Laboratory Activity 3c – Principles of Serologic Reactions Flashcards
Antibodies that aggregate cellular antigens
Agglutinins
Antibodies that form precipitates with soluble antigens
Precipitins
Antibodies that neutralize toxins
Antitoxins
Antibodies that cause dissolution of cell membrane
Lysins
Cause the destruction of RBC in the presence of the complement
Hemolysins
Cause destruction of cells of gram-negative bacteria under certain conditions
Bacteriolysins
Cause destruction of other cell types under appropriate conditions in the presence of the complement
Cytolysins
– assays involving antibody-antigen reactions are called immunoassays
IMMUNOASSAYS
soluble antibody reacts with insoluble antigen or soluble antigen reacts with an insoluble antibody
Agglutination reaction
are made soluble by combining with latex particles, RBCs, dyes or liposomes
Reactants
occurs when particles in suspension clump together due to an antibody-antigen reaction
Agglutination
Involves the interaction of antibody with a multivalent antigen (particulate): results in the cross-linking of various antigen particles by the antibody
Agglutination
Demonstrate the presence of antigen-antibody reactions by the visible aggregation of antigen-antibody complexes
Agglutination
These tests are simple to perform and are often the most sensitive test method
AGGLUTINATION METHODS
Performed with slide, tube or microtiter technique
AGGLUTINATION METHODS
Particulate antigen + antibody ® clumping
AGGLUTINATION
antigen binds with Fab sites of 2 antibodies forming bridges between antigens
Lattice formation
The process by which particulate antigens, such as cells, aggregate to form large complexes when specific antibody is present
Direct agglutination
Febrile agglutinins, Salmonella and Shigella serotyping
Examples of Direct agglutination
Antibodies + surface antigens of bacteria in suspension -> visible agglutination
Direct Bacterial Agglutination
Antibody bound to latex beads + antigen -> visible agglutination results when antigen binds to latex-bound antibody
Latex Agglutination
An antigen-antibody reaction that results in the clumping of red blood cells
Hemagglutination
One solid aggregate, clear background
4+
Several large aggregates, clear background
3+
Medium-sized agglutinates, clear background
2+
Small agglutinates, turbid background
1+
Tiny agglutinates, turbid background
w+
No agglutination or hemolysis
0
ABO typing
Examples of Hemagglutination
A reaction in which soluble antigens are bound to latex beads, bentonite, or charcoal -> the particles are agglutinated by the corresponding antibody
Passive agglutination
Rheumatoid factor
Examples of Passive agglutination
A reaction in which soluble antigens are adsorbed onto RBCs (i.e., proteins coupled to RBCs using bisdiazotized benzidine) -> RBCs are agglutinated by the corresponding antibody
Passive hemagglutination
Cold agglutinins
Examples of Passive hemagglutination
A reaction in which carrier particles coated with antibody clump together due to combination with antigen
Reverse passive agglutination
Rapid tests for identification of bacteria
Examples of Reverse passive agglutination
An agglutination reaction based on competition between particulate antigen (reagent) and soluble antigen (specimen) for limited sites on a reagent antibody
Agglutination inhibition
Detection of illicit drugs
Examples of Agglutination inhibition
A test for detecting antibodies to certain viruses that agglutinate RBCs -> in the presence of antibody, the virus is neutralized and hemagglutination does not occur
Hemagglutination inhibition
Rubella antibody
Examples of Hemagglutination inhibition
An agglutination reaction in which bacteria are used as the carrier for the antibody
Coagglutination
Rapid tests for identification of bacteria
Examples of Coagglutination
Detection of non-agglutinating antibody by coupling with 2nd antibody (antihuman globulin [AHG])
Antiglobulin-mediated agglutination
Direct and indirect antiglobulin test
Examples of Antiglobulin-mediated agglutination
Principle: Soluble antigen combines with soluble antibody to produce visible insoluble complexes
Precipitation
Clumping together of particles to form visible masses over a narrow range of antigen concentration
Flocculation
Similar with precipitation except that the precipitin appears as a fleecy mass or clump
Flocculation
Principle: Soluble antigens react with specific antibody to form precipitate of fine particles
Flocculation
Application: Venereal Disease Research Laboratory (VDRL) tests, Rapid Plasma Reagin (RPR)
Flocculation
Principle: Light scattering by immune complexes is measured -> scattering of light is proportional to the size and amount of immune complexes formed
Nephelometry
Application: Immunoglobulins, complement, C-reactive protein
Nephelometry
Measures the decrease in light intensity in a solution containing immune complexes
Turbidimetry
Principle: Measurement of light transmitted through a suspension of particles -> the formation of immune complexes decreases the amount of light passing through a suspension -> the more immune complexes formed and the larger they are, the greater the decrease in light able to pass through
Turbidimetry
Type of Diffusion
Single
Double
if only one reactant (usually antigen) is moving
Single
if both antigen and antibody are moving through the medium
Double
Type of Dimension
Single
Double
if the reaction in a medium have only one effective dimension for antigen and antibody migration (i.e., up and down)
Single
if the reaction in a circular holes (i.e., wells) cut in a gel on a flat surface diffuses from the wells radially
Double
Principle: Known Antibody fixed in agar + Unknown Antigen (overlaid) -> Precipitin lines
Single linear diffusion (SLD) or Oudin technique
Application: Used to detect multiple antigen-antibody reactions
Single linear diffusion (SLD) or Oudin technique
Principle: Known Antibody fixed in agar + Unknown Antigen (well cut in agar plate) ® Precipitin ring
Single radial diffusion (SRD)/ Fahey or Mancini method
diameter of precipitin ring at 24 hours (Read before it reaches the maximum at 6-12 hours)
Fahey method
48
Mancini method
Principle: Antigen diffuses out of well in gel containing antigen -> Precipitin ring forms -> Diameter proportional to concentration of antigen
Radial immunodiffusion (RID)
Application: Immunoglobulins, complement No longer commonly performed except for low-volume testing of IgD and IgG
Radial immunodiffusion (RID)
Principle: Antigens and antibodies diffuse out from wells cut in gel and precipitin lines where they meet
Ouchterlony technique/Double Immunediffusion
Ouchterlony technique/Double Immunediffusion
Three basic reaction patterns:
a) Identity
b) Non-identity
c) Partial identity
a single smooth arc of precipitation forms between the antigens and antibodies
Identity
two separate lines of precipitation cross each other
Non-identity
two precipitating lines meet, forming a spur
Partial identity
Application: Fungal antigens, extractable nuclear antigens
Ouchterlony technique/Double Immunediffusion
Common errors include overfilling of wells, irregular well punching, unlevel incubation area, gel drying, increased room temperature, and antigen or antibody contamination by bacteria or fungi
Ouchterlony technique/Double Immunediffusion
Principle: antigens and antibodies are placed in wells that are directly opposite one another in a gel -> an electrophoretic charge is applied to drive the reactants toward each other -> precipitin band forms where they meet
Countercurrent immunoelectrophoresis (CIE)
Application: Bacterial antigens
Countercurrent immunoelectrophoresis (CIE)
Principle: Proteins are separated by electrophoresis, then subjected to double diffusion with reagent antibodies placed in a trough cut in the agar ® shape, intensity, and location of the precipitin arcs develop are compared with those of a normal control
Immunoelectrophoresis (IEP)
Application: Serum proteins including immunoglobulins
Immunoelectrophoresis (IEP)
Principle: Proteins are separated by electrophoresis -> cellulose acetate strip impregnated with antiserum is placed on the separated proteins -> the antiserum diffuses into the gel and antigen-antibody complexes precipitate
Immunofixation electrophoresis (IFE)
Application: Identification of immunoglobulins in monoclonal gammopathies, Bence-Jones proteins
Immunofixation electrophoresis (IFE)
Principle: An electrical charge is applied to an RID assay -> height of the rocket-shaped precipitin band is proportional to the concentration of antigen
Rocket electrophoresis
Application: Immunoglobulins, complement, alpha-fetoprotein
Rocket electrophoresis
The substance being measured in an immunoassay
Ligand
An immunoassay that uses radioisotope as the label
Isotopic
An immunoassay that uses something other than a radioisotope as the label
Nonisotopic
An immunoassay in which the patient ligand and the labeled reagent ligand compete for a limited number of binding sites on a reagent antibody
Competitive
An immunoassay in which the reaction does not involve competition for binding sites
Noncompetitive
An immunoassay in which the reaction does not involve competition for binding sites
Noncompetitive
An immunoassay in which a separation step is required to remove free reactant from bound reactant
Heterogenous
An immunoassay in which a separation step is not required
Homogenous
Horseradish peroxidase (HRP)
Fluorescein
Luminol
125I
b-D-galactosidase
Rhodamine
Acridium ester
3H
Alkaline phosphatase (ALP)
Dioxetane phosphate
14C
Labels: 125I, 131I, 3H, 14C
Radioimmunoassay (RIA)
Detection: Radioisotopes emit radioactivity
Radioimmunoassay (RIA)
Principle: Radiolabeled ligand and unlabeled ligand in the specimen compete for binding sites on reagent antibody -> the amount of labeled ligand bound is determined by count per minute (CPM) on a scintillation counter
Radioimmunoassay (RIA)
Results: CPM are proportional to the concentration of the ligand in the specimen
Radioimmunoassay (RIA)
Employs a sorbent for allergen insolubilization. Homologous antibodies of all immunoglobulin classes may be bound to this allergen-sorbent.
Radioallergosorbent Test (RAST)
Principle: Performed by incubating specific allergen-coated particles (i.e., sorbent) with the patient’s serum in a tube ®the tube is centrifuged, and the sorbent is washed to remove all IgE molecules except those specific for the allergen -> a radiolabeled anti-IgE antibody is then allowed to incubate with the complexes followed by centrifugation, washing, and counting
Radioallergosorbent Test (RAST)
Application: RIA method specifically designed to measure antigen-specific IgE
Radioallergosorbent Test (RAST)
Indirect RIST
Direct RIST
Radioimmunosorbent Test (RIST)
(1) Anti-IgE covalently coupled to cross-linked dextran particles (Sephadex), (2) radiolabeled IgE and (3) patient’s serum are all incubated in one tube
(2) The radiolabeled IgE and patient’s IgE compete for the antibody receptor sites of the anti-IgE bound to Sephadex particle
(3) Following incubation, the tube is centrifuged and washed three times with buffer
(4) Decantation after the last wash leaves a pellet of complexes consisting of Sephadex bead + anti-IgE + IgE
(5) Emission of gamma rays per unit time from the radiolabeled IgE is counted
Indirect RIST – Procedure
(1) Anti-IgE is rendered insoluble by being coupled to Sephadex beads and incubated with patient’s serum in a tube
(2) The tube is centrifuged and washed to remove any free antigen
(3) Radiolabeled anti-IgE is added followed by incubation, centrifugation, and washing to remove any unattached labeled reagent
(4) Decantation after the last wash leaves a pellet of complexes consisting of Sephadex bead + anti-IgE + IgE
(5) Emission of gamma rays per unit time from the radiolabeled anti- IgE is counted
Direct RIST – Procedure
Application: competitive binding technique used to quantitate total IgE
Radioimmunosorbent Test (RIST)
Principle:
(1) Radiolabeled IgE, IgE in patient’s serum and anti-IgE antibodies are all incubated in one tube
(2) Soluble IgE + anti-IgE complexes are formed from competitive binding
(3) A second anti-antibody directed to the anti-IgE is added (i.e., anti-anti-IgE) to the tube to promote precipitation or insolubilization of the IgE + antiIgE complex
(4) Contents are mixed, incubated, centrifuged and washed
(5) Last wash leaves a complex consisting of anti-anti-IgE + anti-IgE + IgE
(6) Emission of gamma rays per unit time from the radiolabeled IgE is counted
Radioimmunoprecipitation (RIP) assay
Labels: Alkaline phosphatase, horseradish peroxidase, D-galactosidase, glucose-6phosphate dehydrogenase
Enzyme Immunoassay (EIA)
Detection: Enzymes react with substrate to produce color change
Enzyme Immunoassay (EIA)
Principle: Enzyme-labeled ligand and unlabeled patient ligand compete for binding sites on antibody molecules attached to a solid phase
Enzyme-linked immunosorbent assay (ELISA)
Application: used to detect antibodies to viruses (HIV, HAV, HCV, EBV)
Enzyme-linked immunosorbent assay (ELISA)
Principle: Antibody in the specimen attaches to a solid-phase antigen ® after incubation and washing to remove unbound antibody, an enzyme-labeled antiglobulin is added ® this second antibody reacts with the Fc portion of the patient antibody bound to the solid phase ® following another wash, substrate is added
Indirect or non-competitive ELISA
Principle: The antigen in the specimen is sandwiched between an antibody attached to a solid phase and enzyme-labeled antibody
Sandwich enzymemultiplied immunoassay or capture assay
Application: Antigens must have multiple determinants; used to measure immunoglobulins, hormones, proteins and detect tumor markers, viruses, parasites, fungi
Sandwich enzymemultiplied immunoassay or capture assay
Principle: The antigen in the specimen and an enzyme-labeled antigen compete for binding sites on reagent antibody -> when the enzyme-labeled antigen binds to antibody, enzyme activity is inhibited
Enzyme-multiplied immunoassay technique (EMIT)
Application: Used for determination of low molecular weight analytes not readily measured by other methods, e.g., hormones, therapeutic drugs, drugs of abuse
Enzyme-multiplied immunoassay technique (EMIT)
Labels: Commonly used fluorochromes include fluorescein isothiocyanate (FITC), Rphycoerythrin, quantum red, tetramethyl-rhodamine isothiocyanate, rhodamine B isothiocyanate, Texas red, phycocyanin, acridine orange, and propidium iodide
Fluorescence Immunoassays (FIA)/ Immunofluorescence assay (IFA)
absorbs maximally at 490-495 nm; emits green color at 517 nm
Fluorescein isothiocyanate (FITC)
absorbs at 550 nm: emits bright red light at 580-585 nm
Tetra methyl rhodamine isothiocyanate (TRITC)
derived from algae, porphyrins, and chlorophylls: exhibits red fluorescence over 600 nm
Phycobiliproteins
Detection: Fluorochromes absorb energy from light source; convert to a longer wavelength (lower energy)
Fluorescence Immunoassays (FIA)/ Immunofluorescence assay (IFA)
Common methods include direct and indirect IFA
Fluorescence Immunoassays (FIA)/ Immunofluorescence assay (IFA)
Principle: Conjugated (fluorescent labeled) reagent antibody reacts with an antigen in a clinical sample to form an antigen-antibody complex
Direct immunofluorescence
Analytes: Bacterial, viral antigens
Pataba ka haaaaa!!! HUHUHU
Direct immunofluorescence
Principle: Antigen reacts with unlabeled antibody forming an antigen-antibody complex that is then complexed with a labeled antihuman antibody, creating an antibody-antigen-antibody “sandwich”
Indirect immunofluorescent assays
Analytes: Fluorescent antinuclear antibody (FANA), fluorescent Treponemal antibody (FTA)
Indirect immunofluorescent assays
Principle: This is an indirect assay in which the detection system is modified by using a biotin-labeled antibody followed by avidin-labeled fluorochrome ® This extra step increases the specificity and sensitivity of the assay
Biotin-avidin immunofluorescence
Principle: Based on the change of polarization of fluorescent light emitted from a labeled molecule when it is bound by antibody
Fluorescence Polarization Immunoassay
a. Unlabeled ligand in the specimen and fluorogenic ligand compete for sites on reagent antibody.
• Free-labeled ligand rotates _______________ and emits little polarized fluorescence.
• Bound labeled ligand rotates more _______________and emits more polarized fluorescence.
Fluorescence Polarization Immunoassay
b. The higher the concentration of bound labeled ligand, the more polarized fluorescence.
Fluorescence Polarization Immunoassay
c. The amount of polarized fluorescence is _______________ proportional to the concentration of the ligand in the specimen.
Fluorescence Polarization Immunoassay
Analytes: Therapeutic drugs, hormones
Fluorescence Polarization Immunoassay
Principle: Unlabeled ligand in the specimen and fluorogenic ligand compete for sites on reagent antibody -> free-labeled ligand produces fluorescence; bound-labeled ligand does not produce fluorescence
Substrate-labeled fluorescent immunoassay (SLFIA)
Result: Fluorescence is _______________ proportional to the concentration of the ligand in the specimen
Substrate-labeled fluorescent immunoassay (SLFIA)
Label: Luminol, acridium esters, ruthenium derivatives, nitrophenyl oxalates
Chemiluminescence Immunoassay (ChLIA)
Principle: Chemiluminescent substance -> oxidation using H2O2 or enzyme -> produces intermediates at higher energy -> the intermediates spontaneously return to their original state -> giving off energy in the form of light
Chemiluminescence Immunoassay (ChLIA)
Detection: Chemiluminescent molecules produce light from chemical reaction
o Emission of light is caused by a chemical reaction producing an excited molecule that decays back to the original ground state measured using an illuminometer
Chemiluminescence Immunoassay (ChLIA)
Used to detect the presence of complement-fixing antibodies (IgM and IgG) in patient serum against soluble antigens
COMPLEMENT FIXATION
Serum sample must be heated at ______ for __________ to inactivate native complement
Di ko alam beh sorry
• Bacteriolytic test system
• Hemolytic indicator system
Indicator Requirements
Principle:
• Patient serum is incubated with antigen and complement -> if the corresponding antibody is present in the serum, it forms a complex with the antigen and the complement -> when sensitized RBCs are added, there is no free complement to lyse them
COMPLEMENT FIXATION
If the complement fixation test is positive
no further testing is required
If the complement fixation test is negative
Rice test must be performed
(1) The patient’s serum is mixed with 1 unit of antigen and 1 unit of complement and is incubated. If the serum contains a non-complement-fixing antibody, it will bind some or all of the antigen but none of the complement
Rice Test
(2) A known complement-fixing antibody specific for the antigen is added. Since the antigen was in short supply, to begin with, most or all of it has been bound by antibodies in the patient’s serum. There is little or no antigen with which the complement-fixing antibody can unite; hence the complement remains free.
Rice Test
(3) Finally, sensitized sheep red cells are added.
o The free complement attaches to the antibody causing lysis of red cells -> indicative of a noncomplement-fixing antibody
o If a patient’s serum contains no antibody, both antigen and complement will remain free -> Addition of known complement-fixing antibody results in an antigen-antibody-complement complex -> when sensitized sheep red cells are added, there is no more free complement with which the hemolysin (i.e., antibody attached on the red cell surface) can combine -> hence, the red cells do not lyse
Rice Test
