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.