Serum, in a biological context, refers to the clear, pale-yellow liquid portion of blood that remains after the blood has clotted and all blood cells and clotting factors have been removed. It contains proteins, electrolytes, antibodies, antigens, and hormones, among other substances. Heat inactivation is a common laboratory procedure involving the heating of serum to a specific temperature for a set duration to reduce the activity of certain components. This process prepares the serum for various downstream applications in research and diagnostics.
Why Serum Undergoes Heat Inactivation
The primary reason for heat inactivating serum is to neutralize the complement system. This system, a group of around 20 proteins in serum, is part of the innate immune system. These proteins can initiate reactions leading to cell lysis or interfering with laboratory assays. Inactivating this system prevents unintended cell destruction in cell culture or non-specific reactions in immunological tests.
The complement system can cause issues in sensitive cell lines, potentially leading to cell damage or death. For example, if antibodies in the serum bind to cultured cells, active complement could be triggered, resulting in cell lysis. Heat inactivation can also reduce the activity of certain heat-labile enzymes that might interfere with experimental results.
The Process of Heat Inactivation
The practical steps for heat inactivating serum involve precise temperature control and timing. Typically, serum is heated to 56°C for a duration of 30 minutes. This specific temperature and time are chosen to effectively inactivate the complement system while minimizing damage to other beneficial serum components, such as growth factors and vitamins.
To perform heat inactivation, a circulating water bath or a heat block is commonly used to ensure even heat distribution. It is important to pre-warm the water bath to the target temperature of 56°C before placing the serum bottles inside. The serum bottles should be immersed in the water bath up to the level of the serum, ensuring the serum itself reaches the target temperature. Gentle agitation, such as swirling the bottles every 5 to 10 minutes, is recommended throughout the process to prevent proteins from coagulating at the bottom and to promote uniform heating.
After the 30-minute heating period at 56°C, the serum bottles should be removed immediately from the water bath. Rapid cooling, often in an ice bath, is then applied to prevent prolonged exposure to elevated temperatures, which could further degrade serum components. Proper technique is important because excessive heating or prolonged duration can lead to increased formation of precipitates, gelling of serum proteins, and a reduction in the serum’s growth-promoting properties.
Common Uses of Heat Inactivated Serum
Heat-inactivated serum finds its primary application in cell culture and immunological assays. It is used in cell culture for sensitive cell lines, where active complement could lead to cell damage or inhibit growth. For instance, embryonic stem cells, insect cells, and smooth muscle cells may benefit from heat-inactivated serum due to their sensitivity to complement activity.
In immunological assays and diagnostic tests, heat-inactivated serum is preferred to eliminate interference from the complement system. Complement proteins can bind to antibody-antigen complexes, which might skew results in assays designed to detect specific immune responses or target molecules.