Searching for microbes Part IX. Neutralisation reaction Ondřej Zahradníček To practical of VLLM0421c Contacts to me: 777 031 969 zahradnicek@fnusa.cz ICQ 242-234-100 Content of this slideshow Tale • Once there was a killer toxin, and the toxin wanted to kill a red blood cell • That toxin had in also character of an antigen, that chalenges the body to produce antibodies • And when the toxin prepared for killing 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. What to learn from the tale • Today, we have neutralisation reaction • This reaction is important in viruses and bacterial toxins, that can be directly neutralized by a corresponding antibody • The whole bacterium is rarely neutraized like that • Majority of neutralisation application is in virology. An exception is the most common serological reaction at all – ASO reaction Neutralisation reaction – principle 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 Examples of neutralisation reactions Individual neutralisation reactions ASO • Principle: The antibody blocates the haemolytical effect of the toxin (streptolyzin O) on the RBC. Positive is blocation of haemolysis (as in CFT, but for a different reason) • The microtitration plate is composed of a positive control and seven patient • The titer above 250 is supposed to be risky for an autoimmune disease. Course of serum dillution – ASO 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. • So: Potato-like shape = negative response. Dense round target = positive response • Example of use: We can read HIT results for tick-borne encephalitis. In each patient an accute and a reconvalescent serum is evaluated Interpretation of accute vs. reconvalescent sera is of course the same as in any other serological reaction Remember: • HIT is not an agglutination reaction, it is neutralisation of viral agglutination • HIT differs from ASO reaction mostly by the fact, that the RBCs are not haemolyzed, but agglutination. But the fact, that a specific antibody blocates the reaction is valid in both of the • HIT for detection of antibodies against tick borne encephalitis (unlike ASO) is again a typical „indirect diagnostic“ HIT for tick-borne encephalitis: example of a clinical situation • We have several patients with suspicion for tick borne encephalitis, already tested using complementfixing test (see in J08 practical) • Now we have decided to use an independent test to check the results VNT (do not confuse with TNT J ) • Virus Neutralisation Test • Cell culture uses to be dammaged by a virus. The dammage is visible as a change of colour from original yellow to changed red (pH is changed) • Antibodies, if present, may prevent this viral action on the cell culture, so the colour remains yellow • Titre = last well with unchanged colour VNT – clinical situation • Patient R. S., 35 years, has chronical pain in chest. Cardiological examination showed suspition for inflamation of heart muscle (myocarditis) • As coxackieviruses are common causative agent of myocarditis, it was decided to perform test of antibodies against these viruses VNT – example of use in coxsackieviruses • The whole panel belongs to one patient examination. Odd rows = accute serum, even rows = reconvalescent rows. Every two rows = one coxsackievirus (B[1] to B[6]) • 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. • 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, but it is more likelly that the coxsackievirus with the lower titre has a cross-reaction only ASO and its importance What is the antistreptolyzin O and why we attempt to detect it • After every streptococcal infection antibodies are produced, often including antibodies against streptococcal toxin – streptolysin O. • Nevertheless, sometimes after infection the antibodies increase instead of decreasing. Antibodies are bound to some structures of the host organism (autoimmunity), so a „circulus vitiosus“ starts to run • In such a situation, paradoxically the antibodies are worse than the pathogen that challenged the antibody response to protect us. Remember: • 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 • Indication for ASO examination: suspicion for so named „late sequellae“ of streptococcal infection: accute glomerulonephritis, or rheumatoid fever Rheumatic Fever Accute glomerulonephritis Acute glomerulonephritis II ASO examination principle: haemolysis neutralisation The End Coxsackieviruses: survey of family Picornaviridae • Family Picornaviridae contains mostly following viruses important for humans: • enteroviruses, (name shows their way of transmission, but they cause infection mostly outside intestine!) further classified into – polioviruses – viruses of poliomyelitis – coxsackieviruses and echoviruses – newer enteroviruses 68, 69, 70 and 71 • rhinoviruses – viruses of common cold • virus of hepatitis A Coxsackieviruses – more info • There exist coxsackieviruses A1–A22, A24 and B1–B6 • Diagnostic can be done by virus isolation on newborn mice or tissue cultures • Indirect diagnostic is difficult because of cross-reactions; nevertheless, it is used in coxsackieviruses of B group in suspicion for myocarditis Coxsackieviruses – pathogenicity • CNS: aseptic meningitis (majority of types) • herpangine (A types, mostly A4) • hand-foot-mouth disease (A16) • respiratory infections (all types) • myocarditis and other muscle disease (B types) • lymphadenitis (all types) • relation of some types of diabetes mellitus (B group)?