III Week 5

Immunofluorescence and Immunohistology: * Purpose * Mechanism

Antibodies can be used to detect the cellular localisation of structures or proteins • Same idea as immunohistochemistry, but the label is different • A fluorescent molecule is used and a particular microscope is needed Immunofluorescence • Antibodies labeled with a fluorescent dye such as fluorescein (green triangle) are used to reveal the presence of their corresponding antigens in cells or tissues. • The stained cells are examined using a microscope that exposes them to blue or green light to excite the fluorescent dye. • The excited dye emits light at a characteristic wavelength, which is captured by viewing the sample through a selective filter • Applied widely in biology to determine the location of molecules in cells and tissues. • Different antigens can be detected in tissue sections by labeling antibodies with dyes of distinctive color • The fluorescent dye can be covalently attached directly to the specific anti- body; however, the bound antibody is more commonly detected by fluorescently labeled anti-immunoglobulin, a technique known as indirect immunofluorescence. • The dyes chosen for immunofluorescence are excited by light of one wavelength, usually blue or green, and emit light of a di erent wavelength in the visible spectrum. • By using selective filters, only the light coming from the fluorochrome used is detected in the fluorescence microscope Immunohistology • the microscopic study of tissues with the aid of antibodies that bind to tissue components and reveal their presence.

1. Why are the antibodies able to cross-link the human erythrocytes?

Antibodies can cross-link antigen as they have at least two antigen binding sites and because of flexibility in the hinge region, antibodies can bridge the gap between antigens.

Coombs Test: * Use * Direct * Indirect * Mechanisms

Coombs Test • Uses anti-immunoglobulin Ab to detect the Abs that cause hemolytic disease of the newborn (when mothers make IgG antibodies specific for the rhesus antigen expressed on the RBCs of her fetus - this occurs when Rh-negative others are exposed at delivery to Rh-positive featal RBCS bearing the paternally inherited Rh antigen • During subsequent pregnancies, these Ab cross the placenta to the fetus - coat the fetal RBCs which are then destroyed by phagocytes in the liver, causing hemolytic anemia • Abs against Rh Ags don't agglutinate - can be hard to recognize Direct Coombs Test • Addition of anti-human Ig antibodies to washed foetal RBC will agglutinate if maternal antiRh antibodies were present • Looking for Abs that the mother has made against her fetal RBCs • Maternal IgG Ab bound to fetal RBCs can be detected after washing the cells to remove unbound Ig and then adding anti-human Ig Ab against the washed fetal RBCs agglutinates any cells to which any maternal Abs were bound • Called direct because it directly detects the Ab bound to the surface of RBCs Indirect Coombs Test • Maternal serum with is incubated with Rh+ RBC. If anti-Rh antibodies are present in maternal serum this will be agglutinated in the presence of anti- human Ig antibodies • Just test the mum's serum, not the baby's blood • Used to detect nonagglutinating anti-Rh antibody in maternal serum • Serum is incubated with Rh-positive RBCs, which bind the Rh-Ab, after which the Ab-coated cells are washed to remove any unbound Ab and are then agglutinates with anti-immunoglobulin Ab • Allows Rh incompatibilities that might lead to hemolytic disease of newborn to be detected - allows the disease to be prevented

What is an indirect EIA?

Enzyme Immunoassay for Ab detection - indirect EIA • Most common form of the ELISA test - Indirect ELISA test - If you have no antibodies against that particular Ag, there is no colour change 1. Microwells containing dengue antigen 2. Human test serum 3. Enzyme conjugate: anti-human IgM (μ chain antibody) conjugated to peroxidase (enzyme label) 4. Substrate/chromogen solution

FACS: * Use * Procedure

Flow Cytometry (FACS) Cells can be identified by dyes coupled antibodies to cell-surface antigens • Laser-based way of identifying individual cells • Look in detail at what is on cell surface and what is on the cell • Used routinely, but also in larger laboratories • Used a lot for cancer and infectious disease diagnoses • A flow cytometer equipped to separate the identified cells is called a fluorescence-activated cell sorter (FACS). ese instruments are used to study the properties of cell subsets that are identified by using monoclonal antibodies against cell-surface or intracellular proteins. • Flow cytometers allow individual cells to be identified by their cell-surface antigens and to be sorted. 1. Cells to be analyzed by flow cytometry are first labeled with fluorescent dyes (top panel). - Direct labeling uses dye-coupled antibodies specific for cell- surface antigens (as shown here), while indirect labeling uses a dye-coupled immunoglobulin to detect unlabeled cell-bound antibody. 2. The cells are forced through a nozzle in a single- cell stream that passes through a laser beam (second panel). Photomultiplier tubes (PMTs) detect the scattering of light, which is a sign of cell size and granularity, as well as emissions from the different fluorescent dyes. 3. This information is analyzed by computer (CPU). - By examining a large number of cells, the proportion of cells with a specific set of characteristics can be determined and levels of expression of various molecules on these cells can be measured.

What can lymphocyte proliferation assays be used for?

Measures the ability of lymphocytes to proliferate (in vitro), when stimulated by a mitogen (a chemical that signals all lymphocytes non-specifically), antigen, or foreign molecule. Can assess ability of lymphocytes from patients with suspected immunodeficiencies to proliferate in response to a nonspecific stimulus

Why are monoclonal antibodies important and how are they generated?

Monoclonal antibodies are identical antibodies, specific to a particular epitope , which have been produced by cultured and clonally identical rodent hybridoma cells. WHAT DOES THIS MEAN? ¥ Blood contains many different types of plasma cells (different parental lineages) & each plasma cell as the ability to produce ONE TYPE of antibody against a particular epitope. ¥ Therefore a sample of blood contains a number of different types of plasma cells, and a collection of the personal antibodies that they produce POLYCLONAL ANTIBODIES ¥ In 1975 George Kohler and Cesar Milstein pioneered a method to produce MONOCLONAL ANTIBODIES. HOW DID THEY DO THIS? 1. Inject rodent with epitope that you want to make antibodies against (promote adaptive immune response) 2. Plasma cells (spleen) + myeloma cells (cultured cancer cells) +polyethylene glycol hybridoma cells (FUSED) 3. Mixture of hybridoma cells are cultured & drugs are added which kill the unfused myeloma cells. Plasma cells have a limited life span die. 4. Cultured hybridoma cells have different B cell lineages - therefore will each produce different antibodies. 5. SELECTION OF HYBRIDOMAS that produce desired antibody: add to microtitre wells and test for ability to produce specific antibody. (Add epitope) 6. Hybridoma cells that produce antibodies of interest are isolated and purified.

2 approaches to diagnosing diseases by immunoassays...

Two general approaches to diagnosing diseases by immunoassays: 1. testing for specific antigens 2. testing for antigen-specific antibodies - previous exposure to infectious agent, allergen, autoantibodies - immunoassays can detect Ag or Ab - Ab tells us whether the patient has been exposed to the Ag previously

1. Outline the importance of understanding the nature of primary and secondary immune responses in the diagnosis of infectious diseases?

We can use haemagglutination assays to determine whether a patient has previously been exposed to the pathogen of concern, which will be shown with a greater HA titer value than normal as a result of higher basal levels of antibody. We can also determine whether a patient is producing an adequate immune response to the pathogen they are infected with. This will be indicated by increasing HA titer values as antibody levels are increasing as a result of an adaptive immune response currently occurring. In immunodeficient patients, there may not be an increase in antibody levels in response to the pathogen they are infected with, therefore their HA titer will remain low.

Why is it important to know the characteristics of Ig classes and subclasses?

• In diagnoses, need to know the functions of the different antibodies • Looking for IgE - when diagnosing allergy

What occurs in affinity maturation?

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T cell proliferation Assays: * Use * Procedure

• This assay is used to determine the antigen specific proliferation of CD4 T cells • Antigen-specific T-cell proliferation is used frequently as an assay for T-cell responses. - T cells from mice or humans that have been immunized with an antigen (A) proliferate when they are exposed to antigen A and antigen- presenting cells but not when cultured with unrelated antigens to which the hosts have not been immunized (antigen B). - Proliferation can be measured by incorporation of 3H-thymidine into the DNA of actively dividing cells. Antigen-specific proliferation is a hallmark of specific CD4 T-cell immunity.

How do you interpret immunoassays by using Ab to determine exposure to Ag? (refer to image)

• IgG & IgM Ab negative - No exposure or too early to detect in acute phase • IgG antibody positive, IgM Ab negative - Past exposure to infective agent • IgG & IgM Ab positive - Current or recurrent infection • IgG Ab negative, IgM antibody positive - Very early acute phase or false positive IgM • May want to measure relative amounts of IgM vs IgG - IgG produced after class switching - May get paired testing to see what is happening in the patient over time • Most infectious agents induce Abs within 2 weeks of infecting an individual, but some may take up to 2 months or even longer • For diagnosis of an acute infection a significant rise in antibody titre must be demonstrated in paired sera taken 2 weeks apart • Acute sera - when they are diagnosed • Convalescent sera - later on in the disease process

Laboratory guidance and diagnostic testing...

• There are a lot of tests that need to be done in order to make a clinical diagnosis • A single lab test is not usually enough e.g. Over time course of infection of HepB, need laboratory guidance in how to help these patients

ELISPOT Assays: * Definition * Purpose * Use

• A modified ELISA in which antibodies are bound to the plastic surface of culture wells and used to capture cytokines produced by individual T cells • Human IFN-g ELISPOTs are obtained using a anti-human IFN-g antibody for capture. • human PBMC were restimulated in the microwell of an ELISPOT plate that was pre-coated with anti-human IFN-g. • anti-human IFN-g antibody conjugated with HRP was used to detect the captured IFN-g. • Spots were visualized using substrate/chromogen • This time, put the cells on in the well - these secrete cytokines. If these are recognized by the Abs, they are captured and form a spot • T cells stimulated with antigen o Allowed to settle on plastic culture plate coated with antibodies against cytokine to be assayed • If an activated T cell secretes the cytokine, it's captured by the antibody on the plastic plate • After a period, cells are removed o Second antibody against the cytokine added to the plate • Circle of bound cytokines around each activated T-cell o Count the spots! • Knowing the number of T cells originally added to the plate = calculating frequency of T cells secreting that cytokine. • Modification of ELISA antigen-capture assay • Measures the frequency of T-cell responses and provides information about the cytokines produced • Populations of T cells are stimulated with Ag of interest and are then allowed to settle on a plastic plate coated with Abs against the cytokine to be assayed • If activated T cell is secreting that cytokine, the cytokine is captured by the Ab on the plastic plate • After time, the cells are removed and a second Ab against the cytokine is added to reveal a spot of bound cytokine surrounding the position of each activated T cell - By counting each spot and knowing the number of T cells originally added to the plate, can calculate frequency of T cells secreting that particular cytokine • Can be used to detect specific Ab secretion of B cells (using Ag-coated surfces to trap specific Ab and labelled anti-Ig to detect the bound Ab

Common immunodiagnostic and screening test formats

• Agglutination/haemagglutination tests - Ag binding to Ab • Precipitation tests - Ag binding to Ab (e.g. CRP test - Ag is CRP, antibodies bind to this and the complex preceipitates) • Enzyme-Linked Immunosorbent Assay (ELISA) - Very commonly used - can be used to test for Ab or Ag • Immunofluorescence antibody test (IFA) (don't use an enzyme, but a fluorescent molecule - need a particular microscope for this) or immunohistochemistry (IHC) - detecting Ag in tissues using specific Ab against those Ag; e.g. detecting cells in tissue sections, tumour cells, immune cells in situ • Immunochromatography e.g. snap-tests, point-or-care - Can be done in GP surgery or out in the field - plastic kit - get a colour change - Can test for infections, autoimmunity and allergy

Humoral Immunity:

• An antigen can be defined as a compound which when administered can provoke an immune response • After a lag period there is an antibody response which will decline with time • Following a second dose of antigen there is a shorter lag period, a larger immune response and the antibody persist for a longer period • Antibody responses are specific to a particular antigen • Humoral immunity can be transferred using serum

Anti-immunoglobulin Abs * purpose * definition

• Anti-immunoglobulin antibodies are B cell-generated immunoglobulins that recognize other immunoglobulins. • Anti-immunoglobulin antibodies may bind to either the constant region or to the variable region. Those that bind to the constant region of human IgG are referred to as rheumatoid factors. Anti-immunoglobulin antibodies that bind to variable domains are referred to as anti-idiotypic antibodies HOW DO WE KNOW WHEN AN ANTIBODY IS BOUND TO THE OUTSIDE OF A CELL? One way = use a flourescently LABELLED (b) antibody (a) specific for the bound immunoglobulin. (Anti-immunoglobulin antibody) Anti-immunoglobulin antibodies are a type of antibody that has been labelled with a fluorescent tag, which recognise and bind to immunoglobulins. They may recognise general features shared by all immunoglobulin molecules that are bound, they may be specific for heavy/light chains or individual isotypes. ¥ Anti-isotope antibody: Immunoglobulin is used as an antigen, to create an adaptive immune response produce antibodies (against foreign antibodies) ¥ Causes a type of AIA to be made that recognise all immunoglobulins OF THE SAME ISOTYPE that the injected antibody came from. AIAs are very important in measuring the function of the adaptive immune system especially in hypersensitivity/allergic reactions.

Why are antibody titres important?

• Antibody titres are sometimes used to evaluate how significant a positive antibody level is • These titers involve diluting the sample - creating and testing serial (increasing) dilutions. The highest dilution that is still positive is reported • This is still used to report some antibody levels, especially in the case of autoimmune conditions • "Antibody titre" is a term that is also sometimes used generically to refer to antibody concentrations. • Determine this by the last well where you get a reaction (e.g. colour change in ELISA or agglutination in haemagglutination tests)

Protein Microarray: * Purpose * Procedure * Applications

• Arrangement of purified proteins at high spatial density on glass slides • Most commonly used version is an Ab microarray • Can be used to determine protein-protein interactions, targets of biologically active small molecules • Technology involved is similar to DNA microarray - instead of DNA externally synthesized, purified proteins are attached to the solid surface like glass slide or nano wells Applications: • Can be used in identifying protein-protein interactions, identifying transcript ion factors, substrate for particular enzymes, etc.

Importance of anti-immunoglobulin Abs...

• B cell-generated immunoglobulins that recognize other immunoglobulins. • Anti-immunoglobulin antibodies may bind to either the constant region or to the variable region. Those that bind to the constant region of human IgG are referred to as rheumatoid factors. Anti-immunoglobulin antibodies that bind to variable domains are referred to as anti-idiotypic antibodies

Point of Care Tests

• Bedside/clinic tests • Ag or Ab • E.g. HIV tests • Require confirmatory tests, lab tests Ab can react with Ag embedded in the piece of filter paper • Quick and simple diagnostic - get lines • Needs to be backed up by proper lab tests • Can embed with Ag or Ab in the filter paper

What is Sandwich or capture ELISA? * Use?

• Can be used to quantify cytokines (secreted products) • Instead of coating plate with Ag, can coat it with Ab - These are able to bind antigen with high affinity, and thus concentrate it on the surface of the plate, even with antigens that are present in very low concentrations in the initial mixture. A separate labeled antibody that recognizes a different epitope from that recognized by the immobilized first antibody is then used to detect the bound antigen. • Use to detect Ags, Abs, cytokines, other proteins • Needs matched pairs of Abs for this type of ELISA; not always available so may need to use another ELISA type

Why order a Laboratory Test?

• Diagnosis or monitoring - malignancy, infection, autoimmunity, transplants (matching donors and recipients), dermatology, immune deficiencies, atherosclerosis, asthma/allergy etc - detect Ab or Ag • Some tests essential for diagnosis • Some are useful for investigating disease further • Some only research interest (e.g. mechanisms of disease)

Production of a Hybridoma...

• Don't need to know detail about how they are made

ELISA: * definition * Uses * How it is done

• Enzyme-linked immunosorbent assay identifies a particular substance (or rather, the concentration of an antigen) through the use of antibodies and colour change. It is similar to radioimmunoassays (RIAs), though ELISAs are focused on detecting viruses rather than hormone levels. • The intensity of the colour change provides an indication about either the concentration of primary antibody provided, or the presence (and quality) of antigen used. A spectrometer is used to give an indication about the colour strength of the solution. • • Enzyme-linked immunosorbent assay • Various configurations • Commonest form is when you put on the bottom of the well the Ag - Then come in with the primary Ab and then come in with secondary Ab which has an enzyme label to it • ELISA can be used to detect Ag or Ab - Look at the shades of the different colour • Used for serum, secretions e.g. CSF, urine, faeces, semen, cell culture supernatant, sputum, tissue exudate • Uses: surveillance, evaluation of vaccine efficacy • Direct binding assay for Ab (or Ag) • Frequently used in viral diagnostics • Can determine the amount of specific antigen in a sample • An enzyme is chemically linked to the Ab • Serum containing Ag is attached to the plastic wells of a multiwall plate • Labelled Ab is added to the wells • Unbound Ab and proteins are washed away • Binding is detected by a reaction that converts a substrate into a reaction product of a different colour 1. Microwells containing dengue antigen 2. Human test serum 3. Enzyme conjugate: anti-human IgM (μ chain antibody) conjugated to peroxidase 4. Substrate/chromogen solution

Isolation of Lymphocytes by Ficoll Gradient * Purpose * Procedure

• Ficoll allows us to separate the PBMCs (peripheral blood mononuclear cells - lymphocytes, monocytes • Human lymphocytes can be isolated most readily from peripheral blood by density centrifugation over a step gradient consisting of a mixture of the carbohydrate polymer Ficoll-Hypaque and the dense iodine-containing compound metrizamide. 1. A step gradient is made by preparing a solution of Ficoll-Hypaque at a precise density (1.077 g/liter for human cells) and placing a layer of this solution at the bottom of a centrifuge tube. 2. A sample of heparinized blood mixed with saline (heparin prevents clotting) is carefully layered on top of the Ficoll-Hypaque solution. 3. Following centrifugation for about 30 minutes, the components of the blood have separated based on their densities. - The upper layer contains the blood plasma and platelets, which remain in the top layer during the short centrifugation. - Red blood cells and granulocytes have a higher density than the Ficoll-Hypaque solution and collect at the bottom of the tube. - The resulting population, called peripheral blood mononuclear cells (PBMCs), collects at the interface between the blood and the Ficoll-Hypaque layers and consists mainly of lymphocytes and monocytes ϖ Although this population is readily accessible, it is not necessarily representative of the lymphoid system, because only recirculating lymphocytes can be isolated from blood. • • Isolation of Lymphocytes (in general): - Done so their behavior can be analysed in vitro - Can be done by density-gradient fractionation, antibody-coated magnetic beads, etc. - - Human lymphocytes can be isolated most readily from peripheral blood by Ficoll-Hypaque density gradient centrifugation. This is based on the density differences between mononuclear cells and other elements found in the blood sample. Mononuclear cells and platelets collect on top of the Ficoll-Hypaque layer because they have a lower density. Red blood cells and granulocytes have a higher density than Ficoll-Hypaque and collect at the bottom of the Ficoll-Hypaque layer.

Tests used for the characterisation of lymphocytes (5)

• Flow cytometry • ELISPOT assays • Lymphocyte proliferation assays • Target cell killing - More a research-type assay • DNA microarrays or DNA chip

Outline how B cells switch from IgM to high affinity IgG...

• Germinal centres are formed when activated B cells enter lymphoid follicles • Germinal centre B cells undergo V-region somatic hypermutation and cells with mutations that improve affinity for antigen are selected • Affinity and specificity of BCR continually refined - affinity maturation - This changes as the infection progresses - those produced later in the infection have a greater affinity (affinity maturation) Isotype Switching • Naïve B cells express surface IgM (and IgD), IgM is the first Ab secreted • Switching to IgG, IgA and IgE requires CD4 T cell help and is directed by cytokines • Genes encoding the IgM Heavy chain constant region are replaced (switched) by constant chain genes for IgG, IgA or IgE. • The constant region confers functional specialisation of the Ab • Constant-mu region is the first constant chain attached to the variable region, but this can be swapped for other C gene fragments later on in development

Western Blotting (immunoblotting) * Purpose * Procedure

• Identification of a given protein in a cell lysate • Test sera for the presence of antibodies to specific proteins • Blot the serum onto particular paper, get colour changes in the end • Same idea as ELISA, just done in a different way • Used for identifying presence of a given protein in a cell lysate • Avoids problem of having to label large quantities of cells with radioisotopes • Unlabeled cells placed in detergent to solubilize cell proteins and lysate is run on SDS-PAGE to separate the proteins • Size-separated proteins are then transferred from gel to a stable support such as a nitrocellulose membrane • Specific proteins are detected by treatment with Abs able to react with SDS-solubilised proteins • Bound Ab are revealed by anti-Ig Abs labelled with an enzyme An Immunoblot or Western blot is used to detect specific protein molecules from among a mixture of proteins. Procedure: 1. Prepare the protein sample by mixing it with sodium dodecyl sulfate. 2. The protein molecules are separated according to their sizes by gel electrophoresis. Smaller proteins move further towards the gel. 3. The proteins are transferred from the gel onto a blotting membrane. 4. The membrane is incubated with primary antibody, which specifically binds to the protein of interest. 5. Unbound primary antibody is washed away. 6. Membrane is incubated again with secondary antibody that specifically recognizes and binds to the primary antibody. 7. The secondary antibody is linked to a reporter enzyme that produces colour or light, which allows it to be easily detected and imaged. Western Blot: • Synonym of immunoblotting • Comparable technique for detecting specific DNA sequences is called Southern blotting • Northern blots for size-separated RNA and Western blots for size-separated proteins • Often used to test sera for the presence of Ab against specific proteins (e.g. detecting Abs against different constituents of HIV)

Function of a lymphocyte proliferation assay

• In adaptive immunity, antigen-specific lymphocytes have to proliferate • Ployclonal mitogens induce mitosis in lymphocytes of many different specificities or clonal origins - T and B cells are stimulated by different types of these - Trigger same growth response mechanisms as Ag - Lymphtocytes enter from G0 into G1 phase • Can be measured by incorporation of H-thymidine into DNA • Can assess ability of lymphocytes from patients with suspected immunodeficiencies to proliferate in response to a nonspecific stimulus • Can use FACS - lymphocytes are incubated with a fluorescent dye which enters the cell and once in the cytosol, becomes covalently coupled to lysine residues on cellular proteins - Each time the cell divides, CFSE is cut in half - this fluorescence can be detected, each of which representing cells that have undergone a fixed number of divisions • Capable of detecting 7-8 cell divisions • Used to determine lymphocyte activation and cell mediated immune responses. - measures the ability of lymphocytes to proliferate (in vitro), when stimulated by a mitogen (a chemical that signals all lymphocytes non-specifically), antigen, or foreign molecule. - Mitogens are non-specific to lymphocytes, therefore will activate both T and B cells, and cause proliferation Some foreign antigens may only activate B cells which will then activate T cells (then able to measure lymphocyte proliferation of T cells) **In the example of thymidine, DNA synthesis is measured by adding a radioisotope-labelled DNA precursor (thymidine) to the cell culture medium. The amount of the substance taken up by the dividing cells is correlated to the level of cellular proliferation. The use of the MTT assay is a similar method that measures the activity of the cellular enzymes thought the observation of the activity of the dye in the cell culture medium.

Techniques for measuring immune responses in clinical immunology...

• Innate immune responses vs antigen-specific immune responses - Can look at T and B cells along with innate cells 1) Serological assays - Serum/plasma other body fluid (may be tissue exudate or synovial fluid, etc.) - Measurement of : ϖ Acute phase proteins, complement, cytokines ϖ Immunoglobulins (isotypes/sub-isotypes), Ab to microbial Ag, anti-globulin (e.g. Rheumatoid factors - Abs against self), autoantibodies (Abs against Abs), immune complexes, allergy IgE - e.g. detecting whether they have been exposed to dengue, stage of infection, etc. 2) Cell enumeration and phenotyping (e.g. T cell vs B cell, CD4 T vs CD8 T) - can determine the characteristics of the cell 3) Cell function and physiology (e.g. cytolytic assays, cell death, lymphocyte proliferation, cytokine production 4) Transplant matching: Histocompatibility testing, MLR, cytotoxicity assays - matching donors and recipients - Some are DNA-based, flow cytometry-based, etc. 5) DNA or RNA analysis (detection of different alleles to different things) 6) In vivo assays (e.g. tuberculin test, allergy skin tests) - this is done in the patient

Titre: * Definition * Purpose

• Level of specific Ab present in a sample is defined as the titre of the Ab • relative measurement/amount not absolute concentration of Ab (not in ng/mL) • Titre = reciprocal of the highest sample dilution giving an unequivocally positive reaction in a serological test • You dilute a sample 1:2 - dilute out the Ab you are interested in measuring - IMAGE - Serum A has much more Ab because it is diluted out further - We take an Ag and bind it to the serum - Assay allows us to quantify the amount of Ab Gives more information than simply a "yes" or "no" qualitative result

Monoclonal Antibodies: * Uses * How they are produced

• MAb • Means that every Ab in that reagent are identical - are all made from a single B cell clone in the lab • Use these to detect the particular Abs you are interested in • Used as reagents for a variety of immunoassays • Abs generated in natural immune response or immunization are a mixture of molecules in specificity and affinity • It can be beneficial to have a homogenous population of Abs of known Ag specificity • antibodies that are made by identical immune cells that are all clones of a unique parent cell. Monoclonal antibodies can have monovalent affinity, in that they bind to the same epitope (the part of an antigen that is recognized by the antibody). Production of Monoclonal Antibodies 1. Mice are immunized with antigen A and given an intravenous booster immunization 3 days before they are killed, in order to produce a large population of spleen cells secreting specific antibody. 2. Spleen cells die after a few days in culture. 3. To produce a continuous source of antibody they are fused with immortal myeloma cells by using polyethylene glycol (PEG) to produce a hybrid cell line called a hybridoma. 4. The myeloma cells are selected beforehand to ensure that they are not secreting antibody themselves and that they lack the enzyme hypoxanthine:guanine phosphoribosyl transferase (HGPRT); without this enzyme, unfused myeloma cells are sensitive to the hypoxanthine-aminopterin-thymidine (HAT) medium, which is used to select hybrid cells. The HGPRT gene contributed by the spleen cell allows hybrid cells to survive in the HAT medium, and only hybrid cells can grow continuously in culture, because of the malignant potential contributed by the myeloma cells combined with the finite life-span of unfused spleen cells. 5. Unfused myeloma cells and unfused spleen cells therefore die in the HAT medium, as shown here by cells with dark, irregular nuclei. Individual hybridomas are obtained by single cell dilution and then screened for antibody production, and single clones that make antibody of the desired specificity can be isolated and grown. 6. The cloned hybridoma cells are grown in bulk culture to produce large amounts of antibody. As each hybridoma is descended from a single cell, all the cells of a hybridoma cell line make the same antibody molecule, which is thus called a monoclonal antibody.

More specialist assays - specific diagnostic/reference labs

• Precipitation • Agglutination • Haemagglutination • ELISA/RIA • Immunofluorescence/IHC • Immunoblotting (western blotting)

Microarrays (DNA, RNA, Protein) - definition and purpose

• Simultaneous assessment in expression of a multitudes of genes or gene products under different conditions and between different cells • The intensity and color of each spot encode information on a specific gene / protein from the tested sample DNA and protein microarrays are important in determining which genes are active (and are being transcribed into RNA and proteins), and which genes are inactive (and are therefore condensed to prevent transcription and translocation) within a particular cell-type. However, one must note that although all cell types have the same DNA, the epigenome (interface between DNA and environment) has the ability to condense genes preventing transcription, or to 'unravel' condensed genes and make them more available for transcription. Therefore, microarray profiles may change between cells, and throughout a lifetime of a person. DNA microarrays look for: Ð expression of genes, Ð transcription factor binding sites Ð sequence mutations/deletions Ð BUT tell little about the proteins they encode for Protein microarrays identify: Ð Protein-protein interactions Ð Protein-phospholipid interactions Ð Small molecule targets Ð Substrates of proteins kinases. Gene chips test for a large number of genes. They have 'probes' or 'complementary DNA strands' which will BIND to the gene (which is FLURORESCENTLY LABELLED) by a process called hybridization. (Complementary strands of DNA will pair together, and bond strongly to one another by hydrogen bonding. Remember A-T (double bond), G-C (triple bond).) If there is a lot of complementary binding, there will be a strong bond between the two strands (and if less complementary binding...weaker bonds etc.). After gene chip is washed only STRONGLY paired strands will remain hybridized. Genes which are active, and thus present, will fluoresce.

Affinity Chromatography: * Use * Procedure

• Specificity of an Ag:Ab binding interactions can be exploited for the purification of a specific Ag from a complex mixture or for the purification of specific Abs from antiserum containing a mixture of different Abs - For purification of an antigen, antigen-specific antibodies are bound, often covalently, to small, chemically reactive beads, which are loaded into a column. The antigen mixture is allowed to pass over the beads. The specific antigen binds; all the other components in the mixture can then be washed away. The specific antigen is then eluted, typically by lowering the pH to 2.5 or raising it to greater than 11. Antibodies bind stably under physiological conditions of salt concentration, temperature, and pH, but the binding is reversible because the bonds are noncovalent. • Affinity chromatography can also be used to purify antibodies from complex antisera by using beads coated with specific antigen. e technique is known as affinity chromatography because it separates molecules on the basis of their affinity for one another. Affinity chromatography is a method of separating the molecules belonging to a biochemical mixture by taking advantage of the very specific interactions that occur between an antibody and antigen, ligand and receptor, or enzyme and substrate. This process is often used to try and concentrate a solution, or to identify the components of an unknown solution The solution or antiserum is transferred into a column with a gel matrix. This gel, usually agarose, has had molecules added to it that will attract particular components of the solution or antiserum (e.g.: antigens covalently bound to the gel will attract the appropriately specific antibody). When the solution or antiserum is allowed to run over the beads the targeted component will remain entrapped in the gel, though the rest will be allowed to freely flow over it. Later, the targeted component (antibodies) can be removed by lowering the pH below 2.5, or raising it above 11.0. This is possible because the bonds formed between the antigen and antibody (receptor and ligand; enzyme and substrate) are non-covalent. The term "affinity chromatography" is used (appropriately) as this process is based on one molecule's affinity for another. Later, the targeted component (antibodies) can be removed by lowering the pH below 2.5, or raising it above 11.0. This is possible because the bonds formed between the antigen and antibody (receptor and ligand; enzyme and substrate) are non-covalent. The term "affinity chromatography" is used (appropriately) as this process is based on one molecule's affinity for another.

Importance of Forward and Side Scatter in FACS

• The level of light scattering in forward direction is proportionate to cell size (FSC) • Cells include granules, compartmented nuclei and other organelles leading to side scattering of light. • Side scattering depends on cell structure (SSC) • Scatter alone is not adequate to distinguish lymphocytes - need to use antibodies to pull apart these different populations

DNA Microarray: * Procedure * Applications

• Two dimensionally arranged series of DNA oligonucleotides also known as probes on solid surface like glass slide or silicone thin film used to study gene expression in different cells. The probes such as cDNA or cRNA attached via surface engineering to solid surface like glass slide or silicone chip Technology: 1. Prepared using robotic machines that arrange hundreds and thousands of gene sequences (probes) on a single microscopic slide 2. To determine which genes are active in a cell, reasercher cshould collect all mRNA from that cell. When a gene is active, the cell prepares mRNA which are complementary to the active gene (DNA). Therefore will bind to the original DNA strand 3. mRNA are labelled with fluorescent dye 4. researchers add these labelled mRNA into the DNA microarray slide 5. mRNA that was present in the cell will bind to its complimentary DNA probes 6. Hybridised mRNS gives out fluorescent signal, which can be measured using special scanners 7. Genes (DNA) which are active will produce many mRNA so will generate bright fluorescent area. Genes that are only a little active will produce less mRNA resulting in a dimmer fluorescent area. No hybridization of mRNA to DNA indicates those genes were inactive in that particular cell Applications: • Can be used to compare genomes, studying effect of drug on gene expression, identify single nucleotide polymorphisms within/between the population, etc.

Antiserum and anti-species antibodies: * Purpose * How they are made

• Used as reagents in immunoassays • Antiserum = serum collected from an animal that has been exposed to a particular Ag and therefore contains Ab against that Ag - Need this to be able to find the Abs in the blood • For most serological tests antiserum is produced by immunising an animal (most commonly rabbit, goat, rodent, horse) with the Ag of interest and collecting blood (serum) from that immunised animal • If you inject an animal with Ab, the Ab becomes the Ag and you can produce Ab against the injected Ab • Antispecies antibodies are often used in immunoassays: produced when antibodies from one species are injected into another species. - E. g. Human Ig injected into a rabbit elicits the production of rabbit anti-human antibodies - Ab that acts specifically with another species' Ig • When the immunising Ag contains numerous epitopes, the responding animal will generate Abs against several of these epitopes = polyclonal • Serum produced by immunising an animal is always "polyclonal" • Ab in serum may be purified and then used as reagent • Antiserum of single Ab specificity = monoclonal Ab, specialised techniques are used to make monoclonal Abs

Serum Agglutination Tests (SAT): * Use * Examples

• Various configurations - tests may detect either Ab or Ag in sample • Agglutination - Ag is particulate e.g. bacteria, RBCs, Ag coated onto latex beads • Presence of agglutinating antibodies in test sera react with particulate specific antigens to form visible clumps i.e. Ab crosslinks antigen • If test is designed to test Ag in a sample, Ab specific for that Ag may be adsorbed to particles • Agglutinate may be visible to naked eye (SAT) or require microscope (MAT) • Very commonly used, blood typing, Coombs test, bacterial id • Can be done easily out in the field - look for clumping • can get a good, quick answer

Haemagglutination: * Use * Basis

• can take normal RBCs and coat them with viral Ags and look for Abs against that virus and look for haemagglutination • Based on the ability of Ab binding to alter the physical state of the Ag to which the Ab binds • RBCs are used as the Ag • When Ab is in the sample - get a lattice formation • When there is no Ab, there is nothing to help the RBCs stick together - they form a button-like pattern • When it forms the button-like pattern, you have reached the button-like titre • Based on ability of Ab binding to alter physical state of Ag to which the Ab binds • Ab bound to Ag can cause the cell to agglutinate (clump) such as with hemagglutination • Used to determine ABO blood group of blood donors and transfusion patients • Agglutination caused by antibodies called anti-A and anti-B that bind to the A or B blood group • Each Ab molecule must have at least 2 identical Ag-binding sites to cause agglutination * • Haemagglutination is the aggregation of RBCs due to cross-linking by antibodies bound to the antigens expressed on the surface of RBCs. When agglutinated, RBCs are brought out of suspension from the colloid solution that is our blood plasma, and form an insoluble lattice structure. • We can use haemagglutination assays to determine whether a patient has previously been exposed to the pathogen of concern, which will be shown with a greater HA titer value than normal as a result of higher basal levels of antibody. We can also determine whether a patient is producing an adequate immune response to the pathogen they are infected with. This will be indicated by increasing HA titer values as antibody levels are increasing as a result of an adaptive immune response currently occurring. In immunodeficient patients, there may not be an increase in antibody levels in response to the pathogen they are infected with, therefore their HA titer will remain low.

Assay used to detect target cell killing by CD8

• detected in a Cr-release assay. Live cells will take up, but do not spontaneously release, radioactively labeled sodium chromate, Na251CrO4. When these labeled cells are killed, the radio- active chromate is released and its presence in the supernatant of mixtures of target cells and cytotoxic T cells can be measured . • In a similar assay, proliferating target cells such as tumor cells can be labeled with 3H-thymidine, which is incorporated into the replicating DNA. - On attack by a cytotoxic T cell, the DNA of the target cells is rapidly fragmented and retained in the ltrate, while large, unfragmented DNA is collected on a filter, and one can measure either the release of these fragments or the retention of 3H-thymidine in chromosomal DNA. • These assays provide a rapid, sensitive, and specific measure of the activity of cytotoxic T cells. • Can measure activity in another type of assay - Done in experimental animals - Mice infected with pathogen known to induce strong cytotoxic T cell response (e.g. virus) • Target cells are incubated with the antigenic peptide, which will bind to MHC class I on the target-cell surface. - These cells are then incubated with a low concentration of the uorescent dye CFSE - A control population of cells that is not given the antigenic peptide is incubated with a high concentration of CFSE, allowing these cells to be distinguished from the antigen-bearing target cells. e two cell populations are mixed 1:1 and injected into the experimental animals. Four hours later, spleen cells are recovered from the animals and analyzed by FACS, allowing the specific target-cell lysis to be calculated from the ratio of the two CFSE-labeled cell populations • T cell bioassays provide a rapid sensitive and specific measure of activity of cytotoxic T cells. CD8 T cells (when activated) generally kill any cell that displays MHC class I receptor. CD8 T cell function is determined by T cell bioessay the killing of a target cell by a cytotoxic cell usually detected by a 51 Cr-release assay. • Living cells are used to take up, but not spontaneously release, radioactive Na+ Chromate. When these labeled cells are killed, the radioactive chromate is released and can be measured

Immunohistology

• the microscopic study of tissues with the aid of antibodies that bind to tissue components and reveal their presence.

What is the purpose of antibody tests? What are some examples of these tests carried out around Australia?

• the presence or absence of a particular antibody (qualitative) or for the amount of antibody that is present (quantitative) • 3 most frequently measured are IgM, IgG, and IgE - IgM and IgG often used in combination to diagnose infectious disease • Why are antibody tests done? - Document exposure to an infectious or foreign agent - Evaluate protection level (immune status) against a particular microorganism - e.g. seroconversion determined by amount of antibodies against Hep B - if you don't have an adequate level, may need a booster - Diagnose an autoimmune condition ϖ E.g. looking for Rheumatoid factor - Diagnose the reason for a transfusion reaction or a rejection of a transplanted organ - Diagnose an allergy ϖ Can measure total IgE in a sample to see what the patient is allergic to and to see whether they are having an allergic response - Monitor the course of an infection or autoimmune process • Large diversity of Ab tests offered by pathology labs - Allergy testing - AMA (antimitochondrial antibody) - ANA (antinuclear antibody) - Antiphospholipid antibodies - ASO (antistreptolysin O) - Autoantibodies - Barmah Forest virus - Cardiolipin antibodies - CCP (cyclic citrullinated peptide antibody) - Coeliac disease tests - CMV (Cytomegalovirus) - Diabetes autoantibodies - EBV (Epstein-Barr virus antibodies) - Hepatitis A - Hepatitis B - Hepatitis C - HIV antibody (human immunodeficiency virus) - H. pylori (Helicobacter pylori) - Lyme disease - Monospot for infectious mononucleosis (glandular fever) - Rheumatoid factor - Ross River virus - Rubella - SMA (smooth muscle antibody) - Syphilis - Thyroid antibodies - TORCH test for toxoplasmosis, other, rubella, cytomegalovirus, herpes simplex virus - West Nile virus DON'T NEED TO REMEMBER ALL OF THESE • done routinely around Australia • the ones in red can help diagnose autoimmune diseases