2. Materials Haffkine Biopharmaceutical Corporation Limited (HBPCL), Maharashtra, India, made available the venoms of Indian cobra (CV), Common krait (KV), Russell’s viper (RV), and Saw scaled viper (EV) and ASVA. The venoms were distributed in small aliquots, lyophilized, and stored at -20o C. Adult male Swiss albino mice (18 - 20 g) and New Zealand white male rabbits (2.0 - 3.0 kg) were maintained at HBPCL, Maharashtra, India and used for experimentation. Complete Freund 's adjuvant (CFA), incomplete Freund 's adjuvant (IFA) was purchased from DIFCO, USA and Bentonite powder was purchased from HiMedia, Maharashtra, India. Rabbit anti horse IgG, goat anti rabbit IgG - horseradish peroxidase (HRP) conjugates, Tetra methyl benzidine/Hydrogen
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F. Gao et al., (2013) with minor modifications in dialysis and concentration of antibodies, were obtained using Amicon® Ultra - 4 10K centrifugal filter devices, checked for functionality and cross reactivity with heterogeneous snake venoms as mentioned in sandwich ELISA protocol and stored at 4oC. 3.11 Synthesis of colloidal gold
Gold nanoparticles about 25 nm were prepared using modified citrate reduction method described by Grabar et al. (1995). Briefly, 200 ml of 0.01 % HAuCl4 solution was heated to boiling followed by rapid addition of 2 ml Sodium citrate (1%) with vigorous stirring. The color of the solution changed to blue and subsequently to cherry red. The boiling was continued for 10 min and stirred for additional 15 min; the solution was cooled to room temperature and stored in a dark glass bottle at 4oC.
3.12 Preparation of Colloidal gold …show more content…
An NC membrane provides sites for immobilization of capturing antibodies. The LFA strips were optimized and prepared as mentioned by Posthuma -Trumpie et al. (2008) with some modifications. Briefly, the SSAbs and anti horse antibodies were immobilized on plastic backed NC membrane (10 µm) at test and control lines respectively, with 1 µl/cm using TLC Spotter (Camag Linomat IV, Camag Berlin, Germany). These membranes were dried at 37 o C for 1 hr, blocked with PBS containing 1% BSA for 30 min, followed by washing two times with PBS and drying at 37 o C for 2 hrs. The conjugate pads (PT-R7) were prepared by immersing into colloidal gold probes solution followed by drying for 2 hr. The LFA strips were assembled in such a manner that absorbent pad and conjugate pads were placed towards the control line end and test line end respectively both overlapping the NC membrane. The sample pad (GFB-R7) was placed overlapping conjugate pad opposite to the NC Membrane and the strips were cut into 3 mm width vertically. These strips were housed in the plastic cassette and stored at RT, protected from sunlight and humidity.
3.14 Experimental envenomation detection
Envenomation was simulated in Swiss albino mice by following the protocol of Brunda et al. (2006) with minor modifications. Briefly, Swiss albino mice (20-22 g) were injected subcutaneously with 2 LD50 of CV and RV diluted separately
F. Gao et al., (2013) with minor modifications in dialysis and concentration of antibodies, were obtained using Amicon® Ultra - 4 10K centrifugal filter devices, checked for functionality and cross reactivity with heterogeneous snake venoms as mentioned in sandwich ELISA protocol and stored at 4oC. 3.11 Synthesis of colloidal gold
Gold nanoparticles about 25 nm were prepared using modified citrate reduction method described by Grabar et al. (1995). Briefly, 200 ml of 0.01 % HAuCl4 solution was heated to boiling followed by rapid addition of 2 ml Sodium citrate (1%) with vigorous stirring. The color of the solution changed to blue and subsequently to cherry red. The boiling was continued for 10 min and stirred for additional 15 min; the solution was cooled to room temperature and stored in a dark glass bottle at 4oC.
3.12 Preparation of Colloidal gold …show more content…
An NC membrane provides sites for immobilization of capturing antibodies. The LFA strips were optimized and prepared as mentioned by Posthuma -Trumpie et al. (2008) with some modifications. Briefly, the SSAbs and anti horse antibodies were immobilized on plastic backed NC membrane (10 µm) at test and control lines respectively, with 1 µl/cm using TLC Spotter (Camag Linomat IV, Camag Berlin, Germany). These membranes were dried at 37 o C for 1 hr, blocked with PBS containing 1% BSA for 30 min, followed by washing two times with PBS and drying at 37 o C for 2 hrs. The conjugate pads (PT-R7) were prepared by immersing into colloidal gold probes solution followed by drying for 2 hr. The LFA strips were assembled in such a manner that absorbent pad and conjugate pads were placed towards the control line end and test line end respectively both overlapping the NC membrane. The sample pad (GFB-R7) was placed overlapping conjugate pad opposite to the NC Membrane and the strips were cut into 3 mm width vertically. These strips were housed in the plastic cassette and stored at RT, protected from sunlight and humidity.
3.14 Experimental envenomation detection
Envenomation was simulated in Swiss albino mice by following the protocol of Brunda et al. (2006) with minor modifications. Briefly, Swiss albino mice (20-22 g) were injected subcutaneously with 2 LD50 of CV and RV diluted separately