PROCEDURE

1. Specimen Collection and Handling: Specimens should be clear, non-hemolyzed and non-lipemic. In the case of cell-culture, remove any particulate material by centrifugation and assay immediately or store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. Where as in the case of patient blood, use a serum separator tube and allow samples to clot for 30 minutes, then centrifuge for 10 minutes at 1000 x g. Remove serum and assay immediately or store samples at ≤ -20° C. Avoid repeated freeze-thaw cycles.

2. Preparation of TNF-α Standard dilution: Dissolve the vial content into 1ml deionized water to yield a stock standard 30ng/ml. Allow the standard to equilibrate for at least 15 minutes before making dilutions. For preparing different concentration of TNF-α solution, initially prepare a 1000 pg/mL standard from the stock standard. Vortex to mix. Dilute this stock into different dilutions as per the calculation given in the Table 29.2. and follow as given in the Figure 35. 5.

Table 35.2: Preparation of the TNF-α dilutions
TNF-α concentration (pg/ml)	TNF-α (µl)	Assay Dilution Buffer (ml)	Total volume
1000	300	300	600
500	300	300	600
250	300	300	600
125	300	300	600
62.5	300	300	600
31.25	300	300	600
15.62	300	300	600

 

Figure 35.5: Procedure to prepare the serial dilution of TNF-α.