Antigen antibody reactions

1. Antigen antibody reactions

2. Enzyme linked immunosorbant assay (ELISA)

3. The method depends on conjugation of an enzyme to either Ag or Ab, then substrate is added as a quantitative measure of enzyme activity. • There are 3 ways of ELIZA testing: • Indirect method. • Direct or Double antibody technique (sandwich technique). • Competitive method Non competitive

4. The test is performed in a 8 cm x 12 cm plastic plate which contains 96 wells, each of which are about 1 cm high and 0.7 cm in diameter. 96-well microtiter plates being used for ELISA

5. Principle: • Use of enzyme-labeled immunoglobulin to detect antigens or antibodies. • Signals are developed by the action of the hydrolyzing enzyme on chromogenic substrate. • Optical density measured by micro-plate reader. Examples: • Hepatitis A (Anti-HAV-IgM, anti-HAV IgG) • HIV

6. Types of ELISA

7. Direct ELISA sandwich (double Ab technique): used for detection of Ags. • Known specific Ab is immobilized by adsorption onto a plastic surface. • Clinical sample is added (if Ag present it will bind to the immobilized Ab) • Enzyme-labelled specific Abis added (attach to the fixed Ag if present) • Add the substrate

8. If Not specific • No colour change If Ab specific to Ag change the colour

9. E + wash E E Ag Ab Antibody labelled with enzyme E Change in colour + E E substrate

10. Indirect ELISA: • In this test an enzyme- labelled anti-human Ig is used to detect the presence of specific Abs in patients’ sera. • Known Ag is fixed by adsorption onto a plastic surface. • The serum sample is added ( if specific Ab is present, it will bind the fixed Ag). • Add the enzyme-labelled antihuman Ig. • add the substrate, then quantitatively measure for the degree of color change.

11. Step 1 inactivated HIV antigens Step 2 serum antibodies Step 3 Anti-human Ig coupled to enzyme Step 4 Chromogen or substrate Step 5 ( Measurement )

13. Competitive ELISA • Titre wells coated with antibodies • Unknown antigen competes with enzyme labeled known antigen. • Incubate: till antigen antibody reaction is complete • Wash  remove unbound antigens • Add substrate. • Enzyme + Substrate  Product  measure colour • Colour inversely related to antigen in patient sample. • The less color the more Ag.

14. Radioimmunoassay

15. Radioimmunoassay (RIA) This method is used for the quantification of antigens that can be radioactively labeled. History: • The technique was introduced in 1960 by Berson and Yalow as an assay for the concentration of insulin in plasma. • It represented the first time that hormone levels in the blood could be detected by an in vitro assay.

16. The principle • Is that radioactively labeled antigens competes with the non labeled antigens in a test sample. • For example 125 Iodine (125I), 14 Carbon (14C) or 3 Hydrogen (3H) are used in RIAs for labeling and are called as tracers.

17. Method Used: • Take an antigen labeled with a radioactive material. • Then add primary antibody which complexes with labeled antigen. • Then add an unlabeled antigen, which competes with labeled antigen for binding to primary antibody. • Then we add secondary antibody to the solution which precipitates antigen-antibody complexes. • Then we measure radioactivity of supernatant that indicates free antigen. • And the radioactivity of precipitate indicates bound antigen.

18. The difference between the radioactivity in the control “A” and the unknown sample “B” is proportional to the amount of unlabeled antigens in the unknown sample

20. Gamma Counter for calculating radioactivity

21. Merits: • Radioimmunoassay is widely-used because of its great sensitivity. • Using antibodies of high affinity, it is possible to detect a few picograms (10−12 g) of antigen in the tube. Demerits: • The main drawbacks to radioimmunoassay are the expense and hazards of preparing and handling the radioactive antigen. • 125 Iodine (125I), 14 Carbon (14C) and 3 Hydrogen (3H) emit gamma radiation that requires special counting equipment

22. Uses: • RIA has become a major tool in the clinical laboratory where it is used to assay Plasma levels of: • Most of our hormones • Digitoxin or digoxin in patients receiving these drugs and • Certain abused drugs • To detect presence of hepatitis B surface antigen (HBsAg) in donated blood; • To detect anti-DNA antibodies in systemic lupus erythematosus (SLE).

23. Flow cytometry • Flow cytometers are instruments capable of analyzing properties of single cells from the general population. • The analyzed properties include: • Physical: size, volume, refractive index, viscosity. • Chemical: DNA, RNA, proteins and enzymes content. The data generated can be analyzed statistically by flow cytometry software.

24. Application in microbiology: • Rapid detection & counting of bacteria in pure culture. • Detection of viruses in tissue culture and clinical samples. • Detection of viral Ags on cells of infected person.

25. Flow Cytometry • In this test, the patient's cells are labeled with monoclonal antibody, tagged with a fluorescent dye, such as fluorescein or rhodamine. • Single cells are passed through a laser light beam, cells that fluoresce is counted . The data generated can be anayzed statistically by flow cytometry software to report cellular characteristics such as size , complexity , phenotype & health.

26. Instrument Overview • The primary systems of the flowcytometery are : • The fluidic system , which presents samples to the interrogation point & takes away the waste. • The lasers , which are the light source for scatter & fluorescence . • The optics , which gather & collect the light. • The detectors , which receive the light. • The electronics & Theperipheral computer system which converts the signals from the detectors to the digital data to perform the necessary analyses .