1 BIOMAN 2010 I MMUNOASSAYS Barbara Bielska Northampton Community College Tannersville, PA

2 Antibody-mediated immunity Antibody-mediated immunity is part of acquired or adaptive immunity Antigen Antibody-mediated immunity involves B cells, and T cells (lymphocytes).

3 What is antigen Any substance that can elicit an immune response is said to be immunogenic and is called an immunogen. There is a clear operational distinction between an immunogen and an antigen. An antigen is defined as any substance that can bind to a specific antibody. All antigens therefore have the potential to elicit specific antibodies, but some need to be attached to an immunogen in order to do so. This means that although all immunogens are antigens, not all antigens are immunogenic.

4 Immunogen: adjuvant In immunology, an adjuvant is an agent that may stimulate the immune system and increase the response to a vaccine, without having any specific antigenic effect in itself. The word “adjuvant” comes from the Latin word adjuvare, meaning to help or aid. "An immunologic adjuvant is defined as any substance that acts to accelerate, prolong, or enhance antigen-specific immune responses when used in combination with specific vaccine antigens.“

5 What Is Antibody Antibody: An immunoglobulin, a specialized immune protein, produced because of the introduction of an antigen (immunogen) into the body, and which possesses the remarkable ability to combine with the very antigen that triggered its production. The production of antibodies is a major function of the immune system and is carried out by a type of white blood cell called a B cell (B lymphocyte). Antibodies can be triggered by and directed at foreign proteins, microorganisms, or toxins. The term "antibody" dates to Prior to that time, an "antibody" referred to any of a host of different substances that served as "bodies" (foot soldiers) in the fight against infection and its ill effects. According to

6 What Is Antibody

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8 Immunoglobulins Immunoglobulins – Antibodies IgD surface of many cells (mostly B) IgM surface of B cells IgG blood; the most abundant Ab IgA digestive and respiratory systems IgE skin, digestive and respiratory tract; responsible for allergic reactions

9 IgG: immunoglobulin G

10 Antibodies

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12 Vaccine is made from an antigen isolated or produced from the disease-causing microorganism. The vaccine is injected into the blood stream. The B cells in the blood stream respond to the antigen by producing antibodies. The antibodies bind to the antigen to "neutralize“ or inactivate it. In addition, memory cells are produced and remain ready to mount a quick protective immune response against subsequent infection with the same disease causing agent.

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14 Antibodies: Applications Diagnostic tests Research tool Drug delivery system

15 Immunoassays Diagnostic and research tools Allow to detect very minute amounts of the analytes Highly sensitive and specific Multibillion dollar business Examples: HIV test, hepatitis test, tests for numerous infectious diseases and other disorders (thyroid dysfunction), test for H. pylori, numerous QC tests.

16 Polyclonal and Monoclonal Antibodies Polyclonal Ab recognize the same antigen but different epitopes (antigenic determinants) heterogeneous made by the B cells Monoclonal Ab recognize the same antigen and the same epitope, homogenous clones made by mice hybridomas (tissue culture)

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18 Monoclonal Ab The production of monoclonal anti-bodies was first invented by Cesar Milstein,Georges J. F. Köhler and Niels Kaj Jerne in 1975.

19 HAT medium is often used for preparation of monoclonal antibodies. This process is called Hybridoma technology. Laboratory animals (e.g., mice) are first exposed to an antigen against which we are interested in isolating an antibody. Once splenocytes are isolated from the mammal, the B cells are fused with HGPRT negative, immortalized myeloma cells using polyethylene glycol or the Sendai virus. Fused cells are incubated in the HAT medium. Aminopterin in the medium blocks the de novo pathway. Hence, unfused myeloma cells die, as they cannot produce nucleotides by de novo or salvage pathway.

20 HAT medium is often used for preparation of monoclonal antibodies. Cont’d Unfused B cells die as they have a short lifespan. In this way, only the B cell- myeloma hybrids survive. These cells produce antibodies (a property of B cells) and are immortal (a property of myeloma cells). The incubated medium is then diluted into multiwell plates to such an extent that each well contains only 1 cell. Then the supernatant in each well can be checked for desired antibody. Since the antibodies in a well are produced by the same B cell, they will be directed towards the same epitope, and are known as monoclonal antibodies.

21 HAT Medium (Hypoxanthine Aminopterin Thymidine medium) is a selection medium for mammalian cell culture, which relies on the combination of aminopterin, a drug that acts as a folate metabolism inhibitor by inhibiting dihydrofolate reductase, with hypoxanthine and thymidine, which are intermediates in DNA synthesis—a purine derivative and a deoxynucleoside, respectively. The trick is that aminopterin blocks DNA de novo synthesis, which is absolutely required for cell division to proceed, but the other components provide cells with the raw material to evade the blockage (the "salvage pathway"), provided that they have the right enzymes, which means having functioning copies of the genes that encode them.

22 Therefore, the use of HAT medium for cell culture is a form of artificial selection for cells containing working TK and HGPRT (Hypoxanthine-guanine phosphoribosyltransferase). The protein encoded by this gene is a transferase, which catalyzes conversion of hypoxanthine to inosine monophosphate and guanine to guanosine monophosphate via transfer of the 5-phosphoribosyl group from 5-phosphoribosyl1-pyrophosphate. This enzyme plays a central role in the generation of purine nucleotides through the purine salvage pathway. Mutations in this gene result in Lesch-Nyhan syndrome or gout.

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24 Types of immunoassays ELISA: enzyme linked immuno-sorbent assay Radio-immunoassays Immunoassays with color or fluorescent tags (colloidal gold, polymers, crystals) Basic formats Direct assay Indirect assay Sandwich assay Competitive assay Multiplex assay Rapid assay: lateral flow chromatography

25 Microtiter plate

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27 Direct ELISA The direct ELISA uses the method of directly labeling the antibody itself. Microwell plates are coated with a sample containing the target antigen, and the binding of labeled antibody is quantitated by a colorimetric, chemiluminescent, or fluorescent end-point. Since the secondary antibody step is omitted, the direct ELISA is relatively quick, and avoids potential problems of cross-reactivity of the secondary antibody with components in the antigen sample. However, the direct ELISA requires the labeling of every antibody to be used, which can be a time-consuming and expensive proposition. In addition, certain antibodies may be unsuitable for direct labeling. Direct methods also lack the additional signal amplification that can be achieved with the use of a secondary antibody.

28 Indirect ELISA The indirect, two-step method uses a labeled secondary antibody for detection. First, a primary antibody is incubated with the antigen. This is followed by incubation with a labeled secondary antibody that recognizes the primary antibody. For ELISA it is important that the antibody enzyme conjugate is of high specific activity. This is achieved when the antibody is affinity purified and the enzyme conjugation chemistry preserves antibody specificity as well as enzyme activity.

29 Sandwich ELISA The sandwich ELISA measures the amount of antigen between two layers of antibodies. The antigens to be measured must contain at least two antigenic sites (epitopes), capable of binding to the antibody, since at least two antibodies act in the sandwich. For this reason, sandwich assays are restricted to the quantitation of multivalent antigens such as proteins or polysaccharides. To utilize this assay, one antibody (the “capture” antibody) is purified and bound to a solid phase typically attached to the bottom of a plate well. Antigen is then added and allowed to complex with the bound antibody. Unbound products are then removed with a wash, and a labeled second antibody (the “detection” antibody) is allowed to bind to the antigen, thus completing the “sandwich”. The assay is then quantitated by measuring the amount of labeled second antibody bound to the matrix, through the use of a colorimetric substrate. Major advantages of this technique are that the antigen does not need to be purified prior to use, and that these assays are very specific. However, one disadvantage is that not all antibodies can be used. Monoclonal antibody combinations must be qualified as “matched pairs”, meaning that they can recognize separate epitopes on the antigen so they do not hinder each other’s binding.

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31 ELISA: Enzyme Linked Immunoassay

32 ELISA steps

33 Competitive ELISA When two “matched pair” antibodies are not available for a target, another option is the competitive ELISA. The enzyme may be linked to either the antigen or the primary antibody. In this type of ELISA, there is an inverse relationship between the signal obtained and the concentration of the analyte in the sample, due to the competition between the free analyte and the ligand-enzyme conjugate for the antibody coating the microplate, i.e. the more analyte the lower the signal. Briefly, an unlabeled purified primary antibody is coated onto the wells of a 96 well microtiter plate. This primary antibody is then incubated with unlabeled standards and unknowns. After this reaction is allowed to go to equilibrium, conjugated antigen is added. This conjugate will bind to the primary antibody wherever its binding sites are not already occupied by unlabeled antigen. Thus, the more unlabeled antigens in the sample or standard, the lower the amount of conjugated antigen bound. The plate is then developed with substrate and color change is measured.

34 Competitive immunoassay

35 Competitive immunoassay

36 Multiplex ELISA A logical progression of the widely used microtiter plate ELISA is toward a protein array format that allows simultaneous detection of multiple analytes at multiple array addresses within a single well. Different types of multiplex ELISA have been developed. Generally, multiplex ELISA can be achieved through antibody array, where different primary antibodies are printed on glass plate to capture corresponding antigens in a biological sample such as plasma, cell lysate, or tissue extract. Detection method can be direct or indirect, sandwich or competitive, labeling or non- labeling, depending upon antibody array technologies.

37 Multiplex ELISA protein array

38 Rapid test

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40 Test device component categories Porous materials Sample pads Conjugate pads Membranes Absorbent pads Reagents Capture antibodies and/or antigens (test line and control line) Conjugate ligand (antibody or antigen) Detector particle (e.g., colloidal gold) Blocking agents, detergents, surfactants, stabilizers, buffers, etc. Housing and lamination materials Back laminate (for holding porous components together) Top laminate (optional, to act as a "splash guard" or prevent evaporation and back-migration of detector reagent) Device housing (optional—if used, the housing typically comprises two, snap-fit plastic pieces that envelop the reagent-loaded porous media assembly and facilitate sample addition)

41 Rapid immunoassay

42 Rapid immunoassay

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44 Rapid lateral flow test

45 Pregnancy test

46 Specifications for a pregnancy test Following are the development guidelines given to the research and development manager responsible for designing, developing, and scaling up to manufacturing a hypothetical lateral-flow diagnostic test device for detecting human chorionic gonadotropin (hCG) in urine. The hCG hormone is measured in milli-international units per milliliter (mIU/ml). More than 99% of specimens containing 25 mlU/ml will produce a visible signal at the test line. More than 99% of specimens containing 5 mlU/mL will not produce a visible signal at the test line. The test will have a control line that will always produce a visible signal if the test is performed correctly and if all of the test components are functional. If the sample is positive (contains 25 mIU/ml hCG), the test must produce a visible signal at the test line within 3 minutes of sample addition. The test result must be stable (not change) for at least 30 minutes after the sample is added. The test device will be contained in a plastic housing and designed to be compatible with "in- stream" sampling protocols. The packaged (unused) product must be stable for at least 18 months at ambient conditions (15°– 30°C). Total incremental manufacturing cost per test (excluding licensing and royalties) can not exceed $0.25 US.

47 Rapid test for drugs of abuse

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