Immunochemistry & interpretation of ELISA

Immunoglobulins (or antibodies) are Y-shaped proteins produced by B cells that are essential for the adaptive immune system to fight infection by recognizing and binding to specific antigens, or foreign substances like viruses and bacteria.

1. IMMUNOCHEMISTRY By – Ms. Neha Singh Nursing Tutor CON, KHFH 1

2. The immune system is a line of defence against invasion of the body by foreign organisms. This system can store the information about self. Immunology is a very exciting area and has found applications in preventive, diagnostic and therapeutic aspects of diseases. 2 IMMUNOCHEMISTRY

3. It is a chemical substance which when introduced into the body causes the production of specific antibodies or immune cell function. The antigen should have the following: Immunogenicity: It is the ability to stimulate the formation of specific antibodies. Reactivity: It is a ability of an antigen to specifically bind with the produced antibodies. An antigen that has both the above characteristics is called a complete antigen. 3 ANTIGENS

4. Antigens generally fall under one of the following: proteins, nucleoproteins, lipoproteins, glycoproteins and large-molecular-weight polysaccharides. The molecular weight of antigens is generally greater than 10,000. 4 CHARACTERISTICS OF AN ANTIGEN

5. The antibodies are not formed against the entire antigen. Epitopes are the specific regions on the surface of the antigen called antigenic determinant sites that combine with the antibody. The combination is similar to hormone-receptor and enzyme-substrate unions. Antibodies bind to epitopes via hydrophobic, ionic, van der Waals forces. The number of antigenic determinant sites on the surface of an antigen is called valence. 5 ANTIGENIC DETERMINANT SITES OR EPITOPES

6. Most antigens are multivalent, i.e. they have several antigenic determinant sites. In order to induce antibody formation, at least two determinant sites have to be present, i.e. the antibody has to be bivalent. A hapten or partial antigen is a low-molecular-weight substance (molecular weight: 200-1000), which can bind to antibodies but cannot elicit an immune response on its own. When linked to larger molecules or carriers, it can elicit an immune response. 6 ANTIGENIC DETERMINANT SITES OR EPITOPES

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9. An antibody or immunoglobulin is a protein produced by the body in response to the presence of an antigen. Antibodies are secreted by plasma cells, which develop from lymphocytes (B cells) that are derived from bone marrow. Antibodies are glycoproteins composed of polypeptides (82%-96%) and carbohydrates (4%-18%). They are bifunctional . 9 ANTIBODIES OR IMMUNOGLOBULIN

10. They bind specifically with the antigen. They initiate a variety of secondary phenomena such as complement fixation and histamine release. There are five major classes of antibodies, namely, immunoglobulin M ( IgM ), immunoglobulin D ( IgD ), immunoglobulin G ( IgG ), immunoglobulin E ( IgE )and immunoglobulin A (IgA). All these immunoglobulins share a common structural form. 10 ANTIBODIES OR IMMUNOGLOBULIN

11. Activation of complement Opsonization Prevention of microorganism attachment Neutralization toxins and viruses Restriction of microorganism mobility Agglutination of bacteria 11 FUNCTIONS OF IMMUNOGLOBULIN

12. Basic unit (monomer) This is Y-shaped and made up of four polypeptide chains. Although there are four polypeptide chains, this entire unit is called a monomer. The four polypeptide chains are made up of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain is paired with a light chain. Disulphide bonds The heavy and light chains are held together by disulphide bonds. The disulphide bonds can be interchain or intrachain . 12 STRUCTURE OF IMMUNOGLOBULINS

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14. Variable and constant regions Each polypeptide chain contains an amino terminal part called the variable region(V) and a carboxyl terminal part called the constant region (C). Variable region The variable regions of paired L and H chains are adjacent to each other and form the antigen-binding site. The antigen binds to the antigen-binding sites. 14

15. Constant Region The carboxyl terminal domains of H and L chains have a constant structure. This structure does not differ between the different antibodies. This region is responsible for initiating the antigen-antibody reaction. 15

16. Hyper-variable R egion These are the sub-regions within the variable region that have highly variable amino acid sequence. This region is responsible for antigen binding. Thus, antibodies have two functional domains. Binding domain is made up of variable and hyper-variable regions, which are responsible for binding of the antigen. Effector domain is made up of the constant region and is responsible for initiating the antigen-antibody reaction. 16

17. Hinge Region This is a flexible region present between the two structural domains of the constant region. It allows for movement between the two antibody-binding sites . This region can be acted upon by proteases such as papain and is split up into three fragments, namely, two identical antigen-binding fragments (Fab) and one Fc fragment containing the effector domain. 17

18. Carbohydrate Components Significant amounts of carbohydrates are present in all immunoglobulins in the form of side chains. Their role is uncertain. They may play an important role in the secretion of immunoglobulins by plasma cells and in the biological functions associated with constant regions of heavy chains. 18

19. IgM STRUCTURE Exists as a pentamer . The five monomers are linked by disulphide bonds. There exists an additional single polypeptide chain called J chain. The J chain may be involved in the polymerization of IgM basic units. 19 TYPES OF ANTIBODIES

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21. It is the major antibody produced by B cells. It is the most abundant antibody in foetus . It constitutes approximately 10% of normal immunoglobulins and has the highest molecular weight. It is prominent in primary immune responses to most antigens. It avidly binds viruses and bacteria. It is the most efficient complement-fixing immunoglobulin. It does not cross the placenta. 21 OCCURRENCE AND FUNCTIONS

22. IgG STRUCTURE It is a monomer. Four subclasses of IgG are present (IgG1-IgG4). It contains carbohydrate residues (4%) covalently attached to the H chain. OCCURRENCE AND FUNCTIONS It is the most abundant antibody that constitutes 75% of the total antibodies in serum. It is the only maternal antibody that can pass through the placenta to protect the foetus . It is secreted in the mother's milk and responsible for the protection of newborn during the first few months of life. 22

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24. It is the main immunoglobulin released into blood stream during the later part of primary response and also during secondary immune response. It fixes serum complement and triggers macrophages. It neutralizes toxins and viruses. 24

25. IgA STRUCTURE It can exist as a monomer, dimer or trimer . Each secretory IgA molecule consists of two monomers: one secretory component and a J chain. OCCURRENCE AND FUNCTIONS It constitutes about 15% of total immunoglobulins . It is the predominant immunoglobulin in body secretions such as saliva, tears and nasal mucosa. Secretory IgA provides the primary defence mechanism against some local infections. It does not fix complement. 25

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27. IgD STRUCTURE It is a monomer. Molecular weight is slightly higher than that of IgG . OCCURRENCE AND FUNCTIONS It is present in trace amounts, and 0.2% is present in various body fluids. Its main function is not determined. However, it is present on the surface of human B lymphocytes along with IgM . It may be involved in the differentiation of B lymphocytes. 27

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29. IgE STRUCTURE Exists only in monomeric form. OCCURRENCE AND FUNCTIONS It comprises only 0.004% of the total serum immunoglobulins . It binds with very high affinity to mast cells and stimulates the mast cells to release a number of factors such as histamines to kill various types of parasites, thus causing immediate hypersensitivity. 29

30. Upon combination with certain specific antigens called allergens, IgE antibodies trigger the characteristic reactions, namely, flare skin reactions, asthma and hay fever. It is the host's main defence against helminthic infections. Worm infections are defended by triggering release of enzymes from eosinophils . 30

31. ELISA is a plate based immunoassay technique used to detect and quantify substances such as antigen, antibodies, proteins or hormones. ENZYME-LINKED IMMUNOSORBENT ASSAY

32. Specific antibodies are used to quantify the amount of antigen present in a biological sample. Enzyme-linked immunosorbent assay (ELISA) is able to detect minute quantities (less than nanograms ) of a particular protein. There are many types of ELISA techniques. ENZYME-LINKED IMMUNOSORBENT ASSAY

33. In ELISA, specific antibody is usually coupled to a solid support such as plastic. The sample to be tested is then incubated. If the specific antigen is present, it binds to the antibody . The unbound protein is washed off and then the bound protein is incubated with a secondary antibody . ENZYME-LINKED IMMUNOSORBENT ASSAY

34. A secondary antibody is attached to an enzyme, which catalyses the conversion of a colourless or nonfluorescent substrate into a coloured product. The intensity of the colour or fluorescence produced is then measured to determine the amount of antigen present. Machines are available that scan the wells of plates following ELISA. ENZYME-LINKED IMMUNOSORBENT ASSAY

35. Immobilization of Antigen or Antibody Antibody-Antigen binding Enzyme linked antibody detection Substrate reaction and signal generation Signal measurement and quantification PRINCIPLE OF ELISA

36. Microtitre plate Add antigen (antigen attach to surface) Wash microtitre plate by triphosphate buffer Add enzyme linked antibody Again wash Add colorless substrate (substrate bind with enzyme) Produce color product (chromosomes) DIRECT ELISA

37. Microtitre plate Add antigen (antigen attach to surface) Wash microtitre plate by triphosphate buffer Primary antibody binds to antigen Add enzyme linked secondary antibody binds to primary antibody Again wash Add colorless substrate C olor product USES : Antibody Detection in Serum INDIRECT ELISA

38. Primary Antibody is coated on Microtitre plate Add antigen (antigen attach to surface) Antigen binds to capture antibody Add enzyme linked antibody Again wash Add colorless substrate Color product USES : Antigen Detection SANDWICH ELISA

39. Sample Antigen and inhibitor antigen Fight for binding with Primary antibody Incubate Antibody with sample antigen Mixture added to antigen-coated plate The more antigen in sample shows less color product (less binding of inhibitor antigen) The less antigen in sample shows more color product (more binding of inhibitor antigen) USES : Quantification of Antigen COMPETITIVE ELISA

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