Searching for microbesPart VIII.

Reactions with labelled components

Ondřej ZahradníčekTo practical of VLLM0421c

zahradnicek@fnusa.cz

Content of this slideshow

Classes of antibodies

Reactions with labelled components: survey

Immunofluorescence and RIA

ELISA: principle

ELISA: practical reading

Western blotting

Immunochromatographic tests

Check-up questions

Introduction (tale)

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.

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.

Classes of antibodies

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)

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

Reaction with labelled components: survey

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

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.

Example of positive and negative course

Laboratory antibody

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

Types of labelling agent

• Fluorescent dye is labelling agent in immunofluorescence

• Radioisotope is labelling agent in RIA

• Enzyme is labelling agent in ELISA– Western blotting is 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.

Immuno-fluorescence and RIA

Immunofluorescence

www.biologie.uni-hamburg.de

Immunofluorescence

www.amsbio.com

www.kcl.ac.uk

www.bindingsite.com

Positive result in both direct and indirect IMF looks the same

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.

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)

Radioimmunoassay

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

ELISA: principle

ELISA

www.genprice.com

ELISA

www.cellsignal.com

virology-online.com

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

Examples of component systemblue = 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

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

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.

ELISA antibody detection:2. Negative I (searching IgM, no antibodies)

No antibodies in pacient‘s serum. Conjugate flown out, no change in the well.

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

ELISA: practical reading

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.

ELISA – an example (www.medmicro.info)

ELISA reaction

negative well

positive well

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

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

Example of an ELISA scheme to antibody detection

BL 4 BL 4

K- 5 K- 5

K- 6 K- 6

K+

7 K+

7

K+

8 K+

8

1 9 1 9

2 10 2 10

3 11 3 11IgA IgG

Western blotting

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

Western blotting – principle1: original antigen (mixed)2: decomposition of

antigen by a detergent3: electroforetic division of

antigen

4: „blotting“ of divided antigen to a nitrocelulose membrane

5: ELISA reaction (only some antibodies present)

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

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)

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

One example of immunochromatography test principle

http://www.bl-inc.jp/images/immuno_ge.gif

Principle (only one of possibilities)

+

–

Test area

Control area

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

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

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.

The End

(The picture name: Antibody)

www.twitchfilm.net/archives/003401.html

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

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

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

Check-up questions1. 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?