Immunodiagnostic Techniques Lecture Notes PDF

1Lecture 1- principle of Ag-Ab reactions. by Dr. Hajar Principle of Ag-Ab reactions 2Lecture 1- principle of Ag-Ab reactions. by Dr. Hajar References Mary L. Turgeon (2014). Immunology & serology in laborator...

1Lecture 1- principle of Ag-Ab reactions. by Dr. Hajar Principle of Ag-Ab reactions 2Lecture 1- principle of Ag-Ab reactions. by Dr. Hajar References Mary L. Turgeon (2014). Immunology & serology in laboratory medicine, 5th ed, Elsevier Abbas, A.K (2016). Basic immunology: Functions and disorders of the immune system, 5th ed, Elsevier  Types of infectious diseases: 1. Bacterial (plenty of infectious extracellular and intracellular bacteria). 2. Viral (either DNA or RNA viruses and enveloped or non-enveloped). 3. Fungal (eukaryotes, either unicellular or multicellular molds). 4. Protozoal infections 5. Helminthes Abbreviations used in the lecture: Ag= Antigen, Ab= Antibody, (Ags and Abs are the plural forms), mAb= monoclonal antibodies. Immune response to microorganisms: 1 Immune response to bacteria: A- Extracellular: Ab to capsular polysacchrides, Ab to bacterial enzymes (e.g. ASO), Ab to exotoxins (diphteria or tetanus), complement fixing (CF) Ab by the bacterial endotoxins. B- Intracellular: Cell-mediated immunity (CMI). 2 Immune response to viruses: Viremic viruses: Neutralizing Ab of IgM (recent) IgG (late) and IgA and complement fixing Ab. CMI is the main type of immunity to viral infections 3- Immune response to fungal infections: fungal infections are mainly intracellular so, main immune response is CMI. Post infection immunity: -Viral infection: mostly long time immunity -Bacteria infection: shorter immunity - Protozoa infection: shorter immunity -Helminthes infection: no protective immunity. Diagnosis of infectious diseases: *Epidemiological data (time, locality, vector….etc *Clinical features : (Symptoms and signs) *Laboratory findings Routine examination of blood, urine, feces Bio-chemical examinations Serological Diagnosis Etiological examinations: (Direct exam, Isolation of pathogen) *Molecular biological examinations (PCR, LCR…etc) * Immunological examinations * Endoscope examinations * Image examinations Laboratory Investigation of Microbial infections: Examining specimens to detect isolate and identify pathogens: 1Microscopy 2Culture techniques 3Biochemical reactions 4Serological identification: 5Molecular biology techniques 6- Bacteriophage typing What is the serological diagnosis of infections? - It is the diagnosis of infectious diseases based upon the Antigen-Antibody (Ag-Ab) reactions. - Ag – Ab reaction is the union of an Ag with its specific Ab, best detected when each is in optimal concentration. - The type of such reaction depends upon the nature of both Ag and Ab used. -These reactions are mostly used in the laboratory (In Vitro) to detect either the Ag (Direct diagnosis) or the Ab (indirect diagnosis). Types of Ag-Ab Reactions (In Vitro): 1.Agglutination. 2.Precipitation. 3.Complement fixation Test (CFT). 4.Neutralization. 5.Immunofluorescence Test (IF). 6.Enzyme linked immunosorbent assay (ELISA). 7. Radio immunoassay (RIA). 8.ImmunochromatographY (ICT). 9- Immuno-blotting assays (WESTREN BLOTTING ). Sensitivity of different serologic tests in detecting antibody levels in Ug/mL in the unknownsample: SN Test Sensitivity level (Ug/mL) 1 Gel-diffusion 30 2 Ring Precipitation 18 3 Bacterial agglutination 0.05 4 Complement fixation test 0.05 5 Passive Hemagglutination 0.01 6 Hemagglutination inhibition test 0.005 7 Immunofluorescence 0.005 8 ELISA 0.0005 9 Bacterial neutralization 0.00005 N.B. The lower the level of detection the higher the sensitivity of the test. Application of Ag-Ab Reactions (Serology): 1. Diagnosis of many infectious diseases (direct antigen detection or measuring Immunity to infection) especially for non-culturable or delayed growing microorganisms. 2. Estimation of the Severity or stage of diseases. 3. Determination of the Response to treatment. 4. Epidemiological studies. 5. Diagnosis of congenital infections. 6. Screening donation of blood and tissues. 7. Non-infectious diagnostic applications (Tumors, Autoimmune diseases, endocrinology, …etc.  1.Agglutination Is the visible clumping of particulate (insoluble) Ag with its specific Ab forming visible lattice. The Ab is the divalent agglutinating one either IgG or IgM type. The reaction can be either on slide or tube agglutination. It can be direct or via the use of carrier (Passive or indirect) e.g. Latex, RBCs (hemagglutination), or Staph protein A (Coagglutination). Indirect more easily visible. The reaction can be qualitatively or quantitatively expressed.  Examples of agglutination reactions: 1. Direct slide detection of a bacterial or viral antigens in a lesion or culture. 2. Direct tube agglutination (Classical Widal test) for diagnosis of typhoid fever (replaced now by Latex) 3. Indirect Latex tests commonly used in most microbiology laboratories nowadays. 4. Indirect passive hemagglutination tests e.g. Treponema Pallidum Hemagglutination (TPHA) for diagnosis of syphilis which is more specific. 5. Brucella slide or tube agglutination test (Prozone phenomenon). Lecture 1- principle of Ag-Ab reactions. by Dr. Hajar 14 Hemagglutination Agglutination No Agglutination No Ab  Direct agglutination Passive hemagglutination Ab Ag coated RBC Lecture 1- principle of Ag-Ab reactions. by Dr. Hajar 16 Agglutination Inhibition 5 Widal latex slide agglutination tests for diagnosis of typhoid fever. 6 Weil-Felix Test used for the diagnosis of rickettsial disease e.g., epidemic typhus which is non- specific test using the Heterophil Ag of proteus species. Widal Latex Agg. Latex reactions for detection of Ags or Abs  Why latex test are more common? Easy to manufacture, to use, cheap, clearly visible. No instruments Can be coated with any Ag (Soluble) to detected Ab Can be coated with the Ab to detect the Ag. E. coli O157:H7 Cryptococcal Antigen Latex Pregnancy Test Latex MRSA Latex Toxoplasmosis - Latex Agglutination  Prozone Phenomenon: It is the absence of agglutination in the first tubes containing high concentration of Abs (appearing as being false-negative) and its occurrence in the following tubes containing lower concentrations (higher dilution i.e. > 1/40 or in 1/80). Overcome by the use of higher dilution  Advantages of Agglutination: The most widely used tests for diagnosis and screening. Easy and simple. Very cheap No instrument is required somewhat sensitive Disadvantages of agglutination: low specificity (have false positive results) need confirmatory tests low sensitivity (have false negative results). Results affected by vaccine-induced Ab. Subjective errors in reading. Agglutinating Ab appears late in the infections.  2- Precipitation Tests: It the reaction between soluble Ag and its specific Ab. It can be Precipitation in solution or in gel. Example of Precipitation in solution include the Ring test for detection of infectious Ags in CSF. Another example of precipitation in solution is the slide test for detection of pneumococcal capsule. Precipitation in gel is used for the quantification of Immunoglobulins in patient serum (less common) in form of single, double radial immuno-diffusion or immuno-electrophoresis.  Si n gl e radi al i m m u n o d i f fu s i o n Ri ng of p re ci p it at i on M e a s u r i n g pr ec ip it at io n ri ng The Ab is incorporated into the gel and the Ag to be tested is the one allowed to diffuse in the gel. Diameter of precipitation is proportional to the Ag concentration directly. Double Radial immunodiffusion Explanation of the reaction:  Immuno- electrophoresis  Flocculation tests Flocculation is an antigen-antibody reaction that occurs if the antigen is neither cellular nor soluble, but is an insoluble particulate. The flocculation reaction is a special type of precipitation reaction. Example of the flocculation reaction in current use are the Venereal Disease Research Laboratory (VDRL) test and the rapid plasma reagin (RPR) used for the diagnosis of syphilis. These tests are non-specific (non-treponemal) or called standard test of syphilis (STS).  The antigen used is cardiolipin, a hapten from normal beef heart those cross-reacts with a heterophile (Heterogenetic) antigen of the spirochete of syphilis so to detects antibodies to Treponema pallidum. Cholesterol particles with water-insoluble cardiolipin on their surface are used in the test. Visible aggregates form in the presence of an antibody (reagin) in the serum of patients with syphilis. VDRL has many limitation and not in current use nowadays and the in current use is the RPR. VDRL Slides in \\\\ RPR Test 3- Complement Fixation Test (CFT) It is the detection of complement fixing Abs (IgM or IgG) against the causative organism in the patient serum. The (Guinea pig) complement and the Ag used in the test are added in the lab to pt. serum after serum heating to destroy the internal complement proteins and incubated in Wassermann tubes. Then, an RBCs and Anti-RBCs are added as indicator system to be destroyed by the complement if there is no Ab in the serum. If there is Ab the complement will be fixed in the first reaction so, no hemolysis will occur to certain dilution after which it will occur.  So, no hemolysis mean positive test. The titre is last tube showing no hemolysis. The result are affected by anti-complementary substances in the patient serum e.g. in SLE or heparin therapy. Application of CFTs: Bacterial: Syphilis and chronic gonorrhea. Viral: Influenza. Systemic fungal infections: Systemic candidiasis. The CFTs are laborious and are rarely used nowadays as being replaced by other tests. CFT showing Positive case (no hemolysis). Titre tube number 4. - RBCs Control: Sheep RBCs + anti-sheep RBCS Ab (Indicator RBCs) (NH). - Ag Control: Ag + indicator RBCs + Comp. (Hemolysis). - Serum control: Pt. serum + Ag + indicator sheep RBCs (H). - Complement control: Complement + Indicator RBCs (H).  CFT showing negative case (hemolysis in all tubes)  Anti-complementary case (No hemolysis in all test tubes with hemolysis in the complement control tube (complement is working) no hemolysis in the serum control (some serum factors inhibited the action of the complement). Advantages of CFT: The CF Ab do not persist long (recent infections). Stable antigen used in the test for years (>10 ys). High specificity Quantitative results. Disadvantages of CFT: Low sensitivity (false negative) Time consuming. Not standardized Needs high technical skills Sample contamination giving anticomplentary Still false positive results occur. Still in little use for some viral and protozoal infections  4- Neutralization Tests: It is an Ag-Ab reaction in which the Ab neutralizes the effect of the Ag (bacterial toxin or virus). Example of neutralization test in vitro is the Anti- Streptolysin O titre (ASOT) for the diagnosis of post- Streptococal Complications e.g. Rheumatic fever. Classical ASO test by Neutralization!!! ASO test by Latex (which one is more easier??? 5- Enzyme Linked Immunosorbant Assay [ELISA] Monoclonal antibody (mAb) or recombinant Ag is fixed to a surface of a microtiter plate wells. The test sample (with unknown Ag or Ab) is applied and incubated to react then washed. The bound material is detected by a secondary, enzymatically labeled mAb (Conjugate) which is incubated and then washed to remove unbound. Then a substrate for the enzyme is added later on and the colored reaction is stopped by acid. The color reaction is directly proportionate to the amount of the unknown (Ag or Ab) present and is  Alkaline phosphatase is a commonly used enzyme as it can be covalently couple to mAbs without affecting either the Ag binding capacity of the Ab or inhibiting the activity of the enzyme. Many formats of ELISA are present and used in clinical laboratories. ELISA Formats Indirect ELISA Sandwich ELISA Direct Sandwich Indirect Sandwich Blocking or inhibitory ELISA. Note that the higher the Abs in sample the lower the intensity of OD produced.  Competitive ELISA OD Curve It is to be noted that in the inhibitory or blocking ELISA formats the OD is inversely related to the concentration of the (Standard) or tested material i.e. the higher the concentration the lower the OD and vice versa. ELISA KITS Semi-Automated ELISA (separate washer, Incubator, Reader) ELISA washers (for semi-automated) Stat Fax microplate washer Bio-Tek microplate washer In 50-bed Hospitals of MOH  Manual ELISA readers An automated reader After several incubation and gives a measurement of wash steps, a color reaction optical density (presence occurs if antibody is present of color) for each well Fully Automated ELISA machines (dilution, pipetting, washing, incubation and reading) Evolis (Bio-Rad) Eti- Max (Diasorin)  Advantages of ELISA 1. Simplicity: (a)Reagents added in small volumes. (b)Separation of bound and free reactants is made by simple washing procedures. (c)Passive adsorption of proteins to plastic is easy. (d) Specialized equipment readily available. 2. Reading: (a)Colored end-product can be read by naked eye to see test is working before measurement. (b) Multichannel spectrophotometers is used.  3. Rapidity: (a) Tests can be performed in a few hours. (b) Spectrophotometric reading of results is rapid (96 wells read in 5 s). 4. Sensitivity: Detection levels of 0.01 to 1 μg/mL ideal for most diagnostic purposes. 5. Reagents Commercially available: with great flexibility and open to work in different Equipment. 6. Cost: (a) Startup costs are low. (b) Reagent costs are low. 7. Acceptability: Fully standardized ELISAs in many fields are now accepted as "gold standard” assays. 8. Safety: Safe non-mutagenic reagents are available. Disposal of waste has no problem (unlike radioactive). 9. Availability: ELISAs can be performed anywhere, even in laboratories where facilities are less than state of the art. 10. Kits ELISA kits are widespread and successful.  11. Standardization: Quantification of data allows easier standardization. Main drawbacks of ELISA is lacking the absolute specificity, so, still there are some false-positive results to be confirmed by molecular testing.  6- Radioimmunoassay (RIA) RIA uses radioisotopes as an Ab or Ag conjugates, unlike the enzymes used in ELISA. The isotope iodine-125 (125I- Gamma-emitting isotope) is commonly used as the conjugate because Ab or Ag can be readily iodinated without disrupting their immune specificity. C14 and H3 (Beta-emitting isotopes) can also be used. RIA was used clinically (Endocrinology) to measure serum proteins such as human growth hormone, testosterone and insulin present in humans in extremely small amounts.  Gamma or Beta radioactive counter is used according to the isotope in use. RIA is a very sensitive mean of detecting Ag by either direct or indirect immunoassay. RIA was the first to be introduced in 1950s (Nobel Prize 1975) while ELISA was introduced in 1970s. Due to risk of radiations, RIA is not in common use and is replaced by the more safer ELISA and other serological tests. Principle of Direct RIA Beta Counter Gamma Counter Fully Automated RIA system 7- Immunofluorescence Assay (IFA) It is Ag-Ab reaction where the Ab is labeled with fluorescent dye. The fluorescent dye is either Fluorescin or Rhodamine linked to isothiocyanate (ITC). The reaction is seen by the mean of a special fluorescent microscope. The IFA reaction can be direct to detect specific Ag by the mean of labeling specific Ab. Or indirect by using unlabeled Ab and fluorescently labeled Anti-Ab which is better technique to only label one Anti-Ab and not all specific Abs to plenty of Antigens needed to be detected. IFA has the advantages of detecting Ags in tissues.  The most common example for IFA is Treponema Pallidum Immunofluorescent Antibody test (TP-IFA) and infections caused by Chlamydia species. Advantages of IFT: Fixed slides can persist for long time High sensitivity. Disadvantages of IFT: Subjective errors in reading. Expensive equipment. Direct IFA Indirect IFA IFA (direct and Indirect) Chlamydia trachomatis by IFA Lecture 1- principle of Ag-Ab reactions. by Dr. Hajar 71 Flow Cytometry is commonly used in the clinical laboratory to identify and enumerate cells bearing a particular antigen. Cells in suspension are labeled with a fluorescent tag by either direct or indirect immunofluorescence. The cells are then analyzed on the flow cytometer. In a flow cytometer, the cells exit a flow cell and are illuminated with a laser beam. The amount of laser light that is scattered off the cells as they passes through the laser can be measured, which gives information concerning the size of the cells. In addition, the laser can excite the fluorochrome on the cells and the fluorescent light emitted by the cells can be measured by one or more detectors. Lecture 1- principle of Ag-Ab reactions. by Dr. Hajar 72 Flow Cytometry FACS: Fluoresence-activated Cell sorter Lecture 1- principle of Ag-Ab reactions. by Dr. Hajar 73  8- Immunochromatography (ICT): Immunochromatographic assays, termed also (Rapid Test), (Point-of-care test, POCT) or (lateral- flow dipstick) immunoassays. Based upon Sandwich ELISA assay and run on chromatographic papers or Nitrocellulose membrane by the capillary action. The test strips or cartridge contains two type of fixed monoclonal antibodies one at the test (T) zone which is specific to the Ag or Ab of interest (to be tested for), and the other Ab at the control (C) zone which is anti-antibody to the human IgG for validation of reaction components of the test strip or cartridge.  The 3rd type of mAb is the colloidal gold-labeled (Conjugate) free moving and infiltrating in the strip or cartridge pad and specific to the Ag or Ab of interest (Tested for) and ready to react with the Ag or Ab of interest (if present in sample) and move across the other side of the strip. If the test is positive the Ag or Ab will be trapped giving colored band at test (T) zone and the human (e.g.) IgG will react with the Anti-antibody in the control (C) areas giving colored band. On the other hand, if the sample is negative for the Ag or Ab of interest only colored band will appear at the C zone due to the presence of human immuno- globulins indicating the validity of the test.  If no colored band seen at the control (C), the test is invalid even if there is a band at the Test zone (T). Applications of Rapid tests in clinical diagnosis: 1- plenty and increasing range of usage of the rapid ICT tests including HIV, Hepatitis, malaria, influenza, Pregnancy, filaria, Leishmannia, Trichomoniasis, Chagas disease , Entamoeba histolytica/dispar , Cryptosporidium and Giardia ,..etc. A list of WHO list of tests including the rapid test (RDT) are in this site: http://www.who.int/diagnostics_laboratory/evaluati ons/170316_prequalified_product_list.pdf?ua=1 N.B. All antibodies used are monoclonal (mAb) specific to single epitope. ii- Rapid tests Advantages: Easy to use immediate and fast results (10-20 minutes) so used in screening. Can be used at home or clinic. Inexpensive Some tests are FDA approved Done upon whole blood or saliva or urine. No equipment, refrigeration, No electricity, No multiple timing steps required. Built in controls  Disadvantages of rapid tests. Positive late after infection; after seroconversion. Need confirmation for reactive tests in some cases. Subjective variability in result reading. Qualitative and not quantitative. Less sensitive than ELISA. Strips Both of them Cartridges or cassettes Rapid tests for HIV diagnosis Home HIV Rapid tests. Rapid tests for HBV, HIV, & HCV Malaria Rapid test Influenza Rapid test Giardia Rapid Test Varieties RDT  9- Immuno-blotting assays Western blotting (WB) is analysis and detection of proteins was first developed in 1979. The method is based on building an antibody: protein complex via specific binding of antibodies to immobilized proteins on a membrane and detecting the bound antibody with one of several detection methods. WB is based on the principles of immunochromato- graphy where denaturated proteins are separated into poly acrylamide gel according to the isoelectric point and molecular weight by electrophoresis. Then these separated proteins are transferred (blotted) on a nitrocellulose or Polyvinylidene fluoride (PVDF) membrane.  Specific labeled antibodies are allowed to react to these proteins fixed on the nitrocellulose or PVDF membrane (Ag-Ab reaction). This Ag-Ab reaction then visualized according to the method of labeling of the antibody mainly staining and photography. The photographed films can be kept for long time. Several procedures and reagents are needed for sample preparation, gel electrophoresis, transfer, antibody probing, detection, imaging and analysis. The WB technique (replaced by or /with PCR) are the current confirmatory diagnosis of HIV in our laboratories  Sample preparation WB Steps Gel Electrophoresis Transfer Antibody Probing Detection Imaging Analysis What are the most common serological tests used in our hospital and reference laboratories? 1. Latex tests in the lab are the most common (Widal, Brucella, ASOT, CRP, bacterial and viral identification in the lab…etc). 2. Rapid tests are commonly used in clinics as POCT (HIV, Pregnancy, hepatitis, etc). 3. ELISA are commonly used for viral infections (HIV, HBV, HCV, HSV,..etc) 4. Flocculation tests (RPR) are used for screening syphilis. 5. Syphilis is confirmed by TPHA or ELISA or FTA 6. WB is used as a confirmatory test for HIV. 7. Rarely to non used tests include: RIA, IFA, CFT,  References: Lennox K. Archibald. Rapid Diagnostic Testing of Infectious Diseases. http://www.medscape.com/viewarticle/748139_print. http://www.who.int/diagnostics_laboratory/evaluations/170316_prequalified_pro duct_list.pdf?ua=1 https://www.theglobalfund.org/media/5891/psm_qadiagnosticsmalaria_list_en.p df El-Moamly AA. Immunochromatographic Techniques: Benefits for the Diagnosis of Parasitic Infections. Austin Chromatogr. 2014;1(4): 8. http://www.globalhealthprimer.emory.edu/targets-technologies/rapid-diagnostic- test.html James K. Immunoserology of Infectious Diseases. CLINICAL MICROBIOLOGY REVIEWS, Apr. 1990, p. 132-152. Yang and Ma. Western Blotting and ELISA Techniques. Researcher, 2009;1(2):67-86. GE Healthcare. Western Blotting. Principles and Methods. 28-9998-97 240+ videos of common lab techniques, new technologies and more. See experiments in action: http://www.abnova.com/abvideo/ Some pictures were taken from the internet. Preparation of known antisera in animals Preparation of known antiserum in animals involves inoculating animals with specific known antigens such as a specific strain of a bacterium. After the animal's immune responses have had time to produce antibodies against that antigen, the animal is bled and the blood is allowed to clot. The resulting liquid portion of the blood is the serum and it will contain antibodies specific for the injected antigen. However, one of the problems of using antibodies prepared in animals (by injecting the animal with a specific antigen and collecting the serum after antibodies are produced) is that up to 90% of the antibodies in the animal's serum may be antibodies the animal has made "on its own" against environmental antigens, rather than those made against the injected antigen. The development of monoclonal antibody technique has largely solved Lecture 1- that principleproblem. of Ag-Ab 94 reactions. by Dr. Hajar  Preparation of known antibodies by monoclonal antibody technique. Monoclonal antibodies are antibodies of a single specific type. In this technique, an animal is injected with the specific antigen for the antibody desired. After appropriate time for antibody production, the animal's spleen is removed. The spleen is rich in plasma cells and each plasma cell produces only one specific type of antibody. However, plasma cells will not grow artificially in cell culture. Therefore, a plasma cell producing the desired antibody is fused with a myeloma cell ,a cancer cell from bone marrow which will grow rapidly in cell culture, to produce a hybridoma cell. The hybridoma cell has the characteristics of both parent cells. It will produce the specific antibodies like the plasma cell and will also grow readily in cell culture like the myeloma cell. The hybridoma cells are grown artificially in huge vats where they produce large quantities of the specific antibody. Monoclonal antibodies are now used routinely in medical research and diagnostic serology and are being used experimentally in treating certain cancers and a few other diseases. Lecture 1- principle of Ag-Ab 95 reactions. by Dr. Hajar Thank you