1. Complementfixing test (CFT, topic J08) Neutralisation reaction (J09) Ondřej Zahradníček Practical of Medical Microbiology (VLLM0421c) Contact 777 031 969 zahradnicek@fnusa.czzahradnicek@fnusa.cz ICQ 242-234-100

2. Review: Methods in clinical microbiology Direct methods: detection of a microbe, its part of its product. Microscopy, culture, biochemical identification, antigen analysis. Positivity = it is sure, that the ages in NOW present. Indirect methods: detection of antibodies against the microbe. Positivity = the microbe met the host IN HISTORY (weeks / months / years)

3. Review: Antigen / antibody Antigen = a macromolecule coming from an alien organism: plant, microbe, animal. In microbiology, we are interested in microbial antigens – parts of microbial body, that challenge host body to an antibody response Antibody = an immunoglobuline, formed by the host body as a response to antigen challenge (of course not only by humans, but also by various animals)

4. Review: Two ways how to use it: Antigen detection: laboratory (animal origin) antibodies + pacient‘s sample or microbial strain. Direct method Antibody detection: laboratory antigen (microbial) + pacient‘s serum (or saliva). Indirect method

5. Review: Interpretation Antigen detection: it is a direct method. Positive result means presence of the microbe in the pacient‘s body Antibody detection: it is an indirect method. Some ways how to assess, when the microbe met the body: Amount of antibodies (relative – titre) and its changes during the time (dynamics) Class of antibodies: IgM/IgG (More in J10) (Avidity of antibodies)

6. Dynamics of titre Absolute amount of antibodies is not the most sure information: some patients are poor antibody-producers, etc. Dynamicst of titre: better, means how the response gets changed during the time (usually during two or three weeks) 1 first pacient‘s visit 1 2 after 2 – 3 weeks 2

7. Pair sera and non-pair sera Pair sera = first specimen is kept in the refrigerator until the second comes to the lab, and thed examined together. 4- fold increase is told to be significant under such circumstances Other situations (second specimen is examined separately): an accidental error should be taken into account. So, usually at least 8-fold increase is needed

8. Complement-fixing test (CFT) Complement = one component of immunity reaction For CFT, we use animal (guinea-pig) complement. The patient‘s complement is inactivated before the reaction Complement is not able to get bound to isolated antigen Complement is not able to get bound to isolated antibody Complement is able to get bound to COMPLEX antigen - antibody

9. Use of CFT CFT is used for diagnostics of many (mainly viral) pathogens CFT, like other serological reactions, may be used for antigen detection or antibody detection For simplification, we shall only speak about antibody detection in this practical So, we think about a laboratory antigen being mixed with patient‘s serum

10. Complement – how it does work (Task 1) Sheep RBC + amboceptor without complement  no haemolysis Sheep RBC + complement without amboceptor  no haemolysis Sheep RBC + complement + amboceptor  haemolysis Rabbit RBC + complement + amboceptor  no hemolysis

11. Titration of complement (T 2) For the reaction, we need an amount of guinea-pig complement that is neither too small nor too big That is why we test, what amount of complement is just able to perform haemolysis of a specified amount of red blood cells with amboceptor

12. Titration of complement results Dilution of guinea-pig serum (as a source of the complement): 1 : 32 = haemolysis 1 : 36 = haemolysis 1 : 40 = haemolysis 1 : 44 = no haemolysis Haemolytical unit corresponds to 1 : 40 To obtain 2 units, we have to dilute this serum 1 : 20

13. CFT – principle (Task 3 a, b)

14. Problems existing in CFT Too much complement: false negative results. What to do? Titrate the complement (according to Task 2) Something in serum binding the complement itself (anticomplementarity component): false positive results. What to do? Perform anticomplementarity test without antigen

15. Anticompl. test (Task 3 c, d)

16. Tasks 4 and 5: CFT Task 4: The whole seropanel belongs to one patient. We have six respiratory pathogens, each in two rows (acute speciemen, reconvalescent specimen). We have 7 dilutions of sera (first collumn is the anticomplementarity test) Task 5: The seropanel belongs to a positive control and six patients. We test Ab against tick-borne encephalitis. Mind the anticomplementarity test!

17. Neutralisation reaction: general principle There are many ways, how antibodies do work. One of them is direct neutralising effect This effect is rarely present in whole bacteria. On the other hand, it may be observed in whole viruses, and in bacterial toxins Nevertheless, sometimes antibodies neutralise some characteristic of the whole bacteria, e. g. motility of Treponema in Nelson‘s test

18. Neutralisation schematically Antibody (Ig) prevents an effect of a toxin/virus to a cell / red blood cell Example: ASO (Toxin = streptolysin O, Ig = antistreptolysin O, red blood cell)

19. Task 1 - ASO ASO is not an indirect diagnostics reaction, despite the fact that we search for antibodies. The aim is not to get a pathogen, but to assess the antibodies themselves, as they may be dangerous In ASO, we do not use the geometrical row. The dilutions in individual wells in the panel are written on a paper The titer above 200 is supposed to be risky for an autoimmune disease

20. Task 2: HIT Haemagglutination Inhibition Test: Pay attention, it is NOT an agglutination reaction, it is a neutralisation! Antibody neutralises the aggregation of RBCs due to viruses. Task: Read HIT results for tick-borne encephalitis. Read titers in accute and reconvalescent sera and make a conclusion (accute infection? Or only memory antibodies?)

21. Task 3: VNT (do not confuse with TNT ) Virus Neutralisation Test Cell culture uses to be dammaged by a virus. The dammage is visible as a change of colour (pH is changed) Antibodies, if present, may prevent this viral action on the cell culture

22. Nice day to everybody!