IMMUNOLOGY & SEROLOGY LABORATORY PRECIPITATION & AGGLUTINATION OUTLINE • Introduction • Classifications of Antigen/Antibody Reactions • Precipitation • Agglutination PRECIPITATION & AGGLUTINATION • Precipitation and agglutination are the visible expression of the aggregation of antigens and antibodies through the formation of a framework in which antigen particles or molecules alternate with antibody molecules. • Precipitation is the term for the aggregation of soluble test antigens. It is the combination of soluble antigen with soluble antibody to produce a visible insoluble complex. • Agglutination is the process whereby specific antigens (e.g., red blood cells) aggregate to form larger visible clumps when the corresponding specific antibody is present in the serum. Artificial carrier particles may be needed to indicate visibly that an antigen-antibody reaction has taken place; examples include latex particles and colloidal charcoal. Cells unrelated to the antigen, such as erythrocytes coated with antigen in a constant amount, can be used as biological carriers. Whole bactecrial cells can contain an antigen that will bind with antibodies produced in response to that antigen when it is introduced into the host INTRODUCTION • Sensitization - basic reaction of an antigen and an antibody • Factors that affect antigen/antibody reactions: o Concentration of the reactants ▪ In order for a visible reaction to occur, antigen and antibody should exist in equal proportion (not amount) o Temperature ▪ Optimal temperature 40-45degC (60 above = Proteins start to denature) o Length of incubation ▪ Length of incubation = agglutination/mins, precipitation/hours to days. o pH of the test system: Optimal pH = Neutral (7.0) CLASSIFICATIONS OF ANTIGEN/ANTIBODY REACTIONS • Primary phenomenon (Sensitization) – Initial binding of a single antibody to a single antigen into an antigenic site o Tests that detect this type of reaction are technically difficult, complex, and expensive. (RIA, IFA, EIA) • Secondary phenomenon ( Lattice formation) - Basic antigen/antibody reactions are taken a further step, forming cross links or lattice formation to create large molecules that are easily detectable. o Tests that detect this type of reaction are quick and easy to perform but less sensitive and specific. (Agglutination, Precipitation) • Factors that affect primary phenomenon o Primary union of antigen and antibody are depending on two characteristics, affinity and avidity. o Affinity – The initial force of attraction that an antibody for a specific antigenic epitope or determinant o Avidity – The sum of all attractive forces between an antigen and an antibody. PRECIPITATION • Involves combination of soluble antibody with soluble antigen to produce insoluble complexes. • Precipitation curve o Prozone – Excess antibodies (So much antibody is present that instead of forming cross links, the antibodies just coats the antigen) o Postzone – Excess antigen (So much antigen is present that instead of forming cross links, the antigens just occupy all paratopes) o Zone of equivalence – Zone where antigen and antibody are present in optimal proportions. • Methods in Measuring Precipitation o Optical methods ▪ Turbidimetry ▪ Nephelometry o Immunodiffusion ▪ Single diffusion, single dimension ▪ Single diffusion, double dimension ▪ Double diffusion, single dimension ▪ Double diffusion, double dimension o Electrophoresis OPTICAL METHODS • Methods based on light scattering. • Turbidimetry - Measures the turbidity or cloudiness of a solution by detection of light passing thru the sample. o Soluble ag + antibody = Insoluble complexes (More complexes, more turbid/less light will pass through) • Nephelometry - Measures the turbidity or cloudiness of a solution by detection of light refracted by the sample o Soluble ag + antibody = Insoluble complexes (More complexes, more light will be refracted) (More sensitive)
IMMUNODIFFUSION • Precipitation techniques that use gel instead of a liquid as the medium. • Affected by: o Size of particles ▪ Size = The bigger the particle, the slower it will move. ▪ The smaller the particle, the faster it will move o Temperature ▪ Temperature = Optimal 40-45C ▪ Protein - 60C o Gel viscosity and hydration ▪ Gel = Agar • Single diffusion Single dimension o Oudin method o Antibody is added to agarose gel and placed in a tube, antigen is layered on top of gel and will diffuse down into gel. o If the antibody present reacts with the added antigen a precipitin band will form in the gel. o Most simple immunodiffusion test o Ex. Quantification of antigens (Factor VIII antigen) o Incubation time – 24-48hrs • Single diffusion, double dimension o Fahey and Mancini o Antibody is added to the gel and poured into a plate, wells are cut into the plate. Antigen then is added to the well and will diffuse out radially from the well. o If the antibody present is specific for the antigen added a ring of precipitate will form, the size of the ring is directly proportional to the concentration of the antigen o Radial immunodiffusion o Fahey/Kinetic – Multiple measurements are performed to monitor the activity of the whole test. ▪ Fahey 18hrs incubation o Mancini/Enpoint – Only one measurement is performed (At the end of the test, hence endpoint). ▪ Mancini 48-72hrs incubation ▪ Can also be used for quantitation of antigens. ▪ Example: Salmonella typhi test (Widal) o Sources of errors: ▪ Overfilling and underfilling the well. ▪ Spilling sample outside of the well. ▪ Nicking the well. ▪ Improper incubation time or temperature. • Double diffusion, single dimension o Oakley and Fulthrope o Antibody is incorporated in a gel, above which a column of plain agar. The antigen is then layered on top of the agar medium. o If the antibody present reacts with the added antigen a precipitin band will form in the gel. o Counter immunoelectrophoresis • Double diffusion, double dimension o Ouchterlony o Holes are cut in the agar. One central hole will be made to be surrounded by other wells. Antibody is then added to the central well, antigens are added to the outer wells, the position of the bands formed between the antigens allows for comparison of the antigens to each other. o Results : ▪ Identity – if the bands form an arc. • Fusion of lines at their junction to form an arc (presence of common epitopes) ▪ Partial identity – if the bands fuse but there is a presence of spur. • Heterophile antigens • Demosntrate2 separate reactions (antigen shared no common epitopes) ▪ Non-identity – formation of pattern of lines which cross each other. • Fusion of 2 lines partially (precipitin lines do not form a complete cross) o Used for comparing antigens ELECTROPHORETIC TECHNIQUES • Immunodiffusion combined withelectrical current. • Electrophoresis – Technique which separates molecules according to differences in their electrical charge when they are exposed to an electric current. • A direct current is applied to the gel, the antigen and antibody migrate through the gel, as diffusion takes place precipitin bands are formed. • To speed things up (Active) • Anode attracts anions, Cathode attracts cations • Antibody is positively charged, Antigen is negatively charged. • Techniques: o Rocket electroimmunodiffusion o Immunoelectrophoresis o Countercurrent immunoelectrophoresis o Immunofixation • Rocket immunoelectrophoresis o Laurell technique o One-dimensional electroimmunodiffusion, it is an adaptation of radial immunodiffusion (RID). o A quantitative method for measuring serum proteins. o Height of the rocket is proportional to the concentration of antigen. o Serum proteins – Free hemoglobin, immunoglobulins o Restricted to detection of antigens that move to the positive pole during electrophoresis. o Advantage – Results can be obtained in a few hours.
• Countercurrent immunoelectrophoresis o Double diffusion technique w/ addition of electrophoresis to speed up formation of a visible reaction. o Wells are made on each side of the gel and subjected to electrical field. o Antigens will migrate toward the anode while antibodies, toward the cathode. If the antigen and antibody is specific with each other, a precipitin line will form. • Immunoelectrophoresis (IEP) o A two-step double-diffusion technique which first involves the electrophoretic separation of proteins. o Enhances the semiquantitation of antigens. o Excellent screening test to differentiate serum proteins and detect abnormalities such as in myelomas, Waldenstrom’s macroglobulinemia, malignant lymphomas and other lymphoproliferative disorders. • Immunofixation electrophoresis (IFE) o Similar to IEP except that after electrophoresis is performed the antiserum is applied directly to the surface of the gel. o The most sensitive method used to detect, confirm and characterize monoclonal gammopathies such as those found in plasma cell dyscrasias such as multiple myeloma Waldenstrom’s macroglobulinemia, monoclonal gammopathy of unknown significance, light or heavy chain disease. o The best adaptation of this test is Western Blot test. o More sensitive AGGLUTINATION • The process by which particulate antigen such as cells are aggregated to form large, visible aggregates if the specific antibody is present. • Advantages : easy to carry out, no complicated equipment and can be performed as needed. • Disadvantages : Most reactions are qualitative. (Some can be semiquantitative using titers) • Titer – A way of expressing concentration. (Can be made using serial dilution) • Types of agglutinationr reaction: o Direct agglutination o Passive agglutination o Reverse passive agglutination o Agglutination inhibition o Coagglutination • Direct agglutination o Agglutination of an antigen found naturally on a particle. o Example: ABO blood typing, detection of bacteria present in the serum (Salmonella, Rickettsia) • Indirect or Passive agglutination o The agglutination of inert particles by antibody directed against antigen bound to their surface. o Uses carrier particles. o Passive agglutination routinely used for detecting antibodies to viruses such as cytomegalovirus (CMV), rubella and varicella-zoster. o Antigen + carrier o Latex particle, sheeps RBC, charcoal. • Reverse passive agglutination o Antibody is attached to carrier particle instead of the antigen. o Often used for detection of microbial antigens such as: Group A and B Streptococcus, Staphylococcus aureus, Neisseria Meningitidis, Haemophilus influenzae, Cryptococcus neoformans, Mycoplasma pneumoniae and Candida albicans. o Some of these organisms very difficult to grow and/or quick diagnosis needed so correct treatment can be started. • Agglutination Inhibition o Based on competition between particulate and soluble antigens for limited antibody combining sites. o Reactive samples will not produce a visible result. o Two reagents: Antibody and antigen-carrier complex o • Coagglutination o A type of agglutination wherein bacteria is used as a carrier. o Used to identify Streptococci, Neisseria meningitidis, Neisseria gonorrhea and Haemophilus influenzae. o Staph aureus is the most frequently used carrier. • Hemagglutination o Type of agglutination wherein RBC is used as carrier. o Used to identify viruses (Rubella) and certain bacterial infection. • Summary Active Passive • ABO Blood Typing • Heterophile antibodies • Weil-Felix (Rickettsia) • Mycoplasma pneumoniae (Cold agglutinins) • Phadebact (Gonococci) • Streptococcus MG (milleri) • Syphilis • Rubella • Rheumatoid arthritis • Hepatitis B & C • Human Chorionic gonadotrophin • C reactive protein • Drugs of abuse •