1. Searching for microbes Part VI. Introduction to serology, precipitation and agglutination Ondřej Zahradníček To practical of VLLM0421c Contact to me: zahradnicek@fnusa.cz

2. Content of this slideshow Antigen and antibody Interpretation of the antibody detection Geometric row and titer counting Agglutination and precipitation: overview Agglutination: examples of individual techniques Precipitation: examples of individual techniques Check-up questions Introduction – tale

3. Tale Once a mother bought a toy to her child. The toy was a plastic plate with holes of different shapes, and shapes belonging to the holes were here, too. Once the child cried, as something went wrong. Mum came and told him: „My child, you cannot put a square into a hole for a circle!“ Look, the circle should be here, the square has to be there.

4. Nevertheless, a few days later… …mum came to the child‘s room, and she saw, that the child was successful in putting the circle into the hole for a hexagon. So, the mum realized, that there are some rules, but there are exceptions, too. The same is in the nature – when a shape has its counter-shape, sometimes a counter-shape is able to make a couple with another shape and not the correct one.

5. What to learn from the tale Microbes (but also e. g. plants and animals) have on the surface of their cells antigens. When they meet our body, our body starts to produce antibodies, that are specific to it. The specificity has its limits. Sometimes, we have a cross reactivity, when the antibody reacts also with an alien antigen, only similar to that responsible to its production Sometimes an antibody against a chemical substance formed in the body during the infection is released (e. g. from the vessels); nevertheless, mostly we detect antibodies against true microbial antigen

6. Antigen and antibody

7. Antigen = a macromolecule coming from an alien organism: plant, microbe, animal. (Eventually also from one‘s own body, but the cell containing it is too old, damaged or pathological.) In microbiology, we are interested in microbial antigens – parts or products* of microbial body, that challenge host body to an antibody response Antibody = an immunoglobulin, formed by the host body as a response to antigen challenge (of course not only by humans, but also by various animals) *e. g. toxins

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

9. Two ways how to use interaction between antigen and antibody: Antigen detection: laboratory (animal origin, or from special cells) antibodies + patient‘s sample or microbial strain. Direct method Antibody detection: laboratory antigen (microbial) + patient‘s serum (or saliva). Indirect method

10. There are two subtypes of antigen proof: Direct diagnostics in a specimen: we have the complete specimen and we search for antigen. Example: CSF, we search for common causative agents of purulent meningitis Antigenic analysis: we have already isolated a strain of a specimen and we study its antigen(s). Example: we have a strain of meningococcus and we want to know to which serogroup it belongs (B or C). (Usually it is a diagnostics inside a bacterial species.)

11. Interpretation of the antibody detection

12. Interpretation Antigen detection: it is a direct method. Positive result means presence of the microbe in the patient‘s body Antibody detection: it is an indirect method. Nevertheless, there are some ways how to get the information – when the microbe met the host organism: –Amount of antibodies (relative – titre) and its dynamics (titre dynamics – more in J07) –Class of antibodies: IgM/IgG (More in J08) –(Avidity of antibodies)

13. How to interpret indirect diagnostics Acute infection: large amount of antibodies, mostly class IgM Patient after an infection: small amounts of antibodies, mostly IgG (immunological memory) Chronic infection: various response 1 1 2 2

14. How to perform the reaction „quantitatively“ It is very difficult to assess the amount of antibodies in units like 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   serum contains a lot of antibodies –It reacts only when diluted a few times   only small amounts of antibodies present

15. Geometric progression and titre counting

16. Geometric progression Technically the most simple way, how to dilute patient‘s serum, is the use of geometric progression with coefficient = 2. We start with the undiluted serum, or serum with a certain pre-dilution (e. g. 1 : 5, 1.:.10, 1 : 50 or simillarly) In any case: in every next well, there is double dilution in comparison with the previous, for example, we have a row: 1 : 10, 1 : 20, 1 : 40, 1 : 80, 1 : 160…

17. Counting dilutions in serology Attention, in serology dilution e. g. 1 : 4 means one part of serum and three parts of physiological saline (= total 4 parts)! At „biochemical“ counting (number of parts of serum : parts of dilluent) we would have to use numbers e. g. 1 : 9, 1 : 19, 1 : 39, 1 : 79 (instead 1 : 10, 1 : 20, 1 : 40, 1 : 80). That would be very un-practical.

18. Geometrical row: how to do it a) without predilution of the original serum

19. b) with predilution of the original serum (e. g. here predilution 1 : 100) Of course, the predilution is not always 1 : 100, it can be 1 : 5, 1 : 10, 1 : 20 or any other.

20. Geometric progression „how to do“ At start, we have a „serum“ specimen (replaced by ink), that is – just in this experiment – undiluted In first test tube, we mix it with the same amount of diluent (saline), so that we have dilution 1 : 2 One half of mixture with 1 : 2 dilution is removed to another test tube, and mixed again with the same amount of diluent  1 : 4 One half of 1 : 4 ……  1 : 8 Etc., etc.

21. Titre After serum dilution, we add the antigen In relation with the reaction type, either we can see the reaction result directly (agglutinate, precipitate), or we have to make it visible it by adding some other components (complement, RBCs, etc.) Anyway, after doing all steps, we use to be able to discriminate positive and negative reaction results The highest dilution, where a positive reaction is still visible, is called titre.

22. Titre assessment Titre – the highest positive dilution. If we have two rows, titer = the highest positive dilution of both rows.

23. Not always titers are needed! We never use titers in antigen detection Sometimes we do not assess titers despite the fact that it will be antibody detection. It is because these reactions are screening reactions Example: Every pregnant woman is examined for syphilis, just „for sure“. First couple of tests are screening tests, performed as only qualitative tests. All positive / borderline reactions are confirmed by more specific confirmation reactions.

24. Agglutination and precipitation: overview

25. Precipitation and agglutination – common characteristics Precipitation and agglutination are the two most simple serological reactions, we work here really just with antigen and antibody, without any other components Either we detect antigen using animal (or monoclonal*) antibody, or antibody using laboratory antigen Only in the second example, we count titres! *made by a clonal population of plasmatic cells

26. Precipitation, agglutination, agglutination on carriers Precipitation: Antigens act alone, as macromolecules (colloid antigen) Agglutination: Antigen acts being part of its microbial cell (we work with whole microbes, corpuscular antigen) Agglutination on carriers: Formerly macromolecular antigens are bound to an alien particle – carrier: latex particle, RBC, eventually polycellulose particle

27. Precipitation

28. Agglutination

29. Agglutination on carriers

30. Agglutination: examples of individual techniques

31. Agglutination for antibody detection in a microtitration plate Positive – irregular „potato-pancake shaped“ formation Negative – a small, regular circle Do not forget, that titer = highest dilution with a positive reaction. First well is diluted 1 : 100, second 1 : 200 etc.

32. Demonstration of agglutination reaction in tularemia (from.www.medmicro.info): 1:2 1:4 1:8 First row: Agglutinate visible in 1:2 and 1:4 dilutions, but not 1:8 and higher  the titre is 1:4 Second row: There is no agglutination in any well  no titer, negative reaction

33. Example of a result in Yersinia diagnostics K+ positive, titer = 1 : 200 No 1 negative No 2 posit., titer  1 : 400 No 3 negative No 4 posit., titer = 1 : 200 1:100 1:200 1:400 1:800 Agglutination Sedimentation of free bacteria

34. Example of agglutination on carriers Treponema pallidum haemagglutination (MHA-TP, TPHA in CZ) Here, too, the positive reaction is the „irregular potato- shaped spot“, negative reaction is corpuscular sedimentation on the bottom ot the well. But it is red: it is an agglutination on carrier, the antigen is carried by a red blood cell Today, red blood cells are replaced by polycelulose particles in this test – you can meet abbreviation TPPA

35. Demonstration TPHA (www.medmicro.info) +++ ++ + +/- - - - -

36. Example of slide agglutination to antigen analysis: Testing of an E. coli strain for Enteropathogenous Escherichia coli There are about 12 antigenic types belonging to EPEC group. It would be possible to use 12 individual antisera, but the following approach is more effective: –We use polyvalent sera: nonavalent serum contains antibodies against nine EPEC serotypes, trivalent serum IV contains antibodies against three remaining serotypes. So all twelve serotypes are „covered“. Turbidity = positive –When one of sera (nonavalent and trivalent IV) is „+“, we have to continue using (trivalent and) monovalent sera It is antigen detection  no counting titers!

37. EPEC detection – result

38. Precipitation: examples of individual techniques

39. Precipitaton – flocculation: RRR reaction In this reaction, we try to find antibodies that are positive in syphilis, although they are not antibodies against Treponema pallidum, but against cardiolipin (a stuff present in bodies of syphilis patients) Again, we perform testing only qualitatively. First well is positive control, second well is negative control, and then each patient has one well only. 0.05 ml of serum + 0.05 ml of cardiolipin

40. RRR, RPR, VDRL Non-treponema tests, i. e. detection of non- specific anti-cardiolipin antibodies may be performed in different forms VDRL (Venereal Disease Research Laboratory) is a flocculation (precipitation) reaction on a slide. RRR (rapid reagin reaction), is a modification of VDRL, wells in a panel are used Another similar is reaction called RPR (rapid plasma reagin), where reading is simplified using carbon particles or pigments.

41. Precipitation – microprecipitation in agar The fluid with antigen is placed to the centre. The antigen diffuses through the agar. When the serum contains antibodies, they diffuse against it and on their contact, a precipitation line is formed. + - - - Microprecipitation in agar according to Ouchterlony Antigen 

42. Precipitation – ring precipitation for a antigen detection Step after step, we pour inside the Pasteur pipette: –1) animal serum with antibody –2) four different strain extracts Positivity: a ring formed at contact The picture is only an example! In our practical session, the positive one is not serum No. 1, but one of remaining ones!

43. The End Treponema pallidum (causes syfilis)

44. A note to E. coli Escherichia coli is a bacterium that is normal part of intestinal microflora. On its surface, it has – besides other types – also so named O-antigens (end part of the polysaccharidic chain of the G– bacteria outer membrane) These O-antigens are not the same in all E. coli strains. There exist hundreds of serotypes inside E. coli species Among all these serotypes, only about twelve show elevated pathogenicity in newborns and infants. These serotypes are together called EPEC – enteropathogenous Escherichia coli

45. Indirect diagnostics of syphilis – overview HistoricalBWR – Bordet Wassermann Nontr. ScreeningRRR – Rapid Reagin Test MHA-TP* Treponema ConfirmatoryELISA FTA-ABS (indir. imunofluor.) Western Blotting Historical, or superconfirmation TPIT (Treponema Pallidum Imobilisation Test) = Nelson TPHA – Tr. pasive hemagglutination test TPPA – dtto, RBC replaced by polycelulose

46. Check-up questions 1. What type of specimen 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 antibodies always mean an acute infection? 5. In what situations titers are measured? 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 antibodies against a colloid antigen using agglutination reaction? 9. What does the term "screening reaction" mean? 10. And one more