IMMUNOLOGICAL TECHNIQUES

1. Immunological Techniques By Mubah rafique

2. What are immunological techniques Immunological techniques are the wide varieties of methods and specialized experimental protocols devised by immunologists for inducing, measuring, and characterizing immune responses . These techniques are not restricted to the field of  immunology  , but are widely applied by basic scientists in many other biological disciplines 

3. Immunologic techniques include: ELISA RIA IMMUNOPRECIPITATION WESTERN BLOT

4. An  enzyme-linked immunosorbent assay , also called ELISA or EIA, is a test that detects and measures antibodies in your blood. This test can be used to determine if you have antibodies related to certain infectious conditions. Antibodies are proteins that your body produces in response to harmful substances called  antigens .

5. An ELISA test may be used to diagnose: HIV , which causes AIDS Lyme disease pernicious anemia Rocky Mountain spotted fever rotavirus squamous cell carcinoma syphilis toxoplasmosis varicella -zoster virus, which causes  chickenpox  and  shingles

6. Requirement Microtiter plate Antigen Antibody PBS Buffer BSA blocking protein  

7. Types of ELISA

8. Direct ELISA

9. STEP 1 PBS along with serum is poured in the well of microtiter plate. As the serum contain antigen, when we pour sample in the well antigen get attached with the well surface. This step is called coating.

10. Blocking This is the step in which we use non reactive protein and pour it in the well. This protein will cover or block the space in the well other than where antigen is present. Blocking is done because microtiter plate has ability for binding antibodies and proteins.So we want to bind antibody only with antigen not with surface. Blocking agent is BSA protein.

11. Attachment of enzyme linked antibody Next step is the attachment of enzyme linked antibody. These antibodies are available in markets or we can prepare them. This enzyme linked antibody is specific to antigen,that has enzyme HRP. After attachment of antibody and antigen we will add substrate specific to enzyme.

12. Detection When enzyme and substrate combine color change in the well that shows the presence of antigen. Instensity of color shows severity of infection. If color change does not take place it means there is no infection and there is no antibody.

13. Analysis We a analyse the test by ELISA reader. ELISA plate is placed in reader. This gives quantitative information and qualitative information of antigen.

14. Direct ELISA

15. Indirect ELISA Used for antigen detection. Disease specific microtiter plate is used. In this specific plate antigen is already present and surface is already blocked. So,next sample containing antibody is added. This antibody will attach with antigen.Antibody used is called primary antibody. Then we will use secondery antibody that is also called detection antibody that is enzyme linked.

16. Enzyme attach is HRP or alkaline phosphate. Secondery antibody bind with primary antibody as it is specific to primary antibody. After that specific substrate added and colorful solution is obtained. Then data analysis is performed.

17. Sandwich ELISA First step is serum separation that contain antibodies. If we have some sort of infection then we must have antibodies that will produce in response to antigen. PBS buffer is added to this sample. We will add sample in the well of plate,antibody will attach to the surface. Then blocking step is performed. Here antibody is called capture antibody.

18. Then sample containing antigen is added. After that enzyme linked antibody is added. After that substrate is added reaction takes place and color change occur. Sandwich ELISA is 2 to 3 times more sensitive.

19. Radio immuno assay One of the most sensitive techniques for detecting antigen or antibody is radioimmunoassay (RIA). The technique was first developed in 1960 by two endocrinologists, S. A. Berson and Rosalyn Yalow, to determine levels of insulin–anti-insulin complexes in diabetics. • In 1977, some years after Berson’s death, the significance of the technique was acknowledged by the award of a Nobel Prize to Yalow.

20. Requirement If we want to detect antigen serum A antigen then we need 3 things Anti a antibody Radiolabeled A antigen Unlabeled A antigen

21. Principle of RIA Radioimmunoassay (RIA) involves the separation of a protein (from a mixture) using the specificity of antibody - antigen binding and quantitation using radioactivity.

22. Steps involved in RIA In the basic method of Radioimmunoassay, we use the target antigen which is labeled radioactively and bound to its specific antibodies. We will require a limited and known antibody to be added in a specific amount in Radioimmunoassay. A sample is then added in order to initiate a reaction competitive in nature, of the labeled antigens from the preparation, and the unlabeled antigens from the sample, with the specific antibodies.

23. Steps involved RIA Consequently, unlabeled antigen added to the sample mixture will compete with radiolabeled antigen for the limited supply of antibody. Even a small amount of unlabeled antigen added to the assay mixture of labeled antigen and antibody will cause a decrease in the amount of radioactive antigen bound, and this decrease will be proportional to the amount of unlabeled antigen added.

24. The bound antigens are then separated from the unbound ones, and the radioactivity of the free antigen remaining in the supernatant is measured using a gamma counter.

25. Applications of RIA The test can be used to determine very small quantities (e.g. nanogram ) of antigens and antibodies in the serum. The test is used for quantitation of hormones, drugs, HBsAg , and other viral antigens. Analyze nanomolar and picomolar concentrations of hormones in biological fluids

26. Immuno precipitation Immuno precipitation is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins.

27. Target antigens are usually immuno precipitated from complex solutions, such as cell lysates , the goal being to isolate and eventually detect and measure a specific protein (i.e., the antigen of the specific antibody).  

28. Steps involved in IP First, lyse your cells using some sort of lysis buffer.  Next, add in an antibody that will bind your protein of interest and form a protein-antibody complex. Then drop in some resin which can bind the antibody. These proteins are specifically designed to bind the heavy chains of antibodies so they can easily pull out your protein-antibody complexes.

29. Agarose beads offer higher capacity per bead but magnetic beads are MUCH easier to separate because you can use a magnet to keep them in place. Finally, spin everything down and remove the supernatant. With the remaining bead-antibody-protein conjugates, you can either denature everything and run a  SDS-PAGE western blot .

30. Application of IP Detect proteins of interest. Study protein- protein interact. Identify protein in protein complex. Protein expression in specific tissues.

31. Western blotting A  western blotting  is a laboratory method used to detect specific protein molecules from among a mixture of proteins. This mixture can include all of the proteins associated with a particular tissue or cell type It not only used for proteins but also to find out different chemical reactions inside the cell,protein -protein interaction.

32. Principle of western blotting Western blotting is an Immuno blotting technique which rely on the specificity of binding between a molecule of interest and a probe to allow detection of the molecule of interest in a mixture of many other similar molecules.  In Western blotting, the molecule of interest is a protein and the probe is typically an antibody raised against that particular protein.

33. Steps involved in western blotting technique Extraction of molecule Separation Tranfer Probing Analysis  

34. Step 1 First of all we will treat the cell simply with detergent.it will break down cell membrane.Cytosolic components come out. Then we will do centrifugation that gives us supernatant and pellet.Pellet contain cell debris, membrane , organelles while supernatant contain protein components. We will take supernatant that contain mixture of proteins.

35. Separation of proteins We will separate proteins on the basis of molecular weight.This is done by SDS-PAGE electrophoresis. SDS-PAGE stands for sodium dodicyl sulphate polyacralamide gel electrophoresis. It uses gel acralamide . This type of electrophoresis separate proteins on the basis of molecular weight.

36. Transfer On completion of protein separation by polyacrylamide gel electrophoresis (PAGE), the next step is to transfer the proteins from the gel to a solid support membrane, usually made of a chemically inert substance, such as nitrocellulose or PVDF. Blotting makes it possible to detect the proteins on the membrane using specific antibodies. The proteins transferred from the gels are immobilized at their respective relative migration positions at the time when the electric current of the gel run was stopped.

37. Antibody probing Once your protein samples are separated and transferred onto a membrane, the protein of interest is detected and localized using a specific antibody. Usually, Western blotting protocols utilize an unlabeled primary antibody directed against the target protein and a species-specific, labeled secondary antibody directed against the constant region of the primary antibody.

38. The secondary antibody serves not only as a carrier of the label, but is also a mechanism to amplify the emitted signals, as many secondary antibodies can theoretically bind simultaneously to the primary antibody. This is one of the most effective ways to maximize the potential sensitivity of the assay. For this reason, secondary antibodies are most often polyclonal and can target epitopes on the framework regions of the primary antibody; specificity is thus limited to species and immunoglobulin isotype . 

39. The signal emitted by the labeled secondary antibody is then measured and is proportional to the quantity of protein of interest present on the membrane. With this highly specific immunodetection process, it is possible to reveal the presence of a very low quantity of a specific protein in a complex sample.

40. Imaging The last step in the Western blotting workflow before data analysis is image capture. Enhanced chemiluminescence (ECL) is based on the reaction between an added luminol substrate and horseradish peroxidase (HRP)-labeled antibodies. In the presence of HRP, hydrogen peroxide catalyses the oxidation of luminol , a reaction that results in the emission of light.

41. The light signal can then be detected on X-ray film or by digital imaging with a charge-coupled device (CCD) camera-based imager. When using fluorescence detection, a fluorophore is conjugated to the primary or secondary antibody. Light is emitted by the fluorophore after excitation via a specific wavelength of light. A photomultiplier tube (PMT) or a CCD can be used to collect and convert the emitted light to an electrical signal. The electrical signal is then digitized for image display and analysis.

42. Analysis Detection of signals, using either X-ray film, scanners, or a charge-coupled device (CCD) camera-based imager, results in one or more visible protein bands on the membrane image. The molecular weight of the protein can be estimated by comparison with marker proteins and the amount of protein can be determined as this is related to band intensity (within the limits of the detection system.