1. Introduction to serology, agglutination and precipitation, dilution (topic J07) Ondřej Zahradníček Practical of Medical Microbiology (VLLM0421c) Contact 777 031 969 zahradnicek@fnusa.czzahradnicek@fnusa.cz ICQ 242-234-100

2. 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. Antigen and 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. 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. 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. Nevertheless, there are some ways how to get the information – when the microbe met the body: Amount of antibodies (relative – titre) Class of antibodies: IgM/IgG (More in J10) (Avidity of antibodies)

6. How to get the information Acute infection: large amount of antibodies, mostly class IgM Pacient after an infection: small amounts of antibodies, mostly IgG (immunological memory) Chronical infection: various response 1 1 2 2

7. How to perform the reaction „quantitativelly“ It is very dificult to assess the amount of antibodies in units e. g. mol/l, mg/l etc. But it is possible to use another way: to dilute the patient‘s serum many times. It reacts even when diluted many times   a lot of antibodies in serum It reacts only when diluted a few times   only small amounts of antibodies

8. Task 1: Geometrical row We have a serum specimen, that is undiluted In first test tube, we mix it with the same amount of diluent (saline), so that we have dilution 1 : 2 One halfth of 1 : 2 dilution is removed to another test tube, and mixed again with the same amount of diluent  1 : 4 One halfth of 1 : 4 ……  1 : 8 Etc., etc.

9. Task 2 Titre – the highest positive dilution. If we have two rows, titre = the highest positive dilution of both rows

10. Precipitation, agglutination, agglutination on carriers Precipitation: Antigens act alone, as macromolecules (coloid antigen) Agglutination: Antigen act being part of its microbial cell (we work with whole microbes) Agglutination on carriers: Formerly isolated antibodies are bound to an alien particle – latex or RBC

11. Schematical difference 1 – precipitation 2 – agglutination 3 – agglutination on a carrier 1 2 3

12. Task 3 3a: Determine agglutination titre for antibodies against yersinia Do not forget – titre = highest dillution with still positive reaction. First well is diluted 1 : 2, second – 1 : 4, etc.

13. As we do not actually have testing kits, see the result only using this demonstration: First collumn are controls, the reaction starts in the second collumn 1:2 1:4 1:8

14. Task 3b: Enteropathogenous Escherichia coli There are cca 12 antigenic types belonging to EPEC group We use polyvalent sera: serum A contains antibodies againts several EPEC serotypes, serum B antibodies agaist some more serotypes. Turbidity = positive When one of sera (A or B) is „+“, we have to continue using monovalent sera Do you understand, why it is impossible to asses titres here?

15. Task 4: Treponema pallidum haemagglutination (TPHA) Technically the same as in task 3a, but: Difference 1: done only qualitativelly (no titre evaluation) Diference 2: it is red (as instead of bacteria, red blood cells are used)

16. For technical reasons, demonstrarion only: +++ ++ + +/- - - - -

17. Task 5: precipitation Task 5a: Detection of antibodies that are positive in syphilis, although they are not antibodies against Treponema pallidum, but aganist kardiolipin (a stuff present in bodies of syphilitics) Done only qualitativelly. First well is positive controll, second well is negative controll, and then each patient has one well only. 0.05 ml of serum + 0.05 ml of kardiolipin

18. Demonstration of another type of precipitation Microprecipitation in agar accord. to Ouchterlony + - - -

19. Task 5b – ring precipitation Precipitation for antigen detection: 1) animal serum with antibody 2) four diferent strain extracts 3) positivity: a ring formed at contact

20. Nice day to everybody!