Serology Lab BDS PDF 19.11.24 - Dr. Hafiz Ahmad

SEROLOGY Dr. Hafiz Ahmad DMB 212 (Dental Micro Lab) 19.11.2024  Highly specific. The epitope makes contact with the paratope and the molecules are held together in lock and key fashion. Only surface antigens are involved. Combination is firm and reversible. Primar...

SEROLOGY Dr. Hafiz Ahmad DMB 212 (Dental Micro Lab) 19.11.2024  Highly specific. The epitope makes contact with the paratope and the molecules are held together in lock and key fashion. Only surface antigens are involved. Combination is firm and reversible. Primary reaction: Initial rapid interaction with no visible effects (electrostatic force, hydrogen bonding, Vander Waal’s force). Secondary stage: In most instances the primary reaction is followed by visible demonstrable events like precipitation, agglutination ,etc. Tertiary stage (in vivo only): Destruction of injurious Ags (humoral immunity) Tissue damage (allergy / immune diseases)  Ag-Ab Measurement ❑ Antigen-Antibody reactions in-vitro are known as serological reactions ❑ Unit of measurement : Titre ❑ Definition of titre : The antibody/antigen titre is the highest dilution of the serum which gives an observable reaction with the antigen/antibody in the particular test (amount of antibody present in blood) ❑ Two important parameters of serological tests : 1.Sensitivity : Ability of the test to detect even very minute quantities of antigen or antibody Ability to detect (True positives) Minimal/Absent false negative results 2.Specificity : Ability of the test to detect (True Negatives) and with Minimum/no false positive results DIRECT Vs INDIRECT SEROLOGIC TESTS Direct serologic testing utilizes a known antiserum in order to detect an unknown antigen, either foreign or self. qualitative and quick, screening purposes  Indirect serologic testing Indirect tests are generally more specific, resulting in fewer false-positives. Indirect serologic testing utilizes antibodies from the patient that may be specific for either self or foreign antigen Qualitative or Quantitative - Titre ZONE PHENOMENON ZONE OF EQUIVALENCE POSTZONE PROZONE  Zone Phenomenon Amount of precipitate/agglutinate formed is influenced by the relative proportions of antigens and antibodies To the same amount of antiserum in different tubes increasing quantities of antigens are added (In vitro) Preceding tubes :Precipitation/Agglutination is weak or absent due to antibody excess PROZONE Clinical Importance of Prozone : False negative results in sera rich in antibody ,unless several dilutions are made. Middle tubes: Abundant precipitation /agglutination due to optimal or equivalent proportions of antigen and antibody ZONE OF EQUIVALENCE ❑ Later tubes : Weak or absent precipitation/agglutination due to antigen excess POST ZONE /ZONE OF ANTIGEN EXCESS  Antigen-antibody (serological) reactions Precipitation reactions - Flocculation reactions Agglutination reactions Immunoassays- ELISA, CLIA Immunochromatographic (ICT) assays Immunofluorescence Complement fixation test (CFT) When a soluble Antigen reacts with its soluble Antibody in presence of an electrolyte (NaCl) at an optimal pH(7.4) and temperature (37),the Ag-Ab complex forms an insoluble precipitate.  Precipitation reactions Eg: Flocculation- when instead of sedimenting, the precipitate remains suspended as floccules VDRL test for syphilis (a slide flocculation test) It is an Antigen Antibody reaction in insoluble Antigen with soluble antibody in presence of an electrolyte optimal pH and temperature resulting in visible clumping (aggregation) of the particles. Particles like bacteria, erythrocytes and synthetic latex particles coated with Ag to form particulate antigens. Agglutination reaction with IgM is superior to IgG because IgM has more ag binding sites.  Agglutination reactions e.g. Widal test [for Enteric (typhoid/paratyphoid) fever] - a tube agglutination test Heterophile agglutination tests: - Weil-Felix test [for Rickettsial diseases] - Paul-Bunnell test [for Infectious mononucleosis by EBV] - Cold agglutination test [for Mycoplasma pneumoniae]  BLOOD GROUPING A patient's red blood cells can be mixed with antibody to a blood group antigen to determine a person's blood type. Antigens present on RBCs  ACTIVE AGGLUTINATION Blood grouping and cross matching Rh incompatibility  Coombs Test The direct Coombs is designed to identify maternal anti-Rh antibodies that are already bound to infant RBCs or antibodies bound to RBCs in patients with autoimmune hemolytic anemia The indirect Coombs test is designed to identify Rh- negative mothers who are producing anti-Rh antibodies of the IgG isotype, which may be transferred across the placenta harming Rh-positive fetuses. Principle: CFT is used to detect and quantify antibody in serum that does not form visible precipitate or agglutinate when reacted with antigen until complement is used. Complement can only bind Ag-Ab complexes. When complement takes part in antigen-antibody reactions it is bound or fixed to the Ag-Ab complexes. When these complexes are on bacteria, RBCs or other cells, the complement brings about the lysis of these cells. Complement cannot bind free antibody. Antigen-antibody complex fixes the complement. But the fixation of complement with Ag-Ab complex do not have any visible effect like agglutination and precipitation. So, it is necessary to use indicators system. The indicator system consists of sheep RBC coated with anti-sheep RBC antibody (Amboceptor). Result interpretation: Positive CFT: if no hemolysis is observed, it indicates positive complement fixation test, Antigen- antibody reaction and complement fixation occurred, so NO free complement is available to lyse the RBC. Negative CFT: If hemolysis of RBC observed, it indicates negative complement test. No antibody present in the serum, NO complement fixation occurred, so the complement remains free.  Complement fixation test (CFT) Indicator system: RBC sensitized with hemolysin  Immunochromatographic test (ICT) rapid tests Point-of-care (POC) testing Blood/Serum flows laterally via capillary action and shows band in area of the nitrocellulose strip where Ag/Ab is attached T-Test C-Control ELISA - Enzyme Linked Immunosorbent Assay Advantages: many samples at same time Colored products are easily detected Eg: Hepatitis testing, Swine flu testing  ELISA steps ELISA can be used to detect and quantitate either antigen or antibody. It is carried out in ELISA plates Coating: The first step is to add either antigen or antibody to the wells of the plate—to cover (coat) the surface of wells. Steps in Direct ELISA: (Ab:: Enzyme represents—Antibody and Enzyme linked together) 1: Coat the plate with a serially diluted (usually 2-fold) antigen sample (Ag) 2: Add antibody to which an enzyme is attached (Ab:: Enzyme). Ag::Ab:: Enzyme complex is formed. 3. Add substrate. Enzyme will act on the substrate and color will be produced. Sequence in direct ELISA—Ag + Antibody:: Enzyme + Substrate = Color Sequence in Indirect ELISA--- Ag + Antibody + Anti-Antibody:: Enzyme + Substrate = Color Sequence in Sandwich ELISA—Antibody + Ag + Antibody + Anti-antibody: Enzyme + Substrate = Color  Immunochromatographic test (ICT) Blood/Serum flows laterally via capillary action and shows band in area of the nitrocellulose strip where Ag/Ab is attached Also referred as: rapid tests or Point-of-care (POC) tests Eg: COVID-19 Ag/Ab testing, Influenza, RSV antigen Respiratory Virus Antigen testing LABELLED ANTIBODY ASSAYS…When Ags and Abs react in a way that they do not produce a visible reaction, then their detection is facilitated by labelling the antibodies with specific markers IMMUNOFLORESCENCE…..Antibodies are labelled with fluorochromes and viewed under fluorescent microscope  Immunofluorescence fluorochrome based detection e.g. HCMV pp65 antigenemia assay RADIOIMMUNEASSAY (detection of circulating proteins, hormones and drugs). Antibodies are labelled with radioactive substances and radioactivity of labelled Ab combined with Ag is measured by a gamma-spectrometer  Questions What is the major difference between Precipitation and Agglutination reaction Give examples of each Expand the terms ELISA, CLIA, ICT, VDRL What is the principle of ELISA and different types of ELISA Name the detection system used in complement fixation test What is positive complement fixation test  Reading Ananthnarayan 8thedn.Textbook of Microbiology (pg102 -116) Bailey & Scotts Diagnostic Microbiology 12th edn, Mosby (pg. 147 to 157)