Preparation house in Nagpur and processed for preparation

Preparation of Cysticercus cellulosae antigensMetacestodesof Taenia solium were collected froma naturally infected pig after slaughter from a local slaughter house in Nagpurand processed for preparation of two somatic (scolex antigen (SA) andmembrane-body antigen (MBA)) and one metabolic (excretory secretory antigen(ESA)) antigens.(a)  ScolexAntigen (SA) Scolexantigen (SA) was prepared according to Dhanalakshmi et al. (2005) with some modifications. Scolices were carefullydissected with fine forceps and scalpel andtransferred immediately into coldPBS (pH 7.

2-7.4) containing antibiotics and PMSF (Sigma Aldrich, USA). Thesewere blotted dry, homogenized followed by sonication at 20kHz, 1 mA for 60 seconds and thencentrifugation at 14000 rpm for 60 minutes at 40C in a refrigeratedcentrifuge (Remi, India). Supernatant was used as scolex antigen (SA).

(b)  Membrane-BodyAntigen (MBA)            Thisantigen was prepared from the Taeniasolium cysts after removing cystic fluid and scolices. The protocol was similarto that used for SA.(c)    Excretory-Secretory Antigen (ESA)            Excretory-SecretoryAntigen (ESA) of Cysticercus cellulosaewas prepared according to the method described by D’souza et al. (1999).Ten intact cysts were distributed into tissue cultureflask (25 cc vented cap, Himedia) containing  10 ml of RPMI 1640 media (Himedia) with addedantibiotics 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. Theflasks were placed in a CO2 incubator (Applied Biosystems) at 370C in an atmosphere of 1% CO2.

The mediawere collected after every 24 hours with subsequent replenishment with freshmedia for 7 consecutive days.            Collected media were centrifuged at14000 rpm for 30 minutes at 40C in a refrigerated centrifuge and thesupernatant was saturated (90%) with ammonium sulfate at 40Covernight. The saturated solution was centrifuged at 10000 rpm for 15 minutesat 40C, supernatant was discarded and the precipitate was dissolvedin cold Phosphate Buffer Solution (pH 7.2-7.

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4) and dialyzed against PhosphateBuffer Solution (pH- 7.2-7.4) at 40C. The completion of dialysis wasdetermined 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) at4?C to remove extra PBS and subsequent concentration of the antigen.

The finalvolume was measured, aliquoted and stored at -200C after addition of1µl/ml of 5 mM PMSF (Sigma Aldrich, USA) to be used as ESA. Indirect ELISATheindirect ELISA was performed as per the method described by Shiguekawa et al. (2000) with some modifications. Briefly, polyvinylmicrotitre plates (Nunc, Denmark) were coated with 100 µl of 0.

05 M coatingbuffer (pH 9.6) containing antigen in the concentration of 0.25 µg/well (forSA), 0.

125 µg/well (for ESA) and 0.5 µg/well (for MBA) and incubated at 4oCovernight, washed thrice with PBS-T (PBS containing 0.05 percent Tween-20) followedby blocking with 5% skimmed milk (Himedia, India) in PBS @ 200 µl/well, incubationat 4°C overnight and washing.

Sera were diluted in PBS (pH 7.2) in 1:200, 100µl dispensed into the wells and incubated at 37oC for 2 hours andwashed. Anti-human IgG (whole molecule) HRPO conjugate (Sigma Aldrich, USA) diluted1:10000 in PBS were added @ 100 µl/well and incubated at 37oC for 1hour followed by washing. The enzymatic reaction was carried out by addition of100 µl of substrate solution containing 1 mg/ml of O-phenylene-diaminedihydrochloride (OPD) (Sigma Aldrich, USA) and 0.02% (v/v) H2O2prepared in 0.1M citrate buffer (pH 5.

2) to each well and incubated in dark for10-15 minutes. The reaction was stopped by adding 2N [email protected] 100µl/well and the absorbance read at 492 nm by ELISA reader (Multiskan Go,Thermo Fisher, Finland). Optical Density (OD) values of the sample showingpositive/negative ratio of 2 or more was considered standardized for thatparticular antigen, serum and conjugate concentration. A positive ELISA result was defined as two standarddeviations above the mean OD value of confirmed known negative sera asdescribed by Minozzo et al.

(2008).Enzyme-Linked Immunoelectro TransferBlot (EITB) AssayAllthe sera samples were screened for identification of immunodominant proteins byEITB according to Towbin et al.(1979) with some modifications.             Theantigens were subjected to SDS-PAGE with 12.

5 percent separatinggel with a pre-stained ProteinMolecular Weight Marker (Medium Range) (All Blue) (Merck) comprising of 98, 66,44, 29 and 14 kDa protein bands was used as standard reference. It wastransferred onto the nitrocellulose membrane (Novex Life Technologies,Isreal) using a dry transferapparatus (i-blot dry blot system, Invitrogen) according to the manufacture’sprotocol. Subsequently, the nitrocellulose strips were blocked in 5% skimmedmilk (Himedia, India) in PBS, incubated for 2 hours at 37oC andwashed 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) @ 1:10000for 1 h at 37oC. The strips were developed for 5-7 minutes in substratesolution containing 30% hydrogen peroxide and 3,3?-diaminobenzidinetertahydrochloride (Sigma Aldrich, USA) in PBS. The reaction was stopped bywashing thrice with distilled water and positive reaction was determined byappearance of clearly defined brown colour bands as immunoreactive peptides.