Searching for microbes Part VIII. Complementfixing test Ondřej Zahradníček To practical of VLLM0421c Contacts to me: 777 031 969 zahradnicek@fnusa.cz ICQ 242-234-100 Content of this slideshow Tale • There was a curious park guard. • He wanted to know the true relations between a boy and a girl that used to visit his park. Are they a couple, or they aren‘t? • He knew, there is one only bench in the park. When one wanted to embrass somebody else somewere, one had to do it there. • So, he placed parts of a plant (globules with hooklets, see next slide) on the bench with hope, that the couple would catch them on their clothes The plant However – how to ascertain… • …when both the girl and the boy used another exit? • Then the guard realized, that during a moment his niece and her boy-friend will come to him, and he was sure, that on the way through the park, they will certainly use the bench for embrassing. • And so he made a plan: when his niece and her boy-friend will have globules on them, it means, that the first couple was no true couple, as it did not catch the globules first. What to learn from the tale • Today we have to learn complement fixing test, quite a complicated test. • Not only that we use complement to visualise antigen-antibody complex, but also two more parts of the reaction: the indicator couple (niece and boy-friend). • This couple consists of indicator antigen (sheep RBC) and indicator antibody (amboceptor = rabbit antibody against sheep RBC) Dynamics of titre Interpretation of serological reactions • 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 (titre) and mostly 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. • Dynamics of titre: better, means how the response gets changed during the time (usually during two or three weeks) Why the titer alone is not sufficient • Sometimes a patient with low reactivity has a low titre even in an accute phase Pair sera and non-pair sera • Pair sera = first specimen is kept in the refrigerator until the second comes to the lab (cca 10–14 days), 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 Dynamics of titre – more aspects • A special situation is so named seroconversion – there are no antibodies in the first specimen (it is too soon), but there are antibodies in the second one. Such a finding is more sure than the four-fould increase • Sometimes titer decrease is found instead of increase (a subaccute infection) • Titre value does not correspond to infection activity. Often the highest amount of antibodies comes after the end of disease. Examples of various effects of titre dynamics: • 1 – 2: seroconversion • 3 – 4: titre elevation • 5 – 6: titre decrease Complement and its properties The Complement 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 only able to get bound to the COMPLEX antigen – antibody Complement and its properties Complement fixing test (CFT), its principle CFT principle • Patient serum is mixed with laboratory antigen (or laboratory animal serum with patients specimen in direct CFT). • Complement is added. It binds in positive case (it is only able to bind when a complex Ag-Ig is present) • In the 2nd phase, we add indicator system (sheep RBCs + amboceptor). In positive reaction indicator system remains intact. In negative reaction the indicator system is haemolysed CFT – principle Complement – how it reacts with the indicator system The haemolysis requires presence of sheep (not rabbit) antibodies, amboceptor and complement. One of components missing or replaced à no haemolysis. Sheep RBC + amboceptor without complement à no haemolysis Sheep RBC + complement without amboceptor  no haemolysis Rabbit RBC + complement + amboceptor à no hemolysis Sheep RBC + complement + amboceptor  haemolysis 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 (where we search for antibodies Trouble shooting in CFT Problems existing in CFT • Too much complement: false negative results. What to do? Titrate the complement to asses the proper amount • Something in serum binding the complement itself (anticomplementarity component): false positive results. What to do? Perform anticomplementarity test – like normal course of CFT, but without antigen (A situation like a homeless man sweeping the plant globules from the bench, even when the boy did not come into the park because he was ill) Titration of complement • 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 • Too big amount of complement à false negativity (too many plant globules  some of them remain for niece&boy-friend) Anticomplementarity test Examples of CFT use in practice Clinical situation A • A patient with long term respiratory problems, a few clinical signs, the most probable diagnosis: atypic pneumonia • Atypic pneumonia may be caused by many respiratory viruses, but also several bacteria (Mycoplasma, Chlamydia) • Eventual mycoplasmal/chlamydial etiology would mean effect of antibiotics. In viral etiology antibotics would have no effect Respiratory pathogens • The whole seropanel belongs to one patient. • We have six respiratory pathogens, each in two rows (acute speciemen, reconvalescent specimen). • First collumn = the anticomplementarity test • Then we have seven dilutions of sera, i. e. dilution 1 : 5 in 2^nd collumn, 1 : 10 in 3^rd etc., with coeficient two. Besides viruses, a bacterium Mycoplasma pneumoniae is in the panel, too (difficult culture) Clinical situation B • We have three patients with suspicion for tick-borne encephalitis, all of them with neurologic symptoms and anamnesis of being bitten by a tick • Tick borne encephalitis is a disease quite common in central Europe. Although it has worse course in adults (mostly seniors), people tend to vaccinate rather their childrens and not their parents. Tick-borne encefalitis • We test antibodies again, now againts tick-borne encefalitis. – positive control in the first row – in 2nd and 3rd row the first patient – in 4th and 5th row the second patient – in 6th and 7th row the third patient • Each patient has two rows (accute serum and the reconvalescent one) • In the first collumn, we have anticomplementarity tests again, and then sera dilutions, starting from 1 : 4 (continued: 1 :8, 1 : 16, 1 : 32, 1 : 64 etc.) Clinical situation C • We have several patients that should be screened for presence of antibodies againts toxoplasmosis (Toxoplasma gondii is a tissue parasite, cat is the definitive host) • Seronegativity means that the person never met the infection*. Seropositivity should be studied in more details (one more sampling, eventually ELISA reaction for immunoglobin class assessment) Toxoplasmosis • The seropanel belongs to a positive control (1^st row) and three patients (2^nd to 7^th row) • We search for antibodies against toxoplasmosis. • There are anticomplementarity tests in the first collumn, and then dilution by geometric row starting from 1 : 8. • Each patient has only one row (we do not follow titre dynamics) The End Tick borne encephalitis virus • Tick borne encephalitis often infects children, serious symptoms are rather typical for adults. Despite that adults rarely let themselves vaccinated. In the first phase it has flu-like symptomas, in the second meningeal or cerebral symptomas. Letality of infection is 1–5 %. • It is a typical arbovirus (=arthropode borne virus), rodents are source • Diagnostics is mostly indirect. More flaviviral encephalites and fevers • Besides Central-European tick borne encephalitis we have more tick borne encephalites. Russian spring-summer encephalitis, is another subtype to the Central-European, less related is the scotish „louping ill“ and Omsk haemorrhagic fever. • Also there exist Japanese encephalitis, transmitted by mosquitoes of genus Culex. Related is also West Nile fever, also mosquito transmitted. It is likely that is is present even in Czechia around Lanžhot Virus of tick borne encephalitis Toxoplasma gondii • It is a protozoon; cats are its source, but people having dogs are in higher risk (dogs use to have cat faeces in their fur) • Majority of infections in immunocompetent persons is asymptomatic, or only temporarily enlarged lymphonodes are observed. • Ocular form is dangerous • Infection of foetus is dangerous, too, especially in 1st trimester Toxoplasma gondii Toxoplasma gondii Toxoplasma – life cycle In some persons, toxoplasma retinitis may occur… Letality and mortality • Letality is the ratio between the persons dying for the disease and the total of infected persons • Mortality, on the other hand, is the average number of persons dying for a disease (usually counted per 100 000 inhabitants and one year)