III. Amplified ELISA Procedure for Detection of Botulinal Toxins A, B, E, and F from Culture.

A. Equipment and Materials

1. Microplate, Dynex Immulon ll U-bottom, cat. No. 3655
2. Microtiter pipettors to deliver from 0.1- 2.0, 2-20, and 50-200 µl.
3. Multichannel pipettor, 8 or 12 place 50-200 µl
4. Pipets, disposable 1,5,10 ml
5. Glass test tubes 13X100 mm, 15X150 mm
6. Incubator, 35°C
7. Refrigerated centrifuge
8. Microplate washer
9. Microplate shaker
10. Microplate reader (read 490 and 630 nm reference)
11. Microtiter plate seals
12. Multichannel pipet reservoirs

B. Media and Reagents

1. Tryptone-peptone-glucose-yeast extract broth (TPGY).
2. Cooked meat medium (CMM).
3. 0.05M bicarbonate buffer: 0.8g Na2CO3 + 1.47g NaHCO3 in 500 ml distilled H2O, pH 9.6.
4. 1% Casein buffer: Add 10.0g vitamin-free casein + 7.65 g NaCl, 0.724g Na2HPO4 (anhydrous), 0.21g KH2PO4 to 900 ml H2O, and 3 ml of 1 M NaOH. Heat with stirring to ~ 80°C to dissolve casein. Check pH and adjust to 7.9 with 1 M NaOH, q.s. to 1 liter. Sterilize at 121°C for 20 min. Final pH is ~7.4-7.6.
5. Goat type A or E, rabbit type B, or horse F antitoxin.
6. Goat type A, B, E, or F biotinylated antitoxin
7. Tris buffered NaCl-0.005% Tween 20 (TBST): 6.04g Tris base, 8.76g NaCl, Distilled H2O 900 ml, dissolve Tris and NaCl, pH adjust to 7.5 at 25°C with 2 M HCl, add 50 µl of Tween-20 and q.s. to 1 liter.
8. Extravidin-alkaline phosphatase conjugate (Sigma)
9. Amplified ELISA substrate system (GibCo)
10. 0.3 M H2SO4: dilute concentrated acid (MW 98, specific gravity 1.84, purity 96-98%) by adding 1 ml to 59 ml of distilled H2O.
11. Botulinal complex toxin standards A, B, E, and F. (Metabiologics Inc., Madison, WI)

IV. Detection of Type A, B, E, and F Clostridium botulinum Toxins Using Digoxigenin-labeled IgGs and the ELISA (DIG-ELISA).

A. Equipment and Materials

1. Microplate, Dynex Immulon ll U-bottom, cat. No. 3655
2. Microtiter pipettors to deliver from 0.1- 2.0, 2-20, and 50-200 µl.
3. Multichannel pipettor, 8 or 12 place 50-200 µl
4. Pipets, disposable 1,5,10 ml
5. Glass test tubes 13X100 mm, 15X150 mm
6. Incubator, 35°C
7. Refrigerated centrifuge
8. Microplate washer
9. Microplate shaker
10. Microplate reader (read 450 nm)
11. Microtiter plate seals
12. Multichannel pipet reservoirs

B. Media and Reagents

1. Tryptone-peptone-glucose-yeast extract broth (TPGY).
2. Cooked meat medium (CMM).
3. 0.05M bicarbonate buffer: 0.8g Na2CO3 + 1.47g NaHCO3 in 500 ml distilled H2O, pH 9.6.
   • Capsules to prepare 100 ml volume are available from Sigma.
4. 1% Casein buffer:
   • Add 10.0g vitamin-free casein (Research Organics) + 7.65g NaCl, 0.724g Na2HPO4 (anhydrous), 0.21g KH2PO4 to 900 ml H2O, and 3 ml of 1 M NaOH.
   • Heat with stirring to ~ 80°C to dissolve casein.
   • Check pH and adjust to 7.9 with 1 M NaOH, q.s. to 1 liter.
   • Sterilize at 121°C for 20 min.
   • Final pH is ~7.4-7.6.
   • Casein blocker ready to use product is available from Pierce that gives slightly lower absorbance values than in-house prepared casein buffer. (SRL, Atlanta, GA).
5. Goat type A, B, E, or F digoxigenin-labeled antitoxin (SRL, Atlanta, GA).
6. Phosphate buffered saline with 0.005% Tween 20 wash buffer (PBST).
   • 1.2 g Na2HPO4 (anhydrous), 0.22g NaH2PO4.H20, 8.5g NaCl per liter distilled H2O.
   • Adjust pH to 7.5 Add 50 µl of Tween 20/L PBS. Sterilize at 121°C for 20 min.
   • 10 × PBST: 12.0g Na2HPO4 (anhydrous), 2.2g NaH2PO4.H20, 85.0g NaCl per liter distilled H2O.
   • Adjust pH to 7.5 Add 500 µl of Tween 20/L PBS. 1 × PBST is then prepared by adding 100 ml of 10X PBST to 900 ml of distilled H2O and mixing before use. : 10X PBS is available commercially from GibCo.
7. Anti-digoxigenin HRP poly conjugate (Roche Applied Science).
8. Tetra methyl benzidine (Ultra-TMB) (Pierce).
9. 0.5 M H2SO4.
10. Botulinal complex toxin standards A, B, E, and F. (Metabiologics Inc., Madison, WI)

V. Specific Detection of Clostridium botulinum Types A, B, E, and F Using the Polymerase Chain Reaction (PCR).

A. Equipment and Materials

1. Programmable automatic thermocycler
2. Horizontal gel electrophoresis apparatus
3. Electrophoresis constant-voltage power supply
4. Heating plate
5. Incubators, 35°C
6. Water bath, 37°C and 60°C
7. Freezer, -20 and -70°C
8. Speed Vacuum, optional
9. Microwave
10. Sterile disposable inoculating loops
11. Microcentrifuge tubes, 1.5 and Thin Walled PCR reaction tubes, 0.2 ml or 0.5 ml
12. Variable digital micropipettors (e.g., 0.5-20 µl, 20-200 µl, 100-1,000µl)
13. Aerosol-resistant pipet tips
14. Microcentrifuge
15. UV transilluminator
16. Polaroid camera and Polaroid film 3000 ISO or comparable Gel Documentation System

B. Media and Reagents

Molecular biology grade reagents are recommended and are available from various manufacturers.

1. Tryptone-peptone glucose yeast extract broth (TPGY).
2. Phosphate-buffered saline, pH 7.4 (PBS)
3. Tris EDTA, pH 8.0 (1X TE). 10mM Tris-HCL, 1mM EDTA, pH 8.0 in distilled water
4. Proteinase K- 10 mg Proteinase K/ml 1× TE
5. Lysozyme-10 mg Lysozyme/ml 1 × TE
6. 3 M Sodium Acetate, pH 5.2
7. 95% ethanol
8. 2'-Deoxynucleoside-5'-triphosphates (dATP, dCTP, dGTP, dTTP); stock solution 2.5 mM of each dNTP
9. Taq DNA polymerase (available from various vendors) or Amplitaq® (PerkinElmer)
10. 10 × Reaction Buffer B-500mM KCl, 100 mM Tris-HCl (pH 9.0 at 25°C), 1.0 % Triton X-100
11. 15 mM MgCl2
12. Clostridium botulinum neurotoxin oligonucleotide primers types A, B, E, and F, 10 µM stock solutions (2).
13. Light mineral oil, optional
14. Sterile deionized water, RNase and DNase free
15. 10× TBE (0.9 M Tris-borate, 0.02 M EDTA, pH 8.3)
16. Agarose (nucleic acid electrophoresis grade)
17. Ethidium bromide solution, 10 mg/ml
18. 6× sample loading buffer
19. DNA molecular weight markers (e.g., 123 bp ladder or 100 bp ladder)