Nori Rabbit iNOS ELISA Kit Summary

Alternative names for iNOS: Inducible NOS, nitric oxide synthase 2, NOS2, NOS2A

Standard Curve

Kit Components
1. Pre-coated 96-well Microplate
2. Biotinylated Detection Antibody
3. Streptavidin-HRP Conjugate
4. Lyophilized Standards
5. TMB One-Step Substrate
6. Stop Solution
7. 20 x PBS
8. Assay Buffer

Other Materials Required but not Provided:
1. Microplate Reader capable of measuring absorption at 450 nm
2. Log-log graph paper or computer and software for ELISA data analysis
3. Precision pipettes (1-1000 µl)
4. Multi-channel pipettes (300 µl)
5. Distilled or deionized water

Protocol Outline
1. Prepare all reagents, samples and standards as instructed in the datasheet.
2. Add 100 µl of Standard or samples to each well and incubate 1 h at RT.
3. Add 100 µl of Working Detection Antibody to each well and incubate 1 h at RT.
4. Add 100 µl of Working Streptavidin-HRP to each well and incubate 20 min at RT.
5. Add 100 µl of Substrate to each well and incubate 5-30 min at RT.
6. Add 50 µl of Stop Solution to each well and read at 450 nm immediately.

Background: 

Nitric oxide synthases (NOS) are a family of enzymes catalyzing the production of nitric oxide (NO) from L-arginine. The inducible NOS (iNOS) is encoded by NOS2 gene [1,2] and involved in immune response, binds calmodulin at physiologically relevant concentrations, and produces NO as an immune defense mechanism, as NO is a free radical with an unpaired electron. It is the proximate cause of septic shock and may function in autoimmune disease. As opposed to the critical calcium-dependent regulation of constitutive NOS enzymes (nNOS and eNOS), iNOS has been described as calcium-insensitive, likely due to its tight non-covalent interaction with calmodulin (CaM) and Ca²⁺. While evidence for ‘baseline’ iNOS expression has been elusive, IRF1 and NF-κB-dependent activation of the inducible NOS promoter supports an inflammation mediated stimulation of this transcript. iNOS produces large quantities of NO upon stimulation, such as by proinflammatory cytokines (e.g. Interleukin-1, Tumor necrosis factor alpha and Interferon gamma).^[3] Induction of the high-output iNOS usually occurs in an oxidative environment, and thus high levels of NO have the opportunity to react with superoxide leading to peroxynitrite formation and cell toxicity. These properties may define the roles of iNOS in host immunity, enabling its participation in anti-microbial and anti-tumor activities as part of the oxidative burst of macrophages.^[4] It has been suggested that pathologic generation of nitric oxide through increased iNOS production may decrease tubal ciliary beats and smooth muscle contractions and thus affect embryo transport, which may consequently result in ectopic pregnancy.^[5]

References

1. Stuehr DJ (1999). Biochim. Biophys. Acta. 1411(2–3): 217–30.
2. Knowles RG, Moncada S (1994). Biochem. J. 298(2): 249–58.
3. Green SJ, et al. (1994). Immunol. Lett. 43(1–2): 87–94.
4. Mungrue IN, Husain M, Stewart DJ (2002). Heart Fail Rev. 7(4): 407–22.
5. Al-Azemi M, et al. (2010). Fertil. Steril. 94(3): 833–40.