Te Target Protein “Antigen” K y Epitope Antigen-Antibody Interactions: Antibodylecular association similar to an enzyme-substrate interaction, with an important distinction: it does not lead to an irreversible chemical alteration in either the antibody or the antigen. The association between an anti- body and an antigen involves various noncovalent interac- tions between the antigenic determinant, or epitope, of the antigen and the variable-region (V,)/V;) domain of the an- tibody molecule, particularly the hypervariable regions, or complementarity-determining regions (CDRs). The exquis- ite specificity of antigen-antibody interactions has led to the development of a variety of immunologic assays, which can be used to detect the presence of either antibody or antigen. Immunoassays have played vital roles in diagnosing diseases, monitoring the level of the humoral immune response, and identifying molecules of biological or medical interest. These assays differ in their speed and sensitivity; some are strictly qualitative, others are quantitative. ° i HE ANTIGEN-ANTIBODY INTERACTION IS A BIMO- ANTIGEN. Ws 4-H = ~CH-CAgglutination Reactions ‘The interaction between antibody and a particulate antigen re- sults in visible clumping called agglutination. Antibodies that, produce such reactions are called agglutinins, Agglutination SR ee WEA ets sn PTS —Inert carriers such as latex particles Agglutination reactions now have a wide variety of applications in the detection of both antigens and antibodies including: + blood grouping, + diagnosis of infectious diseases + measure levels of certain therapeutic drugs, hormones, and plasma proteinsAgglutination reactions are similar in principle to precipitation reactions; they depend on the cross linking of polyvalent antigens with the exception that: Precipitation reactions involve soluble antigens, while agglutination involves particulate antigens Pacipitation reactions represent a phase change, while the agglutination reactions manifest as clumping of antigen! antibody complexes ‘Agglutination is more sensitive than precipitation The agglutination reaction has wide spread use in the clinical laboratory due to the following reasons such as they are simple, inexpensive, reliable and the visible manifestation of the agglutination reaction eliminates the need for complex procedures and expensive instrumentation Numerous techniques have been described for agglutination tests, these techniques may be performed using slides, test tubes, or micotiter plates, depending on the purpose of the test.Agglutination is a two-step process that results in the formation of a stable lattice network 1. Sensitization The first reaction involves antigen- antibody combination through single antigenic determinants on the particle surface and is often called sensitization. 2. Lattice formation The second step is the formation of cross- links that form the visible aggregates This represents the stabilization of antigen-antibody complexes with the binding together of multiple antigenic determinants. Each stage of the process is affected by different factors, and it is important to understand these in order to manipulate and enhance end points for such reactionsHemagglutination Is Used in Blood Typing Agglutination reactions are routinely performed _ to type ted blood cells (RBCs). In typing for the ABO antigens, RBCs are mixed on a slide with antisera to the A or B blood-group antigens. I the antigen is present on the cells, they agelutinate, forming a visible clump on the slide. Determination of which antigens are present on donor and recipient RBCs is the basis for matching blood types for transfusions. Demonstration of hemagglutination using antibodies against sheep red blood cells (SRBCs). The contral tube (10) con tains orly SRBC, which settle into a sokd “button.” The experimen tal tubes 1-9 contain a constant number of SRBCS plus serial ‘two-fold dilutions of ant-SRBC serum. The spread pattern in the ex perimental series indicates positive hemagglutination through tube 3, Lousiana State University Medical Center MIP. Courtesy of Harriet Ce Thompson Bacterial Agglutination Is Used To Diagnose Infection A bacterial infection often elicits the production of serum antibodies specific for surface antigens on the bacterial cells. ‘The presence of such antibodics can be detected by bacterial agglutination reactions, Serum from a patient thought to be infected with a given bacterium is serially diluted in an array of tubes to which the bacteria isadded. The last tube showing visible agglutination will reflect the scrum antibody titer of the paticnt. The agglutinin titeris defined as the reciprocal of the greatest serum dilution that elicits a positive agglutina- tion reaction, For example, if serial twofold dilutions of serum are prepared and ifthe dilution of 1/640 shows agelu- ‘ination but the dilution of 1/1280 does not, then the agelu- tination titer of the patient's serum is 640. In some cases serum can be diluted up to 1/50,000and still show agglutina- tion of bacteria. ‘The agglutinin titer of an antiserum can be used to diag- nose a bacterial infection, Patients with typhoid fever, for ex- ample, show a significant rise in the agglutination titer to Salmonella typhi. Agglutination reactions also provide a way to type bacteria, For instance, different species of the bac~ teritim Salmonella can be distinguished by agglutination re~ actions with a panel of typing antisera.TYPES AND USES OF AGGLUTINATION REACTIONS -SOIREGT AcguUTINATION SaAGsen Telouee by antbory bagng (most oes Blade ordub enteensy "3 ("ees 13 + momect agotuTmaTO Biot TEE Tec seem areey moreno ++ PASSIVE AGGLUTINATION, Six prt tox bea to hich ee Btcaly ae Silod: aoereyes ake eo erica (Be Rhoumatoid Factor dotetion) Passive Agglutination Is Useful with Soluble Antigens ‘The sensitivity and simplicity of agglutination reactions can be extended to soluble antigens by the technique of passive hemagglutination. In this technique, antigen-coated red blood cells are prepared by mixing a soluble antigen with red blood cells that have been treated with tannic acid or chromium chloride, both of which promote adsorption of the antigen tothe surface ofthe cells. Serum containing anti- body is serially diluted into microtiter plate wells, and the zen-coated red blood cells are then added to cach well; agglutination is assessed by the size of the characteristic spread pattern of agglutinated red blood cells on the bottom of the well lke the pattern seen in agglutination reactions ramescasons DIRECT Self-Ag © vy 4 AGGLUTINATION $3 AGGLITTINATIONOver the past several years, there has been a shift away from red blood cells to synthetic particles, such as latex beads, as matrices for agglutination reactions. Once the anti- gen has been coupled to the latex beads, the preparation can either be used immediately or stored for later use. The use of synthetic beads offers the advantages of consistency, uniformity, and stability. Furthermore, agglutination reac- tions employing synthetic beads can be rcad rapidly, often within 3 to 5 minutes of mixing the beads with the test sam- ple. Whether based on red blood cells or the more convenient and versatile synthetic beads, agglutination reactions are simple to perform, do not require expensive equipment, and can detect small amounts of antibody (concentrations as low as nanograms per milliliter). -Anigon costes Laps Pane Paton Serum Ades Artgon Arttoay BNDIn Agglutination Inhibition, Absence of Agglutination Is Diagnostic of Antigen ‘A modification of the agglutination reaction, called agglu- tination inhibition, provides a highly sensitive assay for small quantities of an antigen. For example, one of the early types of home pregnancy test kits included latex particles coated with human chorionic gonadotropin (HCG) and antibody to HCG The addition of urine from a pregnant woman, which contained HCG, inhibited agglu- tination of the latex particles when the anti-HCG antibody was added; thus the absence of agglutination indicated pregnancy. Agglutination inhibition assays can also be used to deter- mine whether an individual is using certain types of illegal drugs, stich as cocaine or heroin. A urine or blood sample is first incubated with antibody specific for the suspected drug. ‘Then red blood cells (or other particles) coated with the drug are added. If the red blood cells are not agglutinated by the antibody, it indicates the sample contained an antigen recog- nized by the antibody, suggesting that the individual was NobCG inne: Anteheice AGGLUTINATION of caries iad using the illicit drug. One problem with these tests is that some legal drugs have chemical structures similar to those of illicit drugs, and these legal drugs may cross-react with the antibody, giving a false-positive reaction. For this reason a positive reaction must be confirmed by a nonimmunologic method. Agglutination inhibition assays are widely used in clinical laboratories to determine whether an individual has been exposed to certain types of viruses that cause agglutination of tiviral antibodies, then the antibodies will bind to the virus and interfere with hemagglutination by the virus. This tech- nique is commonly used in premarital testing to determine the immune status of women with respect to rubella virus. The reciprocal of the last serum dilution to show inhibition of rubella hemagglutination is the titer of the serum. A titer greater than 10 (1:10 dilution) indicates that a woman is im- mune to rubella, whereas titer of less than 10 is indicative of a lack of immunity and the need for immunization with the rubella vaccine. + oe 0 in wine: Ait0o seule’ Carriers coated with heG>> [NO AGGLUTINATION of ase Pee text for BOG ‘PREGNANTPrecipitation Reactions Antibody and soluble antigen interacting in aqueous solu- tion form a lattice that eventually develops into a visible pre- that aggregate soluble antigens are called precipitins. Although formation of the soluble Ag-Ab com- plex occurs within minutes, formation of the visible prec {ate occurs more slowly and often takes a day or two to reach completion. Formation of an Ag. both the antibody and a ‘= The antibody must be bivalent; a precipitate will not form with monovalent Fab fragments. lattice depends on the valency of ‘= The antigen must be either bivalent or polyvalent; that is, ‘must have a least two copies of the same epitope, or have different epitopes that react with different antibodies present in polyclonal antisera Experiments with myoglobin illustrate the requirement that protein antigens be bivalent or polyvalent for a precip- itin reaction to occur. Myoglobin precipitates well with spe . Sint eiotin Peseta do Pecoinines ie cto pcm necesImmunofluorescence is a common technique using a fluorescence microscope in labs/institutions that perform biological studies, as it allows scientists to easily identify and differentiate between the antibodies and antigens present in a tissue sample.Immunofluorescence Is an antigen-antibody reaction where the antibodies are tagged (labelled) with a fluorescent dye and the antigen-antibody complex is visualized using ultra-violet fluorescent) microscope. Fluorochromes are dyes that absorb ultra-violet rays and emit visible light. This process is called fluorescence. Commonly used fluorochromes are Acridine Orange, Rhodamine, Lissamine and Calcofluor white. However, these fluorochromes are used for general fluorescence. When fluorescein (FITC) is excited by a blue (wavelength 488nm) light, it will emit a green (520nm) colour. Phycoerythrin (PE) emits an orange (570nm) colour. The fluorochromes commonly used in immunofluorescence are fluorescein isothiocyanate (green) and and tetramethyl rhodamine isothiocyanate (red). The fluorescence can then be quantified using a flow oytometer, array scanner or automated imaging instrument, or visualized using fluorescence or contocal microscopy.Electrons are arranged in discrete energy levels surrounding the atom’s nucleus with each level having a predetermined amount of energy. When an electron absorbs the energy from a photon of light it becomes “excited” and jumps to a higher, less stable energy level. The excited state does not last long, The halt-ife of the excited state is generally less than 10 (8) seconds. The electron loses a small amount of energy as heat and the remainder of the extra energy is ‘ven off in the form of a photon. The emitted fluorescence has a lower ‘energy than the absorbed light, so the wavelength of the emitted light is longer than that of the excitation light (except in the case of multi- photon excitation), Emission Excitation Excitation spectrum: Blue Emission spectrum: Green @ Nucleus ”A range of wavelengths of light can excite the electrons of a fluorochrome. For example, fluorescein will fluoresce when hit by light wth any wavelength between 450 nm and 520 nm. However, the loser the excitation wavelength is to 495 nm, the more fluorescence will be produced. This optimal wavelength is called the excitation peak. Similarly, the light produced by fluorochromes has a range of wavelengths, The emission of light from fluorescein ranges from 490 nm to 630 nm, and the emission peak is approximately 515 nm. Since the phenomenon of fluorescence was first explained by a British scientist, Sir George Stokes, in 1852, the shift in wavelenath from | Waveenath (amy short to long during fluorescence is called “Stokes shift”‘Some fluorochromes have a small Stokes shift while other fluorescent compounds have large Stokes shifts. For example, the fluorochrome fluorescein can be excited by blue-green light, and its Stokes shift is only about 20 nm, which means that the light emitted is green. This contrasts with another fluorochrome, phycoerythrin, which also can be excited by blue-green light, but has a large Stokes shift. Thus, the light emitted is yellow-orange. In immunofiuorescence, a single ‘wavelength can be used to excite several fluorochromes with different ‘Stokes shifts and thereby produce a variety of fluorescent colors shows a single wavelength at 488 nm (Blue line) exciting three ditferent fluorochromes Identified by their absorption curves on the lft of the figure (blue line). Each fluorochrome Is excited at a diferent etficiency and, therefore, the resulting emission will be at different intensities for equivalent fluorochrome concentrations. Knowing the excitation and emission Properties of fluorescent compounds makes it possibie to select ‘combinations of uorochromes that wil work togethor. Howovor, for fluorochrome to be usetul in a biological application it must attach ‘oor be contained within a structure of biological significance. ‘Wavelength (am)The ideal fluorochrome would be a molecule with the following properties: = An absorption peak at an excitation wavelength available on the fluorescence detection instrument (large extinction coefficient at the wavelength of excitation) ® Bright fluorescence (high quantum yield) = Anarrow emission spectrum that falls within one of the instrument's. detection bands * Good photostability and = Fluorescence properties that are not significantly altered by conjugation to an antibody or by the local environment of the sampleFluorochromes can be attached to antibodies which will then bind to sfluorescein, specific chemical structures on or inside celis. Many other chemical “DAPI, and physical properties of fluorochromes determine when and where ipreedlumiodide green fluorescent protein these dyes are useful in various biological assays, For example, some (GFP) of the fluorochromes that bind to DNA, such as Hoechst 33342, can “Texas Red get into living cells, but most DNA-binding fluorochromes cannot get SR OR EE rae bytes, ah past the coll membrane. Those fluorescent dyes that cannot get past Lys tone ffi: Sty het on SLI an intact cell membrane, such as propidium iodide (PI), are often used to distinguish live from dead and dying cells.Types of immunofluorescence: Direct immunofluorescence Q Indirect immunofluorescence Primary Antibody Direct il ” Fluorochrome Indirect Immunofluorescence Secondary AntibodyDirect Immunofluorescence: This technique is used to detect antigen in clinical specimens using specific fluorochrome labeled antibody. The steps involved are; Fixation of smear on the slide, treating with labeled antibody, incubation, washing to remove unbound excess labeled antibody and visualization under fluorescent microscope. When viewed under fluorescent microscope, the field is dark and areas with bound antibody fluoresce green. Direct immunofluorescence Laboled Anti-HSV-2 antibody (KE) Labeled AntiHPV antibody (luoresces green) (luoresces red) Slide containing smear from ccenical ulcer infected with HSV-2 Only antibody against HSV.2 I binds on incubation [nn When observed under fluorescent ie ‘microscope, antigen-antibody complex fuoresces green suggesting presence of HSV-2 antigen EE TS This technique can be used to detect viral, parasitic, tumor antigens from patient specimens or monolayer of cells. Another application is identification of anatomic distribution of an antigen within a tissue or within compartments of a cellAdvantages of direct immunofluorescence include shorter sample staining times and simpler dual and triple labeling procedures. In cases where one has multiple antibodies raised in the same species, for example two mouse monoclonals, a direct labeling may be necessary. Disadvantages of direct immunofluorescence include lower signal, generally higher cost, less flexibility and difficulties with the labeling procedure when commercially labeled direct conjugates are unavailable.Indirect immunofiuorescence: Indirect immunofluorescence is employed to detect antibodies in patient serum. The antigen on smear are made to react with specific unlabeled antibody (raised in mouse) and washed. The unbound antibody gets washed off. The Presence of specific mouse antibody bound to the antigen on smear Is detected by adding another antibody. The ‘second antibody is labeled anti-gamma globulin (rabbit antibody against mouse antibody) antibodies. This antibody binds to Fc portion of first antibody and persists despite washing. The presence of the second antibody is detecting by observing under fluorescent microscope. It is often used to detect autoantibodies. Commonly used in the detection of anti-nuclear antibodies (ANA) found in the serum of patients with SLE. Indirect immunofluorescence Slide containing smear from sm =, cervical ulcer infected with HSV-2 unlabeled antiHSV-2 antibody Antibody binds to antigen as HSV-2 ag is in smear Labeled anti-gamma globulin is added “rn washed Anti-gamma globulin binds to |" anti-HSV-2, remains bound on ‘washing and fluoresces on observation under fluorescent microscope