l Dilute samples 1:1 distribing 50ul of serum into 50ul of dilluent ,Add 50ul of diluted sample to wells.
l Add 50ul of diluted biotinylated anti- porcine insulin to all wells.
l Cover with a plate vover and incubate for 1 hour at 37℃.
l Remove the cover and wash the plate as follows: ⑴ aspirate the liquid from each well, ⑵ dispensse 0.3ml of washing solution into each well. ⑶ Aspirate again the contet of each well after 0.5 minute. ⑷ Repeat steps ⑵ and ⑶ three times.
l Distribute 80ul of strept***idin-HRP solution to all wells, including blank wells.
l Cover and incubate 30 min at 37℃.
l Remove the cover and empty wells, Wash microwell strips according to step, Proceed immediately to the next step.
l Add 50ul Substrate A and Substrate B to each well。Incubate for 10 min at 37℃。
l The enzyme-substrate reaction is stopped by quickly pipetting 50ul of H2SO4. stop reagent into each well, including the blank wells, to completely and uniformly inactivate the enzyme. Results must be red immediately after the addition of H2SO4.
l Read absorbance of each well on a spectrophotometer using 450nm as the primary w***elength and optionally 620nm (610nm to 650nm is acceptable ) as the reference w***elength.
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