immuological technique

1. shaibana
Hemolytic assays

2. • Hemolytic assays have traditionally been used to
assess the functional activity of the complement
system. They provide insight into the integrity of the
entire cascade reaction.
• These tests are particularly useful in the
investigation of suspected complement deficiencies.

3. • Serial dilutions of the sample to be analyzed are incubated
with Ab-sensitized sheep erythrocytes at a defined
temperature.
• Hemolytic assays are performed either in tubes or in agar
plates.
• The results are usually expressed as reciprocal dilutions of
the sample required to produce 50 or 100% lysis (CH50 or
CH100, respectively)

4. • Tests evaluating the functional activity of the
alternative pathway (AH50) use guinea pig, rabbit,
or chicken erythrocytes as target cells.

5. • The total hemolytic complement assay (CH50)
measures the ability of the classical pathway and the
MAC to lyse sheep RBC to which antibodies has been
attached.
• This estimates the standard quantity of serum
required as a complement source to produce lysis of
50% of standard quantity of sensitized sheep red cells.
TOTAL HEMOLYTIC COMPLEMENT
ASSAY(CH50)

6. • The CH50 is an random unit defined as the quantity
of complement necessary for 50% lysis of red cells.
• The CH₅₀ unit is determined under standardized
conditions which depend upon:
– Erythrocyte and antibody concentration;
– Buffering concentration of medium; and
– temperature

7. • As the complements are added to Ab-coated
erythrocytes, an increasing proportion of the cells are
lysed
• Released hemoglobins in the supernatant can be tested
by a photoelectric spectrophotometer.
• The hemolysis level is related with total complement
activity.
PRINCIPLE

8. • When the hemolysis percentage is served as ordinate
and the corresponding serum amount is served as
abscissa, a sigmoid curve can be obtained.
• The relation between the complement amount in the
vicinity of 50% hemolysis and hemolysis level presents a
straight line.
• So the 50% hemolysis point is defined as an end point,
named 50% complement hemolysis (i.e. CH50).

9. Von Krogh equation, which converts the S- shaped
complement titration curve into nearly straight line
X = K ( Y )1/n
( 1-Y )
where X = ml of diluted complement added
Y = degree of percentage lysis
K = constant
n = 0.2 ± 10% under standard E and A conditions.

10. • Materials and equipments
– Barbitone- buffered saline for complement tests
– Sheep erythrocytes (SRBC) in Alsever’s solution
– Serum : this should be either fresh, or guinea-pigs, serum
preserved specially for complements fixation assays
– Horse hemolytic serum ( source of anti-erythrocyte
antibody)
– spectrophotometer
Standardization of erythrocytes

11. • METHOD
1. Dilute the barbitone-buffered saline to working
strength.
2. Wash 4ml of the erythrocytes suspension 3 times in
barbitone- buffered saline.
3. Resuspend the washed erythrocytes in 15 ml of
barbitone-buffered saline.

12. 4. Mix 1ml of erythrocytes with 25ml ammonia
solution to lyse the cells and read absorbance at 541
nm. For a 6% SRBC suspension, in a 1cm cuvette,
absorbance should be 0.48-0.50
5. Mix 15ml barbitone-buffered saline, 0.1ml of horse
hemolytic serum and 15ml of 6% SRBC. Strictly, the
anti-erythrocyte serum should be titred until the
highest dilution still giving full complement fixation
is reached; for most purposes use a 1 : 150 dilution.

13. 6. Incubate at 37°C for 15 min.
7. Sensitized SRBC can be stored overnight at 4°C

14. • METHOD
• Label a series of tubes in duplicate with 1:8, 1:16, 1:32,
1:64 and 1:128.
• Prepare a series of two fold serial dilutions of control and
test serum in BBS each in duplicate
• Start at 1:4 (100μl serum + 300 μl BBS) and transfer 200
μl of sample into the next labelled tube.
ESTIMATION OF CH₅₀ TUBE ASSAY

15. • Mix thoroughly between dilutions and transfer 200
μl to the next dilution with a fresh pipette tip.
• Repeat until all five dilutions are made.
• Discard 200 μl from the final 1:128 dilution.
• Add 200 μl of suspended sensitised SRBC to all
tubes.

16. • Label two separate tubes as BLANK and add 200 μl of
sensitised SRBC + 200 μlBBS. These tubes will measure
spontaneous lysis of the SRBC in the BBS.
• Label another two separate tubes as TOTAL LYSIS and
add 200 μl of sensitised SRBC + 200 μl distilled water.
• Gently mix all tubes.
• Incubate at 37°C for 30 minutes in a waterbath mixing
after 15 minutes.

17. • Centrifuge the samples at 1,500 rpm for 5 minutes to
sediment the RBCs.
• Transfer 100 μl of supernatant from each tube to a
well in a 96 well flat bottom plate.
• Add 100 μl of distilled water to each well.
• Read the absorbance of the samples at 540nm using
a plate spectrophotometer.

19. • Calculations
• Calculate the mean absorbance for each sample
• Subtract the BLANK absorbance (spontaneous lysis)
from all samples
• Calculate the % lysis for each dilution using the
following formula:
% lysis = OD540 (test) – OD540(Blank) ×100
OD540(total lysis) – OD540(Blank)
• Plot the percentage lysis (vertical axis) versus the
serum dilution on the horizontal axis.
• Calculate the dilution required for 50% haemolysis for
the control and test serum

20. •To calculate the 50% lysis, a line is drawn from the 50% percent
value until it intersects with the graph lines and then a vertical line
is drawn down to the dilution

22. • This simple assay, which is analogous to single radial
immunodiffusion, may be used on a routine basis with
reference to a standard serum.
• Antibody-sensitized red cells are incorporated into
molten agarose and the mixture allowed to set.
• Wells are cut in the agarose and filled with either the
sera under test or dilutions of a standard serum.
Estimation of CH100 by assay in agar

23. • The complement diffuses into the agarose and
reacts with and binds to the antibody-coated red
cells.
• Circles of lysis appear, the size of which depend
upon the complement content of the serum.
• MATERIALS AND EQUIPMENT
• Sensitized red cells (made up to 10% v/v)
• Agarose, 2% w/v in barbitone buffer
• Glass plates
• Gel cutter

24. • METHOD
1. Warm 1.5 ml of the barbitone buffer to 56°C in a water
bath.
2. Cool 1.2 ml of molten 2% agarose to 56°C and add to the
barbitone buffer
3. Mix and cool to 45°C in a water bath.
4. Add 0.2 ml of sensitized red-cell suspension and mix
gently.
5. Place the glass plate on a level surface
6. Pour the mixture quickly onto the plate to form a smooth,
even surface.

25. 7. When set, place the plate in a box containing moist
filter paper and chill to 4°C for a few minutes to
harden the agarose.
8. Cut two rows of five wells, approximately 3 mm across
using an gel cutter.
9. Dispense 8 µl samples of the sera under test into
separate wells. Similarly, add four doubling dilutions
of a standard serum to a series of wells.
10. Incubate the plate in a moist box overnight at 4°C.

26. 11. Warm the plate, still in the box, to 37°C for 2 h, to
allow cell lysis to occur.
12. Measure two diameters at right angles across each
well and calculate their mean.
13. Plot the value of the areas (pr2) of the standard
serum dilutions (linear scale) against the log
dilution. Determine the concentration of the
unknown sera as a percentage of the standard by
extrapolation from the standard curve.

27. • very useful screening tool to rule out or to verify
suspected complement deficiency disease.
• Disease associated with reduced serum complement
concentration are,
• Glomerular nephritis
• Serum sickness
• cirrhosis of liver
• myasthenia gravis
• allograft rejection
APPLICATIONS

28. • Disease associated with elevated serum
complement concentration are,
• Gout
• Obstructive jaundice
• Acute rheumatic fever
• Rheumatoid arthritis
• Typhoid fever

29. • The complement alternate pathway (AH50) assay is a
screening test for complement abnormalities in the
alternative pathway.
• The alternate pathway shares C3 and C5-C9
components, but has unique early complement
components designated factors D, B, and P, as well as
regulatory factors H and I.
• This pathway is activated by microbial polysaccharides
and does not require immune complex formation.
AH50

30. • Patients with disseminated infections with pyogenic
bacteria in the presence of a normal CH50 may have
an absent AH50 due to hereditary or acquired
deficiencies of the alternate pathway.
• Patients with deficiencies in the alternate pathway
factors (D, B, P, H, and I) or late complement
components (C3, C5-C9) are unusually susceptible to
recurrent Neisserial meningitis.

31. • Absent complement alternate pathway (AH50) in
the presence of a normal total hemolytic
complement (CH50) suggests an alternate pathway
component deficiency.
• Normal AH50 with absent CH50 suggests an early
(C1, C2, C4) classic pathway deficiency.

32. • Absent AH50 and CH50 suggests a late (C3, C5, C6,
C7, C8, C9) component deficiency or complement
consumption.
• Absent AH50 and CH50 in the presence of a normal
C3 and C4 suggests a late (C5, C6, C7, C8, C9)
component deficiency.

33. • Investigation of suspected alternative pathway
complement deficiency, atypical hemolytic uremic
syndrome, C3 glomerulonephritis, dense-deposit
disease
APLLICATIONS

34. THANK YOU…