Searching for microbes Part VII. Introduction to serology, precipitation and aglutination Ondřej Zahradníček To practical of VLLM0421c Contacts to me: 777 031 969 zahradnicek@fnusa.cz ICQ 242-234-100 Content of this slideshow Tale • Once a mother bougth 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 cicrcle should be here, the square has to be there. Nevertheless, a few days later… • …mum came to the child‘s room, and she saw, that the child was succesfull in puting 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. 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 specifity has its limits. Sometimes, we have a cross reactivity, when the antibody reacts also with an alien antigen, only simillar to that responsible to its production Sometimes, antibodies against an antigen produced in the tissue during the infection, too. Antigen and antibody Antigen and antibody Antigen = a macromolecule coming from an alien organism: plant, microbe, animal. (Eventually also from one‘s own body, but too old, dammaged or pathological cells.) 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) Methods in clinical microbiology • Direct methods: detection of a microbe, its part of its product. Examples: Microscopy, culture, biochemical identification, antigen detection. Positivity = it is sure, that the agens in NOW present. • Indirect methods: detection of antibodies against the microbe. Positivity = the microbe met the host IN HISTORY (weeks / months / years) Two ways how to use interaction between antigen and antibody: Interpretation of the antibody detection 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) How to interprete indirect diagnostics • 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 How to perform the reaction „quantitativelly“ • It is very dificult 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 à  serum contains a lot of antibodies – It reacts only when diluted a few timesà  only small amounts of antibodies present Geometric row and titre counting Geometric row • Technically the most simple way, how to dilute pacient‘s serum, is the use of geometric row with coefficient = 2. • We start with the undiluted serum, or serum with a certain pre-dilution (e. g. 1 : 5, 1.:.10, 1 : 20 and so on) • 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… Counting dilutions in serology Attention, in serology e. g. dilution 1 : 4 means one part of serum and three parts of saline (= total 4 parts)! At „biochemical“ counting (number of parts of serum : parts of diluent) we would have to use numbers e. g. 1 : 9, 1 : 19, 1 : 39, 1 : 79. That would be very un-practical Geometrical row: how to do it a) without predilution of the original serum b) with predilution of the original serum Of course, the predilution is not always 1 : 100, it can be 1 : 5, 1 : 10, 1 : 20 or any other. Geometrical row • At start, 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 à 1 : 4 • One halfth of 1 : 4 ……  1 : 8 • Etc., etc. Titre • After serum dilution, we add the antigen • In relation with the reaction type, either we can se the reaction result directly (aglutinate, precipitate), or we have to visualize it adding other components (complement, RBCs, etc.) • Anyway, we have to be able to discriminate positive and negative reaction results • The highest dilution, where a positive reaction is still visible, is called titre. Titre assessment Not always titres are needed! • We never use titers in antigen detection • Sometimes we do not assess titres despite the fact that it will be antibody detection. It is because these reactions are screening reactions • Example: Every pregnant woman is examinated for syphilis, just „for sure“. First tests are screening tests, performed as only qualitative tests. All positive / borderline reactions are confirmed by more specific confirmation reactions. Agglutination and precipitation: overview 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 antibody, or antibody using laboratory antigen • Only in the second example, we count titres! Precipitation, agglutination, agglutination on carriers • Precipitation: Antigens act alone, as macromolecules (coloid 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 polycelulose particle Precipitation Agglutination Agglutination on carriers Agglutination: examples of individual techniques Agglutination for antibody detection in a microtitration plate Positive – irregular „potato shaped“ formation Negative – a small, regular circle Demonstration of agglutination reaction in tularemia (from.www.medmicro.info): Example of a reult in Yersinia diagnostics K+ positive, titre = 1 : 400 No 1 negative No 2 posit., titre ³ 1 : 800 No 3 negative No 4 posit., titre = 1 : 200 Example of agglutination on carriers Treponema pallidum haemagglutination (TPHA) Here, too, the positive reaction is the „irregular potato-shaped spot“, negative reaction is corpuscullar 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 Demonstration TPHA (www.medmicro.info) 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 – We use polyvalent sera: nonavalent serum contains antibodies againts nine EPEC serotypes, trivalent serum IV contains antibodies agaist three remaining serotypes. Turbidity = positive – When one of sera (nonavalent and trivalent IV) is „+“, we have to continue using (trivalent and) monovalent sera • It is antigen detection à no titres! EPEC detection – result Precipitation: examples of individual techniques Precipitaton – flocculation: RRR reaction • It is 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 syphilis patients) • Again, only qualitativelly. 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 kardiolipin Precipitation – microprecipitation in agar Precipitation – ring precipitation for a antigen detection Step after step, we pour inside the Pasteur pipette: – 1) animal serum with antibody – 2) four diferent strain extracts Positivity: a ring formed at contact The picture is only an example! In your case, the positive one is not serum No. 1, but one of remaining ones! The End 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 (part of the 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 thes serotypes, only about twelve show elevated pathogenicity in newborns and sucklings. These serotypes are togerher called EPEC – enteropathogenous Escherichia coli Indirect diagnostics of syphilis – overview