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    Immunotechniques 1

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    Rekha Govindan

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    Te Target Protein “Antigen” ® ¥ 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, My ia Cr, GHB =C—CH,-0i, on - C—O ay 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 + Types of particles that participate in such reactions: Y ‘oe ts ~€rythrocytes At % Ka Bacterial cells é te Se Inert carriers such as latex BS ‘ = J 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 Pecipitation 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 red 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. If 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 control tube (10) con tains only SRECS, 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. [Louisiana State University Medical Center/AMIP. Courtesy of Haier, C.K Thompson Bacterial Agglutination Is Used To Diagnose Infection AA bacterial infection often clicts 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 scrum dilution that elicits a positive agglutina- tion reaction. For example, if serial twofold dilutions of serum are prepared and if the dilution of 1/640 shows agelu- tination but the dilution of 1/1280 does not, then the agelu- tination titer of the patient's serum is 649. In some cases serum can be diluted up to 1/50,000and still show agghutina- 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~ teriuim Salmonella can be distinguished by agglutination re- actions with a panel of typing antisera. TYPES AND USES OF AGGLUTINATION REACTIONS EE wan Sta yin Pra narra © INPIRE Gagan s panavea Wn a coconcary snibosy recog imtcarigey eons *reraeie btaa + PASSIVE AGGLUTINATION BNE Soe $C tox beac) t nth Hitealy re used agpogate ator the addon amie. Rhoumatoid Factor dotsetion) ‘epee DIRECT Passive Agglutination Is Useful Solf-Ag 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 ry chromium chloride, both of which promote adsorption of ‘the antigen to the surface of the cells. Serum containing anti- L body is serially diluted into microtiter plate wells, and the AGGLUTINATION antigen-coated red blood cells are then added to each well; agglutination is assessed by the size of the characteristic spread pattern of agglutinated red blood cells on the bottom of the well like the pattern seen in agglutination reactions AGGLUTINATION $s 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 becn 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 read 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). “angen comes Lax Parte Paton Serum Ages Artgon Arteody Bring 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, such 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 + AGGLUTINATION of caries, NegtivetetforhCC Nor! aso a 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 red blood cells. If an individual’s serum contains specific an- tiviral antibodies, then the antibodies will bind to the virus and interfere with hemagglutination by the virus. This tech- nigue 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 isim- mune to rubella, whereas a titer of less than 10 is indicative of alack of immunity and the need for immunization with the rubella vaccine, tT ec in wine: Atit0d acute’ [NO AGGLUTINATION oft Pete test for WO so" PREGNANT Precipitation Reactions Antibody and soluble antigen interacting in aqueous solu- tion forma lattice that eventually develops into a visible pre- & ¥ att vo ‘Antigen added Aprecio- tration curve fora system of one antigen and its antibodies. This pot of the amount of antibody precipitated versus increasing antigen concentrations (at constant total antibody) reveals three zones: 2 zone of antibody excess, in which precipitation is inhibited and ant body not bound to antigen can be detected in the supematant: an ‘equivalence zone of maximal precipitation in which antibody and antigen form large insoluble complexes and nether antibody nor antigen can be detected in the supernatant and a zone of antigen ex ‘cess in which precipitation is inhibited and antigen not bound to antibody can be detected in the supernatant Precipitation Reactions in Gels Yield Visible Precipitin Lines ‘Immune precipitates can form not only in solution but also in an agar matrix. When antigen and antibody diffuse toward one another in agar, or when antibody isincorporated into the agar and antigen diffuses into the antibody-containing matrix, a visible line of precipitation will form. As ina precipitation re- action in fluid, visible precipitation occurs in the region of ‘equivalence, whereas no visible precipitate forms in regions of antibody or antigen excess. Two types of immunodiffusion re~ actionscan be used to determine relative concentrations of an- tibodies or antigens, to compare antigens, orto determine the relative purity of an antigen preparation. They are radial im- ‘munodiffusion (the Mancini method) and double immun- diffusion (the Ouchterlony method); both are carried out in a semisolid medium such as agar. In radial immunodiffusion, an antigen sample isplaced ina well and allowed to diffuse into ‘agar containing a suitable dilution of an antiserum. As the antigen diffuses into the agar, the region of equivalence is es- tablished and a ring of precipitation, a precipitin ring, forms around the well Thearea ofthe pre- ‘aprtin ring i proportional tothe concentration of antigen. By ‘comparing the area of the precipitin ring with a standard curve (obtained by measuring the precipitin areas of known coneen- trations ofthe antigen), the concentration ofthe antigen sam- ple can be determined. In the Ouchterlony method, both antigen and antibody diffuse radially from wells toward each, ‘other thereby establishing a concentration gradient. As equiv= alence is reached, a visible line of precipitation, a precipitin line, forms Preeipate Diagrammatic representation of radial immunodiffu- sion (Mancini method) and double immunadiflusion (Ouchterlony ‘method) in a gel. In both cases, large insoluble complexes form in the agar in the zone of equivalence, visible as lines of precipitation (purple regions). Only the antigen (red) difuses in radial immuno. diffusion, whereas both the antibody (blue) and antigen (ted) diffuse in double immunodiffusion Radial Immunodiffusion Double diffusion in two dimension er ito comp oo (perc 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 cytometer, array scanner or automated imaging instrument, or visualized using fluorescence or confocal 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 given 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) Excitation Emission 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 with any wavelength between 450 nm and 520 nm. However, the closer 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 short o 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 immunofluorescence, 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 (Dive tine) exciting three different fluorochromes identitied by thoir absorption curves on the loft of the figure (blue tine). Each fluorochrome Is excited at a diferent efficiency and, theretore, 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 fluorochromes that wil work together. However, for «fluorochrome to be useful in a biological application it must attach 10 or be contained within a structure of biological significance. Wavelength (om) 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 photestability 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 ‘specific chemical structures on or inside cells. Many other chemical and physical properties of fluorochromes determine when and where these dyes are useful in various biological assays. For example, some of the fluorochromes that bind to DNA, such as Hoechst 33342, can Get into living cells, but most DNA-binding fluorochromes cannot get past the cell membrane. Those fluorescent dyes that cannot get past {an intact cell membrane, such as propidium iodide (PI), are often used to distinguish live from dead and dying cells. sfluorescein, “DAPI, =propidium iodide “green fluorescent protein (GFP) Texas Red commen Faroshore in Wel nd Confocal Moony Bytyte aye "et a a ie ae Tit - ty o ot Ay x tthe: ony een “e wee 2 fresparoat Oxy erate moar yan Types of immunofluorescence: Q Direct immunofluorescence Q Indirect immunofluorescence Primary Antibody Direct al Pg 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 Labiled AntiHSV-2 antibody KE) Labeled AntiHPV antibody huoresces green) (Muoresces red) Siide containing smear from a conical ulcer infected with HSV-2 Only antibody against HSV-2 k binds on incubation fn When observed under fluorescent ii ‘microscope, antigon-antibody complex fuoresces green suggesting presence of HSV-.2 antigen 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 immunofiluorescence 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 Fe 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 = =, cervical ulcer infected with HSV-2 unlabeled anti:HSV-2 antibody Antibody binds to antigen as HSV-2 ag is in smear Labeled anti-gamma globulin is added next and washed Anti-gamma globulin binds to |"" anti-HSV-2, remains bound on washing and fluoresces on observation under fluorescent microscope Advantages of indirect immunofluorescence include greater sensitivity than direct immunofluorescence. There is amplification of the signal in indirect immunofluorescence because more than one secondary antibody can attach to each primary Commercially produced secondary antibodies are relatively inexpensive, available in an array of colors, and quality controlled. Disadvantages of indirect immunofluorescence include the potential for cross-reactivity and the need to find primary antibodies that are not raised in the same species or of different isotypes when performing multiple-labeling experiments. Samples with endogenous immunoglobulin may exhibit a high background. Applications of Immunofluorescence In Pathology Some practical applications of immunofluorescence in diagnostic pathology are: ® Analysis of antigens in tresh, frozen or fixed tissues; sub-cellular localization of antigens in tissue culture monolayers; observation of bacterial or parasitic specimens; = Detection and localization of the presence or absence of specific DNA sequences on chromosomes; and = Defining the spatial-temporal patterns of gene expression within cells/tissues. Autofluorescence Biological autofluorescence in mammalian cells due to flavin coenzymes (FAD and FMN: absorption, 450 nm; emission, 515 ‘nm) and reduced pyridine nucleotides (NADH: absorption, 340 nm ‘emission, 460 nm) can be problematic in the detection of fluorescence probes in tissues and cells. Fixation with aldehydes, particularly Glutaraldehyde, can result in high levels of autofluorescence. This can be minimized in fixed cells by washing with 0.1% sodium borohydride in phosphate-butfered saline (6) prior to antibody incubation. Problems due to autofluorescence can be minimized by selecting probes and optical filters that maximize the fluorescence signal relative to the autofluorescence. Other factors that limit IF include the performance of the detection instrument (2. how well the microscope thas been calibrated and set), the specificity ofthe antibodies, and the d and the emited ft ‘specimen preparation. Jedicn ist : Prete Immunoelectrophoresis Combines Electrophoresis and Double Immunodiffusion In immunoelectrophoresis, the antigen mixture is first elec trophoresed to separate its components by charge. Trouighs are then cut into the agar gel parallel to the direction of the electric field, and antiserum is added to the troughs. Antibody and antigen then diffuse toward each other and produce lines of precipitation where they meet in appropy ate proportions (Figure 6-6a). Immunoelectrophoresis is used in clinical laboratories to detect the presence or absence of proteins in the serum. A sample of serum is elec trophoresed, and the individual serum components are identified with antisera specific for a given protein or im- ‘munoglobulin class (Figure 6-6b). This technique is useful in determining whether @ patient produces abnormally low amounts of one or more isotypes, characteristic of certain immunodeficiency diseases. It can also show whether a pa tient overproduices some serum protein, such as albumin, immunoglobulin, or transferrin. The immunoelecteopho retic patter of serum from paticnts with multiple myeloma, for example, shows a heavy distorted arc caused by the large amount of myeloma protein, which is monoclonal Ig and therefore uniformly charged (Figure 6-6b). Because immu- noelectrophoress isa strictly qualitative technique that only detects relatively high antibody concentrations (greater than several hundred g/ml), it utility is limited to the detection of quantitative abnormalities only when the departure from normal is striking, as in immunodeficiency states and im- munoproliferative disorders of an antigen miaure, fist electrophoresed, wi 1e basis of charge. Antiserurn {blu} is then added to troughs on one or both sides ofthe sep antigens and allowed to diffuse; in time, ines of pr n (orange) i separates the component antigens on t! tion (cok where specific antibody and antigen interact. (b) Im: munoelecirophor um fiom a patient with cored arcs} fo ic patterns of human arnount of a monoclonal IgG myeloma. The patient produces a lay (kgf tas co nig) santa A sarple bras naar tie atin vias placed in tie wel ofthe side and elecrophoresed. Then an serum specific forthe indicated antibedy dass of light chain type was placed in the top trough of each slide, At the concentrations of pa nly antelgG and ant:A antibodies produced ines of precipitation. ani), Robert A. Kyle and Terry A. Katzr ent’ Manual of Clnical Immunology, 1987, N. Rose, ed, ASM Press, Wash ington, DC, p. 164) A ‘elated quantitative technique, rocket electrophore- sis, does permit measurement of antigen levels. In rocket clectrophoresis, a negatively charged antigen is elec- trophoresed in a gel containing antibody. The precipitate formed between antigen and antibody has the shape of a rocket, the height of which is proportional to the concen- tration of antigen in the well. One limitation of rocket electrophoresis is the need for the antigen to be negatively charged for electrophoretic movement within the agar matrix. Some proteins, immunoglobulins for example, are not sufficiently charged to be quantitatively analyzed by rocket electrophoresis; nor is it possible to. measure the amounts of several antigens in a mixture at the same time. ELISA(Enzyme Linked ImmunoSorbent assay) The technique was developed in 1971 by Peter Pertmann and Eva Engvall at Stockholm University, Sweden. The Enzyme-Linked Immunosorbent Assay (ELISA for short) is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. to measure the concentration of antibodies or antigens in blood. Principle — use of enzyme-labeled immunoglobulin to detect antigens or antibodies It utilizes two antibodies, one of which is specific to the antigen and the other of which is coupled to an enzyme. This second antibody gives the assay its "enzyme-linked" name, and will cause a chromogenic or fluorogenic substrate to produce a signal. Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool both for determining serum antibody concentrations (such as with the Human Immunodeficiency Virus, HIV test or West Nile Virus) and also for detecting the presence of antigen. ELISA is a plate based assay technique which is used for detecting and quantifying substances such as peptides, proteins, antibodies and hormones. Principle ELISAS are typically performed in 96-well polystyrene plates. The serum is incubated in a well, and each well contains a different (1) (2) (3) (4) (5) serum. A positive control serum and a negative control serum would bbe included among the 96 samples being tested. Antibodies or mm ‘antigens present in serum are captured by corresponding antigen e * or antibody coated on to the solid surface. After some time, the R plate is washed to remove serum and unbound antibodies or | x oN antigens with a series of wash buffer. To detect the bound A antibodies or antigens, a secondary antibodies that are attached to = = 7 an eye such as perrdase or aaa phasphatae ae adéeda, Y Y Y each well. After an incubation period, the unbound secondary antibodies are washed off, When a suitable substrate Is added, the enzyme reacts with it to produce a color, This color produced is ‘measurable as 2 function or quantity of antigens or antbodies present in the given sample. The intensity of color/ optical density is ‘measured at 450nm. The intensity ofthe color gies an indication of the amount of antigen or antibody. ~ ye | Primary antibody. addition a -—, % ‘Chemiuminescent fecomtay Sabstraie antibody edition DU o | « Y { & Streptvidin Blot Duplex -Transcripion —-Transciption Factor == Primary Antibody Second Light Factor Bound t9 Biotin Dupex bod HRP A number of enzymes have been used for ELISA such as alkaline phosphatase, horse radish peroxidase and beta galactosidase. Specific substrate such as ortho- phenyldiamine dihydrochloride (for peroxidase), paranitrophenyl phosphate (for alkaline phosphatase) are used which are hydrolysed by above enzymes to give colored end product. Among the available immunoassays, Elisa is one of the distinguished and widely used types. It is extensively used techniques in microbiology due to the advantages like rapidity or speed in experimentation unlike radioimmunoassay. Further it has greater sensitivity and specificity for even small amount of test samples. The reaction is measurable in both qualitative and quantitative terms. Qualitatively it measures the specific type of substance. While quantitatively, it measures quantity of substance present in the given sample. It finds its application on daily basis in clinical diagnosis, biological, food and medical research. Types of ELISA Frequently there are 3 types of ELISA on the basis of binding structure between the Antibody and Antigen. ELISA TYPES . . (Commonly, HETEROGENEOUS HOMOGENOUS: Used) Direct Elisa Indirect Elisa ‘Sandwich Elisa Heterogeneous: Here the antigens are first added is a solid type. i.e. fixed to the plate and not in liquid form while next antibodies or antigens added are always in liquid phase. Homogeneous: Here the antigens are _first_added_are also (otiguid phase and the next antibodies added on to it are also in liquid phase. Direct Elisa is one wherein there is only one set of antigens and one set of antibodies to react. In this elisa method, antigens from the patient sample fixed to the Elisa plates are made to react with an antibodies sample which is tagged to an enzyme. I.e. directly to the antigen in the test an enzyme linked antibody is added to produce a colour reaction with externally added substrate i.e. Elisa reagent. Ag or Ab + Ab or Ag-(e) -----— Reaction color.. e ubstrate o Direct Assay Indirect ELISA Antibody can be detected or quantitatively determined by indirect ELISA In this technique, antigen is coated on the microtiter well. ‘Serum or some other sample containing primary antibody is added to the microtiter well and allowed to react withthe coated antigen. ‘Any free primary antibody is washed away and the bound antibody to the antigen is detected by adding an enzyme conjugated secondary antibody that binds to the primary antibody. Unbound secondary antibody is then washed away and a specific substrate for the enzyme is added. Enzyme hydrolyzes the substrate to form colored products. The amount of colored end product is measured by spectrophotometric plate readers that can measure the absorbance of all the wells of 96-well plate. Advantages = Increased sensitivity, since more than one labeled antbbody is bound per primary antibody. «= A wide varity of labeled secondary antibodies are avaiable commercially. = Maximum immunoreactity of the primary antibody is retained because its not labeled. «= Versatle because many primary antibodies can be made in ‘one species andthe same labeled secondary antibody can be ocd for detection, weet oie oad «= Flexiblty, since different primary detection antibodies can be used with a single labeled secondary antibody. = Cost savings, since fewer labeled antibodies are required. ® Different visualization markers can be used with the same primary antibody. Disadvantages = Cross-reacthity might occur with the secondary antbody, resulting innonspectic signal 1 Anextra incubation step is required in the procedure. “HRI ‘Sock attoty Exayednied ‘ods angen ‘riod itso sede antany Sardis ail nt ‘oroetadty eye eo CGowedpt, te ate fog tans otialnte rast Age ty Procedure of Indirect ELISA 1. Coat the micro titer plate wells with antigen. 2. Block all unbound sites to prevent false positive results. 3. Add sample containing antibody (e.g. rabbit monoc antibody) to the wells and incubate the plate at 37°c. 4. Wash the plate, so that unbound antibody is removed. 5. Add secondary antibody conjugated to an enzyme (e.g. mouse IgG). 6. Wash the plate, so that unbound enzyme-linked antibc are removed. 7. Add substrate which is converted by the enzyme to prodi colored product. 8. Reaction of a substrate with the enzyme to produce a cal Sandwich ELISA ‘Antigen can be detected by sandwich ELISA. In this technique, Advantages antibody is coated on the microtiter well. A sample containing antigen Is added to the well and allowed to react with the antibody attached to the well, forming antigen-antibody complex. After the well Is washed, a second enzymesinked antibody specific for 2 different epitope on the antigen Is added and allowed to react with the bound antigen. Then after unbound secondary antibody is removed by washing, Finally substrate is added to the plate which is hhydralyzed by enzyme to form colored products. ‘Sandwich ELISA Wash Wash ‘Wash od ae — ‘Antigen binds to antibody ‘= High specificity, since two antibodies are used the antigen 1s specifically coptured and detected. ' Suitable for complex samples, since the antigen does not ‘require purification prior to measurement. Flexibility and sensitivity, since both direct and indirect detection methods can be used. Asecond monoclonal Substrate is added and antibody, linked to converted by enzyme into enzyme, binds to colored product; the rate immobilized antigen of color formation is proportional to the amount of antigen Procedure of sandwich ELISA nN w w a ~ Prepare a surface to which a known quantity of antibody ts bound Add the antigen-containing sample to the plate and incubate the plate at 37°c. Wash the plate, so that unbound antigen is removed. Add the enzyme-linked antibodies which are also specific to the antigen and then incubate at 37°c. . Wash the plate, so that unbound enzyme-linked antibodies are removed. Add substrate which is converted by the enzyme to produce a colored product. Reaction of a substrate with the enzyme to produce a colored product. Competitive ELISA This test Is used to measure the concentration of an antigen in a sample. In this test, antibody is first incubated in solution with a sample containing antigen. The antigen-antibody misture is then added to the microtitre well which is coated with antigen. The more the antigen present in the sample, the less free antibody will be avaliable to bind tothe antigen-coated well. After the well washed, enzyme conjugated secondary antibody specfic for isotype of the primary antibody is added to determine the amount of primary antibody bound to the well The higher the concentration of antigen in the sample, the lower the absorbance. ‘Competitive ELISA antigen to be measured antigen-coated well Advantages 1 High specificity, since two antibodies are used. = High sensitivity, since both dlrect and indirect detection methods can be used, * Suitable for complex samples, since the antigen does not require purification prior to measurement. Incubate was at antibody with js ‘Add enzyme- Add substrate conjugated and measure secondary color antibody Procedure 1. Antibody is incubated with sample containing antigen. 2. Antigen-antibody complex are added to the microtitre well which are pre-coated with the antigen. 3. Wash the plate to remove unbound antibody. 4. Enzyme linked secondary antibody which is specific to the primary antibody is added. 5. Wash the plate, so that unbound enzyme-linked antibodies are removed. 6. Add substrate which is converted by the enzyme into a fluorescent signal. ELISA may be run in a qualitative or quantitative format. Qualitative results provide a simple positive or negative result for a sample. The cutoff between positive and negative is determined by the analyst and may be statistical. Two or three times the standard deviation is often used to distinguish positive and negative samples. In quantitative ELISA, the optical density or fluorescent units of the sample is interpolated into a standard curve which is typically a serial dilution of the target. a Immuno blot analysis Principle — Antigens are separated by Poly Acrylomide Gel Electrophoresis (PAGE) and trans-blotted onto nitrocellulose/nylon membranes — Antibodies in serum react with specific antigens — Signals are detected according to the principles of test systems — Antibodies against microbes with numerous cross- reacting antibodies identified more specifically Examples —T. pallidum, B.burgdorferi, — Herpes simplex virus types 1 and 2 Radioimmunoassay One of the most sensitive techniques for detecting antigen or antibody is radioimmunoassay (RIA). The technique was first developed in 1960 by two endacrinologists, S.A. Berson and Rosalyn Yalow, to determine levels of insulin-anti-in- sulin complexes in diabetics. Although their technique en- countered some skepticism, it soon proved its value for ‘measuring hormones, serum proteins, drugs,and vitamins at, concentrations of 0.001 micrograms per milliliter or less. In 1977, some years after Rerson’s death, the significance of the technique was acknowledged by the award of a Nobel Prize to Yalow. ‘The principle of RIA involves competitive binding of ra- diolabeled antigen and unlabeled antigen to a high-affinity antibody. The labeled antigen is mixed with antibody at a concentration that saturates the antigen-binding sites of the antibody. Then test samples of unlabeled antigen of un- known concentration are added in progressively larger amounts, The antibody does not distinguish labeled from unlabeled antigen, so the two kinds of antigen compete for available binding sites on the antibody. As the concentration of unlabeled antigen increases, more labeled antigen will be displaced from the binding sites. The decrease in the amount of radiolabeled antigen bound to specific antibody in the presence of the test sample is measured in order to determine the amount of antigen present in the test sample, ‘The antigen is generally labeled with a gamma-emitting isotope such as !?"I, but beta-emitting isotopes such as tri- tium PH) are also routinely used as labels. The radiola- beled antigen is part of the assay mixture; the test sample may be a complex mixture, such as serum or other body fluids, that contains the unlabeled antigen. The first step in setting up an RIA is to determine the amount of antibody needed to bind 50%-70% of a fixed quantity of radioactive antigen (Ag’) in the assay mixture. This ratio of antibody to Ag" is chosen to ensure that the number of epitopes presented by the labeled antigen alvvays exceeds the total number of antibody binding sites. Consequently, unlabeled, antigen added to the sample mixture will compete with ra- diolabeled antigen for the limited supply of antibody. Even small amount of unlabeled antigen added to the assay mixture of labeled antigen and antibody will cause a de- crease in the amount of radioactive antigen bound, and this, decrease will be proportional to the amount of unlabeled antigen added. To determine the amount of labeled antigen bound, the Ag-Ab complex is precipitated to separate it from free antigen (antigen not bound to Ab), and the ra- dioactivity in the precipitate is measured. A standard curve can be generated using unlabeled antigen samples of known concentration (in place of the test sample), and from this plot the amount of antigen in the test mixture may be precisely determined. Several methods have been developed for separating the bound antigen from the free antigen in RIA. One method in- volves precipitating the Ag-Ab complex with a secondary anti-isotype antiserum. For example, if the Ag-Ab complex contains rabbit IgG antibody, then goat anti-rabbit IgG will bind to the rabbit IgG and precipitate the complex. Another ‘method makes use ofthe fact that protein A of Staphylococcus ‘aureus has high affinity for IgG. IF the Ag-Ab complex con- tains an IgG antibody, the complex can be precipitated by mixing with formalin-killed S. aureus. After removal of the complex by either of these methods, the amount of free la- beled antigen remaining in the supernatant can be measured in a radiation counter; subtracting this value from the total amount of labeled antigen added yields the amount of la- beled antigen bound. ‘Various solid-phase RIAs have been developed that make it easier to separate the Ag-Ab complex from the unbound antigen. In some cases, the antibody is covalently cross- linked to Sepharose beads. The amount of radiolabeled anti- ‘gen bound to the beads can be measured after the beads have ‘been centrifuged and washed. Alternatively, the antibody can be immobilized on polystyrene or polyvinylchloride wells and the amount of free labeled antigen in the supernatant ‘can be determined in a radiation counter. In another ap- roach, the antibody is immobilized on the walls of mi- ‘rotiter wells and the amount of bound antigen determined. Because the procedure requires only small amounts of sam- ple and can be conducted in small 96-well microtiter plates (slightly larger than a 3% 5 card), this procedure is well suited for determining the concentration of a particular anti- ‘gen in large numbers of samples. For example, a microtiter SE el el hi for the presence of the he- itis virus (Figure 6-9). RIA screening of donor blood has Erp reduced te lcidenc of hepaisBiactlons ne

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