Serology Lecture 1 Introduction PDF

University of Somalia (UNISO) Faculty of Health Science Department of Laboratory Science Subject : Serology Class : ML 21 A, B and C Semester: VI Title: Introduction to Serology Prepared by:
Lecturer Mohamed Ahmed Ali (Aidaruus) 7/6/2024 Aidaruus 1 ...

University of Somalia (UNISO) Faculty of Health Science Department of Laboratory Science Subject : Serology Class : ML 21 A, B and C Semester: VI Title: Introduction to Serology Prepared by:
Lecturer Mohamed Ahmed Ali (Aidaruus) 7/6/2024 Aidaruus 1  Objectives At the end of this chapter, the students should be able to: ◦List different types of immunological tests ◦Describe the application of serology tests ◦Express the immunological techniques are used to detect and measure antigen- antibody reaction 7/6/2024 Aidaruus 2  Introduction  Antibody molecules combine reversibly with antigens to form immune complexes.  The detection and measurements of th es e reaction s form th e bas is of s e r o l o g y, a s u b d i s c i p l i n e o f 7/6/2024 Aidaruus 3  Serology - is the science of measuring antibody or antigen in body fluids.  The immune reaction is the production of antibody (substances) that protect the body against the antigen. T h e r e a r e t i m e s , h o w e v e r, w h e n antibodies are not protective (e.g. Hay fever, rash). 7/6/2024 Aidaruus 4  Application of serology tests Antigen tests  Antigen tests often enable an early diagnosis or presumptive diagnosis of an infectious disease through:-  Identification of a pathogen that has been isolated by culture  Identification of pathogens in different samples of the patients, etc. 7/6/2024 Aidaruus 5 Antibody tests: these tests are used mainly:-  To diagnose a microbial disease w hen t he pathogen or microbial antigen is not present in routine specimen or if present is not easily iso lat e d and id e nt if ie d b y o t he r av ailab le techniques.  To screen donor blood for different infectious diseases  To monitor the effectiveness of a given treatment by measuring antibody titer  To diagnose autoimmune disorders, etc. 7/6/2024 Aidaruus 6  Immunological Techniques T h r e e groups of immunological techniques are used to detect and measure antigen- antibody reaction; these are: ◦Primary binding tests ◦Secondary binding tests and ◦Tertiary binding tests. 7/6/2024 Aidaruus 7 Primary binding tests  Primary binding tests are tests that directly measure the binding of antigen and antibody (i.e.; directly measure or visualize the immune complex).  They are the most sensitive techniques in terms of the amount of detectable antigen or antibody. 7/6/2024 Aidaruus 8  Primary binding tests are performed by allowing antigen and antibody to combine and then measuring or visualizing the amount of immune complex formed. It is usual to use radioisotopes, f luorescent dyes, or enzymes as labels to identify one of the reactants. 7/6/2024 Aidaruus 9 Example:  Enzyme linked Immunosorbent assay (ELISA) tests  Radioimmunoassay (RIA)  Western blotting 7/6/2024 Aidaruus 10 Secondary binding tests  Secondary binding tests are tests that detect and measure the consequences (secondary effect) of antigen-antibody interaction.  These consequences include: ◦Clumping (agglutination) of particulate antigens ◦Precipitation of soluble antigens ◦Activation of the complement system ◦Neutralization of bacteria, viruses, or toxins 7/6/2024 Aidaruus 11 Consequences of Antibody Binding Tertiary binding tests  Tertiary binding tests measure the consequences of immune responses in vivo.  These tests are much more complex than primary and secondary tests, but their results ref lect the practical significance of the immune response. E.g. measurement of the protective effects of antibody. 7/6/2024 Aidaruus 13 University of Somalia (UNISO) Faculty of Health Science Department of Laboratory Science Subject : Serology Class : ML 21 A, B and C Semester: VI Title: Agglutination tests Prepared by:
Lecturer Mohamed Ahmed Ali (Aidaruus) 7/6/2024 Aidaruus 14  Objectives At t h e e n d o f t h i s c h a pt e r, t h e students should be able to: ◦Identify the principle of agglutination tests ◦D e t e r m i n e the phases of agglutination ◦Explain the different methods of agglutination tests. 7/6/2024 Aidaruus 15  Introduction  In district laboratories, agglutination tests are frequently used because compared with other serological tests, they are simpler to perform, require no special equipment, and are usually less expensive.  T h e i n t era c t i on b et w een a n t i b od y a n d a particulate(Insoluble) antigen results in visible 16  Principle of agglutination tests  Agglutination is the visible clumping together of bacteria, cells, or particles, b y an an ti g e n c o m b i n i n g w i th i ts specific antibody.  The resulting clumps are referred to as agglutinates. 7/6/2024 Aidaruus 17  Abs can bind and cross-link cells or particles aggregate formation  Capture microbial invaders  IgM & IgA are the most suitable (IgG in sufficient amounts can agglutinate cells)  Agglutination Test
positive. negative. antigen Antibod y.  Comparative efficiency of Immunoglobulin classes in different serological reactions IgG IgM IgA Precipitation Strong Weak Variable Agglutinatio Weak Strong Moderate n Complemen Strong Weak Negative t Fixation 7/6/2024 Aidaruus 20  Phases of Agglutination  Agglutination is a two-Phase reaction that results in the formation of a stable lattice network  Primary Phase (Sensitization) ◦ Ab reacts with a single antigenic determinant on the surface of Ag.  Secondary Phase (Lattice formation) ◦Ab must be able to bridge the gap between particles so that at least one Fab portion is attached to an antigenic d et ermin an t on each of t w o ad jacen t p ar t icles , is dependent on environmental conditions and the relative concentrations of antigen and antibody. 21 This represents what occurs during stage one of agglutination: Sensitization Stage 1 Antibody molecules attach to their corresponding Antigenic site (epitope) on the red blood cell membrane. There is no visible clumping. 22 This represents what occurs during stage 2 of agglutination: Lattice Formation Stage 2 Antibody molecules crosslink RBCs forming a lattice that results in visible clumping or agglutination. 23  Agglutinin and Agglutinogen  An agglutinin is an antibody that interacts with antigen on the surface of particles such as erythrocytes, bacteria, or latex particles to cause their agglutination.  An agglutinogen is an antigen on the surface of particles such as red blood cells that react with the a n t i b o d y k n o w n a s a g g l u t i n i n to p ro d u c e agglutination.  The most widely known agglutinogens are those of the ABO and related blood group systems. 24  The following examples of agglutination reactions : 1. Rheumatoid factor latex agglutination 2. Bacterial latex agglutination 3. Coombs test 4. Blood typing Agglutination tests can be performed:  A. On slides  B. In tubes  C. In microtitration plates 7/6/2024 Aidaruus 26  A. Slide agglutination tests These are rapid, easily performed techniques that give a reaction in minutes or even seconds.  They are, however, not usually as sensitive as tube or microtitration techniques.  Their specif ic ity depends on the reagent used.  The type of agglutination can be either active or passive. 7/6/2024 Aidaruus 27  I. Active agglutination slide tests  These are tests in which there is a direct agglutination of bacterial antigen with its corresponding antibody.  Example, the slide agglutination of salmonellae, shigellae, or Vibrio cholerae by using specific antibody.  Slide agglutination tests used to identify b a c t e r i a f r o m c u l t u r e s a r e d i f fic u l t t o standardize and control. 7/6/2024 Aidaruus 28  False agglutination (auto- agglutination) may occur due to the organism not emulsifying (mixing) well or the f lu id evaporating.  It is therefore important to check f irst for autoagglutination before adding the antiserum. 7/6/2024 Aidaruus 29 II. Passive agglutination slide tests  These are tests in which the specif ic antibody or known antigen is attached to inert particles or cells.  When the known antigen or antibody combines with its corresponding antibody or antigen in the specimen, the particles or cells are used only to show that an antigen antibody reaction has occurred.  Their role in the reaction is, therefore, passive. 7/6/2024 Aidaruus 30  The substances and cells used as carriers in passive slide agglutination tests include: ◦Latex particles ◦Carbon particles ◦Stabilized staphylococcal cells 7/6/2024 Aidaruus 31 Latex particles  These are polystyrene par ticles that can be coated with either known antigen or specif ic antibody.  An example of a test in which antigen coated particles are used is the antistreptolysin O (ASO) slide test.  Antibody coated latex par ticles are used in several t est s including t he det ect ion of extracellular bacterial antigens in cerebrospinal fluid. 7/6/2024 Aidaruus 32 Carbon particles  These are coated with cardiolipin antigen and used in the rapid plasma reagin (RPR) card test to screen for cardiolipin antibodies in the sera of patient with syphilis. 7/6/2024 Aidaruus 33 Stabilized staphylococcal strains  Most strains of Staphylococcus aureus produce on their outside surfaces a substance called protein A on to which specific antibody can be bound.  Killed staphylococcal cells coated with antibody can be used to ident ify ba ct eria a nd det ect soluble extracellular bacterial antigens in specimens and body fluids.  The term coagglutination (COAG) is used to describe the agglutination of antibody coated staphylococcal cells by antigen. 7/6/2024 Aidaruus 34  COAG techniques are becoming increasingly used since they have been shown to be se nsi t i v e and sp e c i f ic f o r i d e nt i f yi ng important pathogens.  Applications of COAG tests include: ◦The detection of Haemopilus inf luenzae type b, meningococcal, pneumococcal, and cryptococcal extracellualr antigens in cerebrospinal fluid, ◦The identif ic ation of pneumococcal antigen in sputum, and the detection of Salmonella typhi Vi antigen in urine. 7/6/2024 Aidaruus 35  COAG techniques can also be used to detect Streptococcus pyogen and other beta-haemolytic streptococci.  Commercially prepared COAG reagents are expensive, but the reagents can be prepared in a microbiology laboratory at low cost 7/6/2024 Aidaruus 36  B. Tube agglutination tests  In tube tests, agglutination occurs in a larger v o l u m e o f f lu i d a n d t h e r e f o r e , i n a n environment that can be more fully controlled.  Tube tests are usually more sensitive than slide tests.  In this tube agglutination test, serum is diluted serially and then antibody level is measured by adding standard antigenic suspension. 7/6/2024 Aidaruus 37  The temperature and time of agglutination must be correct and a control tube that contains only the antigen suspension must be included to check for auto agglutination of the reagent.  In district laboratories, tube tests are used mainly to detect and measure serum antibodies in the investigation of enteric fever (Widal test), and brucellosis. 7/6/2024 Aidaruus 38  Measurement of antibody in serum (antibody titer)  The antibody level is measured by diluting the serum, usually by using a doubling dilution technique (i.e. 1 in 2, 1 in 4, 1 in 8, 1 in 16, etc).  A standardized antigen suspension is then added.  Following incubation for the required length of time at the correct temperature in a water bath (during which time the bacterial cells settle), the tubes are examined for agglutination.  The last tube to show a clear supernatant with a coarse deposit is the end-point of the test. 7/6/2024 Aidaruus 39  The dilution of the serum at this end- point is known as the titer.  For example, if the end-point is 1 in 64, the antibody titer will be recorded as 64 (1 in 64). 7/6/2024 Aidaruus 40 7/6/2024 Aidaruus 41 7/6/2024 Aidaruus 42  Prozone effect  When testing a serum with a high antibody titer, for example from a patient with acute brucellosis, it is possible for only the higher dilutions (i.e. over 1 in 40 or 1 in 80) to show agglutination.  This is referred to as the prozone reaction, or phenomenon. It is thought to be due to a high level of Ig A (blocking antibody), non-specif ic inhibitory factors or antibody excess.  Diluting the serum appropriately can solve this problem. 7/6/2024 Aidaruus 43 7/6/2024 Aidaruus 44  Note: It is also possible when testing serum for antibodies to brucella species, for the Ig G antibodies present in the serum to combine with the antigen, but not to cause visible agglutination.  The attachment of the antibody to the antigen can be detected by using antihuman globulin, which will agglutinate the antibody antigen complexes. 7/6/2024 Aidaruus 45  Factors affecting agglutination  First stage (Sensitization) ◦Affinity constant of Ab ◦Temperature ◦Ph ◦Incubation time ◦Ag-Ab proportion  The second (Agglutination) ◦The size and physical properties of Ab molecules. ◦Concentration of Ag. site on each cell. ◦The Distance between cells 7/6/2024 Aidaruus 46 Microtitration agglutination tests Prepared by : Lecturer Mohamed Ahmed Ali (Aidaruus) 7/6/2024 Aidaruus 47  Introduction  These techniques are performed in microtitration plates.  They have now replaced several tube agglutination tests since they are more sensitive, more economical, easier to perform, and us ually give quicker results 7/6/2024 Aidaruus 48 Types of microtitration agglutination tests I. Indirect (passive) haemagglutination test (IHA) II. Haemagglutination inhibition antibody test (HIA) III. Reverse passive haemagglutination test (RPHA) 7/6/2024 Aidaruus 49  I. Indirect (passive) haemagglutination test (IHA)  The indirect haemaggutination (IHA) test is a passive agglutination test (see previous text) in which known antigen is coated on treated red cells. 7/6/2024 Aidaruus 50 Carrier red cells  The cells are formalin f ix ed and treated with tannic acid to make the antigen adhere, Antigen coated red cells are referred to sensitized cells.  In the IHA test, the sensitized red cells are added to dilutions of the patient's serum.  If the serum contains the corresponding antibody in suf fic ient concentration, the red cells will be agglutinated and settle to form an even (tight, tough, forceful) covering in the bottom of the well. 7/6/2024 Aidaruus 51  The antibody titer is the highest dilution of serum in which agglutination can be detected.  If the sensitized cells are not agglutinated they will settle and form a red button in the bottom of the well. 7/6/2024 Aidaruus 52 7/6/2024 Aidaruus 53  The specif ic ity and sensitivity of the tests depend on the antigen used and how the cells are prepared.   Some IHA tests give non-specif ic results due to heterophil antibodies present in the patient's serum.  These unwanted antibodies can be removed by absorbing the serum with non-sensitized cells.  Controls should include red cells of the same batch that are not sensitized with antigen to detect any antibody against the red cells. 7/6/2024 Aidaruus 54  Applications of IHA tests include the Treponema pallidum hemagglutination (TPHA) to detect treponemal antibodies an d th e an ti s treptol ys i n O (A SO) titration technique used in the diagnosis of S.pyogens infections. 7/6/2024 Aidaruus 55 7/6/2024 Aidaruus 56 7/6/2024 Aidaruus 57 7/6/2024 Aidaruus 58 II. Haemagglutination inhibition antibody test (HIA) Th is te c h n i q u e i s u s e d to d e te c t antibodies against Arboviruses, Ifluenza viruses, Measles virus, and Rubella virus.  These viruses are able to agglutinate red cells because they possess haemagglutinins on their outer surfaces. 7/6/2024 Aidaruus 59  In the haemagglutination inhibition (HAI) antibody test, the patient's serum is reacted with a suspension of known viral antigen.  If the corresponding antibody is present, it will coat the haemagglutinins (antigens) on the viral par ticles and so prevent haemagglutination when red cells are added. 7/6/2024 Aidaruus 60  Controls must be included to show the agglutinating activity of the antigen and absence of haemagglutination by the serum.  Sera should be treated to destroy non- specific inhibitors of agglutination.  The viral antigen should be titrated. 7/6/2024 Aidaruus 61 7/6/2024 Aidaruus 62 7/6/2024 Aidaruus 63  III. Reverse passive haemagglutination test (RPHA)  This technique is used to identify viruses that do not haemagglutinate.  It is performed by reacting viral specimens with red cells coated with specific viral antibody.  If the corresponding antigen is present, the red cells will be agglutinated.  A control must be included consisting of red cells of the same batch that are not coated with antibody. 7/6/2024 Aidaruus 64 7/6/2024 Aidaruus 65 7/6/2024 Aidaruus 66  Than k You Any Q uest io n  University of Somalia (UNISO)


Faculty of Health Science


Department of Laboratory Science


Subject : Serology


Title: Precipitation Tests




Class : ML 21 A, B and C


Semester: VI





Prepared by:
Lecturer Mohamed Ahmed Ali (Aidaruus) 7/6/2024 Aidaruus 68  Objectives At t h e e n d o f t h i s c h a pt e r, t h e students should be able to: ◦Describe the Precipitation tests ◦Identify the principle of Precipitation tests ◦Explain the mechanisms of immune precipitation ◦Determine the types of precipitation reactions 7/6/2024 Aidaruus 69  Introduction  P re c i p i t a t i o n t e st s/ t e c h n i q u e s a re methods that are used to detect and identify antigens in specimens, extracts, and cultures, and to detect and quantify antibodies in serum.  C omp are d w it h ag g lu t in at ion t e st s, p re c i p i t i n t e c h n i q u e s re q u i re m o re experience in their performance and interpretation. 7/6/2024 Aidaruus 70  Principle  They are based on two soluble reactants that come together to make one insoluble product, the precipitate.  These reactions depend on the formation of lattices (cross-links) when antigen and antibody exist in optimal proportions. 7/6/2024 Aidaruus 71 The mechanisms of immune precipitation  If a suitable amount of a clear solution of soluble antigen is mixed with its antisera and incubated at 37oC, the mixture becomes cloudy within a few minutes, then f lo cculent and within an hour a white precipitate settles to the bottom of the tube.  This process may be analyzed by examining them effect of altering the relative proportion of antigen and antibody. 7/6/2024 Aidaruus 72  Precipitation Curve Equivalence – Ab excess Ag excess Lattice formation  Excess of either component reduces lattice formation and subsequent precipitation. Precipitation reactions differ from agglutination reactions in the size and solubility of the antigen and sensitivity. Antigens are soluble molecules and larger in size in precipitation reactions. Antibodies that aggregate soluble antigens are called precipitins. Precipitation occurs in two media; liquid or gel 7/6/2024 Aidaruus 75  Precipitation curve Zone of antibody excess Prozone precipitation is inhibited and antibody not bound to antigen can be detected in the supernatant Zone equivalence Maximal precipitation in which antibody and antigen form large insoluble complexes and neither antibody nor antigen can be detected in the supernatant; Zone of antigen excess Postzone Precipitation is inhibited & Ag. not bound to Ab. can be detected in the supernatant  Types of Precipitation Reactions P re c i p i t a t i o n re a c t i o n s c a n b e broadly of three types: 1. Precipitation in solution 2. Precipitation in agar  1- Precipitation in solution Ring test, bottom Precipitate and f locculation test are the most precipitation tests in solution. a) Ring test  Precipitation between antigen and antibodies in antiserum solution is marked by the appearance of a ring of precipitation at the junction of two liquid layers.  C-reactive protein (CRP) and streptococcal grouping are the examples of the ring test. 7/6/2024 Aidaruus 79  Precipitate occurs at Test Tube reaction the interface (border) of the two reagents, forming a ring. Capillary Tube reaction Figure 18.4 b) Bottom Precipitate  Occurs when Soluble Ag interact with soluble Ab and form a visible precipitate that give bottom after centrifugation.(Widal test) c) Flocculation test:  Flocculation test may be performed in a slide or tube. VDRL test for detection of reaginic antibodies in syphilis is an example of a slide flocculation test.  In this test, a drop of VDRL antigen suspension is added to a drop of patients’ serum on a cavity slide, and the result is recorded after shaking the slide on a VDRL shaker.  In a positive test, the f loccules ( a small clump of material ) appear, which can be demonstrated well under a microscope. 7/6/2024 Aidaruus 82  Kahn test for syphilis is an example of tube flocculation test. 7/6/2024 Aidaruus 83  2- Precipitation in agar  The precipitation test in agar gel is termed as immunodiffusion test. I m m u n o d i f f u s i o n refers to the movement of antigen or antibody or both antigen and antibody molecules in a support medium by diffusion. 7/6/2024 Aidaruus 84  The rate of diffusion is affected by ◦The size of the particles ◦Temperature ◦Gel viscosity ◦I nteractions between the m atrix and reactants. 7/6/2024 Aidaruus 85  Types of immunodiffusion reactions: I m m u n od i f f u s i on reactions are classified based as follows: ◦Simple (Single) immunodiffusion (Oudin t e c hni q ue ) i n w hi c h o ne o f t he t w o reagents remains f ixed (either the antigen or the antibody) and the other reagent moves ◦Double immunodiffusion (Ouchterlony) technique) in which antigen and antibody 7/6/2024 Aidaruus 86 Single diffusion in one dimension:  Single diffusion in one dimension, as the name suggests, is the single diffusion of antigen in agar in one dimension.  In this method, antibody is included into agar gel in a test tube and the antigen solution is poured over it.  During the co urse o f time, the antigen diffuses downward toward the antibody in agar gel and a line of precipitation is formed. 7/6/2024 Aidaruus 87 7/6/2024 Aidaruus 88  Single diffusion in two dimensions : Single diffusion in two dimensions is also called radial immunodiffusion.  In this method, antiserum solution containing antibody is incorporated in agar gel on a slide or Petri dish.  The antigen is then applied to a well cut into the gel.  When antibody already present in the gel reacts with the antigen, which diffuses out of the well, a ring of precipitation is formed around the wells. 7/6/2024 Aidaruus 89  The diameter of the ring is directly proportional to the concentration of antigen.  The greater the amount of antigen in the well, the farther the ring will be from the well.  However, the test has recently been replaced by more sensitive and automated methods, such as nephelometry and enzyme-linked immunosorbent assays 7/6/2024 Aidaruus 90 7/6/2024 Aidaruus 91 7/6/2024 Aidaruus 92 7/6/2024 Aidaruus 93  Double immunodiffusion (Ouchterlony) technique)  Antigen and antibody diffuse towards each other and where they meet in optimal proportion, a visible line of precipitation forms.  The thickness of the line of precipitation is a semiquantitative measure of the amounts of antigen and antibody that combine. 7/6/2024 Aidaruus 94 7/6/2024 Aidaruus 95  Double gel diffusion techniques can be used in many different ways to detect and identify antigens and antibodies.  Examples of th is type of reaction include, the Elek gel technique used to detect toxigenic strains of Corynebacterium diphtheria and the Bi ken tes t u s ed to detec t toxi n - p r o d u c i n g f ae c al E n tr o tox i g e n i c Escherichia coli (ETEC). 7/6/2024 Aidaruus 96  Than k You Any Q uest io n  University of Somalia (UNISO) Faculty of Health Science Department of Laboratory Science Subject : Serology Class : ML 21 A, B and C Semester: VI Title: ELISA Prepared by:
Lecturer Mohamed Ahmed Ali (Aidaruus) 7/6/2024 Aidaruus 98  Objectives At the end of this chapter, the students should be able to: ◦Define the ELISA ◦Pinpoint the features of ELISA test ◦Know the applications of ELISA ◦Identify the Materials needed in ELISA Testing ◦Have good understanding the Principle of ELISA Test ◦Describe the Methods of ELISA 7/6/2024 Aidaruus 99  Introduction  Enzyme-linked immunosorbent assay (ELISA) te s t i s th e mo s t w i d e l y u s e d ty p e o f immunoassay.  ELISA is a rapid test used for detecting or quantifying antibody (Ab) against viruses, bacteria and other materials or antigen (Ag). 7/6/2024 Aidaruus 100  ELISA is so nam ed because the test technique involves the use of an enzyme system and immunosorbent.  ELISA test is being increasingly used in the detection of antigen (infectious agent) or antibody due to its simplicity and sensitivity.   It has now been widely applied in detection of a variety of antibody and antigens such as hormones, toxins, and viruses. 7/6/2024 Aidaruus 101  Features of ELISA Test  Less costly and Require minimal reagents.  ELISA t e st h as S p e c if ic an d h ig h ly sensitive test  The result of quantitative ELISA tests can be read visually with high level of accuracy. A large number of tests can be done at one time. 7/6/2024 Aidaruus 102  Q u a l i ta ti ve detecti o n o r Q u a n ti ta ti ve measurement of either antigen or antibody.  Wells ca n be co a ted w ith a ntigens o r antibodies.  Can be done by personnel with only minimal training. 7/6/2024 Aidaruus 103  Applications of ELISA  A n a l y s i s o f h o r m o n e s , v i ta m i n s , metabolites, and diagnostic markers.  Therapeutic drug monitoring.  Diagnostic procedures for detecting infection. 7/6/2024 Aidaruus 104 Materials needed in ELISA Testing 1- Pipettes, washer system, ELISA plate reader: ◦ELISA Readers: Readers need to have appropriate filter (650 nm and 450 nm). ◦P ipette: Are a va ila ble a s f ix ed a s w ell a s adjustable volume as well as single channel and multi-channel. ◦Washing system: It can be manual system that washes one row or column at a time or semi automated systems that wash one strip or plate at a time or fully automated systems that can process multiple plates 7/6/2024 Aidaruus 105 2- Reagents needed for the testing: Concluded in the kit (coated plates, sample diluents, controls, wash concentrate, conjugate, substrate, stop solution).  Coated plates: ◦The 96-well plates are made of polystyrene and are coated with either inactivated antigen or antibody. ◦ The function of the plate has to hold the immobilized either antigen or antibody. ◦ Antigen or antibody present in the sample will bind to the plate. ◦ This coating acts as the binding site for the antibodies or antigens in the sample. 7/6/2024 Aidaruus 106  Controls: ◦Negative and positive controls are provided in each kit. ◦The controls help to normalize or standardize each plate. ◦Controls are also used to validate the assay and to calculate sample results. ◦Controls might be pre-diluted and ready to use. (Please refer to kit for specific instructions).  Conjugates: ◦E L I S A c o n j u g a t e s a r e e n z y m e l a b e l e d antibodies that react specifically to plate bound sample analytes. ◦ Unbound conjugates are washed away after incubation and before the addition of substrate. 7/6/2024 Aidaruus 107  Wash Concentrate: ◦It acts as a buffered solution containing detergent to wash unbound material from the plate. ◦(Not all test kits have wash concentrate; in that case distilled water can be used for washing; please refer to kit insert for specif ic instructions)  Stop solution: ◦It stops the enzyme substrate reaction and color development. 7/6/2024 Aidaruus 108 7/6/2024 Aidaruus 109 96-well microtiter plate Fig.: ELISA Reader Fig.: loading the plate  Principle of ELISA Test  Most ELISA methods developed for the detection of antigen or antibody consist of use of corresponding a nt i bo d y o r a nt i ge n, a nd o f t a rge t m o l e c ul e detection/quantitation using an enzyme reaction with its substrate.  The antibody or antigen is f irmly f ixed on solid phase, suc h as pl ast i c sur fac e o f po l yv i nyl pl at e o r polystyrene tube.  Such systems are also called Solid Phase Immunosorbent Assay (SPIA). 7/6/2024 Aidaruus 113  Test sample is added in the microtitre plate, if there is presence of Ag or Ab in the test sample, there will be Ag-Ab reactions (with immobilized Ab or Ag).  Later enzyme labelled antibody is added in the reaction mixture, which will combine with either test antigen or Fc portion of test antibody. 7/6/2024 Aidaruus 114  Substrate is added after the antigen- antibody reaction.  The enzyme catalyzes the substrate to give a color end point (yellow compound in case of alkaline phosphatase).  The intensity of the color is proportional to the amount of antibody or antigen present in the test sample, which can 7/6/2024 Aidaruus 115  Enzyme System  Enzyme labels are used to detect the binding of antigen-antibody complex. It should have high specific reactivity.  The enzyme system consists of ◦An enzyme: horse radish peroxidase, alkaline phosphatase which is labelled or linked, to a specific antibody. ◦A Specific Substrate o-Phenylenediamine dihydrochloride for peroxidase P N itro phe nyl Pho sphate ( PN PP) - f o r Alkaline Phosphatase 7/6/2024 Aidaruus 116  Stages in ELISA 1. The adsorption of either antigen or antibody to the micro-titer plate. 2. The addition of the test sample and subsequent reagents. 3. The incubation of reactants (formation of antigen- antibody complex). 4. The separation of bound and free reactants by washing. 5. The binding of enzyme to the target antigen or antibody. 7/6/2024 Aidaruus 117  Method of ELISA  The techniques of ELISA include: ◦Direct ELISA ◦Indirect ELISA ◦Sandwich ELISA ◦Competitive ELISA 7/6/2024 Aidaruus 118  Direct ELISA A target protein/antigen is immobilized on the surface of microplate wells and incubated with an enzyme-labeled antibody to the target protein.  A fte r was h i n g , th e ac ti v i ty of th e mi c rop l ate we l l - b ou n d e n z yme i s measured. 7/6/2024 Aidaruus 119 7/6/2024 Aidaruus 120  Indirect ELISA A target antigen is immobilized on the surface of microplate wells and incubated with an antibody to the target protein (the primary antibody), followed by a secondary antibody against the primary antibody. After washing, the activity of the microplate well-bound enzyme is measured. 7/6/2024 Aidaruus 121 7/6/2024 Aidaruus 122  The difference between direct ELISA and indirect ELISA is that direct elisa only one antibody is used this single antibody is conjugated directly to the detection enzyme, while the indirect elisa requires two antibodies; a primary antibody and an enzyme-linked secondary antibody that is complementary to the primary antibody. 7/6/2024 Aidaruus 123  Sandwich ELISA.  The Sandwich ELISA is a sensitive and robust method which measures the antigen concentration in an unknown sample.  The antigen of interest is quantif ie d between two layers of antibodies.  capture and the detection antibody. 7/6/2024 Aidaruus 124 7/6/2024 Aidaruus 125  The key advantage of a sandwich ELISA is its high sensitivity; it is 2-5 times more sensitive than direct or indirect ELISAs.  Sandwich ELISA also delivers high specificity as two antibodies are used to detect the antigen. 7/6/2024 Aidaruus 126 https://www.youtube.com/watch?v=4OnLRa7uxuM 7/6/2024 Aidaruus 127  Assignment 1) Radioimmunoassay (RIA) 2) Western blotting 3) Competitive ELISA 4) Serial dilution and determination of End point and Titer 7/6/2024 Aidaruus 128  Than k You Any Q uest io n