UB201L Immunology

Ankur Bhaumik
18895
B1

Lab Report
Quantification of Nitrite using Griess Assay

A) Preparation of nitrite standard reference curve.

12.5 × 5
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑛𝑖𝑡𝑟𝑖𝑡𝑒 𝑖𝑛 1st 𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑠𝑎𝑚𝑝𝑙𝑒 = 𝑚𝑀 = 625 𝜇𝑀
100
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑒𝑎𝑐ℎ 𝑠𝑢𝑐𝑐𝑒𝑒𝑑𝑖𝑛𝑔 𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑖𝑙𝑙 𝑏𝑒 ℎ𝑎𝑙𝑓 𝑜𝑓 𝑡ℎ𝑒 𝑝𝑟𝑒𝑣𝑖𝑜𝑢𝑠 𝑠𝑎𝑚𝑝𝑙𝑒.

Data for preparation of Standard Reference Curve

Concentration of Nitrite OD at 550 nm Adjusted OD (OD-ODblank) at 550 nm

625 3.416 3.3667
312.5 1.8419 1.7926
156.25 1.0092 0.9599
78.125 0.5289 0.4796
39.0625 0.2901 0.2408
19.53125 0.1654 0.1161
9.765625 0.1052 0.0559
4.8828125 0.0759 0.0266
Blank 0.0493 0

• Absorbance/ OD is a unitless quantity

 Reference Curve

𝑆𝑜, 𝑡ℎ𝑒 𝑒𝑞𝑢𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑡ℎ𝑒 𝑙𝑖𝑛𝑒 𝑜𝑏𝑡𝑎𝑖𝑛𝑒𝑑 𝑖𝑠

𝐴𝑑𝑗𝑢𝑠𝑡𝑒𝑑𝑂𝐷 = 0.00541 × 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛(𝜇𝑀) + 0.03801

B) Measurements from Experimental Sample

𝑇ℎ𝑒 𝑂𝐷 𝑜𝑓 𝑡ℎ𝑒 𝑒𝑥𝑝𝑒𝑟𝑖𝑚𝑒𝑛𝑡𝑎𝑙 𝑠𝑎𝑚𝑝𝑙𝑒 𝑖𝑠 1.8514

So, its adjusted OD = 1.8514-0.0493 = 1.8021

C) Determination of nitrite concentration in experimental sample

𝑈𝑠𝑖𝑛𝑔 𝑡ℎ𝑒 𝑒𝑞𝑢𝑎𝑡𝑖𝑜𝑛 𝑚𝑒𝑛𝑡𝑖𝑜𝑛𝑒𝑑 𝑎𝑏𝑜𝑏𝑒 𝑤𝑒 ℎ𝑎𝑣𝑒

1.8021 = 0.00541 × 𝑈𝑛𝑘𝑛𝑜𝑤𝑛𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛(𝜇𝑀) + 0.03801

𝑆𝑜, 𝑢𝑛𝑘𝑛𝑜𝑤𝑛 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑎𝑟𝑖𝑜𝑛 𝑖𝑠 326 𝜇𝑀
D) Inference
• The concentration of nitrite obtained by spontaneous oxidation of NO
produced by macrophages in response to LPS in the unknown sample
was determined to be 𝟑𝟐𝟔 𝝁𝑴.

E) References
• Kuby- Immunology 6th Edition
• Wikipedia