Agglutination Reactions- Molecular Techniques
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Physical attachment of antibody molecules to antigens on particles (e.g., erythrocytes) in agglutination is defined as
Sensitization
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Establishment of cross-links between sensitized particles and antibodies resulting in aggregation is defined as
Lattice formation
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Type of reaction characterized by agglutination of particles with naturally occurring antigen on their surfaces is
Direct agglutination
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Structural nature of antigens involved in direct agglutination reactions is
Antigens normally part of particles
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Diagnostic test examples utilizing direct agglutination include
Widal test, Weil-Felix test, Cold agglutinin test
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Specific bacterial structures detected in the Widal test are
Somatic O antigens and flagellar H antigens of Salmonella
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Diagnostic targets of the Weil-Felix test are
Anti-O (somatic) and Anti-H (flagellar)
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Type of reaction defined as agglutination of particles artificially coated with antigens indicating the presence of antibody in the patient's sample is
Indirect (passive) agglutination
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Carrier particles artificially coated with antigens in indirect passive agglutination include
Latex beads, RBCs, bentonite, charcoal
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Diagnostic antibody targets detected using indirect (passive) agglutination tests include
Rheumatoid Factor (RF), Anti-streptolysin O (ASO), Indirect Hemagglutination (IHA), TPHA
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Type of reaction defined as agglutination of particles artificially coated with antibody indicating the presence of antigen in the patient's sample is
Reverse passive agglutination
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Carrier particles coated with antibodies in reverse passive agglutination are
Particles artificially coated with antibody to detect antigen
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Clinical test examples detecting antigens using reverse passive agglutination include
Rapid Plasma Reagin (RPR), CRP, HBsAg
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Type of reaction defined as agglutination of antibody-coated staphylococcal cells by specific antigen is
Coagglutination
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Organism carrier used in coagglutination assays is
Staphylococcus aureus (using Protein A)
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Diagnostic application of coagglutination tests includes
Rapid detection of bacterial antigens (serotyping N. meningitidis, H. influenzae, S. pneumoniae)
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Type of reaction defined as inhibition of agglutination of antigen-coated particles by reagent antibody in the presence of homologous antigen in patient sample is
Agglutination-inhibition
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Test result interpretation indicating a POSITIVE result in an agglutination-inhibition assay is
No agglutination
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Test result interpretation indicating a NEGATIVE result in an agglutination-inhibition assay is
Agglutination
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Examples of Agglutination inhibition assays
LAI: HCG
HAI: Rubella
HAI: Rubella
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Reaction format where the amount of bound label is inversely proportional to the concentration of the analyte is
Competitive
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Reaction format where the amount of bound label is directly proportional to the concentration of the analyte is
Non-competitive
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Immunoassay format that uses labeled antibody to detect unknown antigen in a patient sample is
Direct
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Immunoassay format that uses labeled antiglobulin to detect patient antibody is
Indirect
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Immunoassay format that uses solid phase-bound antibody and labeled antibody to detect the analyte of interest with multiple determinants is
Sandwich/Capture assay
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Assay format that uses anti-IgM capture antibody to detect IgM antibodies in serum is
IgM antibody capture assay
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Method utilizing biotinylated antibody and avidin- or streptavidin-enzyme conjugate to increase signal intensity and assay sensitivity is
Biotin-avidin-conjugated method
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Assay category requiring a separation technique to remove unbound antigens or antibodies is
Heterogenous
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Assay category that does not require a separation technique and consists only of a liquid phase is
Homogenous
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Labels used in Radioimmunoassay (RIA) include
Radioisotopes (I-125, I-131-gamma; H-3, C-14-beta)
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Detection method used for Radioimmunoassay (RIA) is
Scintillation counting (gamma counter/crystal, beta counter/liquid)
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First type of labeled immunoassay developed is
Radioimmunoassay (RIA)
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Radioimmunoassay variant measuring total IgE is
Radioimmunosorbent test (RIST)
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Radioimmunoassay variant measuring allergen-specific IgE is
Radioallergosorbent test (RAST)
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Chief disadvantage of Radioimmunoassay (RIA) is
Radiation hazard
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Enzyme labels used in Enzyme Immunoassay (EIA) include
Horseradish peroxidase (HRP), Alkaline phosphatase (ALP), β-Galactosidase, Glucose oxidase, G6PD
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Detection method used in EIA following addition of chromogenic substrate is
Colorimetry / Spectrophotometry
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EIA format used for small antigens is
Competitive EIA
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EIA format corresponding to indirect ELISA is
Noncompetitive EIA
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EIA formats used for detection of IgM include
Sandwich or MAC ELISA
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Examples of homogeneous EIA include
EMIT, CEDIA
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Fluorochrome labels used in Fluorescent Immunoassay (FIA) include
FITC (green), Tetramethylrhodamine (red), Cyanine, Phycobiliproteins, β-naphthyl trifluoroacetone, Lucifer yellow, Texas red
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Application of direct FIA is
Antigen detection in tissue or body fluids
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Application of indirect FIA is
Antibody identification
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Fluorescent immunoassay type described as a blocking test whose positive result is no fluorescence is
Inhibition IFA
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Homogenous FIA used to measure therapeutic drugs and hormones is
FPIA (Fluorescence Polarization Immunoassay)
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Labels used in Chemiluminescent Immunoassay (CLIA) include
Acridinium ester, Dioxetane phosphate
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Detection principle parameter measured in CLIA is
Luminosity
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CLIA format used for smaller analytes is
Competitive CLIA
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CLIA format used for larger analytes is
Sandwich CLIA
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Homogenous CLIA assay used in TSH assay is
LOCI
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Label type and detection principle used in Electrochemiluminescent Immunoassay (ECLIA) are
Ruthenium; uses compounds that generate light when a voltage is applied to the electrode
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Labels and detection method used in Rapid Immunoassays (Rapid IA) are
Colloidal gold (gold zone / UPB); lateral flow assay / immunochromatography
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Clinical applications of Rapid Immunoassays include
Identify microbial antigens or antibodies, pregnancy testing, testing for cardiac troponin
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Artifact causing falsely low results when an extremely high antigen concentration fills the majority of binding sites is
Hook effect (prozone phenomenon)
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Interfering antibodies causing false-positive or false-negative results in sandwich assays using mouse capture antibodies are
HAMAs (Human Anti-Mouse Antibodies)
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Endogenous autoantibody causing false-positive results in assays using IgG reagent is
Rheumatoid Factor (RF)
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Procedural error causing false-positive results in ELISA due to unremoved unbound antibody is
Insufficient washing
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Enzymes that recognize and bind to specific nucleotide sequences in DNA to cleave strands are
Restriction endonucleases
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Molecular variation technique useful in forensic, paternity testing, and disease gene detection is
Restriction fragment length polymorphisms (RFLPs)
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First general step in blotting techniques is
Gel electrophoresis to separate target molecules by size
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Second general step in blotting techniques is
Transferring the separated molecules onto a membrane (blotting)
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Third general step in blotting techniques is
Addition of a labeled probe (hybridization)
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Fourth general step in blotting techniques is
Visualization of the bound probe to identify the molecule of interest
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Target molecule, probe type, and key application of Southern blot are
Target: DNA; Probe: DNA or RNA; Application: Genetic disease diagnosis, mutation detection, gene mapping
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Target molecule, probe type, and key application of Northern blot are
Target: RNA; Probe: DNA or cDNA; Application: Gene expression studies, cancer/viral RNA detection
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Target molecule, probe type, and key application of Western blot are
Target: Protein (antibodies); Probe: Protein (secondary antibody / antibody conjugate); Application: Diagnosis of infectious diseases (e.g., HIV) and autoimmune disorders (Lyme disease)
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Target molecule, probe type, and key application of Southwestern blot are
Target: DNA-binding proteins; Probe: DNA; Application: Studying transcription factors, gene regulation
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Target molecule, probe type, and key application of Eastern blot are
Target: Protein modifications (e.g., glycosylation); Probe: Protein (specific ligands/chemicals); Application: Cancer biomarkers, drug development
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Cytogenetic technique using fluorescent-labeled probes to detect target nucleic acids in intact cells is
Fluorescent in situ hybridization (FISH)
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Most widely used nucleic acid amplification method is
Polymerase chain reaction (PCR)
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PCR component representing the DNA region to be amplified extracted from sample or microorganisms is
Template
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Heat-stable DNA polymerase enzyme used in PCR derived from Thermus aquaticus is
Taq polymerase
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Short single-stranded DNA segments designed to hybridize to template strands in PCR are
Primers
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Building blocks from which new DNA strands are synthesized in PCR are
Deoxyribonucleotides (dNTPs)
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Temperature range and action during the Denaturation step of PCR are
94-96°C; separation of template DNA to single strands by breaking hydrogen bonds
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Temperature range and action during the Annealing step of PCR are
50-70°C; binding of primers specific for each target strand sequence
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Temperature range and action during the Extension/Elongation step of PCR are
68-72°C; addition of dNTPs to produce new strands catalyzed by Taq polymerase proceeding in 5'-to-3' direction
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Detection method utilized for visualization of PCR products in Conventional PCR is
Gel electrophoresis
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PCR variation using fluorescent probes (fluorophores) to monitor accumulation of product in real time is
Real-time or quantitative PCR (qPCR)
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Cycle number parameter at which the fluorescence signal generated during qPCR reaches a predefined threshold is
Threshold cycle (Ct)
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PCR variation using reverse transcriptase (RdDp) to synthesize cDNA from RNA template is
Reverse transcription PCR (RT-PCR)
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PCR variation performed to detect multiple target sequences simultaneously using more than one primer pair is
Multiplex PCR
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PCR variation using two separate primer pairs in two sequential reactions to improve sensitivity and specificity is
Nested PCR
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Isothermal amplification methods employing enzymes like reverse transcriptase and RNA polymerase to generate multiple RNA copies include
Transcription-Based Amplification (TMA, NASBA, 3SR)
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Assay using RNA probes complementary to target DNA followed by antibody capture of RNA:DNA hybrid is
Hybrid capture assay
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Clinical diagnostic application of Hybrid capture assay is
Detection of oncogenic variants of HPV
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Signal amplification method using short single-stranded DNA probes to capture target nucleic acid and bind multiple reporter molecules is
Branched DNA (bDNA) amplification
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Clinical diagnostic applications of Branched DNA (bDNA) amplification include
Qualitative and quantitative detection of HBV, HCV, and HIV-1
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Primary clinical purpose of complement fixation tests is
Antibody detection
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Sample requirement before testing in complement fixation is
Heated serum (to inactivate standard complement)
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Two components that make up the test system in complement fixation are
Known target antigen reagent (e.g., beef heart extract, bacterial antigen) and antibody in patient's serum
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Best biological source of complement used in complement fixation is
Guinea pig serum
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Components comprising the indicator system in complement fixation are
Sheep RBCs sensitized with hemolysin
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Best biological source of hemolysin used to sensitize sheep RBCs is
Rabbit serum
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Interpretation of test result when Ag binds to Ab and complement is fixed (No lysis) is
POSITIVE
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Interpretation of test result when Ag remains free, complement is not fixed, and lysis occurs is
NEGATIVE
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Definition of a neutralization reaction in immunology is
Antigen-antibody reaction in which the biological effects of viruses and toxins are neutralized by homologous antibodies
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Basic mechanism involved in Toxin Neutralization Test is
Neutralization of a microbial toxin (bacterial exotoxin) by a specific antibody
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Exotoxin and indicator cells involved in ASO titration are
Neutralization of SLO inhibiting the lysis of indicator red cells
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Specific enzymatic action inhibited by serum antibodies in the Anti-DNase/Streptodornase B test is
Inhibition of DNase activity of streptodornase B by serum antibodies
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Primary diagnostic purpose of Virus Neutralization Test is
Determine if an antibody present in patient serum can neutralize the infectivity of a virus or the ability of a virus to agglutinate red cells
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Mechanism of virus neutralization measured in Plaque Reduction Assay is
Antibodies in patient serum neutralize viruses preventing the formation of plaques (white patches of dead cells)
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Definition of antibody titer in Plaque Reduction Assay is
Reciprocal of the highest dilution showing a 50% reduction in plaques
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Mechanism underlying Viral Hemagglutination Inhibition is
Patient's serum antibodies react with the viruses and inhibit the agglutination of red blood cells
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