Topic J08
Topic J07+J08: Introduction to serology: agglutination,precipitation,
complementfixation test (CFT), neutralisation reactions (ASO, HIT),
dilution and a follow-up of titres
To study: Search WWW for terms: Antigen; Antibody; Serological reactions; Agglutination; Precipitation
Task No. J07/1 and J07/2: not done in this double session
Task No. J07/3 Agglutination
Agglutination is a reaction between antibodies and corpucular antigen. That term „corpuscular“ means that
instead of using molecule of antigent determinant only we use the whole microorganism. It is also possible to
bind an originally coloid antigen to a corpuscle (agglutination on carriers, see Task No. 4)
J07/3a) Agglutination in service of antibody detection
Read a microtitration plate with serum samples, in which antibodies to Yersinia enterocolitica were determined.
In the first well, serum is diluted 1:100 with 2-fold dilution. Antigens for the reaction are plain bacterial cells.
Agglutination is a white map on the boootm of a positive well. Read and draw a result, write a titre.
J07/3b) Agglutination in service of antigen analysis
Using the agglutination reaction, determine whether the given strain of E. coli is enteropathogenic, i.e. it carries
antigens connected with the ability to cause the specific type of diarrhoea. Use a nonavalent serum (containing
antibodies against 9 antigenic types) and a trivalent serum IV (containing antibodies against three more antigenic
types). In case of positivity of a nonavalent serum, we would continue with three trivalent sera (I, II, III) and
monovalent sera; in case of posinitity of trivalent serum IV we would continue with three monovalent sera.
(In the practical session for technical reasons we do not continue, in case of positivity of one of polyvalent
sera we just conclude „it is an EPEC“)
 With a pasteur pipette, place a drop of saline on the slide, and beside it, drop a polyvalent serum containing
antibodies against several enteropathogenic serotypes of E.coli
 Remove about half a loopful of the strain, place part o fit on the slide closely next to the drop of the serum
 Mix the remainder in a drop of saline
 Without flaming the loop, suspend the strain placed on the slide in a drop of the serum
 Rock the slide with circling movements for about one minute, and follow the rise of agglutination
Draw the result and make a conclusion (strike off, what is not true).
Task No. J07/4 Agglutination on carriers
Read a result of TPHA (Treponema pallidum haemagglutination test) in which treponemal antigen is coated on a
surface of erythrocyte and reacts with antibodies. The test is used in diagnostics of syphilis. For this test serum is
not titred and a result is red in a semiquantitative way „1 to 3 crosses“. Draw how are various results look like
and write down numbers of eventual positive sera.
Positive are sera No
Task No. J07/5: Precipitation
Precipitation is a reaction between antibodies and the soluble antigen.
J07/5a) Precipitation in the proof of antibodies
Rapid reagin reaction (RRR) is a nice example of the reaction of that type. RRR is used in the screening syphilis.
It is interesting, that we do not search for antibodies against Treponema pallidum, but antibodies against so
named kardiolipin, that is present in body of patients infected with syphilis.
The following guidelines are only an info, in practice, you have the test already prepared for reading.
 Mark the location of the sera in the wells on a perspex panel
 Add 0.05 ml of negative serum to the first well, and 0.05 ml of positive control serum to the next one
 Then pipette 0.05 ml of the patient’s sample into the next wells
Name _____________________
General Medicine
Date ___. 4. 2010
Page 1
Topic J08
 Between pipetting wash the pipette 3times with 0.05 ml of saline
 Finally, pipette 0.05 ml of cardiolipin antigen into each well containing serum and shake the panel carefully
 Stir about five minutes and then read the precipitation result against a dark background
Draw your result and make a conclusion:
Conclusion:
J07/5b) Precipitation in the proof of antigen
Not performed in this double session.
Task No. 08/1 Examination of function of indicator systém fot the CFT
Complement causes hemolysis of sheep erytrocytes in complex whit amboceptor (= rabbit antibodies against
sheep erythrocytes). Absence of any of needed components leads to absence of haemolysis.
Pipette into tubes individual parts of reaction according to the table. Wash your pipette allways carefully three
times by saline to avoid contamination. Incubate 1hour in a thermostat. Finally evaluate the results.
Note: All amounts are in mililitres (ml)
tube
saline sheep amboceptor
comprabbit Haemolysis
In case of no haemolysis, explain,
ery
lement
ery
(encircle)
why it is absent
1
2
3
4
0.2
0.2
0.2
0.2
0.2
yes – no
0.2
0.2
0.2
0.2
0.2
0.2
yes – no
yes – no
0.2
yes – no
CFT uses exactly measured amount of guinea pig complement, given in hemolytic units (patient‘s conplement is
inactivated by heating of serum). Hemolytic unit is the exact amount of complement which causes hemolysis of
given volume of RBC (in complex with amboceptor).
Task No. 08/2 Schematic analyse of course of CFT, incl. anticomplementarity testing
In following schemes judge where free complement remains free after the fist phase of reaction (encircle what is
true, cross, what is wrong) and add verbal description of the result (haemolysis, sedimentation of RBCs).
Description of
Complement
Result
Result
situation
after Phase 1
description
Course of a
free – bound
positive CFT
Phase 1
Phase 2
Course of a
negative CFT
free – bound
Anticomplementarity
testing, serum is
anticomplementary
free – bound*
*or inactivated
Anticomplementarity
testing, serum is OK
free – bound*
*or inactivated
Note: all schemes describe use of CFT for antibody detection.
LAB Ag = laboratory antigen
PATIENT Ig = patient antibody
C = complement
Amb = amboceptor (rabbit antibody against sheep red blood cells)
Ery = sheep red blood cell
ANTICOMPLEMENT = anticomplementarity causing component
Name _____________________
General Medicine
Date ___. 4. 2010
Page 2
Topic J07–08 (double session due to Easter Monday 5th April 2010)
Task No. 08/3 Assessment of complementfixation antibodies to the most frequent
causative agents of respiratory diseases
Read the titres of CFT in individual patients. Remark anticomplementarity controls in the wells of the first
collumn. CFT starts from the second well with 1:5 dilution. Draw your result, write titre values (in positive
patients) and try to interprete results.
Task No. 08/4: Assessment of complementfixation antibodies to tick-born encephalitis
and Task No. 08/5 Assessment of complementfixation antibodies in toxoplasmosis
Not performed in this double session
Task No. 08/6 ASO – a proof of antistreptolysin O
Principle: Antibodies prevents hemolysin (streptolysin O – i.e. antigen) to hemolyse rabbit RBC.
ASO levels increase after beta-hemolytic streptococci group A (less commonly also other groups) caused
infections. In risk for late sequelaes, ASO increase over 200 I. U. (international units) is seen.
On your serological panel, read ASO values of positive control and seven patients.
Task No. 08/7 HIT – hemagglutination inhibition test
Principle: Antibodies prevents virus to agglutinate erythrocytes. Serum samples taken from patiens with suspect
tick-born encephalitis are examined. Read, draw results and make a clinical conclusion.
Name _____________________
General Medicine
Date 12. 4. 2010
Page 3
Topic J07–08 (double session due to Easter Monday 5th April 2010)
Check-up questions:
1. What type of speciemen is needed for antibody detection?
2. What types of specimens can be used for antigen detection?
3. What does mean the term „antigen analysis“?
4. Does presence of antbodies allways mean an accute infection? Explain.
5. What does the term „titre“ mean? Is it assessed in antibody detection, antigen detection, or both? Explain.
6. What is the difference between precipitation and agglutination reaction?
7. Why it is necessary to perform confirmation in case of a positive RRR reaction?
8. Is it possible to examine antibidies angainst a coloid antigen using agglutination reaction?
9. Which type of errors is caused by
a) anticomplementarity of serum?
b) too big amounts of used complement?
10. What is meaning ot the term „seroconversion“? What can be considered more significant, seroconversion of
4-fold increase of titre? Explain.
11. In which clinical situations ASO diagnostics is rational?
12. Which is a Czech abbreviation for ASO?
13. A bonus question for clever students: Try to find an arrangement for HIT, if it should be used for antigen
detection.
Name _____________________
General Medicine
Date 12. 4. 2010
Page 4