AGGLUTINATION REACTIONS Antibody must be able to bridge the gap
between cells in such a way that one molecule
Agglutination is the visible aggregation of particles can bind to a site on each of two different cells caused by combination with specific antibody. Enhancement of Lattice Formation Reaction takes place on the surface of the LISS decreasing the buffers ionic strength particle, antigen must be exposed and able Albumin 5 to 30% helps to neutralize the to bind with antibody surface charge and allows red cells to A two-step process, involving sensitization approach each other more closely or initial binding followed by lattice formation, or formation of large aggregates. Increase viscosity using enzymes, agitating RBCs, Bacterial cells, inert carriers (latex centrifuging, altering temperature or the Ph particles) multiple antigenic or PEG and Dextran reduce the water determinant sites hydration around cells and allow them to come into closer proximity for antibody to Gruber and Durham (1896) first to report the join together ability of antibody to clump cells, based on observations of agglutination of bacterial cells Bromelin, Papain, Trypsin and Ficin by serum. reduces the surface charge on the RBCs through cleaving of chemical groups and Widal and Sicard detection of antibodies decreasing hydration. occurring in typhoid fever, brucellosis and Ficin cleaves sialoglycoproteins from the tularemia. RBCs surface and may change the external configuration of the membrane to reveal Sensitization - antigenantibody combination more antigenic determinant sites. through single antigenic determinants on the particle surface and is followed by the law of IgGs reacts best at 30 to 370C, IgM reacts mass action and is rapid and reversible. best between 4 to 270C pH optimal 6.5 7.5 The affinity and avidity of an individual antibody determine how much antibody remains attached. TYPES OF AGGLUTINATION REACTIONS: The class of immunoglobulin (IgM is more 1. DIRECT AGGLUTINATION efficient than IgG) 2. PASSIVE AGGLUTINATION If epitopes are sparse or if they are 3. REVERSE PASSIVE AGGLUTINATION obscured by other surface molecules, they 4. AGGLUTINATION INHIBITION are less likely to interact with antibody 5. COAGGLUTINATION Lattice Formation - sum of interactions between Direct Agglutination - occurs when antigens are antibody and multiple antigenic determinants found naturally on a particle. on a particle, is dependent on environmental conditions and the relative concentrations of Patient serum is diluted into a series of tubes antigen and antibody or wells on a slide and reacted with bacterial antigens specific for the suspected disease. Bordet - governed by physicochemical factors such Used in diagnosis of diseases for which the as the milieus ionic strength, pH, and bacterial agents are extremely difficult to temperature cultivate. Fourfold increase in antibody titer over time Haptens that are complexed to proteins; the when paired dilutions of serum samples are haptenprotein conjugate is then attached tested with any of these antigens to a carrier particle. The patient sample is first reacted with a Hemagglutination - agglutination reaction limited amount of reagent antibody that is involves red blood cells specific for the hapten being tested. Examples: ABO typing Indicator particles that contain the same hapten one wishes to measure in the patient Passive/Indirect Agglutination - employs particles are then added. that are coated with antigens not normally found If the patient sample has no free hapten, the on their surfaces. reagent antibody is able to combine with the Carrier particles: carrier particles and produce a visible RBCs possibility of cross-reactivity agglutination. agglutination is a negative (heterophile antibody) Latex inexepensive, relatively reaction, indicating that the patient did not stable, not subject to cross-reactivity have sufficient hapten to inhibit the Gelatin secondary reaction Silicates Either antigen or antibody can be attached Use to detect: to the particles. Rheumatoid Factor The sensitivity of the reaction is governed by Antinuclear antibody the avidity of the antibody itself. It can be a ASO Abs to Trichinella spiralis highly sensitive assay capable of detecting Abs to Treponema pallidum small quantities of antigen. Abs to CMV, Rubella, Varicella-zoster, and HIV-1/2 Hemagglutination Inhibition reactions are the same principle, except RBCs are the There is always a risk of non-specific indicator particles. agglutination caused by the presence of other IgM antibodies Coagglutination - systems using bacteria as the Reverse Passive Agglutination - antibody rather inert particles to which antibody is attached. than antigen is attached to a carrier particle. The antibody must still be reactive and is Staphylococcus aureus is most frequently joined in such a manner that the active sites used, because it has a protein on its outer are facing outward. surface, called protein A, which naturally adsorbs the fragment crystallizable (FC) Adsorption may be spontaneous, or it may portion of antibody molecules. require some of the same manipulation as is used for antigen attachment. These particles exhibit greater stability than latex particles and are more refractory to Agglutination Inhibition - reactions are based on changes in ionic strength. competition between particulate and soluble antigens for limited antibody-combining sites, and a lack of agglutination is an indicator of a positive reaction. Antiglobulin-Mediated Agglutination
1. DIRECT ANTI-GLOBULIN TEST
2. INDIRECT ANTI-GLOBULIN TEST
AHG (Coombs Test) - technique that detects non-
agglutinating antibody by means of coupling with a second antibody. Antibody will react with the FC portion of the human antibody attached to red blood cells. Agglutination Takes place because the antihuman globulin is able to bridge the distance between cells that IgG alone cannot do.
DIRECT ANTI-GLOBULIN TEST (DAT)
- Used to demonstrate in-vivo attachment of antibody or complement to an individuals red blood cells.
Serves as an indicator of autoimmune
haemolytic anemia, HDN, Sensitization of RBCs caused by the presence of drugs, or a transfusion reaction. RBCs are washed to remove any antibody that is not specifically attached and then cells are tested directly with antibody to IgG or complement A positive test indicates that an immune reaction is taking place in that individual.
INDIRECT ANTI-GLOBULIN TEST (IAT)
- Used to determine the presence of a particular antibody in a patient or it can be used to type patient red blood cells for specific blood group antigens.
All reactions are run at 37C to detect
clinically significant antibodies. Used to check for the presence of clinically significant alloantibodies in patient serum.