Searching for microbesPart IX.Part IX.

Neutralisation reactionNeutralisation reaction

Ondřej ZahradníčekOndřej Zahradníček

To practical of VLLM0421c

Contacts to me:Contacts to me:

777 031 969 zahradnicek@fnusa.cz777 031 969 zahradnicek@fnusa.cz

ICQ 242-234-100

Content of this slideshowContent of this slideshow

Neutralisation reaction – principle

Individual neutralisation reactions

ASO and its importance

TaleTale

• Once there was a killer toxin, and the toxin • Once there was a killer toxin, and the toxin wanted to kill a red blood cell

• That toxin had in also character of an • That toxin had in also character of an antigen, that chalenges the body to produce antigen, that chalenges the body to produce antibodies

• And when the toxin prepared for killing the RBC, an antibody, crossed his way, the RBC, an antibody, crossed his way, bound to it and did not allow him killing

• The red blood cell was very happy, and it sedimented to the bottom with other RBCs.sedimented to the bottom with other RBCs.

What to learn from the taleWhat to learn from the tale

• Today, we have neutralisation reaction• Today, we have neutralisation reaction

• This reaction is important in viruses and bacterial toxins, that can be directly bacterial toxins, that can be directly neutralized by a corresponding antibodyneutralized by a corresponding antibody

• The whole bacterium is rarely neutraized like thatlike that

• Majority of neutralisation application is in • Majority of neutralisation application is in virology. An exception is the most common serological reaction at all – ASO common serological reaction at all – ASO reactionreaction

Neutralisation Neutralisation Neutralisation reaction –reaction –reaction –principleprincipleprinciple

Neutralisation reaction: general principleNeutralisation reaction: general principle

• There are many ways, how antibodies • There are many ways, how antibodies do work. One of them is direct neutralising effectneutralising effect

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

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

Neutralisation schematicallyNeutralisation schematically

• Antibody (Ig) prevents an effect of a toxin/virus to a cell / red blood celltoxin/virus to a cell / red blood cell

+ –+ –+ –

Cell in a Toxin or Toxin or Antibody Cell in a Cell in a tissue culture or

Toxin or virus

Toxin or virus

Antibody Cell in a tissue culture or culture or

a red blood cell

culture or a red blood cellblood cell blood cell

Examples of neutralisation reactionsExamples of neutralisation reactions

Task Neutralised Object ReactionTask Neutralised Object Reaction

1 Bacterial toxin RBC ASO1 Bacterial toxin (haemolysin)

RBC

haemolysisASO

2 Virus RBC HIT2 Virus RBC agglutination

HITagglutination

3 Virus Cell metabolic effect

VNT3effect

VNT

Individual Individual Individual neutralisation neutralisation neutralisation reactionsreactionsreactions

ASOASO

• Principle: The antibody blocates the • Principle: The antibody blocates the haemolytical effect of the toxin (streptolyzin O) on the RBC. Positive is blocation of O) on the RBC. Positive is blocation of haemolysis (as in CFT, but for a different haemolysis (as in CFT, but for a different reason)

• The microtitration plate is composed of a • The microtitration plate is composed of a positive control and seven patientpositive control and seven patient

• The titer above 250 is supposed to be risky for an autoimmune disease.for an autoimmune disease.

Course of serum dillution – ASO Course of serum dillution – ASO

Common course (dillution with reometric row, Common course (dillution with reometric row, coefficient 2) would be too rough, we need a more detailed one. In fact, it is a geometric row too, but the detailed one. In fact, it is a geometric row too, but the coefficient is 1,2 and not two as usually

HITHIT• Haemagglutination Inhibition Test: Pay attention, it

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

• So: Potato-like shape = negative response. • So: Potato-like shape = negative response. Dense round target = positive response

• Example of use: We can read HIT results for tick-• Example of use: We can read HIT results for tick-borne encephalitis. In each patient an accute and a reconvalescent serum is evaluatedreconvalescent serum is evaluated

Interpretation of accute vs. reconvalescent sera is of Interpretation of accute vs. reconvalescent sera is of course the same as in any other serological reaction

Remember:Remember:

• HIT is not an agglutination reaction, it is • HIT is not an agglutination reaction, it is neutralisation of viral agglutination

• HIT differs from ASO reaction mostly by the • HIT differs from ASO reaction mostly by the fact, that the RBCs are not haemolyzed, but fact, that the RBCs are not haemolyzed, but agglutination. But the fact, that a specific antibody blocates the reaction is valid in antibody blocates the reaction is valid in both of theboth of the

• HIT for detection of antibodies against tick borne encephalitis (unlike ASO) is again a borne encephalitis (unlike ASO) is again a typical „indirect diagnostic“typical „indirect diagnostic“

HIT for tick-borne encephalitis: HIT for tick-borne encephalitis: example of a clinical situationexample of a clinical situation

• We have several patients with suspicion for tick borne encephalitis, already

• We have several patients with suspicion for tick borne encephalitis, already tested using complementfixing test (see tested using complementfixing test (see in J08 practical)

• Now we have decided to use an independent test to check the resultsindependent test to check the results

VNT VNT (do not confuse with TNT ☺ )

• Virus Neutralisation Test• Virus Neutralisation Test

• Cell culture uses to be dammaged by a • Cell culture uses to be dammaged by a virus. The dammage is visible as a change of virus. The dammage is visible as a change of colour from original yellow to changed red (pH is changed)(pH is changed)

• Antibodies, if present, may prevent this viral • Antibodies, if present, may prevent this viral action on the cell culture, so the colour remains yellowremains yellow

• Titre = last well with unchanged colour• Titre = last well with unchanged colour

VNT – clinical situationVNT – clinical situation

• Patient R. S., 35 years, has chronical • Patient R. S., 35 years, has chronical pain in chest. Cardiological examination pain in chest. Cardiological examination showed suspition for inflamation of heart muscle (myocarditis)muscle (myocarditis)

• As coxackieviruses are common • As coxackieviruses are common causative agent of myocarditis, it was decided to perform test of antibodies decided to perform test of antibodies against these virusesagainst these viruses

VNT – example of use in coxsackievirusesVNT – example of use in coxsackieviruses

• The whole panel belongs to one patient examination. Odd rows = accute serum, even rows = Odd rows = accute serum, even rows = reconvalescent rows. Every two rows = one reconvalescent rows. Every two rows = one coxsackievirus (B1 to B6)

• First collumn has dillution 1 : 5 (then 1 : 10, 1 : 20…)• First collumn has dillution 1 : 5 (then 1 : 10, 1 : 20…)

• Last collumn = controls. When there are six yellow and six red wells here, everything is OK.and six red wells here, everything is OK.

• Titre is the last well with unchanged (yellow) collour.• Titre is the last well with unchanged (yellow) collour.

• When two coxsackieviruses have a significant (at least four-fold) increase of titer, it might be a co-infection, four-fold) increase of titer, it might be a co-infection, but it is more likelly that the coxsackievirus with the lower titre has a cross-reaction onlylower titre has a cross-reaction only

ASO and its ASO and its ASO and its importanceimportanceimportance

What is the antistreptolyzin O and why What is the antistreptolyzin O and why we attempt to detect it

• After every streptococcal infection antibodies are • After every streptococcal infection antibodies are produced, often including antibodies against produced, often including antibodies against streptococcal toxin – streptolysin O.

• Nevertheless, sometimes after infection the • Nevertheless, sometimes after infection the antibodies increase instead of decreasing. Antibodies are bound to some structures of the Antibodies are bound to some structures of the host organism (autoimmunity), so a „circulus vitiosus“ starts to runvitiosus“ starts to run

• In such a situation, paradoxically the antibodies • In such a situation, paradoxically the antibodies are worse than the pathogen that challenged the antibody response to protect us.antibody response to protect us.

Remember:Remember:

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

• Indication for ASO examination: suspicion for so named „late sequellae“ of for so named „late sequellae“ of streptococcal infection: accute streptococcal infection: accute glomerulonephritis, or rheumatoid fever

Rheumatic FeverRheumatic Fever

http://mednote.co.kr

Accute glomerulonephritisAccute glomerulonephritisDiffuse inflammatory cellular infiltration and mesangial hypercellularity (Hematoxylin and Eosin Staining: original

www.ispub.com

hypercellularity (Hematoxylin and Eosin Staining: original magnification X 200)

Acute glomerulonephritis IIAcute glomerulonephritis II

iws.ccccd.edu

ASO examination ASO examination principle: principle: haemolysis haemolysis neutralisation

In Czech Republic, In Czech Republic,

abbreviation ASLO

is used for the is used for the

same thing as

ASO in EnglishASO in English

The EndThe End

http://web.uct.ac.za/depts/mmi/stannard/emimages.html

Coxsackieviruses: survey of Coxsackieviruses: survey of family Picornaviridae

• Family Picornaviridae contains mostly • Family Picornaviridae contains mostly following viruses important for humans:following viruses important for humans:

• enteroviruses, (name shows their way of • enteroviruses, (name shows their way of transmission, but they cause infection mostly outside intestine!) further classified intooutside intestine!) further classified into

– polioviruses – viruses of poliomyelitis

– coxsackieviruses and echoviruses

– newer enteroviruses 68, 69, 70 and 71– newer enteroviruses 68, 69, 70 and 71

• rhinoviruses – viruses of common cold• rhinoviruses – viruses of common cold

• virus of hepatitis A

Coxsackieviruses – more infoCoxsackieviruses – more info

• There exist coxsackieviruses A1–A22, A24 • There exist coxsackieviruses A1–A22, A24 and B1–B6and B1–B6

• Diagnostic can be done by virus isolation • Diagnostic can be done by virus isolation on newborn mice or tissue cultures

• Indirect diagnostic is difficult because of • Indirect diagnostic is difficult because of cross-reactions; nevertheless, it is used in cross-reactions; nevertheless, it is used in coxsackieviruses of B group in suspicion for myocarditismyocarditis

Coxsackieviruses – pathogenicityCoxsackieviruses – pathogenicity

• CNS: aseptic meningitis (majority of types)• CNS: aseptic meningitis (majority of types)

• herpangine (A types, mostly A4)

• hand-foot-mouth disease (A16)• hand-foot-mouth disease (A16)

• respiratory infections (all types)• respiratory infections (all types)

• myocarditis and other muscle disease• myocarditis and other muscle disease(B types)

• lymphadenitis (all types)• lymphadenitis (all types)

• relation of some types of diabetes • relation of some types of diabetes mellitus (B group)?