VMC 221: Veterinary Immunology and Serology (1+1)

MATERIALS

• Phosphate buffered saline as in exercise No.10.
• Coating buffer ( Carbonate-bicarbonate buffer)
  • Disodium Carbonate -1.59 g
  • Sodium bi carbonate - 2.93 g
  • Distilled water 1000 ml
  • Adjust the pH to 9.6 an autoclave at 15 lb  for 15 mts
  • Store at 4 ⁰ C
• Blocking buffer
  • BSA - 0.5g
  • PBS - 100 ml
  • Store at 4 ⁰ C
• Washing buffer
• Tween 20  - 5ml 
• Distilled water - 1000ml
• Conjugate buffer
  • BSA - 0.8 g.
  • PBST - 100 ml
• Substrate buffer (pH 4.5 – 5.5)
  • Di sodium hydrogen phosphate - 7.4 g.
  • Citric acid - 5.0 g.
  • Distilled water - 1000 ml
• Stopping reagent (1N HCl)
  • Conc. HCl - 100 ml
  • Distilled water - 900 ml
• Preparation of working conjugate
  • Pre-assessed working dilution of peroxides conjugated anti immunoglobulin antibody is made using conjugate buffer (say 1:2000)
• Preparation of substrate(To be prepared just before use)
  • Substrate buffer - 10 ml
  • H₂O₂ - 5 ml
  • TMB - 100 ml
• Commercially available TMB H₂O₂ substrate diluted before use as per manufacturer’s instruction.
• 96 well microtitre ELISA plate.
• Micro pipette with tips
• ELISA plate washer
• ELISA reader
• Pipette - 1ml
• Measuring cylinder - 1000 ml