Topic J08: Complementfixation test (CFT) and a follow-up of titres

To study: Web-search: Complement, amboceptor, anticomplementarity

Task No. 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

ery

amboceptor

comp-lement

rabbit

ery

Haemolysis (encircle)

In case of no haemolysis, explain, why it is absent

                                                 1

                                                0.2

                                                0.2

                                                0.2



yes – no


                                                 2

                                                0.2

                                                0.2


                                                0.2


yes – no


                                                 3


                                                0.2

                                                0.2

                                                0.2


yes – no


                                                 4



                                                0.2

                                                0.2

                                                0.2

yes – no


Task No. 2 Assessment of proper amount of complement to be used in CFT (titration)

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).

In four tubes, guinea pig serum is diluted 1:32, 1:36, 1:40 and 1:44. Serum was incubated with
working volume of sheep erythrocytes with amboceptor.

Write, in which dilution is 1 hemolytical unit of complement. It is 1 :  _______

Task No. 3 Schematic analyse of course of CFT, including 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).

Note: all schemes describe use of CFT for antibody detection.

LAB Ag = laboratory antigen

PATIENT Ig = patient antibody

C = complement

Ery = sheep red blood cell

Amb = amboceptor (rabbit antibody against sheep red blood cells)

ANTICOMPLEMENT = anticomplementarity causing component

 Description of  situation

                                                                        Phase 1

                                                      Complement

                                                      after Phase 1

                                                                                                                  Phase 2

                                                                            Result

                                                                                   Result

                                                                                   description

 Course of a

 positive CFT

                                                      free – bound


 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




Task No. 4 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. 5: Assessment of complementfixation antibodies to tick-born encephalitis

Read CFT titres in pair samples of patients. The first dilution is 1:4 in geometric series.
Carefully evaluate controls of anticomplementarity. Draw a result, write titer and try to interpret
results.

Task No. 6 Assessment of complementfixation antibodies in toxoplasmosis

Read CFT titres in sera of patiens with suspected infection caused by Toxoplasma gondii. The first
dilution is 1:8 in geometric series. Carefully evaluate controls of anticomplementarity. Draw a
result, write titer and evaluate only as „positive“ / „negative“ / „anticomplementary

Check-up questions:

1. Why patients own complement is not used for CFT and guinea pig complement is used instead?




2. What is the metod used for patient serum inactivation?




3. Which type of errors is caused by


a) anticomplementarity of serum?


b) too big amounts of used complement?


4. Why 2-fold increase of titre cannot be considered significant? (Think abour error of measuring.)





5. Why it is recomendable to use pair sera when using reactions like CFT?





6. What is meaning ot the term „seroconversion“? What can be considered more significant,
seroconversion of 4-fold increase of titre? Explain.







7. How would result of Task No. 4 influence decision about starting antibiotic therapy? (Answer
during the practical session)