Introduction to serology, agglutination and precipitation,

dilution (topic J07)

Ondřej Zahradníček Practical of Medical Microbiology

(VLLM0421c) Contact 777 031 969 zahradnicek@fnusa.cz ICQ 242-234-100

Methods in clinical microbiology Direct methods: detection of a microbe,

its part of its product. Microscopy, culture, biochemical identification, antigen analysis. Positivity = it is sure, that the ages in NOW present.

Indirect methods: detection of antibodies against the microbe. Positivity = the microbe met the host IN HISTORY (weeks / months / years)

Antigen and antibody Antigen = a macromolecule coming from an

alien organism: plant, microbe, animal. 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)

Two ways how to use it:Antigen detection: laboratory (animal origin) antibodies + pacient‘s sample or microbial strain.

Direct methodAntibody detection: laboratory antigen (microbial) + pacient‘s serum (or saliva).

Indirect method

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 get the information 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

1

1

2

2

How to perform the reaction „quantitativelly“ It is very dificult to assess the amount of

antibodies in units e. g. 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

a lot of antibodies in serum It reacts only when diluted a few times

only small amounts of antibodies

Task 1: Geometrical row 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 1 : 4

One halfth of 1 : 4 …… 1 : 8 Etc., etc.

Task 2

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

Precipitation, agglutination, agglutination on carriers Precipitation: Antigens act alone, as

macromolecules (coloid antigen) Agglutination: Antigen act being part

of its microbial cell (we work with whole microbes)

Agglutination on carriers: Formerly isolated antibodies are bound to an alien particle – latex or RBC

Schematical difference

1 – precipitation

2 – agglutination

3 – agglutination on a carrier

1 2 3

Task 3 3a: Determine agglutination titre for

antibodies against yersinia

Do not forget – titre = highest dillution with still positive reaction. First well is diluted 1 : 2, second – 1 : 4 , etc.

As we do not actually have testing kits, see the result only using this demonstration:

First collumn are controls, the reaction starts in the second collumn

1:2 1:4 1:8

Task 3b: Enteropathogenous Escherichia coli There are cca 12 antigenic types

belonging to EPEC group We use polyvalent sera: serum A contains

antibodies againts several EPEC serotypes, serum B antibodies agaist some more serotypes. Turbidity = positive

When one of sera (A or B) is „+“, we have to continue using monovalent sera

Do you understand, why it is impossible to asses titres here?

Task 4: Treponema pallidum haemagglutination (TPHA)

Technically the same as in task 3a, but: Difference 1: done only qualitativelly

(no titre evaluation) Diference 2: it is red (as instead of

bacteria, red blood cells are used)

For technicalreasons,demonstrarion only:

+++ ++ + +/-

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Task 5: precipitation Task 5a: 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 syphilitics)

Done only qualitativelly. First well is positive controll, second well is negative controll, and then each patient has one well only.

0.05 ml of serum + 0.05 ml of kardiolipin

Demonstration of another type of precipitation

Microprecipitation in agar accord. to Ouchterlony

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Task 5b – ring precipitation

Precipitation for antigen detection: 1) animal serum with antibody 2) four diferent strain extracts 3) positivity: a ring formed at contact

Nice day to everybody!