Isopropylalcohol (1µl of stock solution was mixed per ml of antigen to get the required concentration of 20µg/ml)

Transcription

1 APPENDIX

2 APPENDIX Chromic acid solution Potassium dichromate 500g Sulphuric acid 500ml Double distilled water (DDW) 5 litre Phenyl Methyl Sulfonyl Fluoride (PMSF) Stock solution PMSF 10mg Isopropylalcohol 1 ml (1µl of stock solution was mixed per ml of antigen to get the required concentration of 20µg/ml) Solutions for Sodium do-decyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) Solution A (30% Acrylamide solution) Acrylamide 29.2g (29g) Bisacrylamide 0.8g (1.0g) Distilled water 100 ml Solution B (1.5Mm, Tris HCl buffer, ph 8.8) Tris 18.2g SDS 0.4g HCl 2.0 ml Distilled water 100 ml Solution C (0.5M Tris HCl, ph 6.8) Tris 6.1g SDS 0.4g HCl 4.2 ml Distilled water 100 ml Solution D (10% Ammonium per sulphate) 0.1mg in 1ml distil water (prepare fresh before use) 10% Separating gel Solution A 6.0 ml Solution B 4.5 ml Solution D 0.08 ml

3 TEMED DDW 0.01 ml 7.5 ml Stacking gel (4.5%) Solution A 0.9 ml Solution C 1.5 ml Solution D 0.02 ml TEMED 0.01 ml (N,N, N, N-tetramethylene diamine) DDW 3.6 ml Running buffer (500 ml) Tris 1.5g (25mM) Glycine 7.2g (192mM) SDS 0.5g (0.1%) Make upto 500ml with water. Do not adjust ph with HCl. Staining solution Coomassie Brilliant Blue 0.3g Methanol 40 ml Trichloro acetic acid 20g DW 60 ml Destaining solution Methanol Glacial acetic acid DDW Sample buffer (5x) SDS 2-Mercaptoethanol Glycerol Solution C DDW upto 30.0ml 10.0ml 60.0ml 0.25 g 0.25ml 5.0 ml 2.5 ml 10.0ml Tracking dye Bromophenol blue 0.01g Glycerine 1.0ml DDW 9.0ml Preparation of protein marker

4 Protein marker 10µl Sample buffer 30µl Tracking dye 10µl Standard Protein marker Lysozyme 14.3 kda Soyabean trypsin inhibitor 20.1 kda Carbonic anhydrase 29.0 kda Ovalbumin 43.0 kda Bovine serum albumin 66.0 kda Phosphorylase b 97.4 kda Solutions for Enzyme Linked Immuno Sorbent Assay(ELISA) and Dot- Enzyme Linked Immuno Sorbent Assay(Dot-ELISA) Phosphate buffered saline (PBS ph 7.2) Sodium chloride 8.0g Disodium hydrogen phospate 1.15g Potassium dihydrogen phospate 0.20g Potassium chloride 0.20g DW 1000ml Store at 4 0 C but for not more than two weeks Serum coating buffer Anhydrous sodium carbonate 1.59g Sodium hydrogen carbonate 2.93g Sodium nitrate 0.2g DW 100ml Antigen Coating buffer Solution A (0.2M solution of anhydrous sodium carbonate) Anhydrous sodium carbonate 2.12g DDW 100 ml

5 Solution B (0.2M solution of Sodium hydrogen carbonate) Sodium hydrogen cabonate 1.68g DDW 100ml Working strength (ph 9.6) Stock solution A Stock solution B DDW 1.6ml 3.4ml 20.0ml Solutions and reagents for Enzyme immuno transfer blot (EITB) and Dot- ELISA Washing buffer (0.05% Tween-20, 0.1% Triton-x100 in PBS v/v) Polysorbate-20 (T-20) 50µl Triton-x µl 0.15(M) PBS 100µl Blocking buffer (5% skimmed milk poweder w/v) Skimmed milk powder 5.0g Washing buffer 100ml Transfer buffer for EITB Tris (Hydroxymethyl) aminomethane 5.8g Glycine 2.9g Methanol 0.37g DDW upto 1000ml Substrate chromogen for ELISA Ortho phenylene diamine (OPD) 5.0mg Hydrogen peroxide(30% v/v) 0.05% DDW 12.5ml Substrate chromogen for Dot-ELISA Solution A Acetic acid DDW 6.0ml 500ml Solution B Sodium acetate 8.2g DDW 500ml Acetate buffer (ph 5.0) Solution A 14.8ml Solution B 35.2ml

6 Substrate chromogen buffer (4x) Acetate buffer EDTA Triton-x100 Store at 4 0 C 50.0ml 74.4mg 0.4ml Substrate chromogen working solution Substrate chromogen buffer (1x) 25.0ml Ortho diamine dihydrochloride (ODD) 5.0mg Hydrogen Peroxide (30% v/v) 16.0µl {substrate chromogen(1x) prepared after mixing 10ml of substrate-chromogen buffer (4x) in 30ml DDW} Stopping solution (2M H2S04) H2SO4 DDW 10.8 ml 9.2 ml Solutions for extraction of DNA for Copro PCR Proteinase K stock solution Proteinase K DDW 20.0mg 1.0 ml 1M Tris-HCl stock solution (ph 8.0) Tris (Hydroxymethyl)aminomethane 12.11g DDW 80.0ml (The ph is adjusted to 8.0 using concentrate HCl, DDW is added to make the volume to 100ml. The solution is sterilized by autoclaving) 0.2M EDTA stock solution (ph 8.0) EDTA 7.44g DDW 80.0ml ph adjusted with 10N NaoH. Make up the volume to 100ml. Sterilized by autoclaving. Tris-HCl saturated Phenol (Bangalore Genei) Phenol:Chloroform (1:1)

7 80% alcohol (v/v) Distil alcohol DDW 80.0ml 20.0ml TE Buffer 1M Tris HCl 250µl 0.2M EDTA 1250µl DDW 24.62µl TBE (10x buffer) Tris (Hydroxymethyl) aminomethane 108.0g Boric acid 55.0g 0.5M EDTA (ph 8.0) 40.0ml DDW to final volume 1000ml Agarose for DNA electrophoresis 0.8% Agarose gel 120mg 0.5x TBE 15ml (10x TBE is diluted to twenty times) 1.2% Agarose gel 0.48g 0.5x TBE 40ml (10x TBE is diluted to twenty times) Gel loading dye (6x) Bromophenol blue Xylene Cyanol Sucrose DDW 50mg 50mg 8mg 20ml Ethidium Bromide Ethidium bromide 100mg

8 DDW 10ml Taq Polymerase 20mM Tris HCl (ph 8.0) 100 mm KCl 0.1mM EDTA 1mM DTT 0.5% T % Igepal 50% Glycerol 10x PCR Buffer 100mM Tris (ph 9.0) 500mM Potassoum chloride 15 mm Magnesium chloride 0.1% Gelatin

9 Figure Title Standardization of anti-dog conjugate Standardisation of somatic and E/S antigen Standardization of different dilutions of serum with E/S antigen of E.granulosus Standardization of different dilutions of serum with somatic antigen of E.granulosus OD values of known negative serum samples to determine the cut off value OD Values of positive and negative serum samples with E/S antigen of E.granulosus by ELISA OD Values of positive and negative serum samples with somatic antigen of E.granulosus by ELISA Rf values of standard protein curve to estimate the molecular weights of polypeptides Sensitivity and Specificity of CIEP, Indirect ELISA and EITB with E/S antigen of E.granulosus Sensitivity and Specificity of CIEP, Indirect ELISA and EITB with somatic antigen of E.granulosus Sensitivity and Specificity of LAT, Dot-ELISA and EITB with F/S antigen of E.granulosus

10 OD values :1000 1:2500 1:5000 1:7500 1:100001: :150001:17500 Different dilutions of Conjugate Fig. 1 Standardization of anti-dog conjugate by checker board titration method

11 OD values E/S Ag Somatic Ag Different dilutions of antigen Fig. 2 Standardisation of somatic and E/S antigen of E.granulosus by checker board titration method

12 OD values :50 1:100 1:200 1:300 1:400 1:500 1:600 1:700 Serum Dilutions Fig. 3 Standardization of different dilutions of serum by checker board titration method with E/S antigen of E.granulosus

13 OD values :50 1:100 1:200 1:300 1:400 1:500 1:600 1:700 Serum dilutions Fig. 4 Standardization of different dilutions of serum by checker board titration method with somatic antigen of E.granulosus

14 OD values E/S Ag Somatic Ag No. of known negative serum samples Fig. 5 OD values of Known negative serum samples to determine the cut off value

15 OD values Negative Serium Positive Serium No. of serum samples Fig. 6 OD Values of positive and negative serum samples with E/S antigen of E.granulosus by ELISA

16 OD values No. of serum samples Positive Serum Negative Serum Fig. 7 OD values of positive and negative serum Samples with somatic antigen of E.granulosus by ELISA

17 Rf values Molecular weights (kda) Fig. 8 Rf Values of standard protein curve to estimate the molecular weights of polypeptides

18 Percentage Sensitivity (%) Specificity (%) 20 0 CIEP Indirect ELISA EITB Fig. 9 Sensitivity and Specificity of CIEP, Indirect ELISA and EITB with E/S antigen of E.granulosus

19 Pe rce ntage Sensitivity (%) Specificity (%) 20 0 CIEP Indirect ELISA EITB Fig. 10 Sensitivity and Specificity of CIEP, Indirect ELISA and EITB with somatic antigen of E.granulosus

20 120 Pe rce ntage Sensitivity (%) Specificity (%) 20 0 LAT Dot-ELISA EITB Fig. 11 Sensitivity and Specificity of LAT, Dot- ELISA and EITB with F/S antigen of E.granulosus

21 Plate Title 1. E.granulosus adult worm embedded in the intestinal mucosa of dog 2. Cultivation E.granulosus adult worms in the medium-199 to prepare E/S antigen 3. E.granulosus adult worms recovered from intestine of dog in HBSS to prepare somatic antigen 4. E.granulosus adult worms 5. Anterior end of E.granulosus (40X) 6. D.caninum adult worms recovered from dog intestine 7. Eggs of E.granulosus (10x) 8. Eggs of E.granulosus in the gravid segment 9. T.hydatigena adult worm recovered from dog intestine 10. Egg of Taenia hydatigena (10X) 11. Counter Immunoelectrophoresis (CIEP) with E/S antigen of E.granulosus 12. Counter Immunoelectrophoresis (CIEP) with somatic antigen of E.granulosus 13. Indirect ELISA showing positive reaction for E.granulosus with somatic antigen 14. ELISA plate showing positive reaction for E.granulosus with E/S antigen 15. Protein profile of somatic antigen of E.granulosus by SDS-PAGE 16. Protein profile of E/S antigen of E.granulosus by SDS-PAGE 17. Protein profile of somatic antigen of T.hydatigena by SDS- PAGE 18. Protein profile of somatic antigen of D.caninum by SDS- PAGE 19. Protein profile of F/S antigen of E.granulosus by SDS- PAGE 20. Antibody titre of known positive sera of E/S antigen of E.granulosus by Dot- ELISA 21. Antibody titre of known positive sera of somatic antigen of E.granulosus by Dot- ELISA 22. Antibody titre of known positive sera of F/S antigen of E.granulosus by Dot- ELISA 23. Immunoreactive peptides of E/S antigen of E.granulosus with probed against known positive sera by EITB 24. Immunoreactive peptides of somatic antigen of E.granulosus with probed against known positive sera by EITB Immunoreactive peptides of F/S antigen of E.granulosus with probed 25. against known positive sera by EITB 26. Known positive sera of somatic antigen of E.granulosus showing immunoreactive peptides by EITB 27. Known positive sera of somatic antigen of E.granulosus showing immunoreactive peptides by EITB 28. Known positive sera of F/S antigen of E.granulosus showing

22 immunoreactive peptides by EITB 29. Peptides identified in known positive sera of E.granulosus with somatic antigen of T.hydatigena by EITB 30. Peptides identified in known positive sera of E.granulosus with somatic antigen of D.caninum by EITB 31. EITB with known negative sera 32. Dot- ELISA showing positive reaction for E.granulosus with F/S antigen 33. Latex agglutination test showing positive reaction with F/S antigen of E.granulosus 34. Copro-PCR with Primer Copro-PCR with Primer-2

23 Plate 1. E.granulosus adult worm embedded in the intestinal mucosa of dog Plate 2. Cultivation E.granulosus adult worms in the medium-199 to prepare E/S antigen

24 Plate 3. E.granulosus adult worms recovered from intestine of dog in HBSS to prepare somatic antigen Plate 4. E.granulosus adult worms

25 Plate 5. Anterior end of E.granulosus (40X) Plate 6. D.Caninum adult worms recovered from dog intestine

26 Plate 7. Eggs of E.granulosus (10x) Plate 8. Eggs of E.granulosus in the gravid segment

27 Plate 9. T.hydatigena adult worm recovered from dog intestine Plate 10. Egg of Taenia hydatigena (10X)

28 Plate 11. Counter Immunoelectrophoresis (CIEP) of somatic and E/S antigen of E.granulosus with positive serum Plate 12. Counter Immunoelectrophoresis (CIEP) of E/S and somatic antigen of E.granulosus with hyper immune serum

29 Plate 13. Indirect ELISA showing positive reaction for E.granulosus with somatic antigen (PC- positive control; NC- negative control) Plate 14. ELISA plate showing positive reaction for E.granulosus with E/S antigen (PC- positive control; NC- negative control)

30 Plate 15. Protein profile of somatic antigen of E.granulosus by SDS-PAGE. M: Marker, Lanes: 1 to 8-Partially purified somatic antigen

31 Plate 16. Protein profile of E/S antigen of E.granulosus by SDS-PAGE M-Marker, Lane- 1-7 E/S antigen of E.granulosus

32 Plate 17. Protein profile of somatic antigen of T.hydatigena by SDS-PAGE M-Marker, Lane- 1-6 somatic antigen of T.hydatigena

33 Plate 18. Protein profile of somatic antigen of D.caninum by SDS-PAGE M-Marker, Lane- 1-6 somatic antigen of D.caninum

34 Plate 19. Protein profile of F/S antigen of E.granulosus by SDS-PAGE M-Marker, Lane- 1-4 F/S antigen of E.granulosus

35 Plate 20. Antibody titre of known positive sera of E/S antigen of E.granulosus by Dot-ELISA Horizontal rows : 1 & 2 Known positive sera 3 & 4 Known negative sera 5 Conjugate control 6 Normal sera

36 Plate 21. Antibody titre of known positive sera of somatic antigen of E.granulosus by Dot-ELISA Horizontal rows : 1 & 2 Known positive sera 3 & 4 Known negative sera 5 Conjugate control 6 Normal sera

37 Plate 22. Antibody titre of known positive sera of F/S antigen of E.granulosus by Dot-ELISA Horizontal rows : 1 & 2 Known positive sera 3 & 4 Known negative sera 5 Conjugate control 6 Normal sera

38 Plate 23. Immunoreactive peptides of E/S antigen of E.granulosus probed against known positive sera by EITB

39 Plate 24. Immuno reactive peptides of somatic antigen of E.granulous probed against known positive sera by EITB

40 Plate 25. Immuno reactive peptides of F/S antigen of E.granulosus probed against known positive serum

41 Plate 26. Known positive sera of somatic antigen of E.granulosus showing immunoreactive peptides by EITB

42 Plate 26. Known positive sera of somatic antigen of E.granulosus showing immunoreactive peptides

43 Plate 28. Known positive sera of F/S antigen of E.granulosus showing immunoreactive peptides

44 Plate 28. Known positive sera of F/S of antigen E. granulosus showing immunoreactive peptides.

45 Plate 27. Known positive sera of E/S antigen E.granulosus showing immunoreactive peptides.

46 Plate 31. EITB with known negative sera.

47 Plate 34. Copro-PCR with primers JB 3f and JB 4.5r M DNA Marker 1 & 2 DNA from E.granulosus eggs 3 & 4 DNA from T.hydatigena eggs

48 Plate 35. Copro-PCR with primers Eg 1f and Eg 1r M DNA Marker 1 to 3 DNA from E.granulosus eggs 4 DNA from T.hydatigena eggs

49 Plate 29. Peptides identified in known positive sera of E.granulosus with somatic antigen of T.hydatigena by EITB

50 Plate 30. Peptides identified in known positive sera of E.granulosus with somatic antigen of D.caninum by EITB

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