Practical Immunology
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E-mail:[email protected]
Weifang Medical University
Test Four
Part 1 Hemolytic reaction of complement
Part 2 Detecting the concentration of serum complement
Complement is a system of about 30 kinds of proteins. It is
present in all normal mammalian serum.
Normally, the concentration is stable, dose not increase as a result
of immunization.
Complement plays important roles both in innate immunity and
adaptive immunity.It can destroy different kinds of cells, bacteria,
viruses and directly mediate inflammatory.
It is able to recruit other humoral and cellular effector systems and
induce histamine release from mast cells,
It can direct the migration of leukocytes, phagocytosis and release of
lysosomal constituents from phagocytes.
Pathways of complement activation
Classical pathway
Mannose binding
lectin dependent
Alternative pathway
Activation of C3 and
generation of C5 convertase
Activation of C5
Lytic attack pathway
• Deficiency of complement components always associate with
predisposition to disease or infection.
• Complement defects can be ascertained by the traditional total
hemolytic complement assays (CH50) , radial immunodiffusion,
electroimmunodiffusion or enzyme immunoassays (EIAs) .
Part 1
Hemolytic reaction of complement
Principle:
Sheep red blood cells(SRBC) as antigen combine with antisheep antibody (hemolysin), Complement is activated by
complex of Ag-Ab through classical pathway, ultimately form
a membrane attack complex, which cause SRBC lysis.
Materials:
• Antigen: 2%SRBC suspension
• Antibody: 2 u/ml hemolysin (antibody to SRBC)
• Complement: fresh guinea pig
• Normal saline (N.S)
• 24-well plate; pipettes;
Method:
• Take a 24-well plate, add correct amount of each sample to
each well.
• Put it at room temperature, about 10 minutes, observe the
result.
Well
2%SRBC
Hemolysin
Complement
N.S
1
2
3
4
0.25
0.25
0.25
0.25
0.25
0.25
－
－
0.25
－
0.25
－
0.25
0.5
0.5
0.75
* 0.25ml≈3 drop
Results：
• Well 1 appears hemolysis, the fluid become clarity, the other wells
don’t appear hemolysis because there is no complement in well 2,
no Ab in well 3, no Ab and complement in well 4, and those wells
look cloudy.
• The test is more rapid and convenient for processing a large
number of samples but is less quantitative and reproducible.
Part 2
Detecting the concentration of serum complement
Principle:
• Complement is activated by Ag-Ab. In this test, sheep red blood
cells（SRBC） been preincubated with anti-sheep antibody
（hemolysin） are added with different amount of the complement.
After a suitable incubation time at 37℃, the concentration of
complement are examined according to hemolysis.
• 1 unit of complement is defined as the dilution of complement
which can lyse half of the red cells, that is the CH50 dilution.
• The CH50 assay is the simplest quantitative assay for total
complement in serum or other samples. It depends on the
activation of the classical pathway to lyse SRBCs that have been
sensitized anti-SRBC antibodies.
• Complement activity is quantitated by determining the serum
dilution required to lyse 50% of the SRBCs in the mixture.
• Compare the detected tubes with the standard 50% lysis tube to
determine the CH50.
Materials:
• Detected serum complement (Fresh patient’s serum).
•
•
•
•
2% SRBC suspension (antigen).
Hemolysin (antibody to SRBC).
Standard 50% lysis tube.
Others: normal saline (N.S); small tubes; pipettes; incubator.
Methods:
• Take 7 disposable test tubes, number them.
• Add 0.2ml normal saline (NS) into each tube(1-6).
• Add 0.2ml serum into the first tube. Mix and transfer 0.2ml to the
second tube, till to the 6th. The final 0.2ml from the 6th is discard. The
serum dilutions are expected to be 1:2, 1:4, 1:8, 1:16, 1:32, 1:64.
• Add N.S 0.2ml into detected tubes (1 to 6),0.4ml into 7 tube.
• Add hemolysin 0.1ml and 0.1ml 2%SRBC to tube (1~7).
• Shake the tube rack to mix, incubate tubes at 37℃ for 30
minutes.centrifuge 5 min at 1000r/min, then compare with the
standard tube, observe the results.
1
2
3
4
5
6
7
1:64
control
0.1ml Ag
0.1ml Ab
0.2ml NS
0.2ml C
0.2ml NS
1:2
1:4
1:8
1:16
1:32
Results:
• Tube 1 to 6 are test tubes, the colour of fluid becomes lighter
and lighter, while the RBC deposit on the bottom of tubes is
more and more, which result from decreasing of complement,
7th is hemolysin control. The 7th tube don’t appear hemolysis
because there is no complement in it.
• Measure the optical density (OD) of detected tube and standard
tube,the tube whose OD is the same (or nearest to ) that of standard
tube is end point, its concentration of complement is defined 1 unit
CH50.
• If the forth tube is end point, it’s dilution is 1:16. That means the
forth tube contains 1u CH50, so the serum contains 16u complement.
As 0.2ml serum is used, the CH50 is 16u/0.2ml=16×5ml=80u/ml.
Production of standard tube:
• Take 5%SRBC suspension 1 ml, centrifuge, abandon the fluid, add 0.5ml
distilled water to destroy all the cells.
• Add 0.5ml double dilution N.S.
• Add 5% SRBC suspension 1ml.
• Add 50% hemolysis solution(produced above) 0.1ml to 0.5ml N.S to produce
50% hemolysis standard tube.
CH50
Do not shake!!!