Immunology & Serology (Lec) PDF, National University Manila 2024-2025

IMMUNOLOGY & SEROLOGY [LEC] 2ND TERM | MALACASTE, P. R. NATIONAL UNIVERSITY MANILA - BACHELOR OF SCIENCE IN MEDICAL TECHNOLOGY A.Y. 2024 – 2025 [LESSON 9] PRECIPITATION AND AGGLUTINATION REACTIONS...

IMMUNOLOGY & SEROLOGY [LEC] 2ND TERM | MALACASTE, P. R. NATIONAL UNIVERSITY MANILA - BACHELOR OF SCIENCE IN MEDICAL TECHNOLOGY A.Y. 2024 – 2025 [LESSON 9] PRECIPITATION AND AGGLUTINATION REACTIONS PRECIPITATION o When the value of K is higher, the amount of antigen–  Precipitation involves combining soluble antigen with antibody complex is larger and the assay reaction is soluble antibody to produce insoluble complexes that more visible or easily detectable are visible.  When we say soluble when it is mixed it is not visible, in PRECIPITATION CURVE contrast with insoluble which is visible when mixed. AGGLUTINATION  Agglutination is the process by which particulate antigens such as cells aggregate to form larger complexes when a specific antibody is present. ANTIGEN-ANTIBODY BINDING AFFINITY  Initial force of attraction that exists between a single Fab site on an antibody molecule and a single epitope or determinant site on the corresponding antigen  Opposite charges attract each other and similar charges ZONE OF EQUIVALENCE repel each other, that’s why the  The number of multivalent sites of antigen and photo on the left has higher affinity antibody are approximately equal. than the photo on the right.  Normal ratio = 1:1 o In blood typing the ratio is 2:1 (2 parts of Ab and 1 part AVIDITY of Ag)  “Strength of binding”  Precipitation is the result of random, reversible reactions  Represents the overall whereby each antibody binds to more than one antigen and strength of antigen– vice versa, forming a stable network or lattice. antibody binding and is o Lattice hypothesis is formulated by Marrack the sum of the affinities of all the individual PROZONE AND POSTZONE antibody–antigen PROZONE PHENOMENON combining sites  Excess antibody  A multivalent antibody binds a multivalent antigen and is a o Mnemonic: since “pro” means “fighting for” and our measure of the overall stability of an antigen–antibody antibody is the one who fights antigens, PROzone complex. means there are extra antibodies o Multivalent means there’s multiple attachments  Antigen combines with only one or two antibody molecules  It is the force that keeps the molecules together and no cross-linkages are formed  Igm have greater avidity than IgG because it is a  This results to a false negative result because there are no pentamer lattice formation o Singe IgM is a pentamer it has 10 valence  Remedy: Serum Dilution o Since the antibody is being diluted, they become less LAW OF MASS ACTION and is now able to bind and form lattice formation 𝑏𝑜𝑢𝑛𝑑 𝐴𝑔−𝐴𝑏 𝑐𝑜𝑚𝑝𝑙𝑒𝑥ℎ𝑖𝑔ℎ𝑒𝑟 𝑏𝑖𝑛𝑑𝑖𝑛𝑔/𝑎𝑣𝑖𝑑𝑖𝑡𝑦  𝐾= = (𝑓𝑟𝑒𝑒 𝐴𝑔)(𝑓𝑟𝑒𝑒 𝐴𝑏) 𝑙𝑜𝑤𝑒𝑟 𝑐ℎ𝑎𝑛𝑐𝑒𝑠 𝑜𝑓 𝑑𝑖𝑠𝑠𝑜𝑐𝑖𝑎𝑡𝑖𝑜𝑛 POSTZONE PHENOMENON  All antigen–antibody binding is reversible and is governed  Excess antigen by the law of mass action. This law states that free reactants  Every available antibody site is bound to a single antigen are in equilibrium with bound reactants and no cross-links are formed  The value of K depends on the strength of binding between  This also results to a false negative result because there antibody and antigen are no lattice formation as well o As the strength of binding, or avidity, increases, the tendency of the antigen–antibody complexes to  Remedy: Retest after 1 week-2 weeks dissociate decreases, which increases the value of K. o So that the patient will be able to make more antibody  It means that the relationship of Ag-Ab binding is and increases it inversely proportional to the free Ag and free Ab  The more they are attached to each other, the lesser the chance they will separate OBISPO, F. | MED221 1  LESSON 9: Precipitation and Agglutination Reactions MEASUREMENT OF PRECIPITATION BY LIGHT RADIAL IMMUNODIFFUSION SCATTERING  A single-diffusion technique  In this technique, antibody is uniformly distributed in the support gel and antigen is applied to a well cut into the gel  The area of the ring obtained is a measure of antigen concentration that can be compared with a standard curve obtained by using antigens of known concentration  Radial immunodiffusion has been used to measure IgG and IgA subclasses as well as complement components.  There are two methods for RID: END-POINT METHOD (MANCINI)  Antigen is allowed to diffuse to completion; when equivalence is reached, there is no further change in the ring diameter o It needs completion TURBIDIMETRY  Equivalence occurs between 24 and 72 hours  A measure of the turbidity or cloudiness of a solution  The square of the diameter is then directly proportional to  Reduction in light intensity caused by reflection, absorption, the concentration of the antigen. or scatter.  A graph is obtained by plotting concentrations of standards  The more light is blocked the higher the concentration on the x axis versus the diameter squared on the y axis,  The amount of reduction of light is equivalent to the creating a smooth curve to fit the points amount of the concentration  The major drawback to this method is the time it takes to obtain results NEPHELOMETRY  Measures the light that is scattered at a particular angle KINETIC (FAHEY & MCKELVEY) METHOD o Angle is usually 10°-90°  Uses ring diameter readings taken at about 19 hours before  The amount of light scattered is an index of the equivalence is reached solution’s concentration  It is kinetic o While on the process, you are measuring. You do not RATE NEPHELOMETRY wait for it to end  Many automated instruments use a technique called rate  The diameter is then proportional to the log of the nephelometry for the measurement of serum proteins. concentration and a graph is plotted using semi-log  The rate of scattering increase is measured immediately paper after the reagent antibody is added.  This rate change is directly related to antigen concentration OUCHTERLONY DOUBLE DIFFUSION if the concentration of antibody is kept constant  Both antigen and antibody  Quantification of immunoglobulins such as IgG, IgA, IgM, diffuse independently through and IgE, as well as kappa and lambda light chains, is a semisolid medium in two mainly done by rate nephelometry because other dimensions, horizontally and methods are more labor intensive vertically. o It is a double-diffusion. PASSIVE IMMUNODIFFUSION TECHNIQUES  The position of the precipitin  Agarose, a purified high-molecular-weight complex bands between wells allows for polysaccharide derived from seaweed, is used for this the antigens to be compared purpose. with one another. Several  Agarose helps stabilize the diffusion process and allow patterns are possible: visualization of the precipitin bands  Antigen and antibody are added to wells in the gel and antigen–antibody combination occurs by means of diffusion. SEROLOGICAL IDENTITY (CONTINUOUS)  When no electrical current is used to speed up this  Fusion of the lines at their junction to form an arc process, it is known as passive immunodiffusion. represents serological identity or the presence of a  The rate of diffusion is affected by the size of the particles, common epitope the temperature, the gel viscosity, and the amount of  The (1) and (1) have the same antigen the antibody also hydration. have homogenous diffusion which will merge into the center and makes a continuous arc. OBISPO, F. | MED221 2  LESSON 9: Precipitation and Agglutination Reactions NONIDENTITY (CROSSED-LINES)  The first reaction, called sensitization  A pattern of crossed lines demonstrates two separate o Involves antigen–antibody combination through reactions and indicates that the compared antigens share single antigenic determinants on the particle and is no common epitopes. rapid and reversible.  They don’t have non-identical antigens o Sensitization is affected by the nature of the antigens on the agglutinating particles PARTIAL IDENTITY (SPUR) o The class of immunoglobulin is also important; IgM with a potential valence of 10 is over 700 times  Fusion of two lines with a spur indicates partial identity. more efficient in agglutination than is IgG with a In this last case, the two antigens share a common valence of 2 epitope, but some antibody molecules are not captured  The second step is lattice formation by antigen and travel through the initial precipitin line to o The formation of cross-links that form the visible combine with additional epitopes found in the more complex aggregates antigen. Therefore, the spur always point to the simpler o This represents the stabilization of antigen– antigen antibody complexes with the binding together of  Both antigen are the target of the antibody but there are multiple antigenic determinants some antibody that are not really directed to (3) that’s why  IgM antibodies react best at temperatures between 4°C it extends to the spur and 27°C making them the cold antibody. This is because IgM is a naturally occurring antibody against the ABO blood ELECTROPHORETIC TECHNIQUES groups.  Separates molecules according to differences in their  Antibodies belonging to the IgG class agglutinate best at electric charge when they are placed in an electric field. 30°C to 37°C, that’s why they are called warm antibody.  There is an electric current, making it an active dillution o Achieving visible reactions with IgG often requires the use of enhancement techniques IMMUNOELECTROPHORESIS  Enhancement techniques like centrifugation, LISS  A double-diffusion technique that incorporates (Low Ionic Strength Solution), and incubation to electrophoresis to enhance results. 37°C  Typically, the source of antigen is serum, which is o Rh antibodies are mostly IgG electrophoresed to separate out the main proteins. A  IgG can cross the placenta that’s why when the Rh trough is then cut in the gel parallel to the line of of the mother and the baby is different from each separation other the baby will have HDN (Hemolytic disease  Immunodiffusion takes place in a shorter time and of the newborn) results in a higher resolution than when antibody  Coombs reagent is used. diffuses from a trough.  This reagent is an anti- human immunoglobulin that IMMUNOFIXATION ELECTROPHORESIS enhance agglutination  Similar to immunoelectrophoresis except that after reactions electrophoresis takes place, antiserum is applied directly o It attaches to the antibody to the gel’s surface rather than placed in a trough. of human. o There is no through used in this technique  There are sensitized cells (attachment of antibody to PRINCIPLES OF AGGLUTINATION REACTIONS antigen) and then the lattice  Agglutination is the visible aggregation of particles formation occurs caused by combination with specific antibody  Sometimes IgG is so weak that there are no agglutination o They are larger than precipitation but it sensitize that’s why we need enhancement like  Antibodies that produce such reactions are often called coombs reagent to visualize it more agglutinins o IgM is the most efficient agglutinins TYPES OF AGGLUTINATION REACTIONS  In 1896, Gruber and Durham published the first report DIRECT AGGLUTINATION about the ability of antibody to clump cells  Antigens are found naturally  Widal and Sicard developed one of the earliest on a particle diagnostic tests in 1896 for the detection of antibodies  Widal test is useful in occurring in typhoid fever, brucellosis, and tularemia. diagnosing typhoid fever  Agglutination, like precipitation, is a two-step process that o To detect the titer results in the formation of a stable lattice network. (antibody of the patient)  If an agglutination reaction involves RBCs, then it is called hemagglutination (blood typing) o The best example of this occurs in ABO blood group typing of human RBCs o We use the antigens present on the surface of the RBCs to produce agglutination  This type of agglutination reaction is simple to perform, is relatively sensitive, and is easy to read OBISPO, F. | MED221 3  LESSON 9: Precipitation and Agglutination Reactions  Positive reactions can be graded to indicate the  Viruses can agglutinate RBCs strength of the reaction o When virus is present, spontaneous agglutination occurs because the virus particles link the RBCs together  Presence of patient antibody inhibits the agglutination reaction o If antibody is present, it will attach to the viral particles and prevent agglutination, so a lack of or reduction in agglutination indicates the presence of patient antibody  A viral antigen (reagent) is added to the patient antibody so that the antibody attaches to the antigen and then indicator RBCs are added so that the virus will no longer attach to the RBCs since they are now attached to the antigen.  The RBCs will just float and will not result into agglutination  No hemeagglutination means positive result  When reporting in blood typing, it shouldn’t be positive or negative only but with a specific grading  Slide method only uses the positive and negative interpretation while the tube method is graded that’s why the tube method is the confirmatory method. PASSIVE AGGLUTINATION  Passive, or indirect, agglutination employs particles that are coated with antigens not normally found on their surfaces  A variety of particles, including erythrocytes, latex, and gelatin, are used for passive agglutination   Latex agglutination tests have been used to detect rheumatoid factor, antibodies to Group A Streptococcus antigens (ASO Test), and antibodies to viruses NOTE such as rotavirus,  Particles could be microbeads, latex, RBCs, and bacteria cytomegalovirus,  Protein A of S. aureus CO1 strain rubella, and o All IgG absorbs to Protein A except IgG3 varicella-zoster REVERSE PASSIVE AGGLUTINATION  Antibody rather than antigen is attached to a carrier particle  The antibody must still be reactive and is joined in such a manner that the active sites are facing outward. AGGLUTINATION INHIBITION  Based on competition between particulate and soluble antigens for limited antibody combining sites  A lack of agglutination is an indicator of a positive reaction  Put reagent antibody and then it will bind. After that you will add antigen coated particles to inhibit the antigen coated particles inhibiting the formation of lattice because there is too many antigen HEMAGGLUTINATION INHIBITION  Use the same principle, except RBCs are the indicator particles. OBISPO, F. | MED221 4