1. Searching for microbes Part VIII. Reactions with labelled components Ondřej Zahradníček To practical of VLLM0421c and ZLLM0421c zahradnicek@fnusa.cz

2. Content of this slideshow Classes and avidity of antibodies Reactions with labelled components: survey Immunofluorescence and RIA ELISA: principle ELISA: practical reading Immunoblotting Immunochromatographic tests Check-up questions Introduction (tale)

3. Tale There was a sailorwoman, and she had various object on the board bound so to keep them in the ship. She had her trieder tied to the rescue circle, the rescue circle tied to rescue boat, and the rescue boat to the board. So even the highest wawes were not able to take the objects away. Once the sailor‘s husband came. He wanted to assay the rescue circle. He untied the trieder away and he took the circle from the boat. A wave came – and the trieder was flown away from the board. The original tale about a sailorman and his wife was changed in order to fight with gender stereotypes.

4. What to learn from the tale The principle of reactions of labelled components: We bind one component to another; after each step, the washing out eliminates free (untied) components. This process takes away everything that is not tied A negative reaction = a reaction, where one component of the chain of components bound one to another is missing. The other components are not bound to the surface, and they are washed away.

5. Classes of antibodies, avidity of antibodies

6. Course of antibody answer Class IgM antibodies are formed as first, but they are also first to disappear. They do not go through placenta  in a newborn their presence certifies its own infection Class IgG antibodies are formed later and remain as immunological memory. They are able to slip through placenta. (a newborn may have them from its mother)

7. Other classes of antibodies Class IgA antibodies are important mainly in mucous membrane immunity, so in infections entering the body through mucous membrane (e. g. gastrointestinal). In diagnostics of some pathogens detection of IgA may replace detection of IgM (toxoplasmosis) Class IgE antibodies are present in allergy and in helmint infecions. Specific IgE antibodies against a certain pathogen are rarelly examined Class IgD antibodies are not examined im microbiology

8. Avidity of antibodies Avidity of antiboties is defined as the strength of binding between antigen and antibody. It is usually measured for IgG antibodies. Fresh IgG antibodies have low avidity, older IgG antibodies have high avidity In some diseases (toxoplasmosis, toxocarosis) avidity may show us a difference between „not so fresh, but still active“ infection (IgM present, but high IgG avidity) and „really fresh infection“ (IgM present, IgG avidity is low)

9. Reaction with labelled components: survey

10. Reactions with labelled components Individual components are bound on the previous components, the first of them to the surface. Instead of one component a specimen from pacient is used. The specimen is suspicious to contain the given component. If it is true, the component is bound When all components bind respectivelly, a not-interrupted chain is formed At the end there is a labelling agent

11. Washing out and its importance When also the components that are not bound to the surface would remain, we would not be able to differenciate a positive reaction and a negative one. That is why after each step washing follows. After such a washing, only bound components remain present. When the chain is broken, the part after the missing component is washed out.

12. Example of positive and negative course Laboratory antibody Searched antigen Antigen missing Labelled laboratory antibody (  detection) Labelled laboratory antibody + – It is not bound  it is washed away  it cannot be detected SURFACE (slide, bottom of a well in a serological panel) Patient specimen

13. Types of labelling agent Fluorescent dye is labelling agent in immunofluorescence Radioisotope is labelling agent in RIA Enzyme is labelling agent in ELISA –Immunoblots are a special type of an ELISA, where individual antigens are divided electroforetically When an enzyme is used as a labelling agent, the very last component should be the substrate – so one more component.

14. Immuno- fluorescence and RIA

15. Immunofluorescence www.biologie.uni-hamburg.de

16. Immunofluorescence www.amsbio.com www.kcl.ac.uk www.bindingsite.com

17. Positive result in both direct and indirect IMF looks the same

18. Examples of immunofluorescence (diagnostics of Treponema pallidum) a) Direct imunofluorescence (Surface)-(antigen)-(labelled antibody) b) Indirect imunofluorescence (Surface)-(antigen)-(antibody)-(labelled antibody against human antibodies) Advantage: This reaction uses microscopical slide as surface. This enable us to se the shape of the microorganism.

19. Immunofluorescence reaction schemes A: Treponema pallidum – patient‘s origin B: Labelled antibody against Treponema pallidum (laboratory) C: Treponema pallidum – laboratory origin D: Antibody against Treponema pallidum – patient‘s origin E: Labelled labor. antibody against human antibody (conjugate)

20. Radioimmunoassay www.chbr.noaa.gov darc.tbzmed.ac.ir

21. ELISA: principle

22. ELISA www.genprice.com

23. ELISA www.cellsignal.com virology-online.com

24. ELISA – why used so much In ELISA reaction we have at the end of the whole process an enzymatic reaction. Its intensity is simply described as intensity of colour in a well with the reaction. Very intensive colour = highely positive reaction Simplicity for technique and zero radiation is an advantage in comparison with RIA Possibility of automatisation and not so big need for experience are an advantage in comparison with immunofluorescence

25. Examples of component system blue = component from specimen taken from pacient‘s body Surface-antigen-antibody-labelling agent (D) Surface-antibody-antigen-antibody-labelling agent (D, e. g. detection of HBsAg) Surface-antigen-antibody-antigen-labelling agent (I) Surface-antigen-antibody-conjugate-labelling agent (I) Conjugate is an antibody against human antibody

26. Importance of the conjugate Conjugate is used mostly in indirect reactions (detection of antibodies) It is an antibody that has human antibody (e. g. IgM, IgA or IgG) for an antigen It can be selective against a certain antibody class Use of conjugate is the principle of selective diagnostic of individual immunoglobulin classes

27. ELISA antibody detection: 1. Positive (searching IgM, IgM present) All components bind step by step. An enzymatic reaction leads to colour change in the well.

28. ELISA antibody detection: 2. Negative I (searching IgM, no antibodies) No antibodies in pacient‘s serum. Conjugate flown out, no change in the well.

29. ELISA antibody detection: 3. Negative II (searching IgM, IgG present) Only IgG antibodies in patients serum. Conjugate flown out, no change in the well.

30. ELISA: practical reading

31. ELISA – practical description Usually we have a microtitration plate. Unlike classical serological reactions, each patient has here not the entire row, but one well only. That is because titers are not determined. First wells, foregoing pacient wells, might be: –Bl – blank (for spectrophotometer calibration) –K– and K+ – positive and negative controls –Cut off (c. o., two or three wells) – „cutts off“ the negative results from positive ones sharply, or plus minus 10 %) Producer provides us with „specimens“ with positive (K+, negative (K–) and borderline (c. o.) values of absorbance Each ELISA kit is different, like their producers. Some of them do not have any blank. Some of them do not have cut off wells and the cut off is count as average of negative controls + a constant.

32. ELISA – an example (www.medmicro.info) ELISA reaction negative well positive well

33. Example of ELISA to antigen detection (antigen of Helicobacter pylori) In ELISA reaction, there is an enzymatic reaction at the end of the process. The intensity corresponds with the colour intensity in the well. Collour intensity can be evaluated spectrophotometrically As positive we consider values higher than reference given „cut off“ Usual principle: Surface-antibody-antigen- antibody-enzyme-substrate

34. Example of ELISA to antibody detection In indirect antibody detection using ELISA usually IgM and IgG (or IgA) are assessed separatelly Values higher than „cut off“ are referred as positive (this is not different from antigen detection) Unlike antigen detection, we use to have a „borderline field“. For example, results ranging 90 % to 110 % of cut off vallue are refered as „borderline“, below 90 % as „negative“, above 110 % as „positive“ Common principle: Surface-antigen-antibody- conjugate-enzyme-substrate

35. Example of an ELISA scheme to antibody detection BL4 4 K-5 5 6 6 K+7 7 8 8 1919 2102 3113 IgA IgG

36. Immunoblots

37. Western blotting Language joke (researcher Southern) In fact, it is an ELISA, but antigenic mixture is divided electroforetically to individual determinants So it is more precise, and helpfull mostly in situations, where classical ELISA is problematized by cross-reactions Western blotting is exceptional in the fact that it cannot be used in antigen detection

38. Western blotting – principle 1: original antigen (mixed) 2: decomposition of antigen by a detergent 3: electroforetic division of antigen 4: „blotting“ of divided antigen to a nitrocelulose membrane 5: ELISA reaction (only some antibodies present)

39. Western blot – example (picture from www.medmicro.info)

40. New type immunoblots In new type immunoblots, the antigen is not divided electroforetically Recombinant antigents are placed to different parts of a strip using special technology The reading can be done „normally“, or using a scaner Various different type of immunoblots exist

41. Example of newer immunoblots

42. Example of reading of a Western blot (in Lyme borreliosis) Presence of at least two specific bands (labelled on a pattern)  assessed as positive Exceptions: –in IgG positivity of one band is sufficient, if it is vlsE band (it is highly specific) –in IgM positivity of one band is sufficient, if it is ospC band (it is highly specific)

43. Immunochromatographic tests Immunochromatographic tests are based on binding of individual components, simillarly as previous tests One important difference is that there is no washing here. Some components are bound to surface in specific sites (tests site, control site), other components are bound to a tested component immediatelly and „travel“ together through the porous layer. In positive case, both test and control strip is usually visible; in negative case, control strip is visible only

44. One example of immunochromatography test principle http://www.bl-inc.jp/images/immuno_ge.gif

45. Principle (only one of possibilities) + – Test area Control area

46. Explanation for previous animation In positive case, some labelled antibodies are bound to antigen, and then they remain in test area. Some other molecules come to the control area In negative case, all labelled antibodies come to the control area antibody fixated to a surface labelled antibody labelled antibody + antigen antibody against labelled antibody

47. Advantages of immunochromatography tests They are very quick (tens of minutes) They are very simple  some of them are not performed in a laboratory, but directly at a clinician They are quite precise They can be used for many purposes (including extra-microbiological ones, e. g. pregnancy tests) Disadvantage: they use to be rather expensive, if compared with traditional tests

48. Typical microbiological example Clostridium difficile is an important intestinal pathogen. It is an anaerobic bacterium, living in large intestine, also at normal conditions. At some circumstances (usually use of broad- specter antibiotics, mostly lincosamides) C. d. overmultiplies and its toxins are overproduced Test for both toxins (A/B) production shows positivity by two visible blue strips (Test area, Ctrl area). One strip only (Ctrl area) shows a test that is valid, but negative.

49. The End (The picture name: Antibody) www.twitchfilm.net/archives/003401.html

50. Treponema pallidum A spirochet, causing syfilis Syfilis is a classic sexual disease. It is transmitted sexually only. But it is a systemic disease – in developped stages the whole body is affected (gummata, aortal dissection, neurolues, psychical symptomas) Some subspecies of T. pallidum and some other treponemas cause other diseases (framboesia – yaws, T. pertenue) Some treponemas are non-pathogenous www.primer.ru

51. Borrelia burdorferi sensu lato Another spirochet, causing Lyme disease (after town of Old Lyme in the U. S. A.) and subclassified into Borrelia burgdorferi sensu stricto (= „strict sense of meaning“), Borrelia garinii, Borrelia afzelii and others The disease is characterized by so named erythema migrans and then usually more symptomas follow While in the USA mostly B. b. sensu stricto is common and joint symptomatology is common, in Europe B. garinii and B. afzelii are more common, and the typical disease is neuroborreliosis Besides lyme diseases there exist other species causing recurrent fever (B. duttoni, B. recurrentis) http://www.pasteur.fr

52. Helicobacter pylori Peptic (= gastric / duodenal) ulcus is caused by more causes. Such diseases are called multifactorial diseases. The part of Helicobacter pylori, a spiral rod (not spirochet!), on ulceral disease is still discussed, not only among GPs, but even among specialists. Even healthy persons may have a helicobacter in their stomach. Nevertheless, certain and not negligeable role of this pathogen is sure. http://vietsciences.free.fr

53. Check-up questions 1. What diagnostic value could be a finding of antigen using ELISA in comparison with a finding of antibodies using the same method? (generally) 2. Which clinical sample is usually taken for a proof of antibodies and which should be suitable for a proof of antigen? (Think generally; in particular situations it is necessary to think about pathogenesis of infection) 3. Compare similarities and differencies in ELISA and westernblotting. 4. Why dilution in geometric series usually is NOT used for a proof of antibodies using ELISA? 5. Which is a diagnostic importance of various subclasses of immunoglobulins? 6. What antibodies can pass through placenta? 7. Why individual subclasses of immunoglobulins cannot be detected by “traditional” serological methods (e. g. agglutination, CFT, neutralisation)? 8. Immunochromatographic tests: are they used also in other situation than examination for Clostridium difficile toxin? 9. What is the meaning of „cut off“? 10. What is the name of the device used (besides many other uses) also for reading of ELISA results?