Searching for microbes Part X. Reactions with labelled components 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 sailor, and she had various object on the board bound so to keep them in the ship. She had her trieder bound to the rescue circle, the rescue circle to rescue boat, and the rescue boat to the board. So event the highest wawes were not able to take the objects away. • Once the sailor‘s husband came. He wanted to assay the rescue circle. He bound the trieder away and he bound the circle from the boat. • A wave came – and the trieder was flown away from the board. What to learn from the tale • The principle of reactions of labelled components: We bind one component to another; after each step, the flowing out of components comes. • This process takes away everything that is not bound • A negative reaction = a reaction, where one component of the chain of components bound one to another is missing. The other components are not bound to the surface, and they are flown away. Classes of antibodies Course of antibody answer • Class IgM is formed as first, but it is also first to disappear. They do not come through placenta, in a newborn it certifies its own infection • Class IgG is formed later and remains as memory antibodies. They are able to pass through placenta. Other classes of antibodies • Class IgA is examined in some infections instead of IgM. This class is important mainly in mucous membrane immunity, so in infections entering the body through mucous membrane (e. g. gastrointestinal) • Class IgE are present in allergy and helmint infecions. Specific IgE against a certain pathogen are rarelly examined • Class IgD is not examined im microbiology Reaction with labelled components: survey Reactions with labelled components • Individual components are bound on the previous components, the first of them to the surface. • Instead of one component a specimen from pacient is used. The specimen is suspicious to contain the given component. • If it is true, the component is bound • When all components bind respectivelly, a not-interrupted chain is formed • At the end there is a labelling agent Washing out and its sense • When also the components that are not bound to the surface would remain, we would not be able to differenciate a positive reaction and a negative one. • That is why after each step washing follows. After such a washing, only bound components remain present. • When the chain is broken, the part after the missing component is washed out. Example of positive and negative course Types of labelling agent • Fluorescent dye is labelling agent in immunofluorescence • Radioisotope is labelling agent in RIA • Enzyme is labelling agent in ELISA – Western blotting is a special type of an ELISA, where individual antigens are divided electroforetically When an enzyme is used as a labelling agent, the very last component should be the substrate – so one more component. Immuno-fluorescence and RIA Immunofluorescence Immunofluorescence Positive result in both direct and indirect IMF looks the same Examples of immunofluorescence (diagnostics of Treponema pallidum) a) Direct imunofluorescence • (Surface)-(antigen)-(labelled antibody) b) Indirect imunofluorescence • (Surface)-(antigen)-(antibody)-(labelled antibody against human antibodies) Immunofluorescence reaction schemes Radioimmunoassay ELISA: principle ELISA ELISA ELISA – why used so much • In ELISA reaction we have at the end of the whole process an enzymatic reaction. Its intensity is simply described as intensity of colour in a well with the reaction. Very intensive colour = highely positive reaction • Simplicity for technique and zero radiation is an advantage in comparison with RIA • Possibility of automatisation is an advantage in comparison with immunofluorescence Examples of component system blue = component from specimen taken from pacient‘s body • Surface-antigen-antibody-labelling agent (D) • Surface-antibody-antigen-antibody-labelling agent (D, e. g. detection of HBsAg) • Surface-antigen-antibody-antigen-labelling agent (I) • Surface-antigen-antibody-conjugate-labelling agent (I) Conjugate is an antibody against human antibody Importance of the conjugate • Conjugate is used mostly in indirect reactions (detection of antibodies) • It is an antibody that has human antibody (e. g. IgM, IgA or IgG) for an antigen • It can be selective against a certain antibody class • Use of conjugate is the principle of selective diagnostic of individual immunoglobulin classes ELISA antibody detection: 1. Positive (searching IgM, IgM present) All components bind step by step. An enzymatic reaction leads to colour change in the well. ELISA antibody detection: 2. Negative I (searching IgM, no antibodies) No antibodies in pacient‘s serum. Conjugate flown out, no change in the well. ELISA antibody detection: 3. Negative II (searching IgM, IgG present) Only IgG antibodies in patients serum. Conjugate flown out, no change in the well. ELISA: practical reading ELISA – practical description • Usually we have a microtitration plate. Unlike classical serological reactions, each patient has here not the entire row, but one well only. That is because titers are not assessed. • First wells, foregoing pacient wells, might be: – Bl – blank (for spectrophotometer calibration) – K- and K+ – positive and negative controls – Cut off (two or three wells) – producer provides „specimens“ with just borderline values of absorbance („cutting off“ positive and negative results sharp, or plus minus 10 %) Each ELISA kit is different, like their producers. Some of them do not have any blank. Some of them do not have cut off wells and the cut off is count as average of negative controls + a constant. ELISA – an example (www.medmicro.info) Example of ELISA to antigen detection (antigen of Helicobacter pylori) • In ELISA reaction, there is an enzymatic reaction at the end of the process. The intensity corresponds with the colour intensity in the well. • Collour intensity can be evaluated spectrophotometrically • As positive we consider values higher than reference given „cut off“ • Usual principle: Surface-antibody-antigen-antibody-enzyme-substrate Example of ELISA to antibody detection • In indirect antibody detection using ELISA usually IgM and IgG are assessed separatelly • In our case, IgA is used instead of IgM • Values higher than „cut off“ are again considered to be positive • Often we have a „borderline field“. For example, results ranging 90 % to 110 % of cut off vallue are described as „borderline“, below 90 % as „negative“, above 110 % as „positive“ • Common principle: Surface-antigen-antibody-conjugate-enzyme-substrate Example of an ELISA scheme to antibody detection Western blotting Western blotting • Language joke (researcher Southern) • In fact, it is an ELISA, but antigenic mixture is divided electroforetically to individual determinants • So it is more precise, and helpfull mostly in situations, where classical ELISA is problematized by cross-reactions Western blotting – principle 1: original antigen (mixed) 2: decomposition of antigen by a detergent 3: electroforetic division of antigen 4: „blotting“ of divided antigen to a nitrocelulose membrane 5: ELISA reaction (only some antibodies present) Western blot – example (picture from www.medmicro.info) Example of reading of a Western blot (in Lyme borreliosis) • Presence of at least two specific bands (labelled on a pattern) à assessed as positive • Exceptions: – in IgG positivity of one band is sufficient, if it is vlsE band (it is highly specific) – in IgM positivity of one band is sufficient, if it is ospC band (it is highly specific) The End (A picture name: Antibody) Treponema pallidum • A spirochet, causing syfilis • Syfilis is a classic sexual disease. It is transmitted sexually only. But it is a systemic disease – in developped stages the whole body is affected (gummata, aortal dissection, neurolues, psychical symptomas) • Some subspecies of T. pallidum and some other treponemas cause other diseases (framboesia – yaws, T. pertenue) • Some treponemas are non-pathogenous Borrelia burdorferi sensu lato Helicobacter pylori • Peptic (= gastric / duodenal) ulcus is caused by more causes. Such diseases are called multifactorial diseases. • The part of Helicobacter pylori, a spiral rod (not spirochet!), on ulceral disease is still discussed, not only among GPs, but even among specialists. Even healthy persons may have a helicobacter in their stomach. Nevertheless, certain and not negligeable role of this pathogen is sure.