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14 wells coated with 100 L, of standard and 50 L standard dilution, the blank wells separately considered as testing wells for supernatant in duplicate, 40 L sample dilution were added in the wells, the testing sample were added 10 L in every well the plat closed by closure plate membrane and incubated for 30 minutes in 37C after that wash solution diluted 20-fold with distilled water, the plat uncovered the liquid discarded and washing buffer added in each well, after washing the 50 L HRP conjugate regent were added in each well except blanks, the incubation and washing treated again respectively