1. Searching for microbes Part VIII. Reactions with labelled components
Ondřej Zahradníček
To practical
of aVLLM0421c

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

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 field glasses tied to the rescue circle, the rescue circle tied to rescue boat, and the rescue boat to the board. So even the highest waves were not able to take the objects away.
Once the sailor‘s husband came. He wanted to assay the rescue circle. He untied the field glasses away and he took the circle from the boat.
A wave came – and the field glasses 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 infections. Specific IgE antibodies against a certain pathogen are rarely examined
Class IgD antibodies are not examined in 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 patient is used. The specimen is suspicious to contain the given component.
If it is true, the component is bound
When all components bind respectively, 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 differentiate 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
Patient specimen +
Laboratory antibody
Searched antigen
Labelled laboratory antibody (detection) –
Laboratory antibody
Antigen missing
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)

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

16. Immunofluorescence

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

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

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
darc.tbzmed.ac.ir

21. ELISA: principle

22. ELISA

23. ELISA
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 = highly positive reaction
Simplicity for technique and zero radiation is an advantage in comparison with RIA
Possibility of authomatisation 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 patient'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 patient'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 titres are not determined.
First wells, foregoing patient wells, might be:
Bl – blank (for spectrophotometer calibration)
K– and K+ – positive and negative controls
Cut off (c. o., two or three wells) – „cuts 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 (Inst. for Microbiology)
ELISA reaction
positive well
negative 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.
Colour 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 separately
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 value are referred 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 detectionBL 4 K- 5 6 K+ 7 8 1 9 2 10 3 11
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 helpful mostly in situations, where classical ELISA is less sure because of 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: electrophoretic 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 Inst. for microbiology)

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, similarly 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 immediately 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

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 + antigen
labelled antibody
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 examples
Tests for two respiratory pathogens causing pneumonia – although they are obviously present mostly in the lungs, they also may appear in bloodstream and antigens detected in urine:
Streptococcus pneumoniae (pneumococcus), a typical causative agent of pneumonia (more in P02 practical session)
Legionella pneumophila, another causative agent of pneumonias, mostly in seniors, important role of water aerosols (more in P06 practical session)
Test for A + B toxins and structural antigen of Clostridium difficile – we are going to see it in practical session P07 (in November)

49. The End (The picture name: Antibody)

50. Treponema pallidum A spirochete, causing syphilis
Treponema pallidum
A spirochete, causing syphilis
Syphilis is a classic sexual disease. It is transmitted sexually only. But it is a systemic disease – in developed stages the whole body is affected (gummata, aortal dissection, neurosyphilis, psychical symptomas)
Some subspecies of T. pallidum and some other treponemas cause other diseases (framboesia – yaws, T. pertenue)
Some treponemas are non-pathogenous
More in P09 practical session

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)
More in P09 practical session

52. Helicobacter pylori
Peptic (= gastric / duodenal) ulcus is caused by more causes. Such diseases are called multifactorial diseases.
Helicobacter pylori plays an important role in this disease. Although healthy persons with Helicobacter exist, the importance of Helicobacter is crucial and its eradication is an important part of treatment. More in P04 topic.

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 differences in ELISA and western blotting.
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?