Preparation house in Nagpur and processed for preparation

Preparation of Cysticercus cellulosae antigens

Metacestodes
of Taenia solium were collected from
a naturally infected pig after slaughter from a local slaughter house in Nagpur
and processed for preparation of two somatic (scolex antigen (SA) and
membrane-body antigen (MBA)) and one metabolic (excretory secretory antigen
(ESA)) antigens.

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(a)  
Scolex
Antigen (SA)

Scolex
antigen (SA) was prepared according to Dhanalakshmi et al. (2005) with some modifications. Scolices were carefully
dissected with fine forceps and scalpel andtransferred immediately into cold
PBS (pH 7.2-7.4) containing antibiotics and PMSF (Sigma Aldrich, USA). These
were blotted dry, homogenized followed by sonication at 20kHz, 1 mA for 60 seconds and then
centrifugation at 14000 rpm for 60 minutes at 40C in a refrigerated
centrifuge (Remi, India). Supernatant was used as scolex antigen (SA).

(b)  
Membrane-Body
Antigen (MBA)

            This
antigen was prepared from the Taenia
solium cysts after removing cystic fluid and scolices. The protocol was similar
to that used for SA.

(c)    Excretory-
Secretory Antigen (ESA)

            Excretory-Secretory
Antigen (ESA) of Cysticercus cellulosae
was prepared according to the method described by D’souza et al. (1999).

Ten intact cysts were distributed into tissue culture
flask (25 cc vented cap, Himedia) containing  10 ml of RPMI 1640 media (Himedia) with added
antibiotics and antifungals like penicillin 1000 mg/L, gentamicin 2 mg/L,
cefotaxim 20 mg/L and amphotericin B 2mg/L, procured from a local chemist. The
flasks were placed in a CO2 incubator (Applied Biosystems) at 370C in an atmosphere of 1% CO2. The media
were collected after every 24 hours with subsequent replenishment with fresh
media for 7 consecutive days.

            Collected media were centrifuged at
14000 rpm for 30 minutes at 40C in a refrigerated centrifuge and the
supernatant was saturated (90%) with ammonium sulfate at 40C
overnight. The saturated solution was centrifuged at 10000 rpm for 15 minutes
at 40C, supernatant was discarded and the precipitate was dissolved
in cold Phosphate Buffer Solution (pH 7.2-7.4) and dialyzed against Phosphate
Buffer Solution (pH- 7.2-7.4) at 40C. The completion of dialysis was
determined by addition of Nessler’s reagent (1% BaCl2 solution w/v)
to PBS. The dialysis tubing was taken out and placed over PEG 6000 (Himedia) at
4?C to remove extra PBS and subsequent concentration of the antigen. The final
volume was measured, aliquoted and stored at -200C after addition of
1µl/ml of 5 mM PMSF (Sigma Aldrich, USA) to be used as ESA.

Indirect ELISA

The
indirect ELISA was performed as per the method described by Shiguekawa et al. (2000) with some modifications. Briefly, polyvinyl
microtitre plates (Nunc, Denmark) were coated with 100 µl of 0.05 M coating
buffer (pH 9.6) containing antigen in the concentration of 0.25 µg/well (for
SA), 0.125 µg/well (for ESA) and 0.5 µg/well (for MBA) and incubated at 4oC
overnight, washed thrice with PBS-T (PBS containing 0.05 percent Tween-20) followed
by blocking with 5% skimmed milk (Himedia, India) in PBS @ 200 µl/well, incubation
at 4°C overnight and washing. Sera were diluted in PBS (pH 7.2) in 1:200, 100
µl dispensed into the wells and incubated at 37oC for 2 hours and
washed. Anti-human IgG (whole molecule) HRPO conjugate (Sigma Aldrich, USA) diluted
1:10000 in PBS were added @ 100 µl/well and incubated at 37oC for 1
hour followed by washing. The enzymatic reaction was carried out by addition of
100 µl of substrate solution containing 1 mg/ml of O-phenylene-diamine
dihydrochloride (OPD) (Sigma Aldrich, USA) and 0.02% (v/v) H2O2
prepared in 0.1M citrate buffer (pH 5.2) to each well and incubated in dark for
10-15 minutes. The reaction was stopped by adding 2N H2SO4
@ 100µl/well and the absorbance read at 492 nm by ELISA reader (Multiskan Go,
Thermo Fisher, Finland). Optical Density (OD) values of the sample showing
positive/negative ratio of 2 or more was considered standardized for that
particular antigen, serum and conjugate concentration. A positive ELISA result was defined as two standard
deviations above the mean OD value of confirmed known negative sera as
described by Minozzo et al. (2008).

Enzyme-Linked Immunoelectro Transfer
Blot (EITB) Assay

All
the sera samples were screened for identification of immunodominant proteins by
EITB according to Towbin et al.
(1979) with some modifications.

            The
antigens were subjected to SDS-PAGE with 12.5 percent separating
gel with a pre-stained Protein
Molecular Weight Marker (Medium Range) (All Blue) (Merck) comprising of 98, 66,
44, 29 and 14 kDa protein bands was used as standard reference. It was
transferred onto the nitrocellulose membrane (Novex Life Technologies,
Isreal) using a dry transfer
apparatus (i-blot dry blot system, Invitrogen) according to the manufacture’s
protocol. Subsequently, the nitrocellulose strips were blocked in 5% skimmed
milk (Himedia, India) in PBS, incubated for 2 hours at 37oC and
washed with PBS-T. The strips were incubated with sera samples diluted in PBS @
1:100 for 2 hours at 37oC, washed and  incubated with anti-human IgG (whole molecule)
peroxidase conjugate (Sigma Aldrich, USA) diluted in PBS (pH 7.2-7.4) @ 1:10000
for 1 h at 37oC. The strips were developed for 5-7 minutes in substrate
solution containing 30% hydrogen peroxide and 3,3?-diaminobenzidine
tertahydrochloride (Sigma Aldrich, USA) in PBS. The reaction was stopped by
washing thrice with distilled water and positive reaction was determined by
appearance of clearly defined brown colour bands as immunoreactive peptides.

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