Immunology Semester 2 Students Revised 2022 PDF

UNIVERSITY OF TECHNOLOGY, JAMAICA COLLEGE OF HEALTH AND APPLIED SCIENCE School of Allied Health & Wellness Medical Microbiology 3 Practical MET 3018 IMMUNOLOGY PRACTICAL IMMUNOLOGY OVERVIEW  Devoted to st...

UNIVERSITY OF TECHNOLOGY, JAMAICA COLLEGE OF HEALTH AND APPLIED SCIENCE School of Allied Health & Wellness Medical Microbiology 3 Practical MET 3018 IMMUNOLOGY PRACTICAL IMMUNOLOGY OVERVIEW  Devoted to studying the many mechanism of defense.  Immune mechanisms operate against infectious agents and also against cancers.  Responsible for the rejection of transplanted cells.  Immune responses may be directed against cells of one’s own body (autoimmune diseases) Terms Antigens – Substances that induce an immune response Antibodies – Glycoprotein molecules that react specifically with the antigen that induced them Haptens – small molecules which combine with large “carrier” molecules (e.g. proteins or polysaccharides) to become antigenic and induce an immune response. Agglutination – utilizes insoluble antigens (particulate) soluble antibodies Precipitation –.When antigen comes in contact with a homologous antibody in optimum proportion, they aggregate or precipitate to forma zone of equivalence. Utilizes a soluble antigen Prozone phenomenon – When there is antibody excess Postzone phenomenon – When there is antigen excess Nature of Antigens  Antigens are usually foreign to the host and are recognized by the immune system  They are macromolecules of high molecular weight e.g. proteins, polysaccharides.  Substances less than 10,000 Daltons do not make good antigens.  The immune response to antigens is directed towards a particular chemical group on the large molecule called antigenic determinants. 1|Page Prepared by Alcia Jones Nature of Antibodies  Antibodies are glycoprotein molecules called immunoglobulin  They have specific properties that permit them to recognize antigenic epitopes specifically and react to the antigen.  Immunoglobulins all have a molecular structure consisting of two (2) heavy molecular weight polypeptide chains (H chain) and two (2) light chain (L chain) together to form a single molecule.  There are five (5) classes of immunoglobulin (Ig) IgM, IgG, IgA, IgD and IgE. IgM – First immunoglobulin class produced after administration of an antigen Accounts for about 5% - 10% of circulating immunoglobulins IgG – Accounts for about 80% of the total immunoglobulin in normal immunocompetent people above age 2yrs. The only class that can cross the placenta IgG molecules can neutralize toxins, prevent adherence of viruses and microorganisms to host cells, causes agglutination and precipitation and protects in a number of ways IgA – Accounts for 10% - 13% of antibodies in normal individuals. A small portion found in circulation, however most is associated with mucus membrane in saliva and milk. Secretory IgA in breast milk is important in protecting the newborn and young infants against intestinal pathogens. IgA binds to microorganisms and prevent them from attaching and colonizing the mucus surface IgD – Accounts for less than 1% of all immunoglobulin Functions during the development and maturation of antibody response. Its functions have not been clearly defined. IgE – Barely detectable in normal blood. IgE responsible for many allergic or hypersensitivity reactions. They binds strongly to basophils and mast cells and so are not free in the circulation. Fig 1: Structure of immunoglobulin 2|Page Prepared by Alcia Jones T Lymphocytes (T cells) – Lymphocytes that mature in the thymus gland and are important in cellular immune responses and as auxiliary cells in other responses. B Lymphocytes (B cells) – Lymphocytes that mature in the bone marrow and are important in antibody response. T cells have CD antigens or markers on their surface which enables them to be identified. All T cells carry CD3 markers. Other subgroups are CD4 cells (Helper T cells CD8 cells (Cytotoxic T cells) B cells develop into plasma cells (active antibody producing cells) in the lymphoid tissues. IMMUNOLOGICAL TESTS A. Precipitation Reaction Precipitation can be done in a semisolid or liquid medium. a) Liquid medium – Lancefield grouping of beta haemolytic Streptococcus b) Semi-solid medium e.g. Immunodiffusion – Ouchterlony Technique (Double immunodiffusion) 1. Immunodiffusion Technique for the detection or measurement of antibodies and antigens by their precipitation which involves diffusion through a substance such as agar or gel agarose. Simply, it denotes precipitation in gel. It refers to any of the several techniques for obtaining a precipitate between an antibody and its specific antigen. This can be achieved by: a) Suspending antigen/antibody in a gel and letting the other migrate through it from a well or, b) Letting both antibody and antigen migrate through the gel from separate wells such that they form an area of precipitation. Based on the method employed, immuno-diffusion may be: i. Radial immunodiffusion ii. Ouchterlony Double Diffusion 3|Page Prepared by Alcia Jones  i. Radial Immunodiffusion or Mancini method - also known as Mancini immunodiffusion or single radial immunodiffusion assay Single diffusion (one compound is fixed in place.) A single diffusion technique whereby a solution containing the antigen is placed into wells in a gel or agar surface evenly impregnated with antibody. The diameter of the ring that precipitates around the well as a result of antigen antibody reaction corresponds to the amount of antigen in the solution. Principle of Radial Immunodiffusion Radial immuno-diffusion is a type of precipitation reaction. It is thus based on the principles of the precipitin curve which states that antigen-antibody interact forming visible cross-linked precipitate when the proper ratio of antigen to antibody is present. In the test, antibody is incorporated into agar and poured into a glass plate to form a uniform layer. Circular wells are cut into the agar and antigen is introduced into the wells. Specific antigens to the impregnated antibodies diffuse through the agar in all directions from the well and react with the antibody present forming visible precipitate or a precipitin ring. Ring shaped bands of precipitates from concentrically around the well indicating reaction. The diameter of the precipitate ring formed, corresponds to the amount of antigen in the solution. The concentration of antigen in the sample can be determined by reference to a standard curve based on halo diameter of known concentration of antigen. Result Interpretation of Radial Immunodiffusion Fig. 2: Diagrammatic representation of radial immunodiffusion 4|Page Prepared by Alcia Jones  a. The presence of a precipitin ring around the antigen wells indicate specific antigen-antibody interaction. b. Absence of precipitin ring suggest absence of reaction. c. The greater the amount of antigen in the well, the farther the ring will form from the well. Fig. 3: Diagrammatic representation of the standard curve for the radial immunodiffusion test interpretation ii. Double Immunodiffusion Method Double immunodiffusion is an agar gel immunodiffusion. It is a special precipitation reaction on gels where antibodies react with specific antigens forming large antigen-antibody complexes which can be observed as a line of the precipitate. Both the antibody and antigen are allowed to diffuse into the gel. Antigen and antibody preparations are placed in separate wells that are cut into a thin layer of agar in a petri dish. The reactants diffuse towards each other through the agar until they meet. At optimal antigen antibody proportions, bands of precipitate form. Double immunodiffusion determines antigenic relationship by precipitation pattern. e.g. reactions of identity reactions of non identity reactions of partial identity a. Ouchterlony Technique b. ELEK Test 5|Page Prepared by Alcia Jones  a. Ouchterlony double diffusion or passive double immunodiffusion. In this method, both the antigen and antibody diffuse independently through agar gel in two dimensions, horizontally and vertically. Principle In the test, an antigen solution or a sample extract of interest is placed in wells bore on gel plates while sera or purified antibodies are placed in other remaining wells (Mostly, an antibody well is placed centrally). On incubation, both the antigens in the solution and the antibodies each diffuse out of their respective wells. In case of the antibodies recognizing the antigens, they interact together to form visible immune complexes which precipitate in the gel to give a thin white line (precipitin line) indicating a reaction. In case multiple wells are filled with different antigen mixtures and antibodies, the precipitate developed between two specific wells indicate the corresponding pair of antigen-antibodies. Fig. 4: Diagrammatic representation of the double immunodiffusion test Results a. The presence of an opaque precipitant line between the antiserum and antigen wells indicates antigen-antibody interaction. b. Absence of precipitant line suggests the absence of reaction. c. When more than one well is used there are many possible outcomes based on the reactivity of the antigen and antibody selected. The results may be either of the following: a) A full identity (i.e. a continuous line): Line of precipitation at their junction forming an arc represents serologic identity or the presence of a common epitope in antigens. 6|Page Prepared by Alcia Jones  Fig 5: Identity result obtained from Ouchterlony Technique b) Partial identity (i.e. a continuous line with a spur at one end): The two antigens share a common epitope, but some antibody molecules are not captured by the antigen and traverse through the initial precipitin line to combine with additional epitopes found in the more complex antigen Fig 6: Partial identity result obtained from Ouchterlony Technique c) Non-identity (i.e. the two lines cross completely): A pattern of crossed lines demonstrates two separate reactions and indicates that the compared antigens are unrelated and share no common epitopes. Fig 7: Non-identity result obtained from Ouchterlony Technique 7|Page Prepared by Alcia Jones  3. Immunoelectrophoresis A double diffusion method in which an electric current is used to separate individual antigenic compounds with subsequent addition of antibody. Both antigen and antibody will diffuse towards each other to form separate arcs of precipitate for each agenic compound. Fig 8: Antigenic pattern of immunoeletrophoresis 4. Counterimmunoelectrophoresis (CIE) A variant of the double immunodiffusion where an electric current is used as a migratory force to speed up the reaction of antigen and antibody. Whereas the double diffusion needs 18 – 24hrs. the CIE needs 30 – 90mins... Antigen and antibody are placed in wells and current is applied Antigen – will migrate towards anode while Antibody + will migrate towards cathode. 5. Flocculation Tests A variation of the precipitin tests where the product is macroscopic or microscopic clumps e.g. VDRL Rapid Plasma Reagin (RPR) Test B. Agglutination Assay a. Latex Slide Agglutination Tests Utilizes latex particles (polystyrene) coated (sensitized) with antibody (antigen) which is then mixed with the patient’s serum/body fluid and observed for agglutination b. WIDAL C. Radio Immunoassay (RIA)  Used for measuring serum proteins metabolites 8|Page Prepared by Alcia Jones  drugs e.g. morphine hepatitis B surface antigen microbial agents in serum a. Antibody is coated on a solid phase such as the inner surface of a microtitre well or glass bead. b. This is allowed to react with specimen which is suspected of having antigen. c. Tubes or beads are then washed to get rid of unattached antibody. d. Antibody labeled with radioactive isotopes (e.g. 125I) is added to wells or beads and allowed to react. e. Uncombined labeled antibody is then washed away and any combined labeled antibody is measured using a gamma spectrometer. f. The amount of radioactivity is proportional to the amount of attached labeled antibody, hence the concentration of the antigen. D. Enzyme Linked Immunosorbent Assay This test utilizes an enzyme system to show the specific combination of an antigen with its antibody. (1) Indirect – to detect an assay antibody (2) Double Antibody Technique – to detect antigen. The enzyme system consists of an enzyme labeled or linked to a specific antibody or antigen and a substrate which is added after the antigen-antibody reaction. The substrate is acted upon (hydrolysed) by the enzyme which is attached to the antigen-antibody complex resulting in a colour change The intensity of the colour gives an indication of the amount of the bound antigen or antibody. E. Neutralization Assay AntiStreptolysin O (ASO) Test F. Haemagglutination Inhibition Assay G. Complement Fixation H. Fluorescent Antibody Test ANA FTA-Abs I. Radioimmunoassay (RIA) J. Western Blot Immunoassay 9|Page Prepared by Alcia Jones  K. Immunochromatographic Assay Immunochromatography assays, also called lateral flow dipstick immunoassay or simply strip tests are simple and rapid tests which combines separation of the sample molecules and reagents based on migration on a solid support by capillary flow. The identification and detection procedures are based on the antigen–antibody immune reaction. Principle of immunochromatography is the same as ELISA sandwich method, only difference is in that immunological reaction is carried out on the chromatographic paper by capillary action. For this system, two kinds of specific antibodies against antigen are used. One of the antibodies is immobilized on the chromatographic paper, and the other is labeled with colloidal gold and infiltrated into sample pad. When the liquid sample is dropped on the sample pad, the antigen in the sample forms an immunocomplex with the antibody labeled with colloidal gold. Its complex moves along with the liquid sample, and makes a contact with the antibody immobilized on the membrane, followed by forming an immunocomplex with the immobilized antibody, resulting in generating a colored red purple line. Appearance of red purple line on the membrane indicates the presence of antigen of interest in the sample. Since the liquid of the sample migrates through the membrane very fast, it makes it possible to detect the presence or absence of antigen within 15 minutes. Fig. 9: Basic component of a lateral flow test Step 1: Sample placement - To perform the test, a sample is placed on the sample pad at one end of the strip. The sample may be used alone as is commonly done with urine or serum or whole blood or plasma compatible tests, or it may be mixed with a buffer specific to the test. Fig. 10: lateral flow test - Sample addition Step 2: Molecules solubilized with the addition of the sample, the detector molecules are solubilized. When solubilized the detector molecules mix with and bind to the analyte in the sample (if analyte is present). 10 | P a g e Prepared by Alcia Jones  Fig. 11: lateral flow test – migration of analytes Step 3: Capillary action - Then capillary action draws the fluid mixture up the sample pad and into the membrane. The sample/detector molecule mix continues to move up the membrane until it reaches the analyte capture molecule. In these lines a second (and third) antibody or antigen, immobilized as a thin stripe in the nitrocellulose will then capture the complex if it is positive for the target analyte. The control line should always show as a visible line, otherwise the test is invalid and must be repeated. If the test is positive, a colored (typically pink or purple) line develops along with the control line. Fig. 12: lateral flow test – test and control addition Step 4: Excess absorbed Excess buffer along with any reagents not captured at the test of control line will then move into the absorbent wicking pad. Advantages of immunochromatographic tests: i. Can detect antigen or antibody. ii. Commercially available iii. Easy to perform iv. Limited/no instrumentation v. Single use, rapid test vi. User friendly format vii. Very short time to get test result viii. Long-term stability over a wide range of climates ix. Relatively inexpensive Limitations of immunochromatographic tests: i. Results are qualitative ii. Rapid tests can be less sensitive or less accurate compared to existing tests 11 | P a g e Prepared by Alcia Jones  SEROLOGY Serology is the study of reactions between antigen and antibody. It attempts to quantitate these reactions by keeping one reagent constant and diluting the other. Serological reactions can be used to identify antigens or antibodies, if either of these is known. SEROLOGICAL TESTS 1. Venereal Disease Research Laboratory (VDRL) This is the screening test for syphilis a sexually transmitted disease caused by a spirochete, Treponema pallidum, an extremely slender motile organism with tightly wound coils. Syphilis Occurs in three (3) clinical stages: Primary Syphilis  T. pallidum grows and multiplies in the genitilia (site of inoculation)  Three week after infection, a painless red ulcer called a chancre appears.  The chancre heals within four to six weeks whether or not treatment is given. Secondary Syphilis  This appears two to ten weeks after the primary syphilis.  Symptoms includes aches and pains sore throat malaise weight loss fever rash that involves the palms and soles.  Symptoms due to the reaction of the T. pallidum with specific antibodies to form immune complexes  Infectious lesions occur on the skin and mucous membrane in various locations, especially the mouth.  This stage last for weeks to months, as long as one year.  About 50% of untreated cases never progress beyond this stage. Tertiary Syphilis  Occurs after a latent period from 10 – 25 years.  The organism may be present in any part of the body.  Complications includes: blindness, central nervous system involvement, cardiovascular abnormalities, insanity and death. 12 | P a g e Prepared by Alcia Jones  Congenital Syphilis  During pregnancy T. pallidum crosses the placenta and infect the fetus  About two out of every five infected fetus are lost through miscarriage ore still births.  The remainder is born with congenital syphilis.  Some babies will develop secondary syphilis and others will develop characteristic deformities of their face, teeth and other body parts later in childhood. VDRL Test During all stages of syphilis serological tests are helpful in the diagnosis. The most common screening test is the VDRL (a flocculation test) This test does not employ an antigen from T pallidum and so is called a non-treponemal test Based on the fact that the particles of lipid antigen (Beef heart cardiolipin) remain dispersed with normal serum, but forms visible clumps when combined with reagin (a distinct antibody like substance developed by the spirochetes (Treponema pallidum.) This is a useful screening test; however the ability to detect this disease depends on the stage of the disease. In primary syphilis theVDRL test is positive 60% of the time. In secondary syphilis it is positive 70 – 90% of the time and in tertiary 60%. Several conditions can cause false positive results. These include: HIV, Lymes disease, Mycoplasma pneumoniae, Malaria, Systemic Lupus Erythematosis and Yaws. Principle: The VRDL test is composed of cardiolipin, cholesterol and lecithin. This non treponemal test measures antibody like substances (reagin) by the presence of flocculation a) Specimen collection and preparation 1) Obtain blood and allow to clot 2) Separate serum from clot 3) Inactivate serum at 56oC for 30mins. 4) Cool serum to room temperature for testing. If CSF is collected this specimen is not inactivated. Control sera: Pools of positive sera, weakly positive sera and negative sera are prepared and stored in sealed tubes at -20oC. 13 | P a g e Prepared by Alcia Jones  b) Preparation of antigen 1) Pipette 0.4mls. of VDRL buffered saline in a VDRL antigen emulsion bottle. 2) Ensure the entire bottom of the bottle is coated. 3) Add 0.5mls of VDRL antigen while rotating the bottle. 4) Continue the rotation for 10 seconds. 5) Add 4.1mls of VDRL buffered saline slowly 6) Stopper the bottle and shake. 7) Allow to stand for 10mins before use. c) Qualitative test 1) Add 50l of serum to the ring of a VDRL slide. Cover the entire ring. 2) Add 50l of the antigen. 3) Mix using a rotator for 4mins at 180rpm 4) Observe microscopically (X10) for flocculation (clumping). Report: Reactive, Weakly reactive or Non-reactive d) Quantitative test 1) Prepare a twofold dilution of the inactivated serum in saline. 2) Test each reactive serum as above. 3) Report results Report the lowest dilution that is reactive as the titre of the serum Advantage 1) Useful for preliminary testing of large number of people. 2) Useful in evaluating the success of treatment since the titre of the antibody like protein generally falls four fold within three months when treatment is effective. 3) Fairly inexpensive. 4) Can be performed with a minimal amount of skill and equipment. Disadvantage Test is not specific Needs to be confirmed using a treponemal test 14 | P a g e Prepared by Alcia Jones  Non-reactive Weakly reactive Reactive Fig. 13: VDRL reactions 2. Rapid Plasma Reagin (RPR) Test  This is another commonly used non-treponemal test used to screen for syphilis.  Antigen used is similar to that used in the VDRL test. The tester antigen is prepared from a modified Venereal Disease Research Laboratory (VDRL) antigen suspension containing choline chloride to eliminate the need to heat inactivate serum, ethylenediaminetetraacetic acid (EDTA) to enhance the stability of the suspension, and finely divided charcoal particles as a visualizing agent.  This test like the VDRL is a flocculation reaction. Principle of RPR Test The Rapid Plasma Reagin tests are macroscopic, non-treponemal flocculation card test that detect antibodies produced against antigens released by damaged host cells in patients suffering from syphilis. In the test, the RPR antigen is mixed with unheated or heated serum or with unheated plasma on a plastic-coated card. The antigen used for detection contains 0.03% cardiolipin, 0.21% lecithin, and 0.9% cholesterol in addition to choline chloride, EDTA and charcoal particles. If antibodies are present, they combine with the lipid particles of the antigen, causing them to agglutinate. The charcoal particles coagglutinate with the antibodies and show up as black clumps against the white card. If antibodies are not present, the test mixture is uniformly gray. Advantage 1) Antigens are either carbon containing or dyed to enable the reaction to be read macroscopically on a card. 2) Serum does not have to be inactivated. 3) Plasma or serum can be used. 4) Small amount of specimen is required. 5) Antigen is ready for immediate use. 15 | P a g e Prepared by Alcia Jones  6) Test is easy to perform. 7) Inexpensive 8) Slightly more sensitive than VDRL. Disadvantage 1) False positive occurs more often than with VDRL. 2) Not used for CSF specimen (neurosyphilis). Other non treponemal serological tests include: a) Toluidine red unheated serum test (TRUST). b) Reagin Screen Test (RST). 3. Fluorescent Treponemal Antibody (FTA) – Absorption This test is used to detect antibody directed specifically to the bacteria that causes syphilis Treponema pallidum.. It is used to confirm whether a positive screening test for syphilis reflects a true infection with syphilis. It can also be done when either primary or tertiary syphilis is suspected and the screening test is negative. Principle: The FTA- Abs test is an indirect fluorescent antibody technique. The patient’s serum which has been diluted in sorbent is layered on a slide to which T. pallidum has been fixed. If the patient serum contains antibody, the antibody will coat the treponeme. FITC- labeled antihuman globulin is added and combines with the complex formed resulting in FITC stained spirochetes that are visible by fluorescence microscopy FITC – Fluorescein Isothiocynate. Sorbent – Used to remove group anti-treponemal antibodies from the serum being investigated and does not react with the antibody specific for T. pallidum. Fig. 14 apple green fluorescence seen in FTA 16 | P a g e Prepared by Alcia Jones  4. Microhaemagglutination T. pallidum (MHA-TP) This test like the FTA-Abs is used to detect antibody directed specifically to the bacteria that causes syphilis Treponema pallidum.. It is used to confirm whether a positive screening test for syphilis reflects a true infection with syphilis. Principle MHA-TP test utilize erythrocytes from a turkey or sheep that are coated with the treponemal antigen (sensitized red cells). The patient’s serum which has been diluted in sorbent is mixed with the sensitized red cells. The presence of the specific antibody causes the red cells to agglutinate and form a flat red mat or layer across the bottom of the microtitre well in which the test is performed. If the specific antibody is absent the red cell settles as a small red button (or ring) at the bottom of the well Known positive and negative controls should always be included. Known negative serum will check for autoagglutination of red cells if it is positive. 5. WIDAL Test Widal test is an agglutination test which detects the presence of antibodies in patient’s serum produced against the causative agents of enteric fever (Salmonella typhi and Salmonella paratyphi A, B and C). The Widal test utilizes antigen-antibody interactions to detect specific antibodies in the serum sample of typhoid patients. The antigens used are derived from the bacterial cells of Salmonella typhi and Salmonella paratyphi. Salmonella typhi possesses O antigen on the cell wall and H antigen on flagella. Similarly, Salmonella paratyphi A and Salmonella paratyphi B also possess O antigen on their cell wall and but have AH and BH antigen on their flagella respectively. On infection, these antigens stimulate the body to produce specific antibodies which are released in the blood. These specific antibodies against the antigens can be detected in the patient’s serum after 6 days of infection (fever). Stained Salmonella suspensions are used to detect, identify and quantitate specific antibodies in sera. They are standardized smooth suspension of killed bacteria which have been stained to facilitate reading of the test. O antigen – somatic antigen (stained pink) gives granular agglutination H antigen – flagellar antigen (stained blue) gives a floccular agglutination Principle of Widal Test Individual infected with Salmonella typhi or S. paratyphi produce antibodies against either somatic (O) antigens and/or flagellar antigens (H). These produced antibodies in serum, if exposed to bacterial suspension carrying homologous antigens, result in agglutination. The agglutination reaction can be seen as visible clumping. 17 | P a g e Prepared by Alcia Jones  In the test, the patient’s serum is mixed with killed bacterial suspension of Salmonella carrying specific O, H, AH and BH antigens and observed for agglutination reaction. If the patient’s serum contains specific antibodies against the antigens, clumping is evident which indicates positive test. Absence of agglutination indicates a negative result. Method a) Tube Agglutination Test 1) Make 1 (one) row of serial dilution for each antigen, using.025% saline as diluent. Tube# 1 2 3 4 5 6 7 8 Diluent mls. 1.9 1.0 1.0 1.0 1.0 1.0 1.0 1.0 Patient’s Serum mls. 0.1 ---------1ml serial dilution ----------- 0 Final dilution 1:20 1:40 1:80 1:160 1:320 1:640 1:1280 Mix the content in tube 1 and transfer 1ml to tube 2. Repeat for each tube up to but not including tube 8. Discard 1ml from tube 7. 2) Add 1 drop of the appropriate suspension to each tube of a given row using a Pasteur pipette. 3) Mix and incubate at 37oC. 4) Examine for agglutination. O positive – there is a granular agglutination H positive – there is a floccular appearance. Negative reaction and saline control suspension remain unchanged, and shows a typical swirl when flicked. Result Interpretation of Widal Test The titre is the dilution of the serum in the last tube showing agglutination. Significant titre ≥1:80 H antigen – past infection (last longer) O antigen – recent infection In current infection both the H and O antigens are positive. The Vi antigen is an extra somatic antigen which is looked for in carriers 6. AntiStreptolysin O Test Certain bacteria including the Streptococci produce extra cellular toxins and enzymes during growth. Antibodies formed in response to these antigens can be detected. 18 | P a g e Prepared by Alcia Jones  Streptolysin O is an extra cellular metabolite of Streptococcus A, C and G. The corresponding antibody AntiStreptolysin O is measured. This is used in conjunction with other tests to establish a diagnosis of Rheumatic fever and Streptococcal glomerulonephritis. AntiStreptolysin O test is a neutralization test Principle: A neutralization test which uses the ability of the patient’s serum to neutralize the erythrocyte lysing capability of Streptolysin O. Streptolysin O will lyse red cells. When Streptolysin O is added to a serial dilution of the patient’s serum, if Antistreptolysin O is present a complex is formed. When red blood cells are added the Streptolysin O is not available to lyse the cells so they fall to the bottom of the well as a button If the serum does not contain AntiStreptolysin O, no complex is formed and so the Streptolysin O is available to lyse the red blood cells. Method Make serial dilutions of the patient serum Add standard amount of commercially prepared Streptolysin O Add red cells Positive – buttoning Negative – lysis In our population  200 TODD units Significant 160 TODD units Doubtful significant  80 TODD units Insignificant 7. Ouchterlony This test is a classical method of detecting soluble antigens (i.e. antigen in solution). Principle Antigen and antibody preparations are placed in separate wells that are cut into a thin layer of agar in a petri dish. The reactants diffuse towards each other through the agar until they meet, where they produce a visible band of precipitin. This point is called the zone of equivalence. The precipitation pattern formed will determine reactions of identity reactions of non-identity reactions of partial identity 19 | P a g e Prepared by Alcia Jones  Fig 15: Ouchterlony test results Fig 16: Observations and results of the ouchterlony test 8. Antinuclear Antibody (ANA) Test Antinuclear antibody or ANA is an antibody that is formed against normal proteins in cell nucleus. This test is used to detect auto antibodies in patient’s sera. Principle The Antinuclear Antibody Test is an immunofluorescence test. Hep2 cells are added (fixed) unto a glass slide. The patient’s serum is then added. If specific autoantibodies are present they will react with the nuclear DNA present in the Hep2 cells. Antihuman globulin labeled with a fluorescein dye (Fluorescein Isothiocynate) is added and combines with the complex formed resulting in FITC stained complex that are visible by fluorescence microscopy An apple green fluorescence is observed if positive 20 | P a g e Prepared by Alcia Jones  Fig. 17 ANA positive Fig. 18 ANA negative 9. Enzyme Linked Immunosorbent Assay (ELISA) The enzyme system consists of an enzyme labeled or linked to a specific antibody or antigen and a substrate which is added after the antigen-antibody reaction. The substrate is acted upon (hydrolyzed) by the enzyme which is attached to the antigen- antibody complex resulting in a colour change The intensity of the colour gives an indication of the amount of the bound antigen or antibody The antigen, to which the antibodies bind, if present in the patient’s serum, is either attached to the inside of the wells of a microtitre plate, adherent to a filter matrix, or bound to the surface of beads or plastic paddles. Advantage a) Can be easily performed on many samples at the same time. b) The coloured end product is detected by instrument, thus removing the human subjectivity. Disadvantage a) Test needs special equipment. b) The fairly long reaction time. Fig. 19: ELISA test 21 | P a g e Prepared by Alcia Jones  10. Western Blot Immunoassay Used in the detection of very specific antibodies. Used as the confirmatory test for HIV Principle Method is based on the electrophoretic separation of proteins of an infectious agent in a two dimensional agar gel matrix.. A suspension of the organism against which antibodies are being sought is mechanically or chemically disrupted, and the solubilized antigen suspension is placed at one end of a polyacrylamide (polymer) gel. Using electric current the proteins are separated on the gel forming bands. These protein bands are transferred from the gel to a nitrocellulose membrane, which is treated to immobilize the proteins. The membrane is cut into thin strips, each carrying the pattern of protein bands. When the patient serum is layered over the strip, antibodies (if present) will bind to each of the protein components represented by a band on the strip. The pattern of antibodies present can be used to determine if whether the patient is infected with the agent. 11. Automation a) Turbitimer An instrument used to determine serum proteins using specific antibodies. Proteins IgG, IgM C3 etc. contained in human serum sample form immune complexes with specific antibodies in the reagent. The turbidity generated in the reaction is measured photometrically by the machine. b) FACS Count machine 22 | P a g e Prepared by Alcia Jones