Agglutination Techniques PDF

Agglutination Definition – the visible clumping of antigen-bearing cells, microorganisms or particles in the presence of specific...

Agglutination Definition – the visible clumping of antigen-bearing cells, microorganisms or particles in the presence of specific antibodies. Or antibodies attached on the carrier can LJLangaman agglutinate in the presence of antigen in specimen. Immunology-Serology Particle/carriers may be RBCs (hemagglutination), bacterial cells (bacterial agglutination) or inert particles such as latex (latex agglutination) or charcoal coated with antigen or antibody. Applications Agglutination Technique of agglutination technique Platform: Detection: Advantages: Slide agglutination Qualitative (positive, negative) Easy to carry out Tube agglutination Semi-quantitative (titer) No complicated equipment needed Microwell Quantitative (concentration) Can be performed as needed (mostly commercially prepared) Available in pre-package kits with controls Reactions are QUALITATIVE, i.e., positive or negative Titers can be performed to give semi-quantitative results Reaction errors can occur Types of agglutination techniques Direct agglutination - When antigens on the surface of carrier are naturally occurring (A antigen on RBC, bacterial antigen on its surface) Passive agglutination - When antigens are passively/artificially attached on the surface of carrier (RBC, latex) Reverse passive agglutination - When antibodies are passively attached to the surface of carrier (latex, beads, RBC)  140 PART II The Theory of Immunologic and Serologic Procedures Amount and avidity of an antigen conjugated to the Human Chorionic Gonadotropin carrier Pregnancy tests are designed to detect minute amounts of Conditions of the test environment (e.g., pH, protein human chorionic gonadotropin (hCG), a glycoprotein hor- concentration) mone secreted by the trophoblast of the developing embryo 140 PART II The Theory of Immunologic and Serologic Procedures Tube Test Agglutination Agglutination tests are easy to perform and, in some cases, that rapidly increases in the urine or serum during the early are the most sensitive tests currently available. It is important stages of Chorionic pregnancy. Gonadotropin Slide Test Agglutination -- Semi Quantitative Method Amount and avidity of an antigen conjugated to the to note that quality results are dependent on the proper train- Human This glycoprotein hormone consists of two noncovalently carrier Pregnancy tests are designed to detect minute amounts of ing of the person performing the assay and adherence to linked subunits, alpha (α) and beta (β). The α unit is identical to -- Qualitative and semi-quantitative Method Conditions of the test environment (e.g., pH, protein strict quality control regulations (e.g., positive and negative concentration) human chorionic gonadotropin (hCG), a glycoprotein hor- that found in luteinizing hormone (LH), follicle-stimulating mone secreted by the trophoblast of the developing embryo control sera). Agglutination-type tests have a wide range of hormone (FSH), and thyroid-stimulating hormone (TSH). Agglutination Tube tests are easy to perform Agglutination Tests: and, in some cases, that rapidly increases in the urine or serum during the early applications in the clinical diagnosis of noninfectious The β subunit has a unique carboxy-terminal region. Using are the most sensitive tests currently available. It is important stages of pregnancy. immune disorders and infectious disease. antibodies made against the β subunit will cut down on Latex agglutination — to noteDetermine that quality results thearepresence dependent on&thetiter properof antibody train- This glycoprotein hormone consists of two noncovalently ing of the person performing the assay and adherence to linked subunits, alpha (α) and beta (β). The α unit is identical to strict LATEX quality control regulations (e.g., positive and negative AGGLUTINATION that found in luteinizing hormone (LH), follicle-stimulating control sera). Agglutination-type tests have a wide range of hormone (FSH), and thyroid-stimulating hormone (TSH). Titer: In latex applications agglutination procedures (Box 10-1), antibody mole- in the clinical diagnosis of noninfectious The β subunit has a unique carboxy-terminal region. Using cules can be bound to the surface of latex beads. Many antibody immune disorders and infectious disease. antibodies made against the β subunit will cut down on is defined molecules can be bound as the reciprocal to each latex particle,of the highest increasing the dilution of serum showing potential number of exposed antigen-binding sites. If an anti- LATEX agglutination. AGGLUTINATION gen is present in a test specimen such as C-reactive protein, the antigen In latex will bind to theprocedures agglutination combining(Box sites of the antibody 10-1), antibodyexposed mole- on the cules cansurface be bound of the latex to the beads,offorming surface latex beads.visible Many cross-linked antibody aggregates of latex beads and antigen (Fig. 10-2). In some pro- A molecules can be bound to each latex particle, increasing the cedures (e.g., pregnancy testing, rubella antibody potential number of exposed antigen-binding sites. If an anti- testing), latex particles gen can be is present in coated with antigen. a test specimen such as In C-reactive the presence of serum protein, the antibodies, antigen willthese bind to particles agglutinate the combining sitesinto large of the visible exposed antibody clumps. on Procedures the surface based of theon latex latex agglutination beads, must be forming visible performed cross-linked under standardized conditions. The amount of aggregates of latex beads and antigen (Fig. 10-2). In some pro- antigen-antibody A binding (e.g., cedures is influenced pregnancybytesting,factorsrubella such as pH, osmolarity, antibody and testing), latex ionic concentration of the solution. A variety particles can be coated with antigen. In the presence of serum of conditions can produce false-positive antibodies, these particlesor false-negative agglutinate into reactions large in agglutination visible clumps. testing (see Table Procedures based10-4). on latex agglutination must be performed Coagglutination under and liposome-enhanced standardized conditions. testing are varia- The amount of antigen-antibody tions of islatex binding agglutination influenced (Fig.such by factors 10-3). as Coagglutination pH, osmolarity, and uses 1/100 1/200 1/400 antibodies ionic bound toof concentration a particle to enhance the solution. the visibility A variety of agglu- of conditions can tination.false-positive produce It is a highly specific method but or false-negative may not reactions in be as sensitive agglutination as latex(see testing agglutination Table 10-4). for detecting small quantities of antigen. Coagglutination and liposome-enhanced testing are varia- tions of latex agglutination (Fig. 10-3). Coagglutination uses B PREGNANCY TESTING antibodies bound to a particle to enhance the visibility of agglu- Figure 10-1 Agglutination patterns. A, Slide agglutination of bacteria The principle tination. It is aofhighly antigen and antibody specific methodinteraction but may not hasbebeen applied as sensitive with known antisera or known bacteria. Left, Positive reaction; right, nega- to latex as pregnancy testing since agglutination the first agglutination for detecting small quantities testsofwere devel- antigen. tive reaction. B, Tube agglutination. Left, Positive reaction; right, negative oped in the 1960s. These assays have replaced animal testing. reaction. (From Barrett JT: Textbook of immunology, ed 5, St Louis, 1988, Mosby.) B PREGNANCY TESTING Table 10-1 Examples of Carriers Figure 10-1 Agglutination patterns. A, Slide agglutination of bacteria The principle of antigen and antibody interaction has been applied with known antiseraResult or known bacteria. Left, Positive reaction; right, nega- Type (Reagent) Type of Assay Principle to pregnancy testing since the first agglutination tests were devel- tive reaction. B, Tube agglutination. Left, Positive reaction; right, negative Passive Agglutination- oped Latexinparticles the 1960s. These assays have replaced C-reactive animal protein (CRP)testing. A suspension of reaction. If JT: (From Barrett polystyrene latex CRP particles of uniform size is present Textbook in the serum, of immunology, an antigen- ed 5, St Louis, 1988, Mosby.) antibody reaction takes place. This reaction causes a change in the uniform appearance Passive Agglutination Table 10-1 Examples of Carriers is coated with the IgG fraction of an antihuman Result of the latex suspension and a clear agglutination results. Type (Reagent) Type of Assay Principle CRP-specific serum. Latex particles C-reactive protein (CRP) A suspension of If CRP is present in the serum, an antigen- Stabilized sheep Rheumatoid factor (RF) RF acts like antibodies If gamma globulin reaction is attached place.to a particular Converting a precipitating test to an agglutinating test. polystyrene latex antibody takes This reaction erythrocytes sensitized against gamma particles globulin of uniform size carrier a(e.g., causes RBCs change in or thelatex particles), uniform the appearance with rabbit gamma that is acts with coated as the antigen. the IgG ofreaction the latexof suspension RF with gammaand aglobulin clear Chemically link soluble antigen to carriers such as LATEX or RBC. globulin suspended in buffer solution fraction of an antihuman CRP-specific serum. becomes a visible agglutination agglutination. results. Presence of antibody in serum specimen will cause the particles Stabilized sheep erythrocytes sensitized Rheumatoid factor (RF) RF acts like antibodies against gamma globulin If gamma globulin is attached to a particular carrier (e.g., RBCs or latex particles), the to agglutinate (i.e. Widal test to detect anti-typhoid antibody in with rabbit gamma that acts as the antigen. reaction of RF with gamma globulin Rheumatoied Factor (IgM) globulin suspended becomes a visible agglutination. patient’s serum) in buffer solution Latex Human IgG (Intact Ab) Reverse Passive Agglutination- Quantitative Method -- ELISA Antibody is attached to the particulate carrier. One of the most commonly used laboratory tests that involves latex particle agglutination is the procedure for C-Reactive Protein (CRP) utilizing the reverse passive agglutination. CRP Latex Latex Anti CRP-Ab Anti CRP-Ab  Flocculation Is categorized as agglutination technique with the use of charcoal as inert particle that allows naked eye observation of reaction instead of using microscope (past technique). This technique is applied in tests for Syphilis which utilizes cardiolipin, lecithin, cholesterol (CLC) that reacts with reagin in patient’s serum. Charcoal produces visible aggregate due to limited space created by the interaction of CLC and reagin. (Tests: VDRL, RPR)