Lecture No. 3 Chapter 10 agglutination methods.pptx

Lecture No. 3 CHAPTER 10 Agglutination Methods By Gheyath K. Nasrallah, PhD, MT(AAB) 1  Learning Objectives Types of precipitation reactions (will be discussed in details in the lab) Describe the principles...

Lecture No. 3 CHAPTER 10 Agglutination Methods By Gheyath K. Nasrallah, PhD, MT(AAB) 1  Learning Objectives Types of precipitation reactions (will be discussed in details in the lab) Describe the principles of agglutination. Identify and compare the characteristics of agglutination methods Explain methods for enhancing agglutination. Describe the characteristics of heamagglutination, complement fixation test. Discuss the principles of pregnancy testing, including sources of error.  Discuss principles and applications of 2  Types of immunoassays (Ag-Ab reaction) a. Agglutination reaction: - Soluble antibody reacts with insoluble antigen or soluble antigen reacts with insoluble antibody. - Reactants are made insoluble by combining with latex particles RBCs, dyes, or Iiposomes. b. Precipitation reaction: a. Soluble antigen and soluble antibody react to form an insoluble product (precipitate), such as b. Double gel diffusion, radial immunodiffusion, immunoelectrophoresis, immunofixation, nephelornetry, and turbidimetry. d. Labeled reaction: A label producing a measurable end product is attached to an antibody or antigen. 1-Fluorochrornes: fluorescent immunoassay 2- Enzymes: ELISA 3-Chemiluminescent molecules 4- Radionuclides: radioimmunoassay 3 Classification of Agglutination Reactions 1- Passive and reverse passive agglutination 2. Direct agglutination 3- Viral hemagglutination and other types 4  Definitions  Coagulation: Precipitation and agglutination: (Figure 10-1). aggregation of antigens and antibodies through the formation of a framework in which antigen particles or molecules interact with antibody molecules  Precipitation is the term for the aggregation of soluble test antigens and Abs. 5 Type of Agglutination: Passive agglutination 1) Passive agglutination: insoluble particle bound to Ag Any Antigen Viral, CRP, hormone, Drug L: Latex, colloidal, charcoals, gelatin, resin beads, and RBCs(insoluble particles) 6  Type of agglutination 2) Reverse passive agglutination: insoluble particle can be coated (conjugated or sensitized) with Ab Could be 1- Colloidal Latex particle Charcoals can be coated latex, gelatin, resin with Ab beads, and RBCs Conjugated Visible aggregate or sensitized latex 7  Reverse passive agglutination How about AB blood grouping? Passive or Reverse agglutination? 8  Type of agglutination Co-agglutination and liposome-enhanced testing: Are variations of latex agglutination uses Ags bound to a particle [such as Liposome (phospholipid bilayer)] Liposome: enhances the visibility of agglutination. It is a highly sensitive but no very specific Why? Liposomes are lipids that fuse to each other which could give us the appearance of agglutination hence giving us false – or false + results Liposomes are going to give a false positive result because of nonspecific aggregation Nonparticles always aggreate but we can use buffers that prevent this aggregation Aggregation=false positive results 9 Box 10-1 Immunologic Assays Performed by Latex Particle Agglutination C-reactive protein Immunoglobulin G rheumatoid factors Immunoglobulin M rheumatoid factors Rubella antibody –uses ELISA now adays 10 Human Chorionic Gonadotropin (HCG)  A glycoprotein hormone secreted by the trophoblast of the developing embryo  Peak levels are reached approximately 2 to 3 months after the last menstrual period (LMP).  Two subuntis: α and β  α-subunit amino acid sequence unique carboxy-terminal reg present in other hormones  β- is unique for HCG  Rapidly increases in the urine or serum during early stages of pregnancy  Mab to β- subunit increase specificity  We use monoclonal antibodies in 11 Trophoblast http://ons/thumb/7/72/Blastocyst_Engl ish.svg/608px-Blastocyst_English.svg. png 12  Pregnancy Testing Protocols  Principle latex particles coated with anti-hCG antibodies for detection of HCG in urine  Specimen:  The first morning urine specimen is required because it contains the highest concentration of hormone.  It should have a specific gravity of at least 1.018? Why?  It may be refrigerated for up to 2 days or frozen at −20° C for at least 1 year.  If turbidity or precipitation is present after thawing, filtering or centrifuging is 13 Positive results Technical Sources of Error  Reagents should never be expired  Latex reagent must be well shaken. Why?  Agglutination should be read within 3 minutes to avoid erroneous results caused by evaporation. 14 False-Negative Results  Testing before reaching detectable levels of hCG If its low in urine we take the sample from serum False-Positive Results  Kit uses MAb against α unit or polyclonal AB  A patient has been given an hCG injection to trigger ovulation- in ovulation  Disease: Chorioepithelioma, hydatidiform mole (mass in the uterus)  Excessive ingestion or of aspirin- 15 The quality of test results depends on the following factors: Time of incubation with the Ab source (i.e., patient serum). Amount and avidity of an Ab conjugated to the carrier. (latex coated with IgG or IgM) Concentration of Ag or Ab Conditions of the test environment (e.g., pH, osmolality, and ionic concentration of the solution) 16  Types of agglutinations 3) Direct agglutination: This method uses antigens naturally occurring on a particle to demonstrate agglutination (e.g., RBCs in type and crossmarch) 17  Types of agglutinations 4) indirect agglutination: 18  Types of agglutinations 4) Viral hemagglutination: This is a naturally occurring process in which a virus (e.g., influenza virus) will agglutinate RBCs by binding to surface receptors. Sunken rbcs Negati Positiv ve e 19  Passive (Direct) Hemagglutination (PHA) except: RBCs  Same as latex agglutination (instead of latex particle ) are conjugated with Ag or AB.  Ag- cross linking with RBCs: chemicals such as tanic acid, chromic chloride, and gulteraldehyde.  performed in tube or multi-well plate  Negative results: red blood cells precipitate  positive results: red blood cell diffused  Secondary Ab [Anti human glubulin (AHG)] can be used to enhance agglutination hCG-sensitize 20 RBCs 21  HA inhibition (indirect HA) Incuba te + Anti- HBsAg Anti-HsBAg- HBsAg from the sensitized RBC from kit patient sera This is could be any Ag, like +ve HBeAg, HEV, results Rubella 22  HA inhibition (indirect HA) + HBsA g Free-+ patien Incuba te t sera HBsAg Anti-HsBAg- from sensitized RBC the kit from the kit -ve results 23  HA inhibition (negative results) Influenzae virus Neutralization assay ? 24 https://www.google.com/search? rlz=1C1CHBD_enQA717QA717&biw=1680&bih=913&tbm=isch&sa=1&ei=RXOWW7 7YE8PmvgTpxYCwAg&q=membrane+attack+complex+RBC&oq=membrane+attack +complex+RBC&gs_l=img.3...21567.23993.0.25042.0.0.0.0.0.0.0.0..0.0....0...1c.1.64.img..0.0.0....0.T9ajwopLHtc#imgrc=KKAMvLzIkSwI-M: 25 Complement Fixation test 26  FLOCCULATION TEST (RPR) Will be discussed in the Lab 27  Types of precipitation reactions: covered in the lab 1- Fluid-phase precipitation: a. Turbidimetry b. Nephelometry 2- Precipitation reactions in agar gel: o Double immunodiffusion (Ouchterlony technique) o Countercurrent immunoelectrophoresis (CIE) o Immunofixation electrophoresis o Rocket immunoelectrophoresis 28  Mechanisms of Agglutination Agglutination is influenced by a number of factors and is believed to occur in two stages: 1.Sensitization  Ag-Ab a chemical reaction  Unfavorable conditions leads to elusion of Ab from the Ag. 2. Lattice formation: Agglutination 29  Sensitization Factors that influence Ag-Ab association, include: A.Particle charge (zeta potential) B.Antibody type C.Ag-Ab ratio D.Antigenic determinants E.pH F.Temperature 30  Sensitization Factors that influence Ag-Ab association, include: A. Particle Charge  zeta potential: diff between outside and inside of the particle charge  Zeta potential:net negative surface charge of Inert particles such as latex, RBCs, and bacteria  Zeta potential can be affected by salt concentration because Na+ and Cl− ions in a solution have a shielding effect  Reduce ionic strength can 31 enhance Ag-Ab reactions  Table 10-4 Techniques to Reduce Zeta Potential To bring the 2 molecules together Low ionic strength saline (LIS) or albumin : reduce ionic strength of the environment 32  Sensitization B. Antibody Type  IgM are more efficient at agglutination than IgG?  AHG can increase False IgG negative False concentration positive Ag on phenomen postzone aggregation phenomenon equivalence prozone zone of s C. Ag-Ab ratio - 33 34  Sensitization D. Antigenic Determinants 1.No. of Ag determinant : A blood group antigen has more than 1.5 million sites/RBC Kell blood group antigen has about 3500 to 6000 sites/RBC If we are looking for 2. Placement of Ag determinant: a scarce ag Steric hindrance: Ab uptake by cell surface we add more ab. antigens However , If two PS:Antigenic determent putting too much close ag or ab is too muchto each other: Abs if the ab because it cou l d cause antigenic amount are will compete hindrance and can not bind to any sufficient then we35 add less ab  Sensitization E- pH.  Optimum pH of 6.5 to 7.5.  At a neutral pH, high electrolyte concentrations act to neutralize net negative charge of particles. F. Temperature: differs for different antibodies.  IgM are cold reacting 4°-22° C,  IgG are warm reacting at 37° C. 36  Lattice Formation  cross-links between sensitized particles (e.g., erythrocytes) and Abs resulting in aggregation  is a much slower process than the sensitization phase  Cross-linking is influenced by factors such as the zeta potential. Methods of Enhancing Agglutination Centrifugation Treatment with proteolytic enzymes Use of colloids Antihuman globulin (AHG) 37 Graded Agglutination Reactions Pseudoagglutinat ion, or false appearance of clumping, may rarely. It is where particles will aggregate with each other. 38  Microplate Agglutination Reactions  Large number of tests on a single plate, which eliminates time-consuming steps such as labeling test tubes.  The U-shaped used most often in immunohematology  The microplate is centrifuged for an immediate reading. 39  Summary Immunoassays Assays involving antibody-antigen reactions are called immunoassays. a. Precipitation reaction: Soluble antigen and soluble antibody react to form an insoluble product (precipitate), such as double gel diffusion, radial immunodiffusion, immunoelectrophoresis, immunofixation, nephelornetry, and turbidimetry. b. Agglutination reaction: Soluble antibody reacts with insoluble antigen or soluble antigen reacts with insoluble antibody. Reactants are made insoluble by combining with latex particles, RBCs, dyes, or Iiposornes. c. Labeled reaction: A label producing a measurable end product is attached to an antibody or antigen. Labels include fluorochrornes, enzymes, chemiluminescent molecules, and radionuclides. Titration: Testing serial dilutions of patient sera provides semiquantitative results (titer). A fourfold rise in titer between 40 an acute and convalescent sample is considered clinically  AGGLUTINATION REACTIONS General Information I. Definition: Agglutination occurs when particles in suspension clump together due to antibody-antigen interaction. 2. IgM and IgG antibodies participate in agglutination reactions. Because IgM has more antigen binding sites, it agglutinates more quickly. Comparison of agglutination and precipitation a. Agglutination uses an antigen or antibody attached to a particle (insoluble), whereas precipitation uses soluble antigens and antibodies. b. Agglutination and precipitation reactions use antigens with at least two antigenic determinants (epitopes). c. In agglutination and precipitation reactions, antigen excess can result in a postzone reaction, whereas antibody excess can result in a prozone reaction. d. Agglutination reactions take minutes to hours, whereas precipitation reactions may take hours to days. e. Methods that utilize agglutination reactions are qualitative or semiquantitative, whereas precipitation methods give qualitative, semiquantitative, or quantitative results. 41 Classification of Agglutination Reactions 1.Direct agglutination: This method uses antigens naturally occurring on a particle to demonstrate agglutination (e.g., RBCs in type and crossmarch). 2. Viral hemagglutination: This is a naturally occurring process in which a virus (e.g., influenza virus) will agglutinate RBCs by binding to surface receptors. 3. Passive and reverse passive agglutination a. Passive agglutination: A technique in which soluble antigen is attached to a particle, producing agglutination with a specific soluble antibody b. Reverse passive agglutination: A technique in which an antibody is attached to a particle, producing agglutination with a specific soluble antigen c. Particles used include latex, gelatin, resin beads, and RBCs. 42  Complement Fixation test A. Principle 1. Complement fixation (CF) assays are sometimes used to detect antibody in patient sera. The serum is mixed with a specific known antigen. If antibody to the antigen is present, an immune complex forms. Complement is added, and if an immune complex is present, it will bind the complement. 2. After the addition of sensitized RBCs, hemolysis is a negative reaction (complement is not available), and no hemolysis is a positive reaction, meaning antibody was present in the patient sample. J. When the antibody and antigen combine in this technique, the complement present in the system also combines with the antigen-antibody complexes and no free complement is available to cause lysis of the sensitized indicator RBCs. 4. If antibody is absent, then complement is free to attach to the sensitized indicator RBCs and causes lysis. B. Applications I. CF can be used to detect antibodies to viruses, Rickettsia. and43