Precipitation and Agglutination PDF

lOMoARcPSD|46248096 PRECIPITATION REACTION Involves combination of SOLUBLE ANTIGEN with SOLUBLE ANTIBODY to produce insoluble complexes that are visible Affinity Initial force of attraction that exists between a single Fab site on a...

lOMoARcPSD|46248096 PRECIPITATION REACTION Involves combination of SOLUBLE ANTIGEN with SOLUBLE ANTIBODY to produce insoluble complexes that are visible Affinity Initial force of attraction that exists between a single Fab site on an antibody molecule and a single epitope or determinant site of the corresponding antigen 1. Ionic bond 2. Hydrophobic bond 3. Hydrophilic bond 4. Van der Waals force Avidity Sum of all attractive forces between and antigen and an antibody. It represents the overall strength of antigen– antibody binding Law of mass action All antigen–antibody binding is reversible and is governed by the law of mass action. This law states that free reactants are in equilibrium with bound reactants PRECIPITATION CURVE A. Pro-zone – there is an antibody excess, can cause false negative reaction Remedy : perform dilution B. Post-zone- there is an antigen excess, can cause false negative reaction Remedy : repeat the test after a week C. Zone of equivalence- optimum precipitation, in which the number of multivalent sites of antigen and antibody are approximately equal. TYPES OF PRECIPITATION REACTION I.PRECIPITATION INA FLUID MEDIUM 1. Turbidimetry Is a measure of the turbidity or cloudiness of a solution. A detection device is placed in direct line with an incident light, collecting the light after it has passed through the solution. This device measures the reduction in light intensity caused by reflection, absorption, or scatter. The amount of scatter is proportional to the size, shape, and concentration of molecules present in solution. It is recorded in absorbance units, a measure of the ratio of incident light to that of transmitted light. The measurements are made using a spectrophotometer or an automated clinical chemistry analyzer I.K AYTONA Page 51 Downloaded by a ([email protected])  lOMoARcPSD|46248096 2. Nephelometry Measures the light that is scattered at a particular angle from the incident beam as it passes through a suspension. The amount of light scattered is an index of the solution’s concentration. Nephelometers typically measure light scatter at angles ranging from 10 degrees to about 90 degrees. Quantification of immunoglobulins such as IgG, IgA, IgM, and IgE, as well as kappa and lambda light chains, is mainly done by rate nephelometry IgD cannot be measured II.PRECIPITATION BY PASSIVE IMMUNODIFFUSION When no electrical current is used to speed up this process, it is known as passive immunodiffusion. Support medium: Agar, Agarose, and Gel. The rate of diffusion is affected by the size of the particles, the temperature, the gel viscosity, and the amount of hydration 1. Oudin Single Diffusion In single diffusion, antibody was incorporated into agarose in a test tube. The antigen was layered on top, and as the antigen moved down into the gel, precipitation occurred and moved down the tube in proportion to the amount of antigen present 2. Radial Immunodiffusion In this technique, antibody is uniformly distributed in the support gel, and antigen is applied to a well cut into the gel. As the antigen diffuses out from the well, antigen– antibody combination occurs in changing proportions until the zone of equivalence is reached and a stable lattice network is formed in 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 Mancini/ End point Method Fahey and McKelvey/ Kinetic method Antigen is allowed to diffuse to completion and when Measurements taken before the point of equivalence is equivalence is reached, there is no further change in reached. ring diameter Antigen not allowed to diffuse completely. Reading time: IgG – 24 Hours Reading time: 18 Hours IgM – 50 to 72 Hours (2-3 days) d2 = Antigen concentration d = Log of concentration *The square of the diameter is then directly *The diameter is then proportional to the log of the concentration and a graph is plotted using semi-log paper proportional to the concentration of the antigen A graph is obtained by plotting concentrations of The diameter is plotted on the x axis and the standards on the x axis versus the diameter squared on concentration is on the y axis, which automatically gives a the y axis, creating a smooth curve to fit the points log value. I.K AYTONA Page 52 Downloaded by a ([email protected])  lOMoARcPSD|46248096 3. Ouchterlony Double Diffusion A qualitative gel precipitation technique in which both antigen and antibody diffuse out from wells cut in the gel. The pattern obtained indicates whether or not antigens are identical Ouchterlony plates are set up with a central well surrounded by four to six equidistant outer wells. Antibody that is multispecific is placed in the central well and different antigens are placed in the surrounding wells to determine if the antigens share identical epitopes Diffusion takes place radially from the wells. After an incubation period of between 12 and 48 hours in a moist chamber, precipitin lines form where the moving front of antigen meets that of antibody and the point of equivalence is reached. Ouchterlony double diffusion is still used to identify fungal antigens such as Aspergillus, Blastomyces, Coccidioides, and Candida. Serological Identity Partial Identity Non-Identity Formation of smooth curve/arc Formation of spur Cross lined pattern The 2 antigens are identical The antigens share a common The antigens share no identical epitope determinants III.PRECIPITATION BY ELECTROPHORESIS Diffusion can be combined with electrophoresis to speed up or sharpen the results. Electrophoresis separates molecules according to differences in their electric charge when they are placed in an electric field. Direct current is forced through the gel, causing antigen, antibody, or both to migrate. As diffusion takes place, distinct precipitin bands are formed. 1. ROCKET IMMUNOELECTROPHORESIS ✓ One-dimension electroimmunodiffusion, an adaptation of radial immunodiffusion, was developed by Laurell ✓ It is a combination of RID + electrophoresis ✓ Antibody is distributed in the gel, and antigen is placed in wells cut in the gel, just as in RID ✓ The height of the rocket, measured from the well to the apex, is directly in proportion to the amount of antigen in the sample. 2. IMMUNOELECTROPHORESIS ✓ A double-diffusion technique that incorporates electrophoresis current to enhance results. Introduced by Grabar and Williams ✓ Typically, the source of the antigens is patient serum, which is electrophoresed to separate out the main protein fractions; then a trough is cut in the gel parallel to the line of separation. Antiserum is placed in the trough, and the gel is incubated for 18 to 24 hours. ✓ Interpretation: precipitation lines or arcs can be compared in shape, intensity, and location to that of a normal serum control to detect abnormalities. Any abnormal changes in the shape, size, location, and intensity of the arcs or line indicates an abnormal result. I.K AYTONA Page 53 Downloaded by a ([email protected])  lOMoARcPSD|46248096 3. IMMUNOFIXATION ELECTROPHORESIS ✓ Described by Alper and Johnson, is similar to immunoelectrophoresis except that after electrophoresis takes place, antiserum is applied directly to the gel’s surface rather than placed in a trough. ✓ This method is especially useful in demonstrating those antigens present in serum, urine, or spinal fluid in low concentrations ✓ One of the best-known adaptations of this technique is the Western blot Immunofixation electrophoresis. A complex antigen mixture such as serum proteins is separated by electrophoresis. An antiserum template is aligned over the gel. Then protein fixative and monospecific antisera, IgG, IgA, IgM, κ, and λ are applied to the gel. After incubating for 30 minutes, the gel is stained and examined for the presence of paraproteins. Precipitates form where specific antigen–antibody combination has taken place. The example illustration in the right side shows a patient that has an IgG monoclonal antibody with λ chains In western blot A mixture of HIV antigens is placed on a gel and electrophoresed to separate the individual components. The components are then transferred to nitrocellulose paper by means of blotting or laying the nitrocellulose over the gel so that the electrophoresis pattern is preserved. Patient serum is applied to the nitrocellulose and allowed to react. The strip is then washed and stained to detect precipitin bands. It is simpler to visualize the reaction on the nitrocellulose, and in this manner, antibodies to several antigens can be detected. SOURCES OF ERROR IN ELECTROPHORESIS 1.Applying current in the wrong direction 2.Incorrect pH of the buffer 3.Incorrect electrophoresis time 4.Inappropriate concentration of antigen and antibody 5.Amount of current applied CHARACTERISTIC OF DIFFERENT PRECIPITATION METHOD Oudin’s Test Single diffusion, Single Dimension Oakley and Fulthrope (Modified Oudin) Double diffusion, Single Dimension Rocket immune electrophoresis Single diffusion, Single Dimension Radial immunodifusion Single diffusion, Double Dimension Ouchterlony Double diffusion, Double Dimension Countercurrent Immunoelectrophoresis Double diffusion, Single Dimension I.K AYTONA Page 54 Downloaded by a ([email protected])  lOMoARcPSD|46248096 AGGLUTINATION A process by which particulate antigens such as cells aggregate to form larger complexes when a specific antibody is present STEPS OF AGGLUTINATION Step 1 Sensitization phase - The first reaction involves antigen–antibody combination through single antigenic determinants on the particle surface Step 2 Lattice formation - representing the sum of interactions between antibody and multiple antigenic determinants on a particle, is dependent on environmental conditions and the relative concentrations of antigen and antibody. GRADING OF AGGLUTINATION Grade Description Cells Supernate 0 No agglutinates Dark, turbid, homogenous W+ Many tiny agglutinates Dark, turbid Many free cells May not be visible without microscope 1+ Many small agglutinates Turbid (25%) Many free cells 2+ Many medium-sized agglutinates Clear (50%) Moderate number of free cells 3+ Several large agglutinates Clear (75%) Few free cells 4+ One large, solid agglutinate Clear (100%) No free cells TYPES OF AGGLUTINATION REACTION 1. Direct Agglutination Antigens are found naturally on a particle. Example: Kauffmann and white scheme (WIDAL TEST) for salmonella serotyping If an agglutination reaction involves red blood cells, then it is called hemagglutination. Example: ABO blood group typing of human red blood cells, test for Febrile agglutinins such as Widal 2. Passive agglutination / Indirect agglutination Antigens is attached to a carrier particle such as latex, bentonite, charcoal and RBC Agglutination occurs if patient Antibody is present Example: Detection of rheumatoid factor, ASO, antinuclear antibody, and antibodies to Trichinella I.K AYTONA Page 55 Downloaded by a ([email protected])  lOMoARcPSD|46248096 3. Reverse passive Antibody rather than antigen is attached to a carrier particle. Agglutination occurs if patient Antigen is present This type of testing is often used to detect microbial antigens Example: CRP and Haptoglobin detection 4. Coagglutination Uses bacteria as the inert particles to which antibody is attached. Staphylococcus aureus is most frequently used, because it has a protein on its outer surface, called protein A, which naturally adsorbs the fragment crystallizable (FC) portion of antibody (IgG except IgG3) molecules. 5. Agglutination Inhibition Reactions are based on competition between particulate and soluble antigens for limited antibody-combining sites, and a lack of agglutination is an indicator of a positive reaction. Typically, this type of reaction involves haptens that are complexed to proteins; the hapten–protein conjugate is then attached to a carrier particle. Classic example is the human chorionic gonadotropin (hCG) test Hemagglutination inhibition Red blood cells are the indicator particles This type of testing has been used to detect antibodies to certain viruses, such as rubella, mumps, measles, influenza, parainfluenza, HBV, herpesvirus, respiratory syncytial virus, and adenovirus. RBCs have naturally occurring viral receptors. When virus is present, spontaneous agglutination occurs because the virus particles link the RBCs together. Presence of patient antibody inhibits the agglutination reaction 6. Antiglobulin –Mediated Agglutination / Coomb’s test /AHG Detects NON-AGGLUTINATING antibody by means of coupling with a second antibody Direct Antiglobulin Test Indirect Antiglobulin Test In vivo RBC SENSITIZATION In vitro RBC SENSITIZATION Investigation of HDN Crossmatching Investigation of HTR Antibody detection Diagnosis of AIHA Antibody identification Diagnosis of Drug induced hemolytic anemia RBC antigen phenotyping such as Du / Weak D typing TYPES OF AHG PREPARATION a. Polyclonal antibody- prepared by conventional technology (uses Rabbits, Goats, etc. as source of antibody) b. Monoclonal antibody- Prepared by hybridoma technology (uses Mice as source of antibod ) Poly specific AHG Contains anti- IgG and Anti-c3d Monospecific AHG Contains anti- IgG or Anti-c3d I.K AYTONA Page 56 Downloaded by a ([email protected])  lOMoARcPSD|46248096 Specimen: EDTA WHOLE BLOOD DAT Specimen: Serum (red tap) IAT O CHECK CELLS ✓ Group O RH positive RBC, sensitized with IgG (ANTI-D) ✓ It is added to NEGATIVE AHG tests to validate the negative reaction AHG RESULT AFTER ADDITION OF INTERPRETATION REASONS REACTION CHECK CELLS Negative NO agglutination Invalid test (false negative) 1.AHG reagent was neutralized and inactivated 2.Expired AHG reagent 3. AHG reagent was not added/Omitted Negative Agglutination Valid test (true negative) The test was done properly and the is no sensitization of patient’s antigen or RBC QUANTITATIVE AGGLUTINATION REACTIONS 1. Sol Particle Immunoassays (SPIA) 2. Disperse Dye Immunoassays (DIA) 3. Immunoassay by Particle Counting (IMPACT) I.K AYTONA Page 57 Downloaded by a ([email protected])  lOMoARcPSD|46248096 INSTRUMENTATION Particle Counting Immunoassay (PACIA) PACIA involves measuring the number of residual non-agglutinating particles in a specimen. These particles are counted by means of a laser beam in an optical particle counter similar to the one that is designed to count blood cells. PACIA looks at residual nonagglutinating particles by means of nephelometry These particles are counted by means of a laser beam in an optical particle counter similar to the one that is designed to count blood cells For this type of reaction, small latex particles with a diameter of smaller than 1 μm are used As agglutination occurs, clumps of antigens increase in size; these large clumps are not counted. The amount of unknown antigen in a patient specimen is therefore indirectly proportional to the number of unagglutinated particles PACIAs have been used to measure several serum proteins, therapeutic drugs, tumor markers, and certain viral antigens. ERRORS IN AGGLUTINATION False Positive False Negative Overcentrifugation Under centrifugation Contaminated glasswares, slides or reagents Inadequate washing of cells Autoagglutination Reagents not active Saline stored in glass bottle Delay in testing procedure Presence of cross reactivity Prozone/Post zone phenomenon Presence of rheumatoid factor Incorrect incubation temperature Presence of heterophile antibody Insufficient incubation time Delay in reading a slide test Failure to add antiglobulin reagent I.K AYTONA Page 58 Downloaded by a ([email protected])