Detailed Protocol for BET

Test serial no.Assay tube (in duplicate)LAL reagent water (μL)Control standard endotoxin 4λ (μL)Test sample* (μL)LAL reagent (μL)
1Negative water control100100
2Positive water control 2λ5050100
4PPC 2λ5050100
5PPC 2λ with calcium chloride5050100
  • * Repeated at MVD and MVD/2—that is, 1:66 and 1:33 dilutions.

  • These tubes were set up for all 99mTc radiopharmaceuticals (99mTc-DTPA, 99mTc-DMSA-III, 99mTc-DMSA-V, 99mTc-ECD, 99mTc-mebrofenin, 99mTc-methoxyisobutylisonitrile, and 99mTc-MDP) and their cold kits.

  • Calcium chloride at final concentration of 300 μM was incorporated in assay tube of test sample at final dilution.

  • Tests 3 and 4 were also performed initially for 60 mM stock solution of calcium chloride and saline (0.9% sodium chloride) to ensure absence of endotoxin in stock solutions.