1. Infectious Diseases 1 Chanaka Lakshan, [BS/15/10/15], BMS-10, Dpt. of Biomedical Science, School of Health Science, BCAS-City Campus.

2. Contents Complementary Fixation Test Haemagglutination Inhibition Test PCR – Polymerase Chain Reaction Western Blot Coomb’s Test 2

3. Complementary Fixation It was extensively used in syphilis serology after being introduced by Wasserman in 1909. It is used to detect the presence of specific antibodies in the patient’s serum. Complement is a protein (globulin) present in normal serum. Complement proteins are heat labile and are destroyed by heating at 56°C for 20 – 30 min in a process called heat inactivation. Complement binds to Ag-Ab complex When the Ag is complexed with Ab on surface of cell, Complement causes lysis of cell.

4. 4 Procedure

5. Results & Interpretation Positive test The available complement is fixed by Ag-Ab complex and no hemolysis of sheep RBCs occurs. So the test is positive for presence of antibodies. If the complement is fixed on test system(Ag + Ab), then there will be no lysis of sheep erythrocytes, thus denoting a positive test. No lysis of sheep red blood cells (positive CFT) indicates the presence of antibody in the test serum Negative test No Ag-Ab reaction occurs and the complement is free. This free complement binds to the complex of sheep RBC and it’s antibody to cause hemolysis, causing the development of pink color. If the complement is available not bound to test system, it will be free to combine with indicator system resulting in hemolysis denoting a negative test. lysis of sheep red blood cells (Negative CFT) indicates the absence of antibody in the serum

6. 6 Applications Advantages 1.Large variety of antigens can be used 2.Simple interpretation Disadvantages 1.Demand on equipment & reagents is large 2.Some components needed to be fresh (RBC, complement) 3.Less sensitive than some serological tests (ELISA) Wasserman test  for Syphilis, for viral fungal, Chlamydial & protozoal diseases

7. Haemagglutination Inhibition Test Hemagglutination phenomenon is used for diagnosis of infection produced by Orthomyxoviruses, paramyxoviruses, and the abroviruses-togaviruses (including rubella), flaviviruses, and bunyaviruses. The presence of virus in infected cell cultures can be detected by hemagglutination; the identity of the virus or of antibodies in a patient’s serum can be determined by specific inhibition of that hemagglutination. Typing of the isolate is done by Hemagglutination Inhibition (HAI). Reagents and conditions for the test vary by virus. The basis of the HAI assay is that antibodies to that particular virus will prevent attachment of the virus to RBC. Hemagglutination is Inhibited when Antibodies are Present. By adding specific antibodies to the virus it is possible to block this interaction and detect the virus. If antibodies to the virus are specific, Hemagglutination will not be Observed. VIRUSE SERUM Negative HAI Positive HAI

8. 8 Agglutination 1.Direct  soluble Ab results in clumping due to interaction with relevant Ag 2.Passive  Initially Ab or Ag adsorbed to latex beads. Then mixed with the serum. If relevant Ag or Ab is present, agglutination will occur. Haemagglutination  RBCs are clumped by haemagglutinating viruses Haemagglutination inhibition  done to check for the Abs against haemagglutinating viruses in the patient’s serum sample Procedure

9. 9 Interpretation Applications To check for the presence of Abs in patient’s serum for influenza, measles, mononucleosis, mumps viruses etc. Used for serotyping Used to measure Ab titer If the serum contains no antibodies that react with influenza virus, then hemagglutination will be observed in all wells. If antibodies to the virus are present, hemagglutination will not be observed until the antibodies are sufficiently diluted.

10. 10 Advantages 1.Sensitive for Ab detection 2.Can be used to measure Ab titer/quantitative analysis Disadvantages 1.Time consuming 2.Prozone phenomenon 3.Contamination issues 4.Suspected causative agent should be available & handling them is dangerous

11. Polymerase Chain Reaction The PCR method was invented in 1983 by a California biotech researcher - Karry Mullis. PCR is the abbreviation for "Polymerase Chain Reaction”. This is a laboratory technique used to make multiple copies of a segment of DNA. It makes genome sequencing, detection of genetic diseases, the study of fossil DNA, detection of GMOs and has served the scientific police. PCR is very precise and can be used to amplify, or copy, a specific DNA target from a mixture of DNA molecules. Components 1) DNA Template 2) DNA Polymerase 3) Oligonucleotide primers 4) Deoxynucleotide triphosphates 5) Buffer system

12. Procedure 12

13. 13 Amplification of the interested gene by using PCR is the 1 st part of the identification test. Then the amplified segments needed to be compared with the known DNA segments. Gel electrophoresis is done for this. There after, the separated bands are compared with the ladder Interpretation

14. 14 Advantages 1.Extremely high sensitivity, it might detect one viral genome per sample volume 2.Easy to set up 3.Turn around time is less Disadvantages 1.Extremely liable for contaminations 2.High degree of operator skill required 3.Not easy to set up a quantitative assay 4.Interpretation of positive result is difficult (in case of some viruses such as CMV, seropositive person might have viruses present in their blood irrespective whether they’ve disease or not) Applications 1.Medical applications – In genetic testing for genetic disease mutations, In tissue typing which is vital for organ transplantation. 2. Infectious disease applications – Checking for HIV (1 viral genome among DNA of 50,000 host cells can be detected), checking for tuberculosis. 3.Forensic applications – genetic fingerprinting in crime activities & in paternity testing. 4.Research applications – In DNA cloning, In studying the patterns of gene expression, In genetic mapping, DNA sequencing.

15. Western Blot It was first described by Towbin, et.al in 1979 and has since become one of the most commonly used methods in life science research. Western blotting is a widely used technique for the detection and analysis of proteins based on their ability to bind to specific antibodies. Western blotting is a accomplished rapidly, using simple equipment and inexpensive reagents, it is commonly used laboratory technique. The specificity of the antibody- antigen interaction enables to a target protein to be identified in the midst of a complex protein mixture.

16. Procedure 16

17. 17 Different types of labeled Abs are used in western blots: 1.Enzyme conjugated Abs – enzyme converts the substrate to a colorful compound, hence detection is done. 2.Metal conjugated Abs – X ray film is obtained for the detection. by enzyme conjugated Abs Western Blot Test Results For HIV-AIDS

18. 18 Interpretation Protein band signal confirms the protein presence & identity Position of band informs about protein molecular weight, confirm expected size Intensity of band informs about protein amount in the sample

19. 19 Advantages 1.Well established & trusted 2.Available in most of the labs 3.Ability to identify & quantify a specific protein in a complex mixture such as a cell extract Disadvantages 1.Lack of standardization 2.Lack of quantitation 3.Requires extensive repeat runs & controls to achieve reliable data Applications 1.The confirmatory HIV test employs western blot to detect anti-HIV antibody in a human serum sample 2.Used for detection & analysis of proteins 3.Used to diagnose various diseases such as viral & autoimmune diseases (but basically used to diagnose HIV)

20. Coomb’s Test It is also known as antiglobulin test. It was discovered by Coombs, Mourant and Race in 1945. The Coombs test tests for antibodies that may stick to the red blood cells and cause red blood cells to die too early. Red cells coated with complement or IgG antibodies do not agglutinate directly when centrifuged. These cells are said to be sensitized with IgG or complement. In order for agglutination to occur an additional antibody, which reacts with the Fc portion of the IgG antibody, or with the C3b or C3d component of complement, must be added to the system. This will form a “bridge” between the antibodies or complement coating the red cells, causing agglutination.

21. 21 How is Coomb’s reagent prepared ? Auto antibodies /serum of human collected  Injected in to an animal  Animal forms Ab against auto antibodies  Purified & collected  Coomb’s reagent

22. Procedure & Interpretation 22

23. 23 Applications Direct test 1)Used to check for the immune mediated hemolytic anemia. It may be autoimmune hemolysis/alloimmune hemolysis (new born) or drug induced immune hemolysis Indirect test 1)Used in blood transfusions for antibody screening & cross matching 2)Used to check IgG antibodies that are likely to pass through placenta & would cause hemolytic disease of the new born Advantages Sensitive for antibody detection Disadvantages Prozone phenomenon (the agglutination doesn’t occur when the Ab conc. is very high) Time taken The reactions are only semi quantitative

24. Results & Interpretation Negative Result: No clumping of cells (no agglutination). This means you have no antibodies to red blood cells. Positive Result: Clumping (agglutination) of the blood cells during a direct Coombs test means that you have antibodies on the red blood cells and that you may have a condition that causes the destruction of red blood cells by your immune system (hemolysis). This may be due to 1) Hemolytic anemia, 2) Chronic lymphocytic leukemia or similar disorder, 3) Erythroblastosis fetalis (hemolytic disease of the newborn), 4) Infectious mononucleosis, 5) Mycoplasmal infection, 6) Syphilis, 7) Systemic lupus erythematosus and 8) Transfusion reaction, such as one due to improperly matched units of blood.

25. References Giri, D. (2016) Complement fixation test: principle, procedure and interpretation, [Online] Available from: http://laboratoryinfo.com/complement-fixation-test/ (Accessed 20 May 2016). LinkedIn corporation (2016) Agglutination, [Online] Available from: http://www.slideshare.net/Raniaaboshady/agglutination-39044141 (Accessed 20 May 2016). LinkedIn corporation (2016) Practical micro, [Online] Available from: http://www.slideshare.net/hayamm/practical-micro (Accessed 20 May 2016). Rao (2012) Coomb’s test, [Online] Available from: http://www.slideshare.net/doctorrao/coombs-test (Accessed 20 May 2016). Slide player (2016) Complement fixation test principle procedure disadvantages, [Online] Available from: http://slideplayer.com/slide/9137017/ (Accessed 20 May 2016). 25