Medical Microbiology - Virology Serology Laboratory Techniques

Low Wei Qi Kenneth TG02


Hello everyone,

this time im going to share another serology technique in detecting viruses total antibody for Influenza A, Influenza B, Herpes Simplex and many others. The test is known as Complement Fixation Test ( CFT). This test uses the properties of complement to lyse cells in the presence of antigen-antibody complexes.

CFT is used to detect the presence of specific antibody in the patient's serum. this test utilizes sheep red blood cells (sRBCS), anti-sRBCS antibody, complement and specific Ag. If Ab is present in the serum, the complement will be completely utilized and sRBC lysis will be minimal. If the Ab is not present in the serum, then complement will bind to anti-sRBC antibody and cause sRBCs to hemolyse.

Sooo here is the STEP BY STEP:

1. 50ul of serum specimen is diluted with 350ul of veronal buffer (VB)
2. diluted specimen is then heat inactivated at 56 degree celsius for 30 minutes
3. after inactivation, 25 ul of VB is added to the microtiter wells except the first row
4. 50 ul of specimen is added to the first well
5. titrate 25 ul of specimen from the first well downwards
6. 25 ul of viral antigen is added to each well
7. 25 ul of complement is added to each well
8. the microtiter plate is then incubate at 2-8 degree celsius overnight

Next day,

1. sRBC is washed using VB for three times
2. 24 ul of sRBC is mixed with 24 ul of hemolysin
3. 50 ul oof sRBS mixture is added to the wells
4. the plate is incubated for 30 minutes at 37 degree celsius
5. after incubation, the plate is centrifuged at 2000rpm/ 4 degree celsius for 10 mins
6. reading of results

Controls involved

Antigen control - using positive and negative antigen

serum control - using serum only

complement control - complement at different concentrations and insensitized cells

Reading of Results

Positive - no hemolysis/ button observed - meaning patient serum contains Ab to virus

Negative - hemolysis/ no button observed - meaning patient serum does not contain AB to virus

for more understanding you can visit

http://www.cehs.siu.edu/fix/medmicro/cfix.htm

thanks,

kenneth