper plasmid. Colonies containing
recombinant bacmids are identified by antibiotic selection and blue/white
screening, since the transposition results in disruption of the lacZα gene. High
molecular weight DNA is prepared from selected E. coli colonies containing
the recombinant bacmid and this DNA are then used to transfect insect cells.
The bacmid DNA is > 135 kb. Verification of the insertion of the
gene of interest is difficult using classical restriction endonuclease digestion
98
analysis. It is better to use PCR to confirm that the gene of interest has
transposed to the bacmid. The pUC/M13 amplification primers are directed at
sequences on either side of the miniattTn7 site within the lacZα-
complementation region of the bacmid (Figure 3.6). If transposition has
occured, the PCR product produced by these primers is 2,300 bp plus the size
of the insert. Alternatively, one can amplify a product using one gene specific
primer and one pUC/M13 primer. Use of two gene-specific primers for PCR
will produce an amplification product whether or not transposition has
occurred.
Figure 3.6 Transposition Region
For PCR, the sense primer is the M13/pUC Forward Amplification Primer
(Forward). The anti-sense primer is the M13/pUC Reverse Amplification
Primer (Reverse). If transposition has occured, the PCR product produced
by these primers is 2,300 bp plus the size of the insert {SXP -1(~ 600bp)}.
In case of bacmid alone the PCR product produced by these primers is
300 bp.
99
The presence of the insert was confirmed by both lysate-PCR of the
transformants, using gene-specific primers as well as by restriction digestion
of the clone (Figure 3.7). The pFastbacSXP-1 was transferred to DH10Bac
E.coli. strain for site specific transposition of SXP into bacmid. Recombinant
bacmid was confirmed with PCR amplification using M13 universal primers
(Figure 3.11). Mixed (blue and white) colonies showed ~ 2.9 kb and 300 bp
amplified product from bacmid extracted from DH10BAC E.coli strain
transform with recombinant pFastBacSXP-1 whereas pure (white) colony
showed ~ 2.9 kb product and used for transfection to Sf21 insect cells.
Recombinant bacmid was further confirmed with combination of gene
specific and M13 universal primers (Figure 3.12).
Figure 3.7 Lysate PCR amplification of pFastBacSXP-1
1.0% agarose gel showing positive clones with product size of ~600 bp. The
Clones were selected for further analysis. Lane 1: 100 bp ladder, Lanes 2:
Positive Control (pRSETBSXP-1), Lane 3: Negative control, Lane 4 to 8:
Colony lysate.
1kb
500bp ~ 600bp
100
Figure 3.8 pFastBacSXP-1 clone and pFastBac 1 vector
pFastBacSXP-1 clone and pFastBac 1 vector in 0.8% agarose gel shows
difference between vector and clone. Lane 1: 1kb ladder, Lane 2: pFastBac
1 vector, Lane 3 & 4: pFastBacSXP-1
Figure 3.9 Restriction Digestion of pFastBacSXP-1 using BamHI and
EcoRI
Lane 1: 1kb ladder, Lane 2: Vector single digest with EcoR1, Lane 3:
pFastBacSXP-1, single digest with EcoR1, Lane 4: Vector double digest
with BamH1 and EcoR1, Lane 5: pFastBacSXP-1 double digest with
BamH1 and EcoR1, Lane 6: 100 bp ladder
4kb
1kb
4kb
1kb 1kb
500bp
1 2 3 4 5 6
101
Figure 3.10 PCR amplification of pFastBacSXP-1
The PCR products were resolved on 1.0% agarose gel. A product of ~600
bp was amplified from the positive clones. Lane 1: 1kb ladder, Lane 2:
Negative control, Lane 3 &4: pFastBacSXP-1
Figure 3.11 PCR amplification of recombinant bacmid with SXP gene
using M13 universal primer
The PCR products were resolved on 1.0% agarose gel. Mixed (blue and
white) colonies showed ~2.9 kb and 300 bp amplified bands from bacmid
extracted from DH10BAC E.coli. strain transformed with recombinant
pFastBacSXP-1whereas pure recombinant colony showed ~2.9kb band.
Lane 1: 1kb ladder, Lane 2 to 4: Recombinant Bacmid
1 2 3 4
1kb
500bp
~ 600bp
1 2 3 4 5
3kb
250bp
Mixed colonies Pure
1 2 3 4 5
102
Figure 3.12 PCR amplification of recombinant Bacmid using M13 and
gene specific Primers
The PCR products were resolved on 1.0% agarose gel. Lane 1: 1kb ladder,
Lane 2: Recombinant Bacmid with M13 primer, Lane 3: Recombinant
Bacmid with M13 forward and SXP reverse primer, Lane 4: Recombinant
Bacmid with M13 reverse and SXP forward primer, Lane 5: Recombinant
Bacmid with SXP primers, Lane 6 – 100 bp ladder
3.3.2 PCR Amplification of cDNA from Transfected Insect Cells with
Recombinant Bacmid
Insect cells (Sf21) were transfected with recombinant bacmid and
transfected cells were harvested after 72 hours. RNA was extracted from cells
by using RNAZOL and converted into cDNA using MMLV Reverse
Transcriptase by standard protocols. The cDNA was checked by the presence
of message level of SXP-1 gene in the transfected Sf21 cells by doing PCR
with SXP gene specific forward and reverse primers. Figure 3.13A shows the
amplification of cDNA from transfected cells with a PCR product of ~ 600bp
with SXP gene-specific primers.
1kb
500bp
3kb
1kb
1 2 3 4 5 6
103
Figure 3.13 A) PCR amplification of cDNA from transfected cells with
WbSXP-1 primers
The PCR products were resolved on 1.0% agarose gel. Lane 1: 100bp
ladder, Lane 2: Negative Control, Lane 3: Control cDNA from normal
untransfected Sf21 cells, Lane 4: Positive control, Lane 5: cDNA from
transfected Sf21 cells
B) cDNA from transfected Sf21 cells
Lane1: 1kb ladder, Lane 2: cDNA from transfected insect cells
3.3.3 Expression of rBAC-WbSXP-1 from Insect Cells Infected with
Recombinant Baculovirus
Expression of rBAC-WbSXP-1 was studied in insect cells infected
with recombinant baculovirus with 5 multiplicity of infection (MOI) in serum
free TC100 insect cell medium. Since basal media did not support the
excellent growth Sf21 cells under serum free conditions, cells grown in serum
supplemented insect cell medium were collected by centrifugation and
suspended in fresh serum free media. Expressed protein in infected cell
supernatant was collected on 1st
to 5th day and expressed protein was separated
on 12% SDS-PAGE (Figure 3.14). Western blot analysis of infected cell
1 2 3 4 5
500bp
1kb 500bp
1kb
250bp
750bp
3kb
A B
1 2
~ 600bp
104
supernatants 1st
to 5th
day post infection was carried out using monoclonal
antibody (1F6H3) raised against rWbSXP-1. Result of western blot confirms
the secretion of expressed SXP-1 protein in infected cell supernatant detected
on day 3-5 post infection (Figure 3.15). Moreover, there was no expression on
day 1 and weakly detected on day 2 post infection. Expressed extracellular
fractions of SXP-1 protein were collected from day 1-5 post infection at 24-h
intervals and quantitated by sandwich ELISA. The viable cell density of Sf21
cells after infection was slightly reduced on 2nd
day post infection, while on
the 3rd
day there was sharp decline in the viability of the cells (Figure 3.16).
Result showed the secretion of expressed SXP-1 on day 2 and 3 post infection
with slight increase on day 4. Maximum concentration of expressed SXP-1
quantitated in infected cell supernatant was 8.25µg/ml/106
(Figure 3.17).
Figure 3.14 Secretion of the rBAC-WbSXP from Sf21 cells infected with
recombinant baculovirus in serum free medium
Infected cell supernatant with recombinant baculovirus at 5 MOI and
collected at different time point. Protein fractions was separated on 12%
SDS-PAGE and silver stained. Lane1: Protein marker, Lane 2: Infected
supernatant at 1st day
, Lane3: 2nd
day, Lane4: 3rd
day, Lane 5: 4th day,
Lane6: 5th day
1 2 3 4 5 6
14.4
18.4
25
35
45
66.2
kDa
105
Figure 3.15 Western blot analysis of rBAC-WbSXP-1 expression in
infected insect cell supernatant
Infected cell supernatant with recombinant baculovirus were collected from
1st day to 5
th day. Proteins were separated on 12% SDS-PAGE, transferred
to NC membrane and probed with 2E12E3 monoclonal antibody. Result
confirms the secretion of SXP-1 from 2nd
day post infection. Lane 1:
Infected supernatant at 1st day
, Lane 2: 2nd
day, Lane 3: 3rd
day, Lane 4: 4th
day, Lane 5: 5th day, Lane 6: Protein marker
Figure 3.16 The effect of recombinant baculovirus (MOI 5) infection on
Sf21 viable cell density in monolayer culture
Time course of infection of recombinant baculovirus and effect on Sf21
viable cell density after infection was analysed. The initial 1× 10-6/ ml Sf21
viable cells were infected with recombinant baculovirus and viable cell
density was analysed upto day 5 post infections.
1 2 3 4 5 6
14.4
18.4
25
35
45
66.2
kDa
116
106
Figure 3.17 Time course of SXP-1 protein expression and secretion in Sf21
culture supernatant infected with recombinant baculovirus
in monolayer culture
Extracellular fractions of SXP-1 protein were collected at 24-h intervals for
5 days and quantitated by sandwich ELISA using monoclonal and
polyclonal antibodies. Affinity purified rBACWbSXP-1 protein was used as
a standard.
3.3.4 Growth Profile of Sf21 Cells in Shake Flask
To optimize growth of Sf21 in suspension, cells were grown in TC-
100 insect cell medium with and without serum supplementation. 250-ml
Erlenmeyer flask with 100 ml of TC-100 medium inoculated with 1 × 105
viable cells/ml and were used in standard growth conditions (280C; 100 rpm;
TC-100 medium with or without 10% FBS). Medium was supplement with
0.1% Pluronic F-68 to protect the cells from mechanical shear forces in
suspension culture. In serum supplemented medium the maximum cell density
achieved was 4.5 million/ml on 5th
day. Medium without serum supplement
did not support cell growth. Cells in the mid-log phase (1.5-2 million/ml or 3rd
day) were used for infection with recombinant baculovirus (Figure 3.18).
107
Figure 3.18 Shake flask growth profile of Sf21 in TC-100 medium with
and without serum
Growth of Sf21 cells were observed in TC-100 medium under serum and
serum free condition. Medium was supplement with shear force protectant
0.1% Pluronic F-68. In serum supplemented medium the maximum cell
density achieved was 4.5 million/ml. Medium without serum supplement
did not support cell growth.
3.3.5 Expression Profile SXP-1 from Infected Sf21 Cells in Shake
Flask
Insect cells in mid-log phase were centrifuged and suspended in
serum free medium. Suspended insect cells were infected with recombinant
baculovirus with 5MOI and incubated in shaker incubator at 280C at 90 rpm.
Expression profile was analyzed from day 1 to 5 which was found to be
similar to that of monolayer culture. SXP-1Protein expression was observed
on post-infection day 2 and concentration reached at a plateau around day 3
(Figure 3.19). Maximum concentration of expressed SXP-1 quantitated in
108
infected cell supernatant was 8.5µg/ml/106. The viable cell density of infected
Sf21 cells in shake flask was slightly reduced on 2nd
day post infection, while
on the 3rd
day there was sharp decline of viable cells (Figure 3.20).
Figure 3.19 Time course of shake flask expression profile of SXP-1 from
Sf21 culture supernatant infected with recombinant
baculovirus
Extracellular fractions of SXP-1 protein were collected at 24-h intervals for
5 days and quantitated by sandwich ELISA using monoclonal and
polyclonal antibodies. Affinity purified rBACWbSXP-1 protein was used as
a standard. SXP-1 expression was observed on post-infection day 2 and
concentration reached at a plateau around day 3 post infection.
109
Figure 3.20 The effect of recombinant baculovirus (MOI 5) infection on
Sf21 viable cell density in shake flask
Time course of infection of recombinant baculovirus and effect on Sf21
viable cell density after infection was analysed. The initial 1× 10-6/ ml Sf21
viable cells were infected in serum free medium with recombinant
baculovirus and viable cell density was analysed upto day 5 post infection.
3.3.6 Purification of rBAC-WbSXP-1 by IMAC
The recombinant protein was purified by Immobilised Metal
Affinity Chromatography (IMAC). The column was washed with different
concentrations of imidazole and the histidine-tagged recombinant protein was
finally eluted at 250mM imidazole concentration (Figure 3.21). The fraction
was dialysed against 0.1x PBS and concentrated by lyophilisation. The pure
protein was immunoblotted with anti-antibodies as shown in Figure 3.22. In
order to study the immunoreactivity of the purified recombinant protein
expressed in baculovirus expression system (rBAC-WbSXP-1) Western
blotting was carried out using monoclonal antibodies namely 2E12E3, 1F6H3
and 3G12F7 and rabbit anti SXP-1 polyclonal raised against rWbSXP-1. The
pre- and post-translationally modified forms of recombinant protein are
distinctly detected by antibodies.
110
Figure 3.21 The purified recombinant rBAC-WbSXP-1 by IMAC
Protein fractions was separated on 12% SDS-PAGE and stained with
coomassie brilliant blue. The post translational modification of rBAC-
WbSXP is apparent from apparent molecular size heterogeneity (doublet).
Lane1: Protein marker, Lane 2: Infected cell supernatant, Lane 3:
Uninfected cell supernatant, (Serum free medium), Lane 4: IMAC Purified
rBAC-WbSXP-1
Figure 3.22 Western blot analysis of rBAC-WbSXP-1
rBAC-WbSXP-1 showed immunoreactivity with Monoclonal and
polyclonal antibodies specific to rWbSXP-1. The recombinant protein is
shown by arrow mark. Lane 1: Protein Marker, Lane 2: Rabbit anti-SXP
Polyclonal antibody, Lane 3 to 5: Monoclonal antibodies raised against
rWbSXP-1
rBAC-WbSXP-1
1 2 3 4
14.4
18.4
25
35
45
66.2
kDa
rBAC-WbSXP-1
1 2 3 4 5
18.4
25
35
45 kDa
111
The purified rBAC-WbSXP protein was immunoblotted with MF
positive, CP, EN and NEN serum as shown in Figure 3.23. Results confirm
anti-SXP antibodies in MF positive serum whereas CP, EN and NEN showed
no reactivity with rBAC-WbSXP protein. The SXP-1 protein expressed in
E.coli GJ1158 shown similar result in western blot and distinctively reacted
only with the MF sera of W. bancrofti or B. malayi infection and no reactivity
was observed with CP, EN and NEN sera (S. Janardhan et al, 2010). The
western blot analysis of SXP-1 expressed in baculovirus expression system
showed distinct pre- and post translationally modified forms with MF positive
sera.
To analyze the glycosylation of pure SXP-1 (rBAC-WbSXP-1)
expressed in baculovirus expression system, PAS staining was carried out and
SXP-1 expressed in GJ1158 E.coli (rWbSXP-1) was used as control (Figure
3.24). Results showed presence of glycosylated rBAC-WbSXP-1 in SDS-
PAGE whereas the control E.coli expressed rWbSXP-1 protein did not show
glycosylation. The protein sample was deglycosylated with N-glycosidase
(PNGase F). The untreated and treated proteins were separated by SDS-
PAGE and revealed by Western blot analysis using MAb (1F6H3). The shift
in the electrophoretic mobility confirmed the deglycosylation of rBAC-
WbSXP-1(Figure 3.25). Western blot analysis of rBAC-WbSXP-1 and Bm mf
crude antigen was done with MAb (1F6H3) to compare the post-
translationally modified SXP in rBAC-WbSXP-1 and native SXP in Bm mf
crude antigen. It was observed that the native SXP in Bm mf crude antigen
was recognized by MAb (1F6H3) at 25kDa which is at the same range of
post-translationally modified SXP-1 expressed in baculovirus expression
system (Figure 3.26).
112
Figure 3.23 Western blot analysis of rBAC-WbSXP-1 with clinical sera Expressed rBAC-WbSXP-1 protein was separated on SDS-PAGE,
transferred to nitrocellulose membrane and probed with 1:100 diluted
pooled (pool of 10 sera) W. bancrofti MF, CP, EN and NEN sera. The pre-
and post translationally modified forms of recombinant protein is distinctly
detected by bancroftian MF sera. The recombinant protein is shown by
arrow mark. Lane M=Molecular weight marker is shown on the left side.
a
Figure 3.24 PAS staining of purified rBAC-WbSXP-1 expressed in
Baculovirus expression system PAS staining of purified rBAC-WbSXP-1 and pure rWbSXP-1was done
to check the glycosylation in purified protein. Lane 1: Protein Marker,
Lane 2: Control E.coli rWbSXP-1 (non-glycosylated protein), Lane 3:
rBAC-WbSXP-1
M MF CP EN NEN
18.4
25
35
45
66.2
kDa
1 2 3
14.4
18.4
25
35
45
66.2
kDa
116
113
Figure 3.25 Comparison of recombinant rBAC-WbSXP-1 protein with
(+) or without (-) PNGase-F treatment
Western blot profile with MAb (1F6H3) showed deglycosylation of rBAC-
WbSXP-1 protein treated with PNGase-F.
Figure 3.26 Western blot analysis of rBAC-WbSXP-1 and Bm mf crude
antigen with MAb 1F6H3
Western blot profile of Bm mf crude antigen showed immunoreactivity with
MAb 1F6H3 at 25kDa which is at the same size of post-translationally
modified SXP-1 expressed in baculovirus expression system. The SXP-1
protein in Bm mf crude antigen is shown by arrow mark. Lane 1 – Protein
Marker, Lane 2 – Pure rBACWbSXP-1 protein, Lane 3 – Bm mf crude
antigen
PNGase-F (+) (-) kDa
35
45
25
18.4
14.4
M
1 2 3
18.4
25
35
45
66.2
kDa
114
3.3.7 ELISA Profile for rBAC-WbSXP-1 with Monoclonal and
Polyclonal Antibodies
The reactivity of pure rBAC-WbSXP-1 was analysed with
monoclonal and polyclonal antibodies raised against rWbSXP-1. The result
showed that 1F6H3, 2E12E3, 3G12F7 MAbs and polyclonal were reactive
with inset cell expressed SXP protein whereas 3E4F1 and 3E3D7 showed no
reactivity (Figure 3.27).
Figure 3.27 Reactivity of rBAC-WbSXP-1 with Monoclonal and
polyclonal antibodies for rWbSXP-1
100 ng of rBAC-WbSXP coated on microtitre wells was incubated with
monoclonal and polyclonal antibodies for rWbSXP-1 and the bound
antibodies detected by secondary goat anti-mouse Ig HRP conjugate-
TMB/H2O2
system. MAbs 1F6H3, 2E12E3, 3G12F7 and polyclonal
antibodies showed distinctly high reactivity compared to other monoclonal.
SP2/0 cell supernatant was used as control.
3.3.8 Reactivity of rBAC-WbSXP-1 with Patient’s Sera
The reactivity of pure SXP-1 expressed in baculovirus expression
system was tested with filarial patient serum where non endemic normal sera
115
used as control. Total of 23 MF positive sera, 16 CP, 19 EN and 5 NEN sera
were checked for reactivity with SXP-1expressed in baculovirus expression
system. The serum samples were considered positive for the respective
assays if it showed optical density (OD) greater than the mean OD of the
NEN samples plus three SD (mean NEN OD+3SD). Result showed the
presence of anti-SXP antibody in all MF serum as compared to other clinical
sera in which two chronic pathology clinical samples and one endemic normal
showed higher absorbance than cut-off value (Figure 3.28). The rBAC-
WbSXP-1 based antibody assay with clinical sera showed 100% reactivity
(23/23) with MF positive samples compared to control samples which showed
12.5% reactivity (2/16) for chronic pathology and 5.25 % (1/19) with endemic
normal samples Table 3.1.
Figure 3.28 Reactivity of rBAC-WbSXP-1 purified from baculovirus
eukaryotic expression system with Patient Sera by ELISA
100ng of rBAC-WbSXP coated on microtitre wells was incubated with
1:100 dilution of microfilarial (MF), chronic (CP), endemic normal (EN)
and non-endemic normal (NEN) sera and the bound antibodies detected by
secondary goat anti-human Ig HRP conjugate-TMB/H2O2 system. MF sera
showed distinctly high reactivity compared to other sera.
116
Table 3.1 Reactivity of rBAC-WbSXP-1 with different clinical groups of
bancroftian Filariasis
Clinical groups
( No. of samples)
No. of positive
samples ( % positive
reactivity)
Mean SD
Mf carriers (23) 23 (100%) 0.993 ± 0.203
Chronic pathology (16) 2 (12.5%) 0.323 ± 0.068
Endemic normal‟s (19) 1 (5.25%) 0.251 ± 0.052
Non-endemic normal‟s (5) 0 (0%) 0.241 ± 0.045
3.3.9 Isotype ELISA for rBAC-WbSXP-1 with Patient Sera
The antibody response to rBAC-WbSXP-1 was subclass restricted.
ELISA was done to determine the antigen specific isotype responses in
patient sera with NEN control (Figure 3.29).
MF
CP ENNEN
0.0
0.2
0.4
0.6
0.8
1.0 IgG1
IgG2
IgG3
IgG4
IgM
IgE
IgA
Isotype ELISA for rBAC-WbSXP with Patient Sera
Ab
sorb
ance
at
450
nm
Figure 3.29 Isotype ELISA for rBAC-WbSXP-1 with clinical sera
Isotype ELISA was performed to identify antibody subclass in human
clinical samples elicited by rBAC-WbSXP-1 antigen. The microtiter plates
were coated with the 100 ng of rBAC-WbSXP-1 antigen, and the human
clinical samples were used as primary antibody. Result showed that MF
positive patient samples had significantly elevated level anti-WbSXP-1
IgG4 and IgM levels compared to other clinical group.
117
Results showed that sera from MF positive patient had significantly
elevated level anti-WbSXP-1 IgG4 and IgM levels compared to other clinical
group. The IgM levels against SXP-1 were significantly higher specifically in
individuals with circulating mf.
3.4 DEVELOPMENT OF RAPID FLOW THROUGH TEST KIT
FOR THE DETECTION OF ANTIBODIES IN LYMPHATIC
FILARIASIS USING RECOMBINANT BACWbSXP-1
A rapid-format, simple and qualitative flow through immune
filtration test has been developed for the identification of total IgG antibodies
to recombinant filarial antigen BAC-WbSXP-1 expressed in baculovirus
expression system. This test system employs colloidal gold-Protein-A as
antibody capture reagent. The appearances of typical positive and negative
test results are shown in Figure 3.30.
(A) (B)
Figure 3.30 Interpretation of rapid flow through immuno filtration test
(A) Positive test showing two red magenta spots indicating both
control (C) and test sample (T) positive for filarial antibodies;
(B) Negative test showing only one red magenta spot on control
(C) Negative for filarial antibodies.
The control spot containing goat anti-human IgG antibody serves as
a control to ensure the stability of the test device, and it must give a positive
reaction (a red magenta colour spot at the control area) after the completion of
118
test. Failure of appearance of the control spot indicates a defective test kit.
The test spot contains the recombinant antigen rBAC-WbSXP-1, which
develops only when the serum contains anti filarial IgG antibodies specific to
the antigen. Thus the final result in a positive test is the appearance of two
magenta coloured spots in both control and test areas. When a negative serum
is used, binding with the recombinant antigen does not occur, thus only the
control spot will develop.
3.4.1 Comparative Evaluation of Enhanced Rapid Antibody
Detection Kit Format
The performance and efficacy of improvised rapid antibody
detection kit using rBAC-WbSXP-1 was studied by screening with MF
positive serum samples obtained from different geographical locations and
compared with the rapid flow through immune filtration test kit developed
using E.coli expressed rWbSXP-1 (Table 3.2).
Table 3.2 Performance of rapid antibody detection kit
The efficiency of rapid diagnostic kit using rBAC-WbSXP-1 was studied and
compared with rapid diagnostic kit using rWbSXP-1.
Scale of Antibody
reactivity in Rapid format
MF Positive Samples
rBAC-WbSXP-1 kit
MF Positive Samples
rWbSXP-1 kit
Total tested 20 20
Very strong reaction (++++) 6 4
Strong reaction (+++) 13 14
Moderate reaction (++) 1 2
Weak reaction (+) 0 0
Sensitivity (%) 100 100
Performance of rapid diagnostic kit was further evaluated using the
positive cases showing weak, moderate, strong, very strong antibody reaction.
119
Reactivity of the test with serum samples was determined with spot intensity
of kit (Figure 3.31). Both test forms showed 100% sensitivity towards MF
positive serum samples. Rapid diagnostic kit with rBAC-WbSXP-1 showed
30% (6/20) very strong reactivity for MF positive sampled and better
sensitivity compare to rapid kit with rWbSXP-1, which showed 20% (4/20)
very strong reactivity.
A B
C D
Figure 3.31 Scale of antibody reactivity in rapid diagnostic kit developed
using rBAC-WbSXP-1
A. Very strong reaction, B. Strong reaction, C. Moderate reaction, D. Weak
reaction
3.5 COMPARISON OF REACTIVITY OF rBAC-WbSXP-1 AND
rWbSXP-1 WITH PATIENT SERA
The immunorectivity of SXP-1 protein was expressed in E.coli.
(rWbSXP-1) and baculovirus expression system (rBAC-WbSXP-1) was
compared with human clinical sera belonging to different clinical group, viz.,
microfilareamics (MF), chronic pathology (CP), endemic normal (EN) and
non-endemic normal (NEN) (n=10). The statistical analysis was done using
graph pad 5.0. The immunorectivity was expressed as mean ± SD. The result
120
showed high reactivity of rBAC-WbSXP-1(mean=1.122± 0.254) compared to
rWbSXP-1(mean= 0.911±0.205) against MF sera (Figure 3.32) and less
reactivity with CP and EN. Reactivity of antigen was compared with pooled
sera of all clinical group (n=10) and showed the high reactivity of rBAC-
WbSXP-1 compared to rWbSXP-1 against MF sera (Figure 3.33). Earlier
studies proved that the antibody response to WbSXP-1in W.bancrofti infected
individuals was restricted to the IgG4 subclass (Rao et al 2000). The level of
anti- rBAC-WbSXP-1 IgG4 and anti-rWbSXP-1 IgG4 antibodies in clinical
sera was compared. Results confirm high level of anti- rBAC-WbSXP-1 IgG4
antibodies than anti-rWbSXP-1 IgG4 in MF positive clinical sera (Figure
3.34). Both antigens did not show any reactivity with other clinical groups.
rBAC-W
bSXP
rWbSXP
rBAC-W
bSXP
rWbSXP
rBAC-W
bSXP
rWbSXP
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0 MF CP EN
P value < 0.0001
Ab
so
rban
ce a
t 450 n
m
Figure 3.32 Comparison of reactivity of rBAC-WbSXP-1 and rWbSXP-1
with clinical sera
100 ng of pure rBAC-WbSXP-1and rWbSXP-1 coated on microtitre wells
was incubated with the 1:100 dilution of microfilarial (MF; n=10), chronic
(CP; n=10), endemic normal (EN; n=10) sera and the bound antibodies
detected by secondary goat anti-human Ig HRP conjugate-TMB/H2O2
system.
121
MF
CP
ENNEN
0.0
0.5
1.0
1.5
2.0rBAC-WbSXP
rWbSXP
Ab
so
rban
ce a
t 450 n
m
Figure 3.33 Comparison of reactivity of rBAC-WbSXP-1 and rWbSXP-1
with pooled clinical sera
Reactivity of rBAC-WbSXP-1 and rWbSXP-1 was compared with pooled
clinical sera (n=10) sera. MF sera with rBAC-WbSXP-1 purified from
baculovirus expression system showed high reactivity compared to
rWbSXP-1 expressed in E.coli.
MF
CP
ENNEN
0.0
0.2
0.4
0.6
0.8
rBAC-WbSXP
rWbSXP
Ab
so
rban
ce a
t 450 n
m
Figure 3.34 Comparison of level of anti- rBAC-WbSXP-1 IgG4 and anti-
rWbSXP-1 IgG4 in clinical sera
IgG4 isotype ELISA was done with 100 ng of rBAC-WbSXP-1 and
rWbSXP-1 pure antigen coated on microtitre wells and 1:100 dilution of
pooled clinical sera was used as primary antibody. Result confirms high
level of anti- rBAC-WbSXP-1 IgG4 antibodies than anti-rWbSXP-1 IgG4 in
MF positive clinical sera.
122
3.6 DEVELOPMENT OF MONOCLONAL ANTIBODIES TO
RECOMBINANT FILARIAL ANTIGEN rWbSXP-1
The recombinant filarial antigen WbSXP-1 was expressed in E.coli
GJ1158. The purified protein was used for immunizing mice and hybridoma
development. The rWbSXP-1 protein at a concentration of 1mg/mL was used
for fusion, the cells were screened for the hybridomas in HAT selection
medium. Hybridoma for the development of monoclonal antibodies resulted
in several antibody secreting clones. Initially clones were screened through
ELISA and those binding to rWbSXP-1 were selected (Figure 3.35).
Figure 3.35 Primary screening of hybrids
Primary screening of hybrids from 96 well plates to select the clones for
further analysis and scale up.
Every well had at least two hybrid clones which were apparent by
the colour change of the culture medium. The plates were observed and
screened for clones secreting antibodies by ELISA on recombinant WbSXP
123
coated (1 μg/well) plate. The results showed that 16 clones secreted specific
antibodies to recombinant filarial antigen, the selected clones were further
screened with rWbSXP-1 and B. malayi mf crude antigen for reactivity. Of
these 9 clones secreted specific antibodies to recombinant and mf antigen and
selected for further expansion (Figure 3.36).
Figure 3.36 Secondary Screening of hybrids from 24 well plates
Screening of hybrids from 24 well plates to select the clones for further
analysis and scale up.
3.6.1 Scale-Up of the Clones
The clones secreting antibodies with positive reactivity (ELISA) to
recombinant and mf antigen for reactivity were scaled-up to 1ml culture in 24
well plate at 1X HT and retested by ELISA after 3-5 days of growth. The
selected hybrids in 1 mL culture were expanded to 5 ml in culture flasks at
0.5X HT and retested by ELISA after 3-5 days of growth. The cells were
slowly weaned off HT. After 3-4 passages the cells were cloned to
monoclonality by the limiting dilution method.
124
3.6.2 Sub-Cloning: Cloning by Limiting Dilution and Derivation of
Stable Clones
Cloning by limiting dilution was a standard method based on the
Poisson distribution. Dilution of cells to an appropriate number per well
maximized the proportion of wells that could contain a single clone.
Hybridomas to be cloned were diluted to 1 cell/well. This kind of dilution
provides ~ 30-40 % of wells with 1 cell/well as per the poisson statistics. As a
standard procedure, hybridoma that yielded > 90 % antibody positive cultures
upon recloning was considered to be stable. At the end of this cloning
process, the clones were selected and cryopreserved.
3.6.3 Selection of Monoclones
We screened for stable and high secreting clones which showed
good affinity to Wuchereria bancrofti and B.malayi mf antigen and rWbSXP-
1 in ELISA were scaled-up to 1 ml and subsequently to 5 ml culture. Finally,
five clones namely 3E4F1, 2E12E3, 1F6H3, 3E3D7 and 3G12F7 were
selected (Figure 3.37) and cryopreserved for future directions.
Figure 3.37 Screening of the Clones secreting monoclonal antibody for
rWbSXP, Wb and Bm mf crude antigen
125
3.6.4 Characterization of the MAbs
To determine the titers and sensitivity of the antibodies, the MAb‟s were again tested for binding to rWbSXP-1 antigen in ELISA by varying
dilution. To determine this, the selected five clones were tested in ELISA by
varying the dilution of supernatant (Figure 3.38) and different concentration
of rWbSXP-1 (Figure 3.39).
Figure 3.38 Reactivity of MAbs against rWbSXP-1 in ELISA
The assay data plotted are mean value of triplicates +/- deviations. As per
the procedure, the ELISA was performed. The two fold dilution of MAbs
supernatant was used as primary antibody.
Figure 3.39 Reactivity of monoclonal antibodies against rWbSXP using
ELISA
The assay performed with varying concentration of rWbSXP from 1000 ng
to 31.25 ng. The MAbs supernatant was used as primary.
126
3.6.5 Confirmation of MAbs Against rWbSXP-1 in Western Blot
The selected MAbs (3E4F1, 2E12E3, 1F6H3, 3E3D7 and
3G12F7) were further characterized by Western blot. The affinity and
sensitivity of the clones was again confirmed by using the MAbs supernatant
as primary antibody (Figure 3.40).
Figure 3.40 Western blot analysis of hybridoma culture supernatant
against rWbSXP-1
Lane 1: molecular weight marker, Lane 2: 3G12F7 clone, Lane 3: 1F6H3
clone, Lane 4: 3E3D7 clone, Lane 5: 3E4F1 clone, Lane 6: 2E12E3 clone.
From sub-cloning the selected clones (3E4F1, 2E12E3, 1F6H3,
3E3D7 and 3G12F7) were scaled up and found to produce MAb continuously
against rWbSXP-1. Thus it confirmed the MAb‟s γE4F1, βE1βEγ, 1F6Hγ,
3E3D7 and 3G12F7 were more stable and it can be further used for
developing suitable methods for antigen detection of lymphatic filariasis.
3.6.6 Isotyping of Monoclones
The Isotyping of all five MAbs was carried out by the Rapidot-
mouse immunoglobulin isotyping kit. The three clones 3E3D7, 3E4F1 and
2E12E3 belonged to IgM isotypes and 1F6H3 and 3G12F7 are IgG2a.
1 2 3 4 5 6
45
14
18.4
25
66.2
kDa
116
127
3.6.7 Affinity Measurement of Anti-SXP-1 Monoclonal Antibodies
Anti-SXP-1 hybridomas were established by immunization of
BALB/c mice with rWbSXP-1 protein. Hybridomas were screened by the
differential ELISA to identify wells with high-affinity MAbs, and the selected
hybridoma cells were cloned. Affinities of selected MAb clones to SXP-1
protein were measured. The Kd of each MAb was determined by measuring
the rate of binding to the antigen at different protein concentration and was
calculated using the equation derived from Scatchard and Klotz (Friguet et al
1985). The result revealed high affinity of 1F6H3 monoclonal antibody to the
SXP-1 antigen. The Kd values of the 1F6H3 (IgG2a) monoclonal antibody for
SXP-1were two fold lower than 3G12F7, the other IgG2a monoclonal
(Table 3.3). The IgM monoclonal antibodies showed high Kd values and low
affinity to antigen.
Table 3.3 Affinity of anti-SXP-1 monoclonal antibodies (Dissociation
constant of antibody- antigen complex)
Mab Isotype Kd (mol/lit)
1F6H3 IgG2a 5.8 × 10–9
3G12F7 IgG2a 1.51 × 10–7
2E12E3 IgM 6.9 × 10–7
3E4F1 IgM *
3E3D7 IgM *
*Kd values too high to be calculated
3.6.8 Effect of Urea Treatment on Monoclonal Antibodies Reactivity
with rWbSXP-1 and Wb mf Crude Antigen
Monoclonal antibodies were developed for recombinant WbSXP-1
expressed in E.coli and were screened for reactivity with recombinant and
crude mf antigen and five monoclonal antibodies were successfully identified
128
for SXP-1. Urea wash was given in ELISA to measure the specificity and
binding strength of MAbs to their corresponding epitope. Effect of urea
treatment was previously described by Binley et al (1997) on the binding of
gp120 of HIV type1 with panel of monoclonal antibodies. Result of urea
elution showed high avidity index for 1F6H3 with recombinant and
moderately high with mf crude antigen and intermediate avidity index for
2E12E3 clones with recombinant as well as mf antigen, while other clones
showed low avidity index instead of showing high reactivity with
recombinant as well as mf antigen (Table 3.4). SXP polyclonal antibodies
showed high avidity index for both recombinant and mf antigen (Table 3.4).
Low avidity index of high reactive monoclonal antibodies with recombinant
and mf antigen may explain the cross reactivity of monoclonal and low
affinity immune complexes was eluted with the treatment of mild denaturant
(8M Urea).
Table 3.4 Effect of urea elution of MAb reactivity with rWbSXP-1 and
Wb mf crude antigen
MAbs Isotype
Avidity Index
in percentage
(%) ( with
rWbSXP-1)
Avidity Index in
percentage (%)
(with Wb mf crude
antigen)
1. 1F6H3 IgG2a 68.9 48
2. 3G12F7 IgG2a 29 <5
3. 2E12E3 IgM 36.6 31.9
4. 3E4F1 IgM 10.5 12
5. 3E3D7 IgM 8.2 10
6. Rabbit anti-SXP-1
polyclonal
-- 72.8 53.6
129
3.7 DEVELOPMENT OF SANDWICH ELISA (ANTIGEN
DETECTION) USING ANTIBODIES RAISED RECOMBINANT
FILARIAL ANTIGEN (rWbSXP-1)
Sandwich ELISA was standardized for antigen detection with
MAbs and polyclonal antibodies in combination as capture antibody and
detection antibody to detect antigens. Result showed significant difference
between detection of control and rWbSXP-1, when MAbs used as capture
antibody (Figure 3.41). Sandwich ELISA prototype for detecting parasite
SXP-1 was developed for field trial. The response of sandwich ELISA was
checked with MF positive blood samples and used EN and CP and NEN as
control.
3.7.1 Optimization of Various Parameters for the Development of
Sandwich ELISA Using Antibodies Raised to rWbSXP-1
Antigen
Criss cross serial dilution analysis was carried out to determine
optimal reagent concentration to be used in the ELISA. All the three
reactants in this ELISA namely -a primary solid phase coating reagent, a
secondary reagent (rWbSXP-1, rBAC-WbSXP-1 and mf crude antigen) that
binds to the primary reagent and the second antibody that binds to the
secondary reagent were serially diluted and analyzed by criss cross matrix.
Sandwich ELISA was standardized for SXP-1 with MAbs and polyclonal
antibodies in combinations using either one as capture antibody and the
other as detection antibody to detect antigens. Results showed that MAb
can be the better option as capture antibody than rabbit anti SXP
polyclonal antibody. Sandwich ELISA was done for five MAbs as
capture antibody and rabbit anti SXP polyclonal as detection antibody,
while 50 ng of rWbSXP-1and 5µg of Wb mf crude antigen used as
standard and 1µg E.coli. protein was used as control. Two MAbs 1F6H3
130
(IgG2a) and 2E12E3 (IgM) showed the detection of recombinant and
native antigen in sandwich assay and selected for further standardization,
while detection was not significant enough with monoclonal antibodies
3E3D7, 3G12F7 and 3E4F1 (Figure 3.41). Both of MAbs were used
(namely 1F6H3 and 2E12E3) in combination to develop the assay which
showed promising results for antigen detection than when either MAbs
were used as single capture antibody. Native antigen showed the same
pattern of absorbance in sandwich assay as showed with recombinant
antigen (Figure 3.42).
Figure 3.41 Sandwich ELISA with 50 ng of rWbSXP-1 and 5 µg of Wb
mf native antigen
1 µg of SXP MAbs were used as capture antibody and 1:1000 dilution
of rabbit anti SXP polyclonal as detection antibody, while 50 ng of
rWbSXP-1and 5 µ g of Wb mf crude antigen used as standard test
antigen. 1 µg of E.coli host protein was used as control. Two MAbs
1F6H3 and 2E12E3 showed the detection of recombinant and native
antigen in sandwich assay, while other MAbs didn‟t show significant
detection.
131
3.7.2 Sensitivity of the SXP Antigen ELISA Using Recombinant
and Native Wb mf Crude Antigen
ELISA was carried out to find out the minimum detectable
concentration of purified recombinant and mf native crude antigen. A
known amount of the purified rBAC-WbSXP-1 antigen starting from 100
ng to 6.25 ng (Figure 3.42) and for mf crude antigen from 15 µg to
1.9 µg was added to normal sera (Figure 3.43). The serum was added to
the microtitre plate, which was coated with anti SXP monoclonal
antibody and the assay performed as mentioned above. The E. coli
antigen was used as control. It was found that the minimum amount of
recombinant BAC-WbSXP-1 antigen that could be detected was 20 ng/ml
by ELISA and no reactivity to E. coli antigen was observed even at a
higher concentration. SXP-1 present in Wb mf crude antigen was
detected significantly in capture assay.
Figure 3.42 Capture Assay with different amounts (100 ng to 6.25 ng) of
rBAC-WbSXP
The monoclonal antibodies, SXP MAbs 1F6H3, 2E12E3, were selected for
validating capture assay at 1 µg as single and in combinations. The rabbit
anti SXP polyclonal antibody was used at the dilution of 1:1000 for
detection. The cocktail monoclonal (1F6H3 + 2E12E3) showed better
sensitivity compared to the individual.
132
Figure 3.43 Capture Assay with different amounts (15 µg to 1.9 µg) of
Wb mf native antigen
The cocktail monoclonal (1F6H3 + 2E12E3) showed better sensitivity
compared to the individual with Wb mf native antigen.
3.7.3 Determination of the Titers of Anti SXP-1 Polyclonal
Antibodies
ELISA plates (96 wells) were coated with 100 ng /well of
purified rWbSXP-1 antigen and 2 µg of Wb mf antigen and direct ELISA
was performed. The pre and post immune sera were serially diluted
starting from 1:100 to 1:6400. Rabbit anti rWbSXP-1 sera showed
reactivity with the recombinant SXP-1 antigen and mf antigen. Pre-
immune control sera showed no reactivity with recombinant antigen as
well as mf antigen (Figure 3.44).
133
Figure 3.44 Reactivity of rabbit anti SXP-1 polyclonal antibody with
rWbSXP-1 and Wb mf crude antigen in ELISA
100 ng of rWbSXP-1 and 2µg of mf antigens were coated on 96 well
microtiter plates. The two fold dilution of rabbit antiSXP-1polyclonal serum
was used as primary. The assay data plotted are mean value of duplicate +/-
deviations.
3.7.4 SXP Antigen Specific Capture Assay for Detection of
Circulating Filarial Antigens
In order to validate the utility of SXP specific antigen assay in early
diagnosis of filariasis, a sandwich ELISA was performed with different
clinical groups of W. bancrofti infected filarial samples with appropriate
controls.
Figure 3.45 shows the SXP specific antigen levels in the different
clinical groups of bancroftian filariasis. The results were expressed as mean
optical densities at 450 nm for each group ± standard error. Samples were
considered positive for the assay when the optical density was higher than the
134
mean OD + 3SD value of 10 NEN control sera. The cut off line is indicated in
the Figure 3.44 as a parallel line drawn to the X-axis. Based on this criterion it
was observed that the bancroftian MF individuals had higher OD values
above the cut off value compared with other clinical groups.
MF EN CP NEN0.1
0.3
0.5
0.7
0.9
1.1
1.3
1.5P Value < 0.0001
Cut-off
Ab
so
rb
an
ce a
t 4
50
nm
Figure 3.45 Antigen based Sandwich ELISA of monoclonal antibody
(1F6H3+2E12E3) and polyclonal antibody with various
clinical samples
MF: Microfilareamics, EN: Endemic normals, CP: Chronic pathology,
NEN: Non Endemic normal; Sandwich ELISA was performed with
monoclonal (1F6H3+2E12E3) as capture antibody and rabbit polyclonal
anti-rWbSXP-1 antibodies as detection antibodies for capture and detection
of MF antigen. The reactivity to MF serum group is considerably higher
than in control groups; EN & CP. (p<0.05).
In bancroftian filariasis, the MF group exhibited the highest
optical density (OD) mean OD = 0.769 whereas the CP, EN and NEN showed
a mean value of OD=0.259, 0.271 and 0.255 respectively (Table 3.5). Result
showed that MF group had significantly higher OD values than that of control
group (p < 0.05) whereas the OD values of CP group had no significantly
difference than control group. The MF group had the highest percentage of
135
antigen positive reactivity 23/23 (100%) while EN and CP group had the least
antigen reactivity 4/24 (16.66%) and 0/10 (0%) (Table 3.5).
Though the level of antigen in patient samples and its bound state
as immunocomplexes play a major role in detection, it was the MF isolated
from whole blood of patients which helped to determine that WbSXP-1 is a
prospective candidate for the development of antigen based diagnostic assays
for lymphatic Filariasis.
Table 3.5 Evaluation of antigen based capture assay for the detection of
circulating SXP-1 in clinical samples
Clinical groups
( No. of samples)
No. of samples
showing antigen
detection ( %
positive reactivity)
Mean SD
Mf carriers (23) 23 (100%) 0.769 ± 0.14
Endemic normal‟s (β5) 4 (16%) 0.271 ± 0.084
Chronic pathology (10) 0 (0%) 0.259 ± 0.038
Non endemic normal‟s (10) 0 (0%) 0.255 ± 0.045
3.8 DEVELOPMENT OF RAPID DIPSTICK DIAGNOSTIC
ASSAY FOR DETECTION
Rapid dipstick diagnostic assay for filarial antigen detection was
optimized using 2E12E3 and 1F6H3 monoclonal antibodies as capture and
detection antibody and pure recombinant WbSXP-1 protein and
rBACWbSXP-1 was used as standard test antigen. The minimal detection
limit of the dipstick assay, based on the reactivity of dipsticks in samples
containing various concentrations of the WbSXP-1 and rBAC-WbSXP-1
antigen, was 25 ng/ml and 100 ng/ml of sample. Finally clinical samples (MF
136
and EN patient blood samples) were tested as a means to develop field-mode-
rapid diagnostic prototype as the same with recombinant antigens gave
promising sensitivity and specificity (Table 3.6).
Table 3.6 Performance of Antigen detection dipstick kit
The efficiency of dipstick assay was evaluated with MF positive samples and
compared with the capture ELISA.
Clinical groups
( No. of samples)
Capture ELISA
( % positive
reactivity)
Dipstick device
( % positive
reactivity)
Mf carriers (24) 24 (100%) 10 (41.7%)
Endemic normal‟s (β5) 4 (16%) 0 (0%)
Chronic pathology (10) 0 (0%) 0 (0%)
Non endemic normal‟s (10) 0 (0%) 0 (0%)
The samples tested in this fashion showed a moderate sensitivity
(41.7%) and high levels of specificity (100%). An extensive on-the-field trials
(with samples procured and tested immediately) in the future could remedy
and enhance sensitivity if fresh samples are used. Moreover, the reduced
sensitivity may be directly related to the MF load which may in turn result in
low circulating antigens. The development of high efficiency MAb‟s against
eukaryotic expressed protein could enhance the sensitivity of rapid dipstick
diagnostic assay.
Prototype of dipstick device was developed (Figure 3.46) and
assayed as described at the research facility at SPAN Diagnostics Ltd. Briefly,
the prototype contains a test line of capture anti-SXP monoclonal 2E12E3 and
a control line with goat-anti mouse IgG on nitro cellulose membrane. The
sample adsorbent pad contains detection reagent with colloidal gold
conjugated monoclonal anti-SXP antibody (1F6H3).The patient blood sample
137
will be drawn in the adsorbent pad and any native antigen present will bind
with the colloidal gold conjugated monoclonal and will be carried further
across the test and control line. The indication of positive reaction will be
seen as two dark magenta coloured lines in test and control regions
respectively. The negative reaction will be represented as a single magenta
coloured line in the control region.
(A) (B)
Figure 3.46 Dipstick Prototype Device
It contains test and control line with capture anti-SXP monoclonal
(2E12E3) and goat-anti mouse IgG respectively on nitro cellulose
membrane. The sample adsorbent pad contains colloidal gold conjugated
monoclonal anti-SXP antibody (1F6H3) for detection. The test is confirmed
positive with two dark magenta coloured lines in test and control regions
respectively and negative reaction with a single magenta coloured line in
the control region. A. Dipstick device assembly; B. Dipstick prototype
138
Figure 3.47 Dipstick diagnostic test device
The device developed with capture anti-SXP monoclonal (2E12E3) on nitro
cellulose membrane and the sample adsorbent pad contains colloidal gold
conjugated monoclonal anti-SXP antibody (1F6H3) for detection. The test
is confirmed positive with dark magenta coloured lines with the test
samples.