Serum and plasma
11.3 Serum and plasma
Serum is to be preferred to plasma for any immunoassay due to the tendency of the clotting factors in plasma to form spontaneous clots and so mimic or mask antigen–antibody reactions.
11.3.1 Collection of serum
MATERIALS AND EQUIPMENT Blood without anticoagulant Glass containers (test tubes or conical flasks) Low-speed centrifuge
METHOD
1 Collect the blood into a glass container and allow it to clot at room temperature for 1 h.
2 Once the clot has formed, loosen it from the walls of the container to aid retraction.
3 Transfer to 4°C and leave overnight if necessary.
4 Collect the expressed serum and centrifuge at 150 g for 5 min (to sediment the erythrocytes) and then at 350 g for 15 min.
5 Transfer the serum (straw-coloured supernatant) to containers suitable for long-term storage and heat at 56°C for 30 min to destroy the heat-labile components of complement.
TECHNICAL NOTES • Blood clots better in glass than in plastic containers.
• Although heating to destroy the complement components is largely of historical interest, it is still good practice as the unrecognized activation of complement in an antiserum can have far-reaching consequences in some immunoassays.
• Serum may be frozen at –20°C for long-term storage but repeated freezing and thawing should
be avoided. Protein denaturation at room temperature is minimal if serum is sterilized by filtration (0.22-µm pore size). For filter sterilization of volumes of serum greater than 20 ml use a combination of filters; 0.45-µm prefilter pad, 0.22-µm filter. A single 0.22-µm filter will block very rapidly. Alternatively, storage at 4°C is possible after the addition of merthiolate (0.01% w/v, final concentration) as a preservative. Preservation of non-sterile serum for
11.3SERUM AND PLASMA
11.3.2 Serum preparation from plasma
Pooled plasma is frequently the only source of human serum for use as a tissue culture supple- ment or for the isolation of plasma proteins.
The action of the anticoagulant used to prevent clotting can be successfully reversed, but some plasma proteins are degraded during the preparation.
Citrate-dextrose anticoagulant
MATERIALS Plasma with citrate anticoagulant Thrombin solution
1 M calcium chloride solution
METHOD
1 Prepare thrombin solution for use by diluting to 100 IU/ml in calcium chloride solution.
2 Warm plasma to 37°C, add 1/100 volume of thrombin solution and mix vigorously to promote clot formation over 5–10 min.
3 Leave at room temperature for 60 min and collect supernatant.
4 Centrifuge at 20 000 g for 20 min at 4°C and collect the supernatant.
5 Filter sterilize (0.22 µm), if required, and heat inactivate at 56°C for 45 min.
6 Store at –20°C until used. TECHNICAL NOTE
For filter sterilization of volumes of serum greater than 20 ml use a combination of filters; 0.45-µm prefilter pad, 0.22-µm filter. A single 0.22-µm filter will block very rapidly.
Heparin anticoagulant
MATERIALS Plasma with heparin anticoagulant Protamine sulphate Thrombin solution
1 M calcium chloride solution
METHOD
1 Prepare thrombin solution for use by diluting to 100 IU/ml in calcium chloride solution and adding 5 mg/ml protamine sulphate.
2 Warm plasma to 37°C, add 1/100 volume of thrombin solution and mix vigorously to promote clot formation over 5–10 min.
3 Leave at room temperature for 60 min and collect supernatant.
4 Centrifuge at 20 000 g for 20 min at 4°C and collect the supernatant.
5 Filter sterilize (0.22 µm), if required, and heat inactivate at 56°C for 45 min.
6 Store at –20°C until used.
C H A P T E R 1 1: Immunological manipulations in vivo
TECHNICAL NOTE Additional protamine sulphate may be added if a clot does not form.