In vitro antigen antibody reactions

1. In vitro antigen antibody reactions Dr. Mohammad Odibate

2. Antigen-Antibody interactions Anti-HBsAg HBV What are the Lab methods used to detect the presence of viral antigens and their specific antibodies? HBsAg

3. Antigen-Antibody interactions Serology • Is the scientific study of serum and other bodily fluids. • In practice, the term usually refers to the diagnostic identification of antibodies in the serum. Serology can be used To • To know blood type • diagnose infection, autoimmune diseases, Immune deficiency diseases

4. Antigen-Antibody interactions Serological Reactions Immuno-cyto/histo-chemistry Agglutination Precipitation • immunoassays • Radio-immuno assay • ELISA • Western blot • Immunofluorescence Complement fixation Flow cytometry

5. Antigen-Antibody interactions Ag-Ab interaction • union between different complexes the result depend on nature of both Ag and Ab • Precipitation if the antigen is soluble and Ab mostly is IgG • Agglutination; if the antigen is insoluble and Ab mostly is IgM and IgA • Complement fixation if the antigen unites Ab in the presence of complement

6. Antigen-Antibody interactions Precipitation Reactions Amount of precipitate Influenced by • Maximum precipitation occurs when Ags & Abs at optimal or equivalent proportions • Precipitation occur when a lattice (line) formed Soluble antigen + specific antibody = Insoluble Precipitate of Ag-Ab complex

7. Antigen-Antibody interactions Types of Precipitation In gel In solution

8. Antigen-Antibody interactions Types of Precipitation In solution Visible precipitation ring Soluble antigens Specific antibodies

9. Antigen-Antibody interactions Types of Precipitation In solution • fluid with unknown antigen is layered over known antibody if the corresponding antigen is present then line appear in between the 2 layers • Identification of H. influenza b and N. meningiditis in cerebrospinal fluids

10. Antigen-Antibody interactions Types of Precipitation In gel Petri dish Serum from patient suspected having hepatitis Known Antibody against HBsAg

11. Antigen-Antibody interactions Types of Precipitation In gel Petri dish Serum from patient suspected having hepatitis Known Antibody against HBsAg Precipitation line

12. Precipitation in gel Example Patient A Patient D Known Antibody against HBsAg Patient B Patient E Patient C Patient F • Precipitation in gel • Called Immunodiffusion • Advantages of precipitation in gel • Reaction visible as a distinct stable band . • Can be stained & preserved • Number of different Ags can be observed in one gel + + + + + +

13. Antigen-Antibody interactions Types of Precipitation In gel Single radial immunodiffusion Petri dish Serum from patient suspected having hepatitis Anti-HBsAg antibodies Already incorporated In gel

14. Antigen-Antibody interactions Types of Precipitation In gel Single radial immunodiffusion precipitation ring

15. Antigen-Antibody interactions Types of Precipitation In gel Single radial immunodiffusion • Single diffusion in two dimensions • (Radial immunodiffusion) - Ab incorporated in agar gel - Ag. added to wells in agar. - Ag. diffuses radially from the well - Forms precipitation ring around antigen

16. Antigen-Antibody interactions Medical applications Types of Precipitation • to determine the quantity of an antigen by measuring the diameters of circles of precipitin complexes surrounding samples • quantification of the major classes of serum immunoglobin • as well as complement factors, eg C3 and C4. • The test is predominantly used to determine abnormally low levels of one or more serum immunoglobulins, as a screen for presumptive immunodeficiency disease • Failure of passive transfer of immunoglobulins at birth is the most common reason for assessing IgG levels in new born • screening Abs against viruses (Influenza) In gel

18. Antigen-Antibody interactions Agglutination reactions + its Antibody Particulate Ag = clumping/ agglutinated Agglutination is more sensitive than precipitation for the detection of antibodies.

19. Antigen-Antibody interactions Agglutination reactions Qualitative agglutination test Quantitative agglutination

20. Antigen-Antibody interactions Agglutination reactions Qualitative agglutination test Whole cell Whole cell Serum To study the presence of an antigen To study the presence of an antibody

21. Antigen-Antibody interactions Agglutination reactions • 1-Qualitative agglutination test Agglutination tests can be used in a qualitative manner to assay for the presence of an antigen or an antibody. The antibody is mixed with the particulate antigen and a positive test is indicated by the agglutination of the particulate antigen. • Blood grouping, a patient's red blood cells can be mixed with known antibody to a blood group antigen to determine a person's blood type.

22. application Antigen-Antibody interactions Agglutination reactions 1. Slide Agglutination ;Blood grouping & Cross matching. Smooth suspension of blood on 3 slides + drop of antibody (anti-A, anti B and anti RH on each slide. Clumping of blood means it has that antibody specific antigen

23. Antigen-Antibody interactions Agglutination reactions 2-Quantitative agglutination test (on tube)Agglutination tests can also be used to measure the level of antibodies to particulate antigens. In this test, serial dilutions are made of a antibody sample (patient serum) and then a fixed number of particulate antigen is added. Then the maximum dilution that gives agglutination is determined. The maximum dilution that gives visible agglutination is called the titer. The results are reported as the reciprocal of the maximal dilution that gives visible agglutination. • Diagnosis of: Typhoid, Brucellosis

24. Antibody Titer

25. Antibody Titer What is a Titer? • A Titer is a blood test that checks your immune status to vaccinations or diseases you may have received in the past. There are two types of immunity a person can have: 1. Active Immunity • The body’s immune system produces antibodies and cellular immunity which usually lasts for many years to a lifetime. • Develops from surviving infection or by vaccination • Every individual varies in response and production of antibodies 2. Passive Immunity • Immunity transferred from one to another • Develops from mother to infant or by blood product such as immune globulin • This immunity usually only offers temporary protection during a period of weeks to months

26. Antibody Titer When do we use antibody titer? • To monitor the immune status against certain infection and ; • To reduce the costs of vaccination people who are already immune to certain pathogen

27. Antigen antibody reaction Agglutination + HBsAg Specific Ab

28. Procedure of HBV antibody titer HBsAg HBsAg HBsAg HBsAg HBsAg HBsAg HBsAg HBsAg 0.5 0.5 0.5 0.5 0.5 0.5 Saline 0.8 + Serum 0.2 ml 1:5 1:10 1:20 1:40 1:80 1:160 1:320 Dilution 1:720

29. Procedure of HBV antibody titer Titer = 320 Titer = 0 Titer = 5 Titer = 40 1:5 1:10 1:20 1:40 1:80 1:160 1:320 Dilution 1:720

30. Dilution 1:5 1:10 1:20 1:40 1:80 1:160 1:320 1:720 Titer= 40

31. Titer = 0 10 Titer = 5 Titer = Titer = 320

32. Antibody Titer Example Blood withdrawal Needle stick Antibody titer ≥10 Positive HBV Blood withdrawal Needle stick Antibody titer <10 Positive HBV

33. Antigen-Antibody interactions Diagnostic Applications Titer • An antibody titer is a laboratory test that detects and measures the amount of specific antibodies in the blood. • It can indicate if a person has or recently had an infection such as viral hepatitis. • Because vaccines stimulate antibody production against specific diseases, an antibody titer can indicate one's need for a booster shot or whether a recent immunization produced enough of an immune response to provide adequate disease protection. • Sometimes the test is used to diagnose autoimmune diseases, conditions in which one's immune system produces antibodies to the body's own tissues. • Antibodies can also detect small amounts of drugs, toxins and hormones.