Complementfixing test(CFT, topic J08)

Neutralisation reaction (J09)

Ondřej Zahradníček Practical of Medical Microbiology

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

Review: 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)

Review: Antigen / 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)

Review: 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

Review: 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. Some ways how to assess, when the microbe met the body: Amount of antibodies (relative – titre) and

its changes during the time (dynamics) Class of antibodies: IgM/IgG (More in J10) (Avidity of antibodies)

Dynamics of titre Absolute amount of antibodies is not the

most sure information: some patients are poor antibody-producers, etc.

Dynamicst of titre: better, means how the response gets changed during the time (usually during two or three weeks)

1 first pacient‘s visit

1

2 after 2 – 3 weeks

2

Pair sera and non-pair sera Pair sera = first specimen is kept in the

refrigerator until the second comes to the lab, and thed examined together. 4-fold increase is told to be significant under such circumstances

Other situations (second specimen is examined separately): an accidental error should be taken into account. So, usually at least 8-fold increase is needed

Complement-fixing test (CFT) Complement = one component of

immunity reaction For CFT, we use animal (guinea-pig)

complement. The patient‘s complement is inactivated before the reaction

Complement is not able to get bound to isolated antigen

Complement is not able to get bound to isolated antibody

Complement is able to get bound to COMPLEX antigen - antibody

Use of CFT CFT is used for diagnostics of many

(mainly viral) pathogens CFT, like other serological reactions,

may be used for antigen detection or antibody detection

For simplification, we shall only speak about antibody detection in this practical

So, we think about a laboratory antigen being mixed with patient‘s serum

Complement – how it does work (Task 1) Sheep RBC + amboceptor without

complement no haemolysis Sheep RBC + complement without

amboceptor no haemolysis Sheep RBC + complement +

amboceptor haemolysis Rabbit RBC + complement +

amboceptor no hemolysis

Titration of complement (T 2)

For the reaction, we need an amount of guinea-pig complement that is neither too small nor too big

That is why we test, what amount of complement is just able to perform haemolysis of a specified amount of red blood cells with amboceptor

Titration of complement results Dilution of guinea-pig serum (as a

source of the complement): 1 : 32 = haemolysis 1 : 36 = haemolysis 1 : 40 = haemolysis 1 : 44 = no haemolysis

Haemolytical unit corresponds to 1 : 40 To obtain 2 units, we have to dilute this

serum 1 : 20

CFT – principle (Task 3 a, b)

Problems existing in CFT

Too much complement: false negative results. What to do? Titrate the complement (according to Task 2)

Something in serum binding the complement itself (anticomplementarity component): false positive results. What to do? Perform anticomplementarity test without antigen

Anticompl. test (Task 3 c, d)

Tasks 4 and 5: CFT Task 4: The whole seropanel belongs to

one patient. We have six respiratory pathogens, each in two rows (acute speciemen, reconvalescent specimen). We have 7 dilutions of sera (first collumn is the anticomplementarity test)

Task 5: The seropanel belongs to a positive control and six patients. We test Ab against tick-borne encephalitis. Mind the anticomplementarity test!

Neutralisation reaction: general principle There are many ways, how antibodies

do work. One of them is direct neutralising effect

This effect is rarely present in whole bacteria. On the other hand, it may be observed in whole viruses, and in bacterial toxins

Nevertheless, sometimes antibodies neutralise some characteristic of the whole bacteria, e. g. motility of Treponema in Nelson‘s test

Neutralisation schematically Antibody (Ig) prevents an effect of a

toxin/virus to a cell / red blood cell Example: ASO (Toxin = streptolysin O,

Ig = antistreptolysin O, red blood cell)

Task 1 - ASO ASO is not an indirect diagnostics

reaction, despite the fact that we search for antibodies. The aim is not to get a pathogen, but to assess the antibodies themselves, as they may be dangerous

In ASO, we do not use the geometrical row. The dilutions in individual wells in the panel are written on a paper

The titer above 200 is supposed to be risky for an autoimmune disease

Task 2: HIT Haemagglutination Inhibition Test: Pay

attention, it is NOT an agglutination reaction, it is a neutralisation! Antibody neutralises the aggregation of RBCs due to viruses.

Task: Read HIT results for tick-borne encephalitis. Read titers in accute and reconvalescent sera and make a conclusion (accute infection? Or only memory antibodies?)

Task 3: VNT (do not confuse with TNT )

Virus Neutralisation Test Cell culture uses to be dammaged by a

virus. The dammage is visible as a change of colour (pH is changed)

Antibodies, if present, may prevent this viral action on the cell culture

Nice day to everybody!